Howard Y. Chang, MD, PhD

All Publications

• Circular ecDNA promotes accessible chromatin and high oncogene expression. Nature Wu, S., Turner, K. M., Nguyen, N., Raviram, R., Erb, M., Santini, J., Luebeck, J., Rajkumar, U., Diao, Y., Li, B., Zhang, W., Jameson, N., Corces, M. R., Granja, J. M., Chen, X., Coruh, C., Abnousi, A., Houston, J., Ye, Z., Hu, R., Yu, M., Kim, H., Law, J. A., Verhaak, R. G., Hu, M., Furnari, F. B., Chang, H. Y., Ren, B., Bafna, V., Mischel, P. S. 2019

  Abstract

  Oncogenes are commonly amplified on particles of extrachromosomal DNA (ecDNA) in cancer1,2, but our understanding of the structure of ecDNA and its effect on gene regulation is limited. Here, by integrating ultrastructural imaging, long-range optical mapping and computational analysis of whole-genome sequencing, we demonstrate the structure of circular ecDNA. Pan-cancer analyses reveal that oncogenes encoded on ecDNA are among the most highly expressed genes in the transcriptome of the tumours, linking increased copy number with high transcription levels. Quantitative assessment of the chromatin state reveals that although ecDNA is packaged into chromatin with intact domain structure, it lacks higher-order compaction that is typical of chromosomes and displays significantly enhanced chromatin accessibility. Furthermore, ecDNA is shown to have a significantly greater number of ultra-long-range interactions with active chromatin, which provides insight into how the structure of circular ecDNA affects oncogene function, and connects ecDNA biology with modern cancer genomics and epigenetics.

  View details for DOI 10.1038/s41586-019-1763-5

  View details for PubMedID 31748743

• Clonal replacement of tumor-specific T cells following PD-1 blockade. Nature medicine Yost, K. E., Satpathy, A. T., Wells, D. K., Qi, Y. n., Wang, C. n., Kageyama, R. n., McNamara, K. L., Granja, J. M., Sarin, K. Y., Brown, R. A., Gupta, R. K., Curtis, C. n., Bucktrout, S. L., Davis, M. M., Chang, A. L., Chang, H. Y. 2019

  Abstract

  Immunotherapies that block inhibitory checkpoint receptors on T cells have transformed the clinical care of patients with cancer1. However, whether the T cell response to checkpoint blockade relies on reinvigoration of pre-existing tumor-infiltrating lymphocytes or on recruitment of novel T cells remains unclear2-4. Here we performed paired single-cell RNA and T cell receptor sequencing on 79,046 cells from site-matched tumors from patients with basal or squamous cell carcinoma before and after anti-PD-1 therapy. Tracking T cell receptor clones and transcriptional phenotypes revealed coupling of tumor recognition, clonal expansion and T cell dysfunction marked by clonal expansion of CD8+CD39+ T cells, which co-expressed markers of chronic T cell activation and exhaustion. However, the expansion of T cell clones did not derive from pre-existing tumor-infiltrating T lymphocytes; instead, the expanded clones consisted of novel clonotypes that had not previously been observed in the same tumor. Clonal replacement of T cells was preferentially observed in exhausted CD8+ T cells and evident in patients with basal or squamous cell carcinoma. These results demonstrate that pre-existing tumor-specific T cells may have limited reinvigoration capacity, and that the T cell response to checkpoint blockade derives from a distinct repertoire of T cell clones that may have just recently entered the tumor.

  View details for DOI 10.1038/s41591-019-0522-3

  View details for PubMedID 31359002

• Massively parallel single-cell chromatin landscapes of human immune cell development and intratumoral T cell exhaustion. Nature biotechnology Satpathy, A. T., Granja, J. M., Yost, K. E., Qi, Y. n., Meschi, F. n., McDermott, G. P., Olsen, B. N., Mumbach, M. R., Pierce, S. E., Corces, M. R., Shah, P. n., Bell, J. C., Jhutty, D. n., Nemec, C. M., Wang, J. n., Wang, L. n., Yin, Y. n., Giresi, P. G., Chang, A. L., Zheng, G. X., Greenleaf, W. J., Chang, H. Y. 2019; 37 (8): 925–36

  Abstract

  Understanding complex tissues requires single-cell deconstruction of gene regulation with precision and scale. Here, we assess the performance of a massively parallel droplet-based method for mapping transposase-accessible chromatin in single cells using sequencing (scATAC-seq). We apply scATAC-seq to obtain chromatin profiles of more than 200,000 single cells in human blood and basal cell carcinoma. In blood, application of scATAC-seq enables marker-free identification of cell type-specific cis- and trans-regulatory elements, mapping of disease-associated enhancer activity and reconstruction of trajectories of cellular differentiation. In basal cell carcinoma, application of scATAC-seq reveals regulatory networks in malignant, stromal and immune cells in the tumor microenvironment. Analysis of scATAC-seq profiles from serial tumor biopsies before and after programmed cell death protein 1 blockade identifies chromatin regulators of therapy-responsive T cell subsets and reveals a shared regulatory program that governs intratumoral CD8+ T cell exhaustion and CD4+ T follicular helper cell development. We anticipate that scATAC-seq will enable the unbiased discovery of gene regulatory factors across diverse biological systems.

  View details for DOI 10.1038/s41587-019-0206-z

  View details for PubMedID 31375813

• Atlas of Subcellular RNA Localization Revealed by APEX-Seq. Cell Fazal, F. M., Han, S. n., Parker, K. R., Kaewsapsak, P. n., Xu, J. n., Boettiger, A. N., Chang, H. Y., Ting, A. Y. 2019

  Abstract

  We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.

  View details for DOI 10.1016/j.cell.2019.05.027

  View details for PubMedID 31230715

• Promoter of lncRNA Gene PVT1 Is a Tumor-Suppressor DNA Boundary Element. Cell Cho, S. W., Xu, J., Sun, R., Mumbach, M. R., Carter, A. C., Chen, Y. G., Yost, K. E., Kim, J., He, J., Nevins, S. A., Chin, S., Caldas, C., Liu, S. J., Horlbeck, M. A., Lim, D. A., Weissman, J. S., Curtis, C., Chang, H. Y. 2018; 173 (6): 1398

  Abstract

  Noncoding mutations in cancer genomes are frequentbut challenging to interpret. PVT1 encodes an oncogenic lncRNA, but recurrent translocations and deletions in human cancers suggest alternative mechanisms. Here, we show that the PVT1 promoter has a tumor-suppressor function that is independent of PVT1 lncRNA. CRISPR interference of PVT1 promoter enhances breast cancer cell competition and growth invivo. The promoters of the PVT1 and the MYC oncogenes, located 55 kb apart on chromosome 8q24, compete for engagement with four intragenic enhancers in the PVT1 locus, thereby allowing the PVT1 promoter to regulate pause release of MYC transcription. PVT1 undergoes developmentally regulated monoallelic expression, and the PVT1 promoter inhibits MYC expression only from the same chromosome via promoter competition. Cancer genome sequencing identifies recurrent mutations encompassing the human PVT1 promoter, and genome editing verified that PVT1 promoter mutation promotes cancer cell growth. These results highlight regulatory sequences of lncRNA genes as potential disease-associated DNA elements.

  View details for PubMedID 29731168

• The chromatin accessibility landscape of primary human cancers. Science (New York, N.Y.) Corces, M. R., Granja, J. M., Shams, S. n., Louie, B. H., Seoane, J. A., Zhou, W. n., Silva, T. C., Groeneveld, C. n., Wong, C. K., Cho, S. W., Satpathy, A. T., Mumbach, M. R., Hoadley, K. A., Robertson, A. G., Sheffield, N. C., Felau, I. n., Castro, M. A., Berman, B. P., Staudt, L. M., Zenklusen, J. C., Laird, P. W., Curtis, C. n., Greenleaf, W. J., Chang, H. Y. 2018; 362 (6413)

  Abstract

  We present the genome-wide chromatin accessibility profiles of 410 tumor samples spanning 23 cancer types from The Cancer Genome Atlas (TCGA). We identify 562,709 transposase-accessible DNA elements that substantially extend the compendium of known cis-regulatory elements. Integration of ATAC-seq (the assay for transposase-accessible chromatin using sequencing) with TCGA multi-omic data identifies a large number of putative distal enhancers that distinguish molecular subtypes of cancers, uncovers specific driving transcription factors via protein-DNA footprints, and nominates long-range gene-regulatory interactions in cancer. These data reveal genetic risk loci of cancer predisposition as active DNA regulatory elements in cancer, identify gene-regulatory interactions underlying cancer immune evasion, and pinpoint noncoding mutations that drive enhancer activation and may affect patient survival. These results suggest a systematic approach to understanding the noncoding genome in cancer to advance diagnosis and therapy.

  View details for DOI 10.1126/science.aav1898

  View details for PubMedID 30361341

• Analysis of RNA conformation in endogenously assembled RNPs by icSHAPE STAR protocols Chen, L., Chang, H. Y., Artandi, S. E. 2021; 2 (2)

  View details for DOI 10.1016/j.xpro.2021.100477

• HiChIRP reveals RNA-associated chromosome conformation. Nature methods Mumbach, M. R., Granja, J. M., Flynn, R. A., Roake, C. M., Satpathy, A. T., Rubin, A. J., Qi, Y., Jiang, Z., Shams, S., Louie, B. H., Guo, J. K., Gennert, D. G., Corces, M. R., Khavari, P. A., Atianand, M. K., Artandi, S. E., Fitzgerald, K. A., Greenleaf, W. J., Chang, H. Y. 2019

  Abstract

  Modular domains of long non-coding RNAs can serve as scaffolds to bring distant regions of the linear genome into spatial proximity. Here, we present HiChIRP, a method leveraging bio-orthogonal chemistry and optimized chromosome conformation capture conditions, which enables interrogation of chromatin architecture focused around a specific RNA of interest down to approximately ten copies per cell. HiChIRP of three nuclear RNAs reveals insights into promoter interactions (7SK), telomere biology (telomerase RNA component) and inflammatory gene regulation (lincRNA-EPS).

  View details for DOI 10.1038/s41592-019-0407-x

  View details for PubMedID 31133759

• Single-cell lineage tracing by endogenous mutations enriched in transposase accessible mitochondrial DNA ELIFE Xu, J., Nuno, K., Litzenburger, U. M., Qi, Y., Corces, M., Majeti, R., Chang, H. Y. 2019; 8

  View details for DOI 10.7554/eLife.45105

  View details for Web of Science ID 000465101100001

• Single-cell lineage tracing by endogenous mutations enriched in transposase accessible mitochondrial DNA. eLife Xu, J., Nuno, K., Litzenburger, U. M., Qi, Y., Corces, M. R., Majeti, R., Chang, H. Y. 2019; 8

  Abstract

  Simultaneous measurement of cell lineage and cell fates is a longstanding goal in biomedicine. Here we describe EMBLEM, a strategy to track cell lineage using endogenous mitochondrial DNA variants in ATAC-seq data. We show that somatic mutations in mitochondrial DNA can reconstruct cell lineage relationships at single cell resolution with high sensitivity and specificity. Using EMBLEM, we define the genetic and epigenomic clonal evolution of hematopoietic stem cells and their progenies in patients with acute myeloid leukemia. EMBLEM extends lineage tracing to any eukaryotic organism without genetic engineering.

  View details for PubMedID 30958261

• RNA structure maps across mammalian cellular compartments NATURE STRUCTURAL & MOLECULAR BIOLOGY Sun, L., Fazal, F. M., Li, P., Broughton, J. P., Lee, B., Tang, L., Huang, W., Kool, E. T., Chang, H. Y., Zhang, Q. 2019; 26 (4): 322-+

  View details for DOI 10.1038/s41594-019-0200-7

  View details for Web of Science ID 000463168900014

• RNA structure maps across mammalian cellular compartments. Nature structural & molecular biology Sun, L., Fazal, F. M., Li, P., Broughton, J. P., Lee, B., Tang, L., Huang, W., Kool, E. T., Chang, H. Y., Zhang, Q. C. 2019

  Abstract

  RNA structure is intimately connected to each step of gene expression. Recent advances have enabled transcriptome-wide maps of RNA secondary structure, called 'RNA structuromes'. However, previous whole-cell analyses lacked the resolution to unravel the landscape and also the regulatory mechanisms of RNA structural changes across subcellular compartments. Here we reveal the RNA structuromes in three compartments, chromatin, nucleoplasm and cytoplasm, in human and mouse cells. The cytotopic structuromes substantially expand RNA structural information and enable detailed investigation of the central role of RNA structure in linking transcription, translation and RNA decay. We develop a resource with which to visualize the interplay of RNA-protein interactions, RNA modifications and RNA structure and predict both direct and indirect reader proteins of RNA modifications. We also validate a novel role for the RNA-binding protein LIN28A as an N6-methyladenosine modification 'anti-reader'. Our results highlight the dynamic nature of RNA structures and its functional importance in gene regulation.

  View details for PubMedID 30886404

• Enhancer Connectome Nominates Target Genes of Inherited Risk Variants from Inflammatory Skin Disorders JOURNAL OF INVESTIGATIVE DERMATOLOGY Jeng, M. Y., Mumbach, M. R., Granja, J. M., Satpathy, A. T., Chang, H. Y., Chang, A. S. 2019; 139 (3): 605–14

  View details for DOI 10.1016/j.jid.2018.09.011

  View details for Web of Science ID 000459158400026

• A Mutation in the Transcription Factor Foxp3 Drives T Helper 2 Effector Function in Regulatory T Cells IMMUNITY Van Gool, F., Nguyen, M. T., Mumbach, M. R., Satpathy, A. T., Rosenthal, W. L., Giacometti, S., Le, D. T., Liu, W., Brusko, T. M., Anderson, M. S., Rudensky, A. Y., Marson, A., Chang, H. Y., Bluestone, J. A. 2019; 50 (2): 362-+

  View details for DOI 10.1016/j.immuni.2018.12.016

  View details for Web of Science ID 000459006700012

• TFAP2C-and p63-Dependent Networks Sequentially Rearrange Chromatin Landscapes to Drive Human Epidermal Lineage Commitment CELL STEM CELL Li, L., Wang, Y., Torkelson, J. L., Shankar, G., Pattison, J. M., Zhen, H. H., Fang, F., Duren, Z., Xin, J., Gaddam, S., Melo, S. P., Piekos, S. N., Li, J., Liaw, E. J., Chen, L., Li, R., Wernig, M., Wong, W. H., Chang, H. Y., Oro, A. E. 2019; 24 (2): 271-+

  View details for DOI 10.1016/j.stem.2018.12.012

  View details for Web of Science ID 000458027300013

• Global DNA methylation remodeling during direct reprogramming of fibroblasts to neurons ELIFE Luo, C., Lee, Q., Wapinski, O., Castanon, R., Nery, J. R., Malin, M., Kareta, M. S., Cullen, S. M., Goodell, M. A., Chang, H. Y., Wernig, M., Ecker, J. R. 2019; 8

  View details for DOI 10.7554/eLife.40197

  View details for Web of Science ID 000455567800001

• TFAP2C- and p63-Dependent Networks Sequentially Rearrange Chromatin Landscapes to Drive Human Epidermal Lineage Commitment. Cell stem cell Li, L., Wang, Y., Torkelson, J. L., Shankar, G., Pattison, J. M., Zhen, H. H., Fang, F., Duren, Z., Xin, J., Gaddam, S., Melo, S. P., Piekos, S. N., Li, J., Liaw, E. J., Chen, L., Li, R., Wernig, M., Wong, W. H., Chang, H. Y., Oro, A. E. 2019

  Abstract

  Tissue development results from lineage-specific transcription factors (TFs) programming a dynamic chromatin landscape through progressive cell fate transitions. Here, we define epigenomic landscape during epidermal differentiation of human pluripotent stem cells (PSCs) and create inference networks that integrate gene expression, chromatin accessibility, and TF binding to define regulatory mechanisms during keratinocyte specification. We found two critical chromatin networks during surface ectoderm initiation and keratinocyte maturation, which are driven by TFAP2C and p63, respectively. Consistently, TFAP2C, but not p63, is sufficient to initiate surface ectoderm differentiation, and TFAP2C-initiated progenitor cells are capable of maturing into functional keratinocytes. Mechanistically, TFAP2C primes the surface ectoderm chromatin landscape and induces p63 expression and binding sites, thus allowing maturation factor p63 to positively autoregulate its own expression and close a subset of the TFAP2C-initiated surface ectoderm program. Our work provides a general framework to infer TF networks controlling chromatin transitions that will facilitate future regenerative medicine advances.

  View details for PubMedID 30686763

• The novel lncRNA lnc-NR2F1 is proneurogenic and mutated in human neurodevelopmental disorders ELIFE Ang, C., Ma, Q., Wapinski, O. L., Fan, S., Flynn, R. A., Lee, Q., Coe, B., Onoguchi, M., Olmos, V., Do, B. T., Dukes-Rimsky, L., Xu, J., Tanabe, K., Wang, L., Elling, U., Penninger, J. M., Zhao, Y., Qu, K., Eichler, E. E., Srivastava, A., Wernig, M., Chang, H. Y. 2019; 8

  View details for DOI 10.7554/eLife.41770

  View details for Web of Science ID 000459171400001

• Coupled Single-Cell CRISPR Screening and Epigenomic Profiling Reveals Causal Gene Regulatory Networks CELL Rubin, A. J., Parker, K. R., Satpathy, A. T., Qi, Y., Wu, B., Ong, A. J., Mumbach, M. R., Ji, A. L., Kim, D. S., Cho, S., Zarnegar, B. J., Greenleaf, W. J., Chang, H. Y., Khavari, P. A. 2019; 176 (1-2): 361-+

  View details for DOI 10.1016/j.cell.2018.11.022

  View details for Web of Science ID 000455410800030

• A Mutation in the Transcription Factor Foxp3 Drives T Helper 2 Effector Function in Regulatory T Cells. Immunity Van Gool, F. n., Nguyen, M. L., Mumbach, M. R., Satpathy, A. T., Rosenthal, W. L., Giacometti, S. n., Le, D. T., Liu, W. n., Brusko, T. M., Anderson, M. S., Rudensky, A. Y., Marson, A. n., Chang, H. Y., Bluestone, J. A. 2019

  Abstract

  Regulatory T (Treg) cells maintain immune tolerance through the master transcription factor forkhead box P3 (FOXP3), which is crucial for Treg cell function and homeostasis. We identified an IPEX (immune dysregulation polyendocrinopathy enteropathy X-linked) syndrome patient with a FOXP3 mutation in the domain swap interface of the protein. Recapitulation of this Foxp3 variant in mice led to the development of an autoimmune syndrome consistent with an unrestrained T helper type 2 (Th2) immune response. Genomic analysis of Treg cells by RNA-sequencing, Foxp3 chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-sequencing), and H3K27ac-HiChIP revealed a specific de-repression of the Th2 transcriptional program leading to the generation of Th2-like Treg cells that were unable to suppress extrinsic Th2 cells. Th2-like Treg cells showed increased intra-chromosomal interactions in the Th2 locus, leading to type 2 cytokine production. These findings identify a direct role for Foxp3 in suppressing Th2-like Treg cells and implicate additional pathways that could be targeted to restrain Th2 trans-differentiated Treg cells.

  View details for PubMedID 30709738

• Functional significance of U2AF1 S34F mutations in lung adenocarcinomas Nature Communications Shahrokh Esfahani, M. 2019; 10

  View details for DOI 10.1038/s41467-019-13392-y

• Satb1 integrates DNA binding site geometry and torsional stress to differentially target nucleosome-dense regions. Nature communications Ghosh, R. P., Shi, Q. n., Yang, L. n., Reddick, M. P., Nikitina, T. n., Zhurkin, V. B., Fordyce, P. n., Stasevich, T. J., Chang, H. Y., Greenleaf, W. J., Liphardt, J. T. 2019; 10 (1): 3221

  Abstract

  The Satb1 genome organizer regulates multiple cellular and developmental processes. It is not yet clear how Satb1 selects different sets of targets throughout the genome. Here we have used live-cell single molecule imaging and deep sequencing to assess determinants of Satb1 binding-site selectivity. We have found that Satb1 preferentially targets nucleosome-dense regions and can directly bind consensus motifs within nucleosomes. Some genomic regions harbor multiple, regularly spaced Satb1 binding motifs (typical separation ~1 turn of the DNA helix) characterized by highly cooperative binding. The Satb1 homeodomain is dispensable for high affinity binding but is essential for specificity. Finally, we find that Satb1-DNA interactions are mechanosensitive. Increasing negative torsional stress in DNA enhances Satb1 binding and Satb1 stabilizes base unpairing regions against melting by molecular machines. The ability of Satb1 to control diverse biological programs may reflect its ability to combinatorially use multiple site selection criteria.

  View details for DOI 10.1038/s41467-019-11118-8

  View details for PubMedID 31324780

• Activation of PDGF pathway links LMNA mutation to dilated cardiomyopathy. Nature Lee, J. n., Termglinchan, V. n., Diecke, S. n., Itzhaki, I. n., Lam, C. K., Garg, P. n., Lau, E. n., Greenhaw, M. n., Seeger, T. n., Wu, H. n., Zhang, J. Z., Chen, X. n., Gil, I. P., Ameen, M. n., Sallam, K. n., Rhee, J. W., Churko, J. M., Chaudhary, R. n., Chour, T. n., Wang, P. J., Snyder, M. P., Chang, H. Y., Karakikes, I. n., Wu, J. C. 2019

  Abstract

  Lamin A/C (LMNA) is one of the most frequently mutated genes associated with dilated cardiomyopathy (DCM). DCM related to mutations in LMNA is a common inherited cardiomyopathy that is associated with systolic dysfunction and cardiac arrhythmias. Here we modelled the LMNA-related DCM in vitro using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Electrophysiological studies showed that the mutant iPSC-CMs displayed aberrant calcium homeostasis that led to arrhythmias at the single-cell level. Mechanistically, we show that the platelet-derived growth factor (PDGF) signalling pathway is activated in mutant iPSC-CMs compared to isogenic control iPSC-CMs. Conversely, pharmacological and molecular inhibition of the PDGF signalling pathway ameliorated the arrhythmic phenotypes of mutant iPSC-CMs in vitro. Taken together, our findings suggest that the activation of the PDGF pathway contributes to the pathogenesis of LMNA-related DCM and point to PDGF receptor-β (PDGFRB) as a potential therapeutic target.

  View details for DOI 10.1038/s41586-019-1406-x

  View details for PubMedID 31316208

• Retinoic acid and BMP4 cooperate with p63 to alter chromatin dynamics during surface epithelial commitment NATURE GENETICS Pattison, J. M., Melo, S. P., Piekos, S. N., Torkelson, J. L., Bashkirova, E., Mumbach, M. R., Rajasingh, C., Zhen, H., Li, L., Liaw, E., Alber, D., Rubin, A. J., Shankar, G., Bao, X., Chang, H. Y., Khavari, P. A., Oro, A. E. 2018; 50 (12): 1658-+

  View details for DOI 10.1038/s41588-018-0263-0

  View details for Web of Science ID 000451434400012

• The RNA Base-Pairing Problem and Base-Pairing Solutions COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Lu, Z., Chang, H. Y. 2018; 10 (12)

  View details for DOI 10.1101/cshperspect.a034926

  View details for Web of Science ID 000451944700003

• Mechanoresponsive stem cells acquire neural crest fate in jaw regeneration NATURE Ransom, R. C., Carter, A. C., Salhotra, A., Leavitt, T., Marecic, O., Murphy, M. P., Lopez, M. L., Wei, Y., Marshall, C. D., Shen, E. Z., Jones, R., Sharir, A., Klein, O. D., Chan, C. F., Wan, D. C., Chang, H. Y., Longaker, M. T. 2018; 563 (7732): 514-+

  View details for DOI 10.1038/s41586-018-0650-9

  View details for Web of Science ID 000450960000045

• Retinoic acid and BMP4 cooperate with p63 to alter chromatin dynamics during surface epithelial commitment. Nature genetics Pattison, J. M., Melo, S. P., Piekos, S. N., Torkelson, J. L., Bashkirova, E., Mumbach, M. R., Rajasingh, C., Zhen, H. H., Li, L., Liaw, E., Alber, D., Rubin, A. J., Shankar, G., Bao, X., Chang, H. Y., Khavari, P. A., Oro, A. E. 2018

  Abstract

  Human embryonic stem cell (hESC) differentiation promises advances in regenerative medicine1-3, yet conversion of hESCs into transplantable cells or tissues remains poorly understood. Using our keratinocyte differentiation system, we employ a multi-dimensional genomics approach to interrogate the contributions of inductive morphogens retinoic acid and bone morphogenetic protein 4 (BMP4) and the epidermal master regulator p63 (encoded by TP63)4,5 during surface ectoderm commitment. In contrast to other master regulators6-9, p63 effects major transcriptional changes only after morphogens alter chromatin accessibility, establishing an epigenetic landscape for p63 to modify. p63 distally closes chromatin accessibility and promotes accumulation of H3K27me3 (trimethylated histone H3 lysine 27). Cohesin HiChIP10 visualizations of chromosome conformation show that p63 and the morphogens contribute to dynamic long-range chromatin interactions, as illustrated by TFAP2C regulation11. Our study demonstrates the unexpected dependency of p63 on morphogenetic signaling and provides novel insights into how a master regulator can specify diverse transcriptional programs based on the chromatin landscape induced by exposure to specific morphogens.

  View details for PubMedID 30397335

• Joint single-cell DNA accessibility and protein epitope profiling reveals environmental regulation of epigenomic heterogeneity. Nature communications Chen, X., Litzenburger, U. M., Wei, Y., Schep, A. N., LaGory, E. L., Choudhry, H., Giaccia, A. J., Greenleaf, W. J., Chang, H. Y. 2018; 9 (1): 4590

  Abstract

  Here we introduce Protein-indexed Assay of Transposase Accessible Chromatin with sequencing (Pi-ATAC) that combines single-cell chromatin and proteomic profiling. In conjunction with DNA transposition, the levels of multiple cell surface or intracellular protein epitopes are recorded by index flow cytometry and positions in arrayed microwells, and then subject to molecular barcoding for subsequent pooled analysis. Pi-ATAC simultaneously identifies the epigenomic and proteomic heterogeneity in individual cells. Pi-ATAC reveals a casual link between transcription factor abundance and DNA motif access, and deconvolute cell types and states in the tumor microenvironment in vivo. We identify a dominant role for hypoxia, marked by HIF1alpha protein, in the tumor microvenvironment for shaping the regulome in a subset of epithelial tumor cells.

  View details for PubMedID 30389926

• Joint single-cell DNA accessibility and protein epitope profiling reveals environmental regulation of epigenomic heterogeneity NATURE COMMUNICATIONS Chen, X., Litzenburger, U. M., Wei, Y., Schep, A. N., LaGory, E. L., Choudhry, H., Giaccia, A. J., Greenleaf, W. J., Chang, H. Y. 2018; 9

  View details for DOI 10.1038/s41467-018-07115-y

  View details for Web of Science ID 000449069600012

• Mechanoresponsive stem cells acquire neural crest fate in jaw regeneration. Nature Ransom, R. C., Carter, A. C., Salhotra, A., Leavitt, T., Marecic, O., Murphy, M. P., Lopez, M. L., Wei, Y., Marshall, C. D., Shen, E. Z., Jones, R. E., Sharir, A., Klein, O. D., Chan, C. K., Wan, D. C., Chang, H. Y., Longaker, M. T. 2018

  Abstract

  During both embryonic development and adult tissue regeneration, changes in chromatin structure driven by master transcription factors lead to stimulus-responsive transcriptional programs. A thorough understanding of how stem cells in the skeleton interpret mechanical stimuli and enact regeneration would shed light on how forces are transduced to the nucleus in regenerative processes. Here we develop a genetically dissectible mouse model of mandibular distraction osteogenesis-which isa process that is used in humans to correct an undersized lower jawthat involves surgically separating the jaw bone, whichelicits new bone growth in the gap. We use this model to show that regions of newly formed bone are clonally derived from stem cells that reside in the skeleton. Using chromatin and transcriptional profiling, we show that these stem-cell populations gain activity within the focal adhesion kinase (FAK) signalling pathway, and that inhibiting FAK abolishes new bone formation. Mechanotransduction via FAK in skeletal stem cells during distraction activates a gene-regulatory program and retrotransposons that are normally active in primitive neural crest cells, from which skeletal stem cells arise during development. This reversion to a developmental state underlies the robust tissue growth that facilitates stem-cell-based regeneration of adult skeletal tissue.

  View details for PubMedID 30356216

• Enhancer connectome nominates target genes of inherited risk variants from inflammatory skin disorders. The Journal of investigative dermatology Jeng, M. Y., Mumbach, M. R., Granja, J. M., Satpathy, A. T., Chang, H. Y., Chang, A. L. 2018

  Abstract

  The vast majority of polymorphisms for human dermatologic diseases fall in non-coding DNA regions, leading to difficulty interpreting their functional significance. Recent work utilizing chromosome conformation capture (3C) technology in combination with chromatin immunoprecipitation (ChIP) has provided a systematic means of linking non-coding variants within active enhancer loci to putative gene targets. Here, we apply H3K27ac HiChIP high-resolution contact maps, generated from primary human T-cell subsets (CD4+ Naive, TH17, and Treg), to 21 dermatologic conditions associated with single nucleotide polymorphisms (SNPs) from 106 genome-wide association studies (GWAS). This "enhancer connectome" identified 1,492 HiChIP gene-targets from 542 non-coding SNPs (p<5.0x10-8). SNP-containing enhancers from inflammatory skin conditions were significantly enriched within the human leukocyte antigen (HLA)-locus, and also targeted several key factors from the JAK-STAT signaling pathway, while non-immune conditions did not. A focused profiling of systemic lupus erythematosus (SLE) HiChIP-genes identified enhancer interactions with factors important for effector CD4+ T-cell differentiation and function, including interferon regulatory factor 8 (IRF8) and members of the Ikaros family of zinc-finger proteins. Our results demonstrate the ability of the enhancer connectome to nominate functionally-relevant candidates from GWAS-identified variants, representing a powerful tool to guide future studies into the genomic regulatory mechanisms underlying dermatologic diseases.

  View details for PubMedID 30315781

• A Chromatin Basis for Cell Lineage and Disease Risk in the Human Pancreas CELL SYSTEMS Arda, H., Tsai, J., Rosli, Y. R., Giresi, P., Bottino, R., Greenleaf, W. J., Chang, H. Y., Kim, S. K. 2018; 7 (3): 310-+

  View details for DOI 10.1016/j.cels.2018.07.007

  View details for Web of Science ID 000445732000007

• Identification of the Human Skeletal Stem Cell. Cell Chan, C. K., Gulati, G. S., Sinha, R., Tompkins, J. V., Lopez, M., Carter, A. C., Ransom, R. C., Reinisch, A., Wearda, T., Murphy, M., Brewer, R. E., Koepke, L. S., Marecic, O., Manjunath, A., Seo, E. Y., Leavitt, T., Lu, W., Nguyen, A., Conley, S. D., Salhotra, A., Ambrosi, T. H., Borrelli, M. R., Siebel, T., Chan, K., Schallmoser, K., Seita, J., Sahoo, D., Goodnough, H., Bishop, J., Gardner, M., Majeti, R., Wan, D. C., Goodman, S., Weissman, I. L., Chang, H. Y., Longaker, M. T. 2018; 175 (1): 43

  Abstract

  Stem cell regulation and hierarchical organization ofhuman skeletal progenitors remain largely unexplored. Here, we report the isolation of a self-renewing and multipotent human skeletal stem cell (hSSC) that generates progenitors of bone, cartilage, and stroma, but not fat. Self-renewing and multipotent hSSCs are present in fetal and adult bones and can also be derived from BMP2-treated human adipose stroma (B-HAS) and induced pluripotent stem cells (iPSCs). Gene expression analysis of individual hSSCs reveals overall similarity between hSSCs obtained from different sources and partially explains skewed differentiation toward cartilage in fetal and iPSC-derived hSSCs. hSSCs undergo local expansion in response to acute skeletal injury. In addition, hSSC-derived stroma can maintain human hematopoietic stem cells (hHSCs) in serum-free culture conditions. Finally, we combine gene expression and epigenetic data of mouse skeletal stem cells (mSSCs) and hSSCs to identify evolutionarily conserved and divergent pathways driving SSC-mediated skeletogenesis. VIDEO ABSTRACT.

  View details for PubMedID 30241615

• Identification of the Human Skeletal Stem Cell CELL Chan, C. F., Gulati, G. S., Sinha, R., Tompkins, J., Lopez, M., Carter, A. C., Ransom, R. C., Reinisch, A., Wearda, T., Murphy, M., Brewer, R. E., Koepke, L. S., Marecic, O., Manjunath, A., Seo, E., Leavitt, T., Lu, W., Allison Nguyen, Conley, S. D., Salhotra, A., Ambrosi, T. H., Borrelli, M. R., Siebel, T., Chan, K., Schallmoser, K., Seita, J., Sahoo, D., Goodnough, H., Bishop, J., Gardner, M., Majeti, R., Wan, D. C., Goodman, S., Weissman, I. L., Chang, H. Y., Longaker, M. T. 2018; 175 (1): 43-+

  View details for DOI 10.1016/j.cell.2018.07.029

  View details for Web of Science ID 000445120000013

• Expression of the transcription factor ZBTB46 distinguishes human histiocytic disorders of classical dendritic cell origin MODERN PATHOLOGY Satpathy, A. T., Brown, R. A., Gomulia, E., Briseno, C. G., Mumbach, M. R., Pan, Z., Murphy, K. M., Natkunam, Y., Chang, H. Y., Kim, J. 2018; 31 (9): 1479–86

  View details for DOI 10.1038/s41379-018-0052-4

  View details for Web of Science ID 000444569400013

• A Chromatin Basis for Cell Lineage and Disease Risk in the Human Pancreas. Cell systems Arda, H. E., Tsai, J., Rosli, Y. R., Giresi, P., Bottino, R., Greenleaf, W. J., Chang, H. Y., Kim, S. K. 2018

  Abstract

  Understanding the genomic logic that underlies cellular diversity and developmental potential in the human pancreas will accelerate the growth of cell replacement therapies and reveal genetic risk mechanisms in diabetes. Here, we identified and characterized thousands of chromatin regions governing cell-specific gene regulation in human pancreatic endocrine and exocrine lineages, including islet betacells, alpha cells, duct, and acinar cells. Our findings have captured cellular ontogenies at the chromatin level, identified lineage-specific regulators potentially acting on these sites, and uncovered hallmarks of regulatory plasticity between cell types that suggest mechanisms to regenerate beta cells from pancreatic endocrine or exocrine cells. Our work shows that disease risk variants related to pancreas are significantly enriched in these regulatory regions and reveals previously unrecognized links between endocrine and exocrine pancreas in diabetes risk.

  View details for PubMedID 30145115

• Genetic determinants of co-accessible chromatin regions in activated T cells across humans NATURE GENETICS Gate, R. E., Cheng, C. S., Aiden, A. P., Siba, A., Tabaka, M., Lituiev, D., Machol, I., Gordon, M., Subramaniam, M., Shamim, M., Hougen, K. L., Wortman, I., Huang, S., Durand, N. C., Feng, T., De Jager, P. L., Chang, H. Y., Aiden, E., Benoist, C., Beer, M. A., Ye, C. J., Regev, A. 2018; 50 (8): 1140-+

  Abstract

  Over 90% of genetic variants associated with complex human traits map to non-coding regions, but little is understood about how they modulate gene regulation in health and disease. One possible mechanism is that genetic variants affect the activity of one or more cis-regulatory elements leading to gene expression variation in specific cell types. To identify such cases, we analyzed ATAC-seq and RNA-seq profiles from stimulated primary CD4+ T cells in up to 105 healthy donors. We found that regions of accessible chromatin (ATAC-peaks) are co-accessible at kilobase and megabase resolution, consistent with the three-dimensional chromatin organization measured by in situ Hi-C in T cells. Fifteen percent of genetic variants located within ATAC-peaks affected the accessibility of the corresponding peak (local-ATAC-QTLs). Local-ATAC-QTLs have the largest effects on co-accessible peaks, are associated with gene expression and are enriched for autoimmune disease variants. Our results provide insights into how natural genetic variants modulate cis-regulatory elements, in isolation or in concert, to influence gene expression.

  View details for PubMedID 29988122

• Discovery of stimulation-responsive immune enhancers with CRISPR activation (vol 549, pg 111, 2017) NATURE Simeonov, D. R., Gowen, B. G., Boontanrart, M., Roth, T. L., Gagnon, J. D., Mumbach, M. R., Satpathy, A. T., Lee, Y., Bray, N. L., Chan, A. Y., Lituiev, D. S., Nguyen, M. L., Gate, R. E., Subramaniam, M., Li, Z., Woo, J. M., Mitros, T., Ray, G. J., Curie, G. L., Naddaf, N., Chu, J. S., Ma, H., Boyer, E., Van Gool, F., Huang, H., Liu, R., Tobin, V. R., Schumann, K., Daly, M. J., Farh, K. K., Ansel, K., Ye, C. J., Greenleaf, W. J., Anderson, M. S., Bluestone, J. A., Chang, H. Y., Corn, J. E., Marson, A. 2018; 559 (7715): E13

  Abstract

  In this Letter, analysis of steady-state regulatory T (Treg) cell percentages from Il2ra enhancer deletion (EDEL) and wild-type (WT) mice revealed no differences between them (Extended Data Fig. 9d). This analysis included two mice whose genotypes were incorrectly assigned. Even after correction of the genotypes, no significant differences in Treg cell percentages were seen when data across experimental cohorts were averaged (as was done in Extended Data Fig. 9d). However, if we normalize the corrected data to account for variation among experimental cohorts, a subtle decrease in EDEL Treg cell percentages is revealed and, using the corrected and normalized data, we have redrawn Extended Data Fig. 9d in Supplementary Fig. 1. The Supplementary Information to this Amendment contains the corrected and reanalysed Extended Data Fig. 9d. The sentence "This enhancer deletion (EDEL) strain also had no obvious T cell phenotypes at steady state (Extended Data Fig. 9)." should read: "This enhancer deletion (EDEL) strain had a small decrease in the percentage of Treg cells (Extended Data Fig. 9).". This error does not affect any of the main figures in the Letter or the data from mice with the human autoimmune-associated single nucleotide polymorphism (SNP) knocked in or with a 12-base-pair deletion at the site (12DEL). In addition, we stated in the Methods that we observed consistent immunophenotypes of EDEL mice across three founders, but in fact, we observed consistent phenotypes in mice from two founders. This does not change any of our conclusions and the original Letter has not been corrected.

  View details for PubMedID 29899441

• Integrative analysis of single-cell genomics data by coupled nonnegative matrix factorizations PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Duren, Z., Chen, X., Zamanighomi, M., Zeng, W., Satpathy, A. T., Chang, H. Y., Wang, Y., Wong, W. 2018; 115 (30): 7723–28

  View details for DOI 10.1073/pnas.1805681115

  View details for Web of Science ID 000439574700050

• Integrative analysis of single-cell genomics data by coupled nonnegative matrix factorizations. Proceedings of the National Academy of Sciences of the United States of America Duren, Z., Chen, X., Zamanighomi, M., Zeng, W., Satpathy, A. T., Chang, H. Y., Wang, Y., Wong, W. H. 2018

  Abstract

  When different types of functional genomics data are generated on single cells from different samples of cells from the same heterogeneous population, the clustering of cells in the different samples should be coupled. We formulate this "coupled clustering" problem as an optimization problem and propose the method of coupled nonnegative matrix factorizations (coupled NMF) for its solution. The method is illustrated by the integrative analysis of single-cell RNA-sequencing (RNA-seq) and single-cell ATAC-sequencing (ATAC-seq) data.

  View details for PubMedID 29987051

• An Activity Switch in Human Telomerase Based on RNA Conformation and Shaped by TCAB1 CELL Chen, L., Roake, C. M., Freund, A., Batista, P. J., Tian, S., Yin, Y. A., Gajera, C. R., Lin, S., Lee, B., Pech, M. F., Venteicher, A. S., Das, R., Chang, H. Y., Artandi, S. E. 2018; 174 (1): 218-+

  View details for DOI 10.1016/j.cell.2018.04.039

  View details for Web of Science ID 000437005800022

• Integrated Single-Cell Analysis Maps the Continuous Regulatory Landscape of Human Hematopoietic Differentiation CELL Buenrostro, J. D., Corces, M., Lareau, C. A., Wu, B., Schep, A. N., Aryee, M. J., Majeti, R., Chang, H. Y., Greenleaf, W. J. 2018; 173 (6): 1535-+

  Abstract

  Human hematopoiesis involves cellular differentiation of multipotent cells into progressively more lineage-restricted states. While the chromatin accessibility landscape of this process has been explored in defined populations, single-cell regulatory variation has been hidden by ensemble averaging. We collected single-cell chromatin accessibility profiles across 10 populations of immunophenotypically defined human hematopoietic cell types and constructed a chromatin accessibility landscape of human hematopoiesis to characterize differentiation trajectories. We find variation consistent with lineage bias toward different developmental branches in multipotent cell types. We observe heterogeneity within common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) and develop a strategy to partition GMPs along their differentiation trajectory. Furthermore, we integrated single-cell RNA sequencing (scRNA-seq) data to associate transcription factors to chromatin accessibility changes and regulatory elements to target genes through correlations of expression and regulatory element accessibility. Overall, this work provides a framework for integrative exploration of complex regulatory dynamics in a primary human tissue at single-cell resolution.

  View details for PubMedID 29706549

• Promoter of IncRNA Gene PVT1 Is a Tumor-Suppressor DNA Boundary Element CELL Cho, S., Xu, J., Sun, R., Mumbach, M. R., Carter, A. C., Chen, Y., Yost, K. E., Kim, J., He, J., Nevins, S. A., Chin, S., Caldas, C., Liu, S., Horlbec, M. A., Lim, D. A., Weissman, J. S., Curtis, C., Chang, H. Y. 2018; 173 (6): 1398-+

  View details for DOI 10.1016/j.cell.2018.03.068

  View details for Web of Science ID 000433989100013

• ATAC Primer Tool for targeted analysis of accessible chromatin NATURE METHODS Yost, K. E., Carter, A. C., Xu, J., Litzenburger, U., Chang, H. Y. 2018; 15 (5): 304–5

  View details for PubMedID 29702633

• Epigenomics: Technologies and Applications. Circulation research Wang, K. C., Chang, H. Y. 2018; 122 (9): 1191–99

  Abstract

  The advent of high-throughput epigenome mapping technologies has ushered in a new era of multiomics where powerful tools can now delineate and record different layers of genomic output. Integrating various components of the epigenome from these multiomics measurements allows the interrogation of cellular heterogeneity in addition to the discovery of molecular connectivity maps between the genome and its functional output. Mapping of chromatin accessibility dynamics and higher-order chromatin structure has enabled new levels of understanding of cell fate decisions, identity, and function in normal development, physiology, and disease. We provide a perspective on the progress of the epigenomics field and applications and anticipate an even greater revolution in our understanding of the human epigenome for years to come.

  View details for PubMedID 29700067

• Epigenomics Technologies and Applications CIRCULATION RESEARCH Wang, K. C., Chang, H. Y. 2018; 122 (9): 1191–99

  View details for DOI 10.1161/CIRCRESAHA.118.310998

  View details for Web of Science ID 000435406500005

• m(6)A mRNA methylation sustains Treg suppressive functions CELL RESEARCH Tong, J., Cao, G., Zhang, T., Sefik, E., Vesely, M., Broughton, J. P., Zhu, S., Li, H., Li, B., Chen, L., Chang, H. Y., Su, B., Flavell, R. A., Li, H. 2018; 28 (2): 253–56

  View details for PubMedID 29303144

• Tissue-selective effects of nucleolar stress and rDNA damage in developmental disorders NATURE Calo, E., Gu, B., Bowen, M. E., Aryan, F., Zalc, A., Liang, J., Flynn, R. A., Swigut, T., Chang, H. Y., Attardi, L. D., Wysocka, J. 2018; 554 (7690): 112-+

  Abstract

  Many craniofacial disorders are caused by heterozygous mutations in general regulators of housekeeping cellular functions such as transcription or ribosome biogenesis. Although it is understood that many of these malformations are a consequence of defects in cranial neural crest cells, a cell type that gives rise to most of the facial structures during embryogenesis, the mechanism underlying cell-type selectivity of these defects remains largely unknown. By exploring molecular functions of DDX21, a DEAD-box RNA helicase involved in control of both RNA polymerase (Pol) I- and II-dependent transcriptional arms of ribosome biogenesis, we uncovered a previously unappreciated mechanism linking nucleolar dysfunction, ribosomal DNA (rDNA) damage, and craniofacial malformations. Here we demonstrate that genetic perturbations associated with Treacher Collins syndrome, a craniofacial disorder caused by heterozygous mutations in components of the Pol I transcriptional machinery or its cofactor TCOF1 (ref. 1), lead to relocalization of DDX21 from the nucleolus to the nucleoplasm, its loss from the chromatin targets, as well as inhibition of rRNA processing and downregulation of ribosomal protein gene transcription. These effects are cell-type-selective, cell-autonomous, and involve activation of p53 tumour-suppressor protein. We further show that cranial neural crest cells are sensitized to p53-mediated apoptosis, but blocking DDX21 loss from the nucleolus and chromatin rescues both the susceptibility to apoptosis and the craniofacial phenotypes associated with Treacher Collins syndrome. This mechanism is not restricted to cranial neural crest cells, as blood formation is also hypersensitive to loss of DDX21 functions. Accordingly, ribosomal gene perturbations associated with Diamond-Blackfan anaemia disrupt DDX21 localization. At the molecular level, we demonstrate that impaired rRNA synthesis elicits a DNA damage response, and that rDNA damage results in tissue-selective and dosage-dependent effects on craniofacial development. Taken together, our findings illustrate how disruption in general regulators that compromise nucleolar homeostasis can result in tissue-selective malformations.

  View details for PubMedID 29364875

• Rapid chromatin repression by Aire provides precise control of immune tolerance NATURE IMMUNOLOGY Koh, A. S., Miller, E. L., Buenrostro, J. D., Moskowitz, D. M., Wang, J., Greenleaf, W. J., Chang, H. Y., Crabtree, G. R. 2018; 19 (2): 162-+

  Abstract

  Aire mediates the expression of tissue-specific antigens in thymic epithelial cells to promote tolerance against self-reactive T lymphocytes. However, the mechanism that allows expression of tissue-specific genes at levels that prevent harm is unknown. Here we show that Brg1 generates accessibility at tissue-specific loci to impose central tolerance. We found that Aire has an intrinsic repressive function that restricts chromatin accessibility and opposes Brg1 across the genome. Aire exerted this repressive influence within minutes after recruitment to chromatin and restrained the amplitude of active transcription. Disease-causing mutations that impair Aire-induced activation also impair the protein's repressive function, which indicates dual roles for Aire. Together, Brg1 and Aire fine-tune the expression of tissue-specific genes at levels that prevent toxicity yet promote immune tolerance.

  View details for PubMedID 29335648

• ChIRP-MS: RNA-Directed Proteomic Discovery. Methods in molecular biology (Clifton, N.J.) Chu, C., Chang, H. Y. 2018; 1861: 37–45

  Abstract

  Comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-ms) is a novel technique for studying endogenous ribonucleoprotein complexes. ChIRP-ms is robust across a wide range of expression level, from abundant housekeeping RNAs (e.g., spliceosomal U RNAs) to relatively lowly expressed RNAs (e.g., Xist). In vivo RNA-protein interactions are chemically cross-linked, and purified using biotinylated antisense oligonucleotides against RNA of interest. Coprecipitated proteins are gently eluted, and identified by mass-spectrometry (for discovery) or by western blotting (for validation).

  View details for PubMedID 30218357

• ChIRP-MS: RNA-Directed Proteomic Discovery X-CHROMOSOME INACTIVATION: METHODS AND PROTOCOLS Chu, C., Chang, H. Y., Sado, T. 2018; 1861: 37–45

  View details for DOI 10.1007/978-1-4939-8766-5_3

  View details for Web of Science ID 000458611600004

• An Activity Switch in Human Telomerase Based on RNA Conformation and Shaped by TCAB1. Cell Chen, L. n., Roake, C. M., Freund, A. n., Batista, P. J., Tian, S. n., Yin, Y. A., Gajera, C. R., Lin, S. n., Lee, B. n., Pech, M. F., Venteicher, A. S., Das, R. n., Chang, H. Y., Artandi, S. E. 2018

  Abstract

  Ribonucleoprotein enzymes require dynamic conformations of their RNA constituents for regulated catalysis. Human telomerase employs a non-coding RNA (hTR) with a bipartite arrangement of domains-a template-containing core and a distal three-way junction (CR4/5) that stimulates catalysis through unknown means. Here, we show that telomerase activity unexpectedly depends upon the holoenzyme protein TCAB1, which in turn controls conformation of CR4/5. Cells lacking TCAB1 exhibit a marked reduction in telomerase catalysis without affecting enzyme assembly. Instead, TCAB1 inactivation causes unfolding of CR4/5 helices that are required for catalysis and for association with the telomerase reverse-transcriptase (TERT). CR4/5 mutations derived from patients with telomere biology disorders provoke defects in catalysis and TERT binding similar to TCAB1 inactivation. These findings reveal a conformational "activity switch" in human telomerase RNA controlling catalysis and TERT engagement. The identification of two discrete catalytic states for telomerase suggests an intramolecular means for controlling telomerase in cancers and progenitor cells.

  View details for PubMedID 29804836

• Expression of the transcription factor ZBTB46 distinguishes human histiocytic disorders of classical dendritic cell origin. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc Satpathy, A. T., Brown, R. A., Gomulia, E. n., Briseño, C. G., Mumbach, M. R., Pan, Z. n., Murphy, K. M., Natkunam, Y. n., Chang, H. Y., Kim, J. n. 2018

  Abstract

  Distinguishing classical dendritic cells from other myeloid cell types is complicated by the shared expression of cell surface markers. ZBTB46 is a zinc finger and BTB domain-containing transcription factor, which is expressed by dendritic cells and committed dendritic cell precursors, but not by plasmacytoid dendritic cells, monocytes, macrophages, or other immune cell populations. In this study, we demonstrate that expression of ZBTB46 identifies human dendritic cell neoplasms. We examined ZBTB46 expression in a range of benign and malignant histiocytic disorders and found that ZBTB46 is able to clearly define the dendritic cell identity of many previously unclassified histiocytic disease subtypes. In particular, all examined cases of Langerhans cell histiocytosis and histiocytic sarcoma expressed ZBTB46, while all cases of blastic plasmacytoid dendritic cell neoplasm, chronic myelomonocytic leukemia, juvenile xanthogranuloma, Rosai-Dorfman disease, and Erdheim-Chester disease failed to demonstrate expression of ZBTB46. Moreover, ZBTB46 expression clarified the identity of diagnostically challenging neoplasms, such as cases of indeterminate cell histiocytosis, classifying a fraction of these entities as dendritic cell malignancies. These findings clarify the lineage origins of human histiocytic disorders and distinguish dendritic cell disorders from all other myeloid neoplasms.

  View details for PubMedID 29743654

• LncRNA Seduction of GOT2 Goes Viral IMMUNITY Chen, Y., Chang, H. Y. 2017; 47 (6): 1021–23

  Abstract

  Mechanisms of viral infection are active areas of investigation. In a recent issue of Science, Wang et al. (2017) reveal an additional function of a host-encoded long non-coding RNA (lncRNA) in regulating viral expression by binding a host metabolic enzyme to enhance its catalytic activity.

  View details for PubMedID 29262346

• Challenges and recommendations for epigenomics in precision health NATURE BIOTECHNOLOGY Carter, A. C., Chang, H. Y., Church, G., Dombkowski, A., Ecker, J. R., Gil, E., Giresi, P. G., Greely, H., Greenleaf, W. J., Hacohen, N., He, C., Hill, D., Ko, J., Kohane, I., Kundaje, A., Palmer, M., Snyder, M. P., Tung, J., Urban, A., Vidal, M., Wong, W. 2017; 35 (12): 1128–32

  View details for PubMedID 29220033

• Mechanistic insights in X-chromosome inactivation. Philosophical transactions of the Royal Society of London. Series B, Biological sciences Lu, Z., Carter, A. C., Chang, H. Y. 2017; 372 (1733)

  Abstract

  X-chromosome inactivation (XCI) is a critical epigenetic mechanism for balancing gene dosage between XY males and XX females in eutherian mammals. A long non-coding RNA (lncRNA), XIST, and its associated proteins orchestrate this multi-step process, resulting in the inheritable silencing of one of the two X-chromosomes in females. The XIST RNA is large and complex, exemplifying the unique challenges associated with the structural and functional analysis of lncRNAs. Recent technological advances in the analysis of macromolecular structure and interactions have enabled us to systematically dissect the XIST ribonucleoprotein complex, which is larger than the ribosome, and its place of action, the inactive X-chromosome. These studies shed light on key mechanisms of XCI, such as XIST coating of the X-chromosome, recruitment of DNA, RNA and histone modification enzymes, and compaction and compartmentalization of the inactive X. Here, we summarize recent studies on XCI, highlight the critical contributions of new technologies and propose a unifying model for XIST function in XCI where modular domains serve as the structural and functional units in both lncRNA-protein complexes and DNA-protein complexes in chromatin.This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'.

  View details for PubMedID 28947655

• Mechanistic insights in X-chromosome inactivation PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES Lu, Z., Carter, A. C., Chang, H. Y. 2017; 372 (1733)

  View details for DOI 10.1098/rstb.2016.0356

  View details for Web of Science ID 000411682500003

• The conserved RNA helicase YTHDC2 regulates the transition from proliferation to differentiation in the germline ELIFE Bailey, A. S., Batista, P. J., Gold, R. S., Chen, Y., de Rooij, D. G., Chang, H. Y., Fuller, M. T. 2017; 6

  Abstract

  The switch from mitosis to meiosis is the key event marking onset of differentiation in the germline stem cell lineage. In Drosophila, the translational repressor Bgcn is required for spermatogonia to stop mitosis and transition to meiotic prophase and the spermatocyte state. Here we show that the mammalian Bgcn homolog YTHDC2 facilitates a clean switch from mitosis to meiosis in mouse germ cells, revealing a conserved role for YTHDC2 in this critical cell fate transition. YTHDC2-deficient male germ cells enter meiosis but have a mixed identity, maintaining expression of Cyclin A2 and failing to properly express many meiotic markers. Instead of continuing through meiotic prophase, the cells attempt an abnormal mitotic-like division and die. YTHDC2 binds multiple transcripts including Ccna2 and other mitotic transcripts, binds specific piRNA precursors, and interacts with RNA granule components, suggesting that proper progression of germ cells through meiosis is licensed by YTHDC2 through post-transcriptional regulation.

  View details for PubMedID 29087293

• m(6)A mRNA methylation controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathways NATURE Li, H., Tong, J., Zhu, S., Batista, P. J., Duffy, E. E., Zhao, J., Bailis, W., Cao, G., Kroehling, L., Chen, Y., Wang, G., Broughton, J. P., Chen, Y., Kluger, Y., Simon, M. D., Chang, H. Y., Yin, Z., Flavell, R. A. 2017; 548 (7667): 338-+

  Abstract

  N6-methyladenosine (m6A) is the most common and abundant messenger RNA modification, modulated by 'writers', 'erasers' and 'readers' of this mark. In vitro data have shown that m6A influences all fundamental aspects of mRNA metabolism, mainly mRNA stability, to determine stem cell fates. However, its in vivo physiological function in mammals and adult mammalian cells is still unknown. Here we show that the deletion of m6A 'writer' protein METTL3 in mouse T cells disrupts T cell homeostasis and differentiation. In a lymphopaenic mouse adoptive transfer model, naive Mettl3-deficient T cells failed to undergo homeostatic expansion and remained in the naive state for up to 12 weeks, thereby preventing colitis. Consistent with these observations, the mRNAs of SOCS family genes encoding the STAT signalling inhibitory proteins SOCS1, SOCS3 and CISH were marked by m6A, exhibited slower mRNA decay and showed increased mRNAs and levels of protein expression in Mettl3-deficient naive T cells. This increased SOCS family activity consequently inhibited IL-7-mediated STAT5 activation and T cell homeostatic proliferation and differentiation. We also found that m6A has important roles for inducible degradation of Socs mRNAs in response to IL-7 signalling in order to reprogram naive T cells for proliferation and differentiation. Our study elucidates for the first time, to our knowledge, the in vivo biological role of m6A modification in T-cell-mediated pathogenesis and reveals a novel mechanism of T cell homeostasis and signal-dependent induction of mRNA degradation.

  View details for PubMedID 28792938

• IL-4R alpha Inhibitor for Atopic Disease CELL Chang, H. Y., Nadeau, K. C. 2017; 170 (2): 222

  Abstract

  Dupilumab is a fully human IgG4 monoclonal antibody directed against the IL-4Rα subunit of IL-4 and IL-13 receptors. It blocks the signaling pathways of IL-4 and IL-13, key cytokines that drive type 2 inflammatory response. In March 2017, dupilumab was approved for use in the treatment of atopic dermatitis (eczema). To view this Bench to Bedside, open or download the PDF.

  View details for PubMedID 28708993

• Long Noncoding RNAs: At the Intersection of Cancer and Chromatin Biology COLD SPRING HARBOR PERSPECTIVES IN MEDICINE Schmitt, A. M., Chang, H. Y. 2017; 7 (7)

  Abstract

  Although only 2% of the genome encodes protein, RNA is transcribed from the majority of the genetic sequence, suggesting a massive degree of cellular functionality is programmed in the noncoding genome. The mammalian genome contains tens of thousands of long noncoding RNAs (lncRNAs), many of which occur at disease-associated loci or are specifically expressed in cancer. Although the vast majority of lncRNAs have no known function, recurring molecular mechanisms for lncRNAs are now being observed in chromatin regulation and cancer pathways and emerging technologies are now providing tools to interrogate lncRNA molecular interactions and determine function of these abundant cellular macromolecules.

  View details for PubMedID 28193769

• The Mammalian Ribo-interactome Reveals Ribosome Functional Diversity and Heterogeneity. Cell Simsek, D., Tiu, G. C., Flynn, R. A., Byeon, G. W., Leppek, K., Xu, A. F., Chang, H. Y., Barna, M. 2017; 169 (6): 1051-1065 e18

  Abstract

  During eukaryotic evolution, ribosomes have considerably increased in size, forming a surface-exposed ribosomal RNA (rRNA) shell of unknown function, which may create an interface for yet uncharacterized interacting proteins. To investigate such protein interactions, we establish a ribosome affinity purification method that unexpectedly identifies hundreds of ribosome-associated proteins (RAPs) from categories including metabolism and cell cycle, as well as RNA- and protein-modifying enzymes that functionally diversify mammalian ribosomes. By further characterizing RAPs, we discover the presence of ufmylation, a metazoan-specific post-translational modification (PTM), on ribosomes and define its direct substrates. Moreover, we show that the metabolic enzyme, pyruvate kinase muscle (PKM), interacts with sub-pools of endoplasmic reticulum (ER)-associated ribosomes, exerting a non-canonical function as an RNA-binding protein in the translation of ER-destined mRNAs. Therefore, RAPs interconnect one of life's most ancient molecular machines with diverse cellular processes, providing an additional layer of regulatory potential to protein expression.

  View details for DOI 10.1016/j.cell.2017.05.022

  View details for PubMedID 28575669

• Transcription coactivator and lncRNA duet evoke Hox genes PLOS GENETICS Wang, K. C., Chang, H. Y. 2017; 13 (6): e1006797

  View details for PubMedID 28662057

• Generation of pure GABAergic neurons by transcription factor programming. Nature methods Yang, N., Chanda, S., Marro, S., Ng, Y., Janas, J. A., Haag, D., Ang, C. E., Tang, Y., Flores, Q., Mall, M., Wapinski, O., Li, M., Ahlenius, H., Rubenstein, J. L., Chang, H. Y., Buylla, A. A., Südhof, T. C., Wernig, M. 2017; 14 (6): 621-628

  Abstract

  Approaches to differentiating pluripotent stem cells (PSCs) into neurons currently face two major challenges-(i) generated cells are immature, with limited functional properties; and (ii) cultures exhibit heterogeneous neuronal subtypes and maturation stages. Using lineage-determining transcription factors, we previously developed a single-step method to generate glutamatergic neurons from human PSCs. Here, we show that transient expression of the transcription factors Ascl1 and Dlx2 (AD) induces the generation of exclusively GABAergic neurons from human PSCs with a high degree of synaptic maturation. These AD-induced neuronal (iN) cells represent largely nonoverlapping populations of GABAergic neurons that express various subtype-specific markers. We further used AD-iN cells to establish that human collybistin, the loss of gene function of which causes severe encephalopathy, is required for inhibitory synaptic function. The generation of defined populations of functionally mature human GABAergic neurons represents an important step toward enabling the study of diseases affecting inhibitory synaptic transmission.

  View details for DOI 10.1038/nmeth.4291

  View details for PubMedID 28504679

• Gpr124 is essential for blood-brain barrier integrity in central nervous system disease NATURE MEDICINE Chang, J., Mancuso, M. R., Maier, C., Liang, X., Yuki, K., Yang, L., Kwong, J. W., Wang, J., Rao, V., Vallon, M., Kosinski, C., Zhang, J. J., Mah, A. T., Xu, L., Li, L., Gholamin, S., Reyes, T. F., Li, R., Kuhnert, F., Han, X., Yuan, J., Chiou, S., Brettman, A. D., Daly, L., Corney, D. C., Cheshier, S. H., Shortliffe, L. D., Wu, X., Snyder, M., Chan, P., Giffard, R. G., Chang, H. Y., Andreasson, K., Kuo, C. J. 2017; 23 (4): 450-?

  Abstract

  Although blood-brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt-β-catenin signaling. Constitutive activation of Wnt-β-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.

  View details for DOI 10.1038/nm.4309

  View details for PubMedID 28288111

• Landscape of monoallelic DNA accessibility in mouse embryonic stem cells and neural progenitor cells. Nature genetics Xu, J., Carter, A. C., Gendrel, A., Attia, M., Loftus, J., Greenleaf, W. J., Tibshirani, R., Heard, E., Chang, H. Y. 2017; 49 (3): 377-386

  Abstract

  We developed an allele-specific assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) to genotype and profile active regulatory DNA across the genome. Using a mouse hybrid F1 system, we found that monoallelic DNA accessibility across autosomes was pervasive, developmentally programmed and composed of several patterns. Genetically determined accessibility was enriched at distal enhancers, but random monoallelically accessible (RAMA) elements were enriched at promoters and may act as gatekeepers of monoallelic mRNA expression. Allelic choice at RAMA elements was stable across cell generations and bookmarked through mitosis. RAMA elements in neural progenitor cells were biallelically accessible in embryonic stem cells but premarked with bivalent histone modifications; one allele was silenced during differentiation. Quantitative analysis indicated that allelic choice at the majority of RAMA elements is consistent with a stochastic process; however, up to 30% of RAMA elements may deviate from the expected pattern, suggesting a regulated or counting mechanism.

  View details for DOI 10.1038/ng.3769

  View details for PubMedID 28112738

• Comparison of SHAPE reagents for mapping RNA structures inside living cells RNA Lee, B., Flynn, R. A., Kadina, A., Guo, J. K., Kool, E. T., Chang, H. Y. 2017; 23 (2): 169-174

  Abstract

  Recent advances in SHAPE technology have converted the classic primer extension method to next-generation sequencing platforms, allowing transcriptome-level analysis of RNA secondary structure. In particular, icSHAPE and SHAPE-MaP, using NAI-N3 and 1M7 reagents, respectively, are methods that claim to measure in vivo structure with high-throughput sequencing. However, these compounds have not been compared on an unbiased, raw-signal level. Here, we directly compare several in vivo SHAPE acylation reagents using the simple primer extension assay. We conclude that while multiple SHAPE technologies are effective at measuring purified RNAs in vitro, acylimidazole reagents NAI and NAI-N3 give markedly greater signals with lower background than 1M7 for in vivo measurement of the RNA structurome.

  View details for DOI 10.1261/rna.058784.116

  View details for PubMedID 27879433

• Single-cell epigenomic variability reveals functional cancer heterogeneity. Genome biology Litzenburger, U. M., Buenrostro, J. D., Wu, B., Shen, Y., Sheffield, N. C., Kathiria, A., Greenleaf, W. J., Chang, H. Y. 2017; 18 (1): 15-?

  Abstract

  Cell-to-cell heterogeneity is a major driver of cancer evolution, progression, and emergence of drug resistance. Epigenomic variation at the single-cell level can rapidly create cancer heterogeneity but is difficult to detect and assess functionally.We develop a strategy to bridge the gap between measurement and function in single-cell epigenomics. Using single-cell chromatin accessibility and RNA-seq data in K562 leukemic cells, we identify the cell surface marker CD24 as co-varying with chromatin accessibility changes linked to GATA transcription factors in single cells. Fluorescence-activated cell sorting of CD24 high versus low cells prospectively isolated GATA1 and GATA2 high versus low cells. GATA high versus low cells express differential gene regulatory networks, differential sensitivity to the drug imatinib mesylate, and differential self-renewal capacity. Lineage tracing experiments show that GATA/CD24hi cells have the capability to rapidly reconstitute the heterogeneity within the entire starting population, suggesting that GATA expression levels drive a phenotypically relevant source of epigenomic plasticity.Single-cell chromatin accessibility can guide prospective characterization of cancer heterogeneity. Epigenomic subpopulations in cancer impact drug sensitivity and the clonal dynamics of cancer evolution.

  View details for DOI 10.1186/s13059-016-1133-7

  View details for PubMedID 28118844

• An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nature methods Corces, M. R., Trevino, A. E., Hamilton, E. G., Greenside, P. G., Sinnott-Armstrong, N. A., Vesuna, S. n., Satpathy, A. T., Rubin, A. J., Montine, K. S., Wu, B. n., Kathiria, A. n., Cho, S. W., Mumbach, M. R., Carter, A. C., Kasowski, M. n., Orloff, L. A., Risca, V. I., Kundaje, A. n., Khavari, P. A., Montine, T. J., Greenleaf, W. J., Chang, H. Y. 2017

  Abstract

  We present Omni-ATAC, an improved ATAC-seq protocol for chromatin accessibility profiling that works across multiple applications with substantial improvement of signal-to-background ratio and information content. The Omni-ATAC protocol generates chromatin accessibility profiles from archival frozen tissue samples and 50-μm sections, revealing the activities of disease-associated DNA elements in distinct human brain structures. The Omni-ATAC protocol enables the interrogation of personal regulomes in tissue context and translational studies.

  View details for PubMedID 28846090

• Gender and Gene Regulation in Human Immunity PRINCIPLES OF GENDER-SPECIFIC MEDICINE: GENDER IN THE GENOMIC ERA, 3RD EDITION Longmire, M. R., Chang, H., Legato, M. J. 2017: 335–40

  View details for DOI 10.1016/B978-0-12-803506-1.00002-4

  View details for Web of Science ID 000439190900026

• Discovery of stimulation-responsive immune enhancers with CRISPR activation. Nature Simeonov, D. R., Gowen, B. G., Boontanrart, M. n., Roth, T. L., Gagnon, J. D., Mumbach, M. R., Satpathy, A. T., Lee, Y. n., Bray, N. L., Chan, A. Y., Lituiev, D. S., Nguyen, M. L., Gate, R. E., Subramaniam, M. n., Li, Z. n., Woo, J. M., Mitros, T. n., Ray, G. J., Curie, G. L., Naddaf, N. n., Chu, J. S., Ma, H. n., Boyer, E. n., Van Gool, F. n., Huang, H. n., Liu, R. n., Tobin, V. R., Schumann, K. n., Daly, M. J., Farh, K. K., Ansel, K. M., Ye, C. J., Greenleaf, W. J., Anderson, M. S., Bluestone, J. A., Chang, H. Y., Corn, J. E., Marson, A. n. 2017

  Abstract

  The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (TH17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.

  View details for PubMedID 28854172

• Lineage-specific dynamic and pre-established enhancer-promoter contacts cooperate in terminal differentiation. Nature genetics Rubin, A. J., Barajas, B. C., Furlan-Magaril, M. n., Lopez-Pajares, V. n., Mumbach, M. R., Howard, I. n., Kim, D. S., Boxer, L. D., Cairns, J. n., Spivakov, M. n., Wingett, S. W., Shi, M. n., Zhao, Z. n., Greenleaf, W. J., Kundaje, A. n., Snyder, M. n., Chang, H. Y., Fraser, P. n., Khavari, P. A. 2017; 49 (10): 1522–28

  Abstract

  Chromosome conformation is an important feature of metazoan gene regulation; however, enhancer-promoter contact remodeling during cellular differentiation remains poorly understood. To address this, genome-wide promoter capture Hi-C (CHi-C) was performed during epidermal differentiation. Two classes of enhancer-promoter contacts associated with differentiation-induced genes were identified. The first class ('gained') increased in contact strength during differentiation in concert with enhancer acquisition of the H3K27ac activation mark. The second class ('stable') were pre-established in undifferentiated cells, with enhancers constitutively marked by H3K27ac. The stable class was associated with the canonical conformation regulator cohesin, whereas the gained class was not, implying distinct mechanisms of contact formation and regulation. Analysis of stable enhancers identified a new, essential role for a constitutively expressed, lineage-restricted ETS-family transcription factor, EHF, in epidermal differentiation. Furthermore, neither class of contacts was observed in pluripotent cells, suggesting that lineage-specific chromatin structure is established in tissue progenitor cells and is further remodeled in terminal differentiation.

  View details for PubMedID 28805829

• Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements. Nature genetics Mumbach, M. R., Satpathy, A. T., Boyle, E. A., Dai, C. n., Gowen, B. G., Cho, S. W., Nguyen, M. L., Rubin, A. J., Granja, J. M., Kazane, K. R., Wei, Y. n., Nguyen, T. n., Greenside, P. G., Corces, M. R., Tycko, J. n., Simeonov, D. R., Suliman, N. n., Li, R. n., Xu, J. n., Flynn, R. A., Kundaje, A. n., Khavari, P. A., Marson, A. n., Corn, J. E., Quertermous, T. n., Greenleaf, W. J., Chang, H. Y. 2017

  Abstract

  The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer-promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases.

  View details for PubMedID 28945252

• Novel Gene Expression Profile of Women with Intrinsic Skin Youthfulness by Whole Transcriptome Sequencing PLOS ONE Xu, J., Spitale, R. C., Guan, L., Flynn, R. A., Torre, E. A., Li, R., Reber, I., Qu, K., Kern, D., Knaggs, H. E., Chang, H. Y., Chang, A. L. 2016; 11 (11)

  Abstract

  While much is known about genes that promote aging, little is known about genes that protect against or prevent aging, particularly in human skin. The main objective of this study was to perform an unbiased, whole transcriptome search for genes that associate with intrinsic skin youthfulness. To accomplish this, healthy women (n = 122) of European descent, ages 18-89 years with Fitzpatrick skin type I/II were examined for facial skin aging parameters and clinical covariates, including smoking and ultraviolet exposure. Skin youthfulness was defined as the top 10% of individuals whose assessed skin aging features were most discrepant with their chronological ages. Skin biopsies from sun-protected inner arm were subjected to 3'-end sequencing for expression quantification, with results verified by quantitative reverse transcriptase-polymerase chain reaction. Unbiased clustering revealed gene expression signatures characteristic of older women with skin youthfulness (n = 12) compared to older women without skin youthfulness (n = 33), after accounting for gene expression changes associated with chronological age alone. Gene set analysis was performed using Genomica open-access software. This study identified a novel set of candidate skin youthfulness genes demonstrating differences between SY and non-SY group, including pleckstrin homology like domain family A member 1 (PHLDA1) (p = 2.4x10-5), a follicle stem cell marker, and hyaluronan synthase 2-anti-sense 1 (HAS2-AS1) (p = 0.00105), a non-coding RNA that is part of the hyaluronan synthesis pathway. We show that immunologic gene sets are the most significantly altered in skin youthfulness (with the most significant gene set p = 2.4x10-5), suggesting the immune system plays an important role in skin youthfulness, a finding that has not previously been recognized. These results are a valuable resource from which multiple future studies may be undertaken to better understand the mechanisms that promote skin youthfulness in humans.

  View details for DOI 10.1371/journal.pone.0165913

  View details for Web of Science ID 000387724300066

  View details for PubMedCentralID PMC5102383

• An inducible long noncoding RNA amplifies DNA damage signaling. Nature genetics Schmitt, A. M., Garcia, J. T., Hung, T., Flynn, R. A., Shen, Y., Qu, K., Payumo, A. Y., Peres-da-Silva, A., Broz, D. K., Baum, R., Guo, S., Chen, J. K., Attardi, L. D., Chang, H. Y. 2016; 48 (11): 1370-1376

  Abstract

  Long noncoding RNAs (lncRNAs) are prevalent genes with frequently precise regulation but mostly unknown functions. Here we demonstrate that lncRNAs guide the organismal DNA damage response. DNA damage activated transcription of the DINO (Damage Induced Noncoding) lncRNA via p53. DINO was required for p53-dependent gene expression, cell cycle arrest and apoptosis in response to DNA damage, and DINO expression was sufficient to activate damage signaling and cell cycle arrest in the absence of DNA damage. DINO bound to p53 protein and promoted its stabilization, mediating a p53 auto-amplification loop. Dino knockout or promoter inactivation in mice dampened p53 signaling and ameliorated acute radiation syndrome in vivo. Thus, inducible lncRNA can create a feedback loop with its cognate transcription factor to amplify cellular signaling networks.

  View details for DOI 10.1038/ng.3673

  View details for PubMedID 27668660

• HiChIP: efficient and sensitive analysis of protein-directed genome architecture. Nature methods Mumbach, M. R., Rubin, A. J., Flynn, R. A., Dai, C., Khavari, P. A., Greenleaf, W. J., Chang, H. Y. 2016; 13 (11): 919-922

  Abstract

  Genome conformation is central to gene control but challenging to interrogate. Here we present HiChIP, a protein-centric chromatin conformation method. HiChIP improves the yield of conformation-informative reads by over 10-fold and lowers the input requirement over 100-fold relative to that of ChIA-PET. HiChIP of cohesin reveals multiscale genome architecture with greater signal-to-background ratios than those of in situ Hi-C.

  View details for DOI 10.1038/nmeth.3999

  View details for PubMedID 27643841

• Molecular and Neural Functions of Rai1, the Causal Gene for Smith-Magenis Syndrome. Neuron Huang, W., Guenthner, C. J., Xu, J., Nguyen, T., Schwarz, L. A., Wilkinson, A. W., Gozani, O., Chang, H. Y., Shamloo, M., Luo, L. 2016; 92 (2): 392-406

  Abstract

  Haploinsufficiency of Retinoic Acid Induced 1 (RAI1) causes Smith-Magenis syndrome (SMS), which is associated with diverse neurodevelopmental and behavioral symptoms as well as obesity. RAI1 encodes a nuclear protein but little is known about its molecular function or the cell types responsible for SMS symptoms. Using genetically engineered mice, we found that Rai1 preferentially occupies DNA regions near active promoters and promotes the expression of a group of genes involved in circuit assembly and neuronal communication. Behavioral analyses demonstrated that pan-neural loss of Rai1 causes deficits in motor function, learning, and food intake. These SMS-like phenotypes are produced by loss of Rai1 function in distinct neuronal types: Rai1 loss in inhibitory neurons or subcortical glutamatergic neurons causes learning deficits, while Rai1 loss in Sim1(+) or SF1(+) cells causes obesity. By integrating molecular and organismal analyses, our study suggests potential therapeutic avenues for a complex neurodevelopmental disorder.

  View details for DOI 10.1016/j.neuron.2016.09.019

  View details for PubMedID 27693255

• HOXC10 Expression Supports the Development of Chemotherapy Resistance by Fine Tuning DNA Repair in Breast Cancer Cells CANCER RESEARCH Sadik, H., Korangath, P., Nguyen, N. K., Gyorffy, B., Kumar, R., Hedayati, M., Teo, W. W., Park, S., Panday, H., Munoz, T. G., Menyhart, O., Shah, N., Pandita, R. K., Chang, J. C., DeWeese, T., Chang, H. Y., Pandita, T. K., Sukumar, S. 2016; 76 (15): 4443-4456

  Abstract

  Development of drug resistance is a major factor limiting the continued success of cancer chemotherapy. To overcome drug resistance, understanding the underlying mechanism(s) is essential. We found that HOXC10 is overexpressed in primary carcinomas of the breast, and even more significantly in distant metastasis arising after failed chemotherapy. High HOXC10 expression correlates with shorter recurrence-free and overall survival in patients with estrogen receptor-negative breast cancer undergoing chemotherapy. We found that HOXC10 promotes survival in cells treated with doxorubicin, paclitaxel, or carboplatin by suppressing apoptosis and upregulating NF-κB Overexpressed HOXC10 increases S-phase-specific DNA damage repair by homologous recombination (HR) and checkpoint recovery in cells at three important phases. For double-strand break repair, HOXC10 recruits HR proteins at sites of DNA damage. It enhances resection and lastly, it resolves stalled replication forks, leading to initiation of DNA replication following DNA damage. We show that HOXC10 facilitates, but is not directly involved in DNA damage repair mediated by HR. HOXC10 achieves integration of these functions by binding to, and activating cyclin-dependent kinase, CDK7, which regulates transcription by phosphorylating the carboxy-terminal domain of RNA polymerase II. Consistent with these findings, inhibitors of CDK7 reverse HOXC10-mediated drug resistance in cultured cells. Blocking HOXC10 function, therefore, presents a promising new strategy to overcome chemotherapy resistance in breast cancer. Cancer Res; 76(15); 4443-56. ©2016 AACR.

  View details for DOI 10.1158/0008-5472.CAN-16-0774

  View details for PubMedID 27302171

• Structural organization of the inactive X chromosome in the mouse NATURE Giorgetti, L., Lajoie, B. R., Carter, A. C., Attia, M., Zhan, Y., Xu, J., Chen, C. J., Kaplan, N., Chang, H. Y., Heard, E., Dekker, J. 2016; 535 (7613): 575-?

  Abstract

  X-chromosome inactivation (XCI) involves major reorganization of the X chromosome as it becomes silent and heterochromatic. During female mammalian development, XCI is triggered by upregulation of the non-coding Xist RNA from one of the two X chromosomes. Xist coats the chromosome in cis and induces silencing of almost all genes via its A-repeat region, although some genes (constitutive escapees) avoid silencing in most cell types, and others (facultative escapees) escape XCI only in specific contexts. A role for Xist in organizing the inactive X (Xi) chromosome has been proposed. Recent chromosome conformation capture approaches have revealed global loss of local structure on the Xi chromosome and formation of large mega-domains, separated by a region containing the DXZ4 macrosatellite. However, the molecular architecture of the Xi chromosome, in both the silent and expressed regions,remains unclear. Here we investigate the structure, chromatin accessibility and expression status of the mouse Xi chromosome in highly polymorphic clonal neural progenitors (NPCs) and embryonic stem cells. We demonstrate a crucial role for Xist and the DXZ4-containing boundary in shaping Xi chromosome structure using allele-specific genome-wide chromosome conformation capture (Hi-C) analysis, an assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) and RNA sequencing. Deletion of the boundary disrupts mega-domain formation, and induction of Xist RNA initiates formation of the boundary and the loss of DNA accessibility. We also show that in NPCs, the Xi chromosome lacks active/inactive compartments and topologically associating domains (TADs), except around genes that escape XCI. Escapee gene clusters display TAD-like structures and retain DNA accessibility at promoter-proximal and CTCF-binding sites. Furthermore, altered patterns of facultative escape genes indifferent neural progenitor clones are associated with the presence of different TAD-like structures after XCI. These findings suggest a key role for transcription and CTCF in the formation of TADs in the context of the Xi chromosome in neural progenitors.

  View details for DOI 10.1038/nature18589

  View details for Web of Science ID 000380856600034

  View details for PubMedID 27437574

  View details for PubMedCentralID PMC5443622

• CD44+ Cells in Head and Neck Squamous Cell Carcinoma Suppress T-Cell-Mediated Immunity by Selective Constitutive and Inducible Expression of PD-L1. Clinical cancer research Lee, Y., Shin, J. H., Longmire, M., Wang, H., Kohrt, H. E., Chang, H. Y., Sunwoo, J. B. 2016; 22 (14): 3571-3581

  Abstract

  Human tumors consist of heterogeneous populations of cells with distinct marker expression and functional properties. In squamous cell carcinoma of the head and neck (SCCHN), CD44 is a well-characterized marker of a resilient subpopulation of cells associated with increased tumorigenesis, radioresistance, and chemoresistance. Evidence indicates that these cells have an immune suppressive phenotype; however, mechanisms have been elusive.Using primary human SCCHN tumor samples and patient-derived xenografts, we examined the phenotypes of subsets of tumor cells and investigated mechanisms regulating their immunogenicity.CD44+ cells in primary human SCCHN were found to have an epithelial-to-mesenchymal (EMT) phenotype and were less immunogenic than CD44- cells when cultured with autologous CD8+ tumor-infiltrating T cells. Selective expression of the programmed death-ligand 1 (PD-L1) was observed on CD44+ cells compared to CD44- cells and was associated with constitutive phosphorylation of STAT3 on CD44+ cells. Importantly, inhibition of STAT3 decreased expression of PD-L1 on CD44+ cells. Interferon-γ (IFNγ) treatment preferentially induced even further PD-L1 expression on CD44+ cells and was associated with enhanced IFNγ receptor expression and phosphorylation of STAT1. Finally, the decreased immunogenicity of CD44+ cells was partially reversed by antibody blockade of the programmed death 1 (PD-1) receptor, indicating that the differences in PD-L1 expression between CD44+ and CD44- cells are biologically and clinically relevant.Our findings provide a mechanism by which long-lived CD44+ tumor-initiating cells can selectively evade host immune responses and provide rationale for targeting the PD-1 pathway in the adjuvant therapy setting of SCCHN.

  View details for DOI 10.1158/1078-0432.CCR-15-2665

  View details for PubMedID 26864211

• A Long Noncoding RNA lincRNA-EPS Acts as a Transcriptional Brake to Restrain Inflammation CELL Atianand, M. K., Hu, W., Satpathy, A. T., Shen, Y., Ricci, E. P., Alvarez-Dominguez, J. R., Bhatta, A., Schattgen, S. A., McGowan, J. D., Blin, J., Braun, J. E., Gandhi, P., Moore, M. J., Chang, H. Y., Lodish, H. F., Caffrey, D. R., Fitzgerald, K. A. 2016; 165 (7): 1672-1685

  Abstract

  Long intergenic noncoding RNAs (lincRNAs) are important regulators of gene expression. Although lincRNAs are expressed in immune cells, their functions in immunity are largely unexplored. Here, we identify an immunoregulatory lincRNA, lincRNA-EPS, that is precisely regulated in macrophages to control the expression of immune response genes (IRGs). Transcriptome analysis of macrophages from lincRNA-EPS-deficient mice, combined with gain-of-function and rescue experiments, revealed a specific role for this lincRNA in restraining IRG expression. Consistently, lincRNA-EPS-deficient mice manifest enhanced inflammation and lethality following endotoxin challenge in vivo. lincRNA-EPS localizes at regulatory regions of IRGs to control nucleosome positioning and repress transcription. Further, lincRNA-EPS mediates these effects by interacting with heterogeneous nuclear ribonucleoprotein L via a CANACA motif located in its 3' end. Together, these findings identify lincRNA-EPS as a repressor of inflammatory responses, highlighting the importance of lincRNAs in the immune system.

  View details for DOI 10.1016/j.cell.2016.05.075

  View details for PubMedID 27315481

• irCLIP platform for efficient characterization of protein-RNA interactions NATURE METHODS Zarnegar, B. J., Flynn, R. A., Shen, Y., Do, B. T., Chang, H. Y., Khavari, P. A. 2016; 13 (6): 489-?

  Abstract

  The complexity of transcriptome-wide protein-RNA interaction networks is incompletely understood. While emerging studies are greatly expanding the known universe of RNA-binding proteins, methods for the discovery and characterization of protein-RNA interactions remain resource intensive and technically challenging. Here we introduce a UV-C crosslinking and immunoprecipitation platform, irCLIP, which provides an ultraefficient, fast, and nonisotopic method for the detection of protein-RNA interactions using far less material than standard protocols.

  View details for DOI 10.1038/NMETH.3840

  View details for PubMedID 27111506

• Age-Dependent Pancreatic Gene Regulation Reveals Mechanisms Governing Human beta Cell Function CELL METABOLISM Arda, H. E., Li, L., Tsai, J., Torre, E. A., Rosli, Y., Peiris, H., Spitale, R. C., Dai, C., Gu, X., Qu, K., Wang, P., Wang, J., Grompe, M., Scharfmann, R., Snyder, M. S., Bottino, R., Powers, A. C., Chang, H. Y., Kim, S. K. 2016; 23 (5): 909-920

  Abstract

  Intensive efforts are focused on identifying regulators of human pancreatic islet cell growth and maturation to accelerate development of therapies for diabetes. After birth, islet cell growth and function are dynamically regulated; however, establishing these age-dependent changes in humans has been challenging. Here, we describe a multimodal strategy for isolating pancreatic endocrine and exocrine cells from children and adults to identify age-dependent gene expression and chromatin changes on a genomic scale. These profiles revealed distinct proliferative and functional states of islet α cells or β cells and histone modifications underlying age-dependent gene expression changes. Expression of SIX2 and SIX3, transcription factors without prior known functions in the pancreas and linked to fasting hyperglycemia risk, increased with age specifically in human islet β cells. SIX2 and SIX3 were sufficient to enhance insulin content or secretion in immature β cells. Our work provides a unique resource to study human-specific regulators of islet cell maturation and function.

  View details for DOI 10.1016/j.cmet.2016.04.002

  View details for PubMedID 27133132

• Medulloblastoma-associated DDX3 variant selectively alters the translational response to stress ONCOTARGET Oh, S., Flynn, R. A., Floor, S. N., Purzner, J., Martin, L., Do, B. T., Schubert, S., Vaka, D., Morrissy, S., Li, Y., Kool, M., Hovestadt, V., Jones, D. T., Northcott, P. A., Risch, T., Warnatz, H., Yaspo, M., Adams, C. M., Leib, R. D., Breese, M., Marra, M. A., Malkin, D., Lichter, P., Doudna, J. A., Pfister, S. M., Taylor, M. D., Chang, H. Y., Cho, Y. 2016; 7 (19): 28169-28182

  Abstract

  DDX3X encodes a DEAD-box family RNA helicase (DDX3) commonly mutated in medulloblastoma, a highly aggressive cerebellar tumor affecting both children and adults. Despite being implicated in several facets of RNA metabolism, the nature and scope of DDX3's interactions with RNA remain unclear. Here, we show DDX3 collaborates extensively with the translation initiation machinery through direct binding to 5'UTRs of nearly all coding RNAs, specific sites on the 18S rRNA, and multiple components of the translation initiation complex. Impairment of translation initiation is also evident in primary medulloblastomas harboring mutations in DDX3X, further highlighting DDX3's role in this process. Arsenite-induced stress shifts DDX3 binding from the 5'UTR into the coding region of mRNAs concomitant with a general reduction of translation, and both the shift of DDX3 on mRNA and decreased translation are blunted by expression of a catalytically-impaired, medulloblastoma-associated DDX3R534H variant. Furthermore, despite the global repression of translation induced by arsenite, translation is preserved on select genes involved in chromatin organization in DDX3R534H-expressing cells. Thus, DDX3 interacts extensively with RNA and ribosomal machinery to help remodel the translation landscape in response to stress, while cancer-related DDX3 variants adapt this response to selectively preserve translation.

  View details for DOI 10.18632/oncotarget.8612

  View details for Web of Science ID 000377741700097

  View details for PubMedCentralID PMC5053718

• Single-cell profiling of lncRNAs in the developing human brain GENOME BIOLOGY Ma, Q., Chang, H. Y. 2016; 17

  Abstract

  Single-cell RNA-seq in samples from the human neocortex demonstrate that long noncoding RNAs (lncRNAs) are abundantly expressed in specific individual brain cells, despite being hard to detect in bulk samples. This result suggests that the lncRNAs might have important functions in specific cell types in the brain.

  View details for DOI 10.1186/s13059-016-0933-0

  View details for PubMedID 27079200

• Stress from Nucleotide Depletion Activates the Transcriptional Regulator HEXIM1 to Suppress Melanoma. Molecular cell Tan, J. L., Fogley, R. D., Flynn, R. A., Ablain, J., Yang, S., Saint-André, V., Fan, Z. P., Do, B. T., Laga, A. C., Fujinaga, K., Santoriello, C., Greer, C. B., Kim, Y. J., Clohessy, J. G., Bothmer, A., Pandell, N., Avagyan, S., Brogie, J. E., van Rooijen, E., Hagedorn, E. J., Shyh-Chang, N., White, R. M., Price, D. H., Pandolfi, P. P., Peterlin, B. M., Zhou, Y., Kim, T. H., Asara, J. M., Chang, H. Y., Young, R. A., Zon, L. I. 2016; 62 (1): 34-46

  Abstract

  Studying cancer metabolism gives insight into tumorigenic survival mechanisms and susceptibilities. In melanoma, we identify HEXIM1, a transcription elongation regulator, as a melanoma tumor suppressor that responds to nucleotide stress. HEXIM1 expression is low in melanoma. Its overexpression in a zebrafish melanoma model suppresses cancer formation, while its inactivation accelerates tumor onset in vivo. Knockdown of HEXIM1 rescues zebrafish neural crest defects and human melanoma proliferation defects that arise from nucleotide depletion. Under nucleotide stress, HEXIM1 is induced to form an inhibitory complex with P-TEFb, the kinase that initiates transcription elongation, to inhibit elongation at tumorigenic genes. The resulting alteration in gene expression also causes anti-tumorigenic RNAs to bind to and be stabilized by HEXIM1. HEXIM1 plays an important role in inhibiting cancer cell-specific gene transcription while also facilitating anti-cancer gene expression. Our study reveals an important role for HEXIM1 in coupling nucleotide metabolism with transcriptional regulation in melanoma.

  View details for DOI 10.1016/j.molcel.2016.03.013

  View details for PubMedID 27058786

• Systematic Characterization of Long Noncoding RNAs Reveals the Contrasting Coordination of Cis- and Trans-Molecular Regulation in Human Fetal and Adult Hearts CIRCULATION-CARDIOVASCULAR GENETICS He, C., Hu, H., Wilson, K. D., Wu, H., Feng, J., Xia, S., Churko, J., Qu, K., Chang, H. Y., Wu, J. C. 2016; 9 (2): 110-118

  Abstract

  -The molecular regulation of heart development is regulated by cis- and trans- factors acting on the genome and epigenome. As a class of important regulatory RNAs, the role of long non-coding RNAs (lncRNAs) in human heart development is still poorly understood. Furthermore, factors that interact with lncRNAs in this process are not well characterized.-Utilizing RNA sequencing, we systematically define the contrasting lncRNA expression patterns between fetal and adult heart. We report that lncRNAs up-regulated in adult versus fetal heart have different sequence features and distributions. For example, the adult heart expresses more sense lncRNAs compared to fetal heart. We also report the co-expression of lncRNAs and neighboring coding genes that have important functions in heart development. Importantly, the regulation of lncRNA expression during fetal to adult heart development appears to be due in part to the coordination of specific developmental epigenetic modifications such as H3K4me1 and H3k4me3. The expression of promoter-associated lncRNAs in adult and fetal heart also appears to be related to these epigenetic states. Finally, transcription factor binding analysis suggests that lncRNAs are directly regulating cardiac gene expression during development.-We provide a systematic analysis of lncRNA control of heart development that gives clues to the roles that specific lncRNAs play in fetal and adult hearts.

  View details for DOI 10.1161/CIRCGENETICS.115.001264

  View details for PubMedID 26896382

• Integrative genomic analysis by interoperation of bioinformatics tools in GenomeSpace NATURE METHODS Qu, K., Garamszegi, S., Wu, F., Thorvaldsdottir, H., Liefeld, T., Ocana, M., Borges-Rivera, D., Pochet, N., Robinson, J. T., Demchak, B., Hull, T., Ben-Artzi, G., Blankenberg, D., Barber, G. P., Lee, B. T., Kuhn, R. M., Nekrutenko, A., Segal, E., Ideker, T., Reich, M., Regev, A., Chang, H. Y., Mesirov, J. P. 2016; 13 (3): 245-?

  Abstract

  Complex biomedical analyses require the use of multiple software tools in concert and remain challenging for much of the biomedical research community. We introduce GenomeSpace (http://www.genomespace.org), a cloud-based, cooperative community resource that currently supports the streamlined interaction of 20 bioinformatics tools and data resources. To facilitate integrative analysis by non-programmers, it offers a growing set of 'recipes', short workflows to guide investigators through high-utility analysis tasks.

  View details for DOI 10.1038/NMETH.3732

  View details for PubMedID 26780094

• 7SK-BAF axis controls pervasive transcription at enhancers. Nature structural & molecular biology Flynn, R. A., Do, B. T., Rubin, A. J., Calo, E., Lee, B., Kuchelmeister, H., Rale, M., Chu, C., Kool, E. T., Wysocka, J., Khavari, P. A., Chang, H. Y. 2016; 23 (3): 231-238

  Abstract

  RNA functions at enhancers remain mysterious. Here we show that the 7SK small nuclear RNA (snRNA) inhibits enhancer transcription by modulating nucleosome position. 7SK occupies enhancers and super enhancers genome wide in mouse and human cells, and it is required to limit enhancer-RNA initiation and synthesis in a manner distinct from promoter pausing. Clustered elements at super enhancers uniquely require 7SK to prevent convergent transcription and DNA-damage signaling. 7SK physically interacts with the BAF chromatin-remodeling complex, recruits BAF to enhancers and inhibits enhancer transcription by modulating chromatin structure. In turn, 7SK occupancy at enhancers coincides with that of Brd4 and is exquisitely sensitive to the bromodomain inhibitor JQ1. Thus, 7SK uses distinct mechanisms to counteract the diverse consequences of pervasive transcription that distinguish super enhancers, enhancers and promoters.

  View details for DOI 10.1038/nsmb.3176

  View details for PubMedID 26878240

• Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE. Nature protocols Flynn, R. A., Zhang, Q. C., Spitale, R. C., Lee, B., Mumbach, M. R., Chang, H. Y. 2016; 11 (2): 273-290

  Abstract

  icSHAPE (in vivo click selective 2-hydroxyl acylation and profiling experiment) captures RNA secondary structure at a transcriptome-wide level by measuring nucleotide flexibility at base resolution. Living cells are treated with the icSHAPE chemical NAI-N3 followed by selective chemical enrichment of NAI-N3-modified RNA, which provides an improved signal-to-noise ratio compared with similar methods leveraging deep sequencing. Purified RNA is then reverse-transcribed to produce cDNA, with SHAPE-modified bases leading to truncated cDNA. After deep sequencing of cDNA, computational analysis yields flexibility scores for every base across the starting RNA population. The entire experimental procedure can be completed in ∼5 d, and the sequencing and bioinformatics data analysis take an additional 4-5 d with no extensive computational skills required. Comparing in vivo and in vitro icSHAPE measurements can reveal in vivo RNA-binding protein imprints or facilitate the dissection of RNA post-transcriptional modifications. icSHAPE reactivities can additionally be used to constrain and improve RNA secondary structure prediction models.

  View details for DOI 10.1038/nprot.2016.011

  View details for PubMedID 26766114

• Decoding the RNA structurome. Current opinion in structural biology Lu, Z., Chang, H. Y. 2016; 36: 142-148

  Abstract

  Structures of RNA molecules are essential for their architectural, regulatory, and catalytic functions. Recent advances in high throughput sequencing enabled the development of methods for probing RNA structures on a transcriptome-wide scale-termed the RNA structurome. Here we review the state-of-the-art technologies for probing the RNA structurome, and highlight insights gained from these studies. We also point out the limits of current methods and discuss potential directions for future improvements.

  View details for DOI 10.1016/j.sbi.2016.01.007

  View details for PubMedID 26923056

• Decoding the RNA structurome CURRENT OPINION IN STRUCTURAL BIOLOGY Lu, Z., Chang, H. Y. 2016; 36: 142-148

  Abstract

  Structures of RNA molecules are essential for their architectural, regulatory, and catalytic functions. Recent advances in high throughput sequencing enabled the development of methods for probing RNA structures on a transcriptome-wide scale-termed the RNA structurome. Here we review the state-of-the-art technologies for probing the RNA structurome, and highlight insights gained from these studies. We also point out the limits of current methods and discuss potential directions for future improvements.

  View details for DOI 10.1016/j.sbi.2016.01.007

  View details for Web of Science ID 000372681200019

  View details for PubMedCentralID PMC4785074

• Unique features of long non-coding RNA biogenesis and function NATURE REVIEWS GENETICS Quinn, J. J., Chang, H. Y. 2016; 17 (1): 47-62

  Abstract

  Long non-coding RNAs (lncRNAs) are a diverse class of RNAs that engage in numerous biological processes across every branch of life. Although initially discovered as mRNA-like transcripts that do not encode proteins, recent studies have revealed features of lncRNAs that further distinguish them from mRNAs. In this Review, we describe special events in the lifetimes of lncRNAs - before, during and after transcription - and discuss how these events ultimately shape the unique characteristics and functional roles of lncRNAs.

  View details for DOI 10.1038/nrg.2015.10

  View details for Web of Science ID 000366398700012

• The histone chaperone CAF-1 safeguards somatic cell identity NATURE Cheloufi, S., Elling, U., Hopfgartner, B., Jung, Y. L., Murn, J., Ninova, M., Hubmann, M., Badeaux, A. I., Ang, C. E., Tenen, D., Wesche, D. J., Abazova, N., Hogue, M., Tasdemir, N., Brumbaugh, J., Rathert, P., Jude, J., Ferrari, F., Blanco, A., Fellner, M., Wenzel, D., Zinner, M., Vidal, S. E., Bell, O., Stadtfeld, M., Chang, H. Y., Almouzni, G., Lowe, S. W., Rinn, J., Wernig, M., Aravin, A., Shi, Y., Park, P. J., Penninger, J. M., Zuber, J., Hochedlinger, K. 2015; 528 (7581): 218-?

  Abstract

  Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.

  View details for DOI 10.1038/nature15749

  View details for PubMedID 26659182

• Leukemia-Associated Cohesin Mutants Dominantly Enforce Stem Cell Programs and Impair Human Hematopoietic Progenitor Differentiation CELL STEM CELL Mazumdar, C., Shen, Y., Xavy, S., Zhao, F., Reinisch, A., Li, R., Corces, M. R., Flynn, R. A., Buenrostro, J. D., Chan, S. M., Thomas, D., Koenig, J. L., Hong, W., Chang, H. Y., Majeti, R. 2015; 17 (6): 1-14

  View details for DOI 10.1016/j.stem.2015.09.017

  View details for Web of Science ID 000366139100010

• Individuality and Variation of Personal Regulomes in Primary Human T Cells CELL SYSTEMS Qu, K., Zaba, L. C., Giresi, P. G., Li, R., Longmire, M., Kim, Y. H., Greenleaf, W. J., Chang, H. Y. 2015; 1 (1): 51-61

  Abstract

  Here we survey variation and dynamics of active regulatory elements genome-wide using longitudinal samples from human individuals. We applied Assay of Transposase Accessible Chromatin with sequencing (ATAC-seq) to map chromatin accessibility in primary CD4+ T cells isolated from standard blood draws of 12 healthy volunteers over time, from cancer patients, and during T cell activation. Over 4,000 predicted regulatory elements (7.2%) showed reproducible variation in accessibility between individuals. Gender was the most significant attributable source of variation. ATAC-seq revealed previously undescribed elements that escape X chromosome inactivation and predicted gender-specific gene regulatory networks across autosomes, which coordinately affect genes with immune function. Noisy regulatory elements with personal variation in accessibility are significantly enriched for autoimmune disease loci. Over one third of regulome variation lacked genetic variation in cis, suggesting contributions from environmental or epigenetic factors. These results refine concepts of human individuality and provide a foundational reference for comparing disease-associated regulomes.

  View details for DOI 10.1016/j.cels.2015.06.003

  View details for Web of Science ID 000209925400012

  View details for PubMedCentralID PMC4522940

• Single-cell chromatin accessibility reveals principles of regulatory variation NATURE Buenostro, J. D., Wu, B., Litzenburger, U. M., Ruff, D., Gonzales, M. L., Snyder, M. P., Chang, H. Y., Greenleaf, W. J. 2015; 523 (7561): 486-U264

  Abstract

  Cell-to-cell variation is a universal feature of life that affects a wide range of biological phenomena, from developmental plasticity to tumour heterogeneity. Although recent advances have improved our ability to document cellular phenotypic variation, the fundamental mechanisms that generate variability from identical DNA sequences remain elusive. Here we reveal the landscape and principles of mammalian DNA regulatory variation by developing a robust method for mapping the accessible genome of individual cells by assay for transposase-accessible chromatin using sequencing (ATAC-seq) integrated into a programmable microfluidics platform. Single-cell ATAC-seq (scATAC-seq) maps from hundreds of single cells in aggregate closely resemble accessibility profiles from tens of millions of cells and provide insights into cell-to-cell variation. Accessibility variance is systematically associated with specific trans-factors and cis-elements, and we discover combinations of trans-factors associated with either induction or suppression of cell-to-cell variability. We further identify sets of trans-factors associated with cell-type-specific accessibility variance across eight cell types. Targeted perturbations of cell cycle or transcription factor signalling evoke stimulus-specific changes in this observed variability. The pattern of accessibility variation in cis across the genome recapitulates chromosome compartments de novo, linking single-cell accessibility variation to three-dimensional genome organization. Single-cell analysis of DNA accessibility provides new insight into cellular variation of the 'regulome'.

  View details for DOI 10.1038/nature14590

  View details for Web of Science ID 000358378900042

• Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells. Nature Grow, E. J., Flynn, R. A., Chavez, S. L., Bayless, N. L., Wossidlo, M., Wesche, D. J., Martin, L., Ware, C. B., Blish, C. A., Chang, H. Y., Pera, R. A., Wysocka, J. 2015; 522 (7555): 221-225

  Abstract

  Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections, and comprise nearly 8% of the human genome. The most recently acquired human ERV is HERVK(HML-2), which repeatedly infected the primate lineage both before and after the divergence of the human and chimpanzee common ancestor. Unlike most other human ERVs, HERVK retained multiple copies of intact open reading frames encoding retroviral proteins. However, HERVK is transcriptionally silenced by the host, with the exception of in certain pathological contexts such as germ-cell tumours, melanoma or human immunodeficiency virus (HIV) infection. Here we demonstrate that DNA hypomethylation at long terminal repeat elements representing the most recent genomic integrations, together with transactivation by OCT4 (also known as POU5F1), synergistically facilitate HERVK expression. Consequently, HERVK is transcribed during normal human embryogenesis, beginning with embryonic genome activation at the eight-cell stage, continuing through the emergence of epiblast cells in preimplantation blastocysts, and ceasing during human embryonic stem cell derivation from blastocyst outgrowths. Remarkably, we detected HERVK viral-like particles and Gag proteins in human blastocysts, indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product, the HERVK accessory protein Rec, in a pluripotent cell line is sufficient to increase IFITM1 levels on the cell surface and inhibit viral infection, suggesting at least one mechanism through which HERVK can induce viral restriction pathways in early embryonic cells. Moreover, Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy, indicating that complex interactions between retroviral proteins and host factors can fine-tune pathways of early human development.

  View details for DOI 10.1038/nature14308

  View details for PubMedID 25896322

• Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells NATURE Grow, E. J., Flynn, R. A., Chavez, S. L., Bayless, N. L., Wossidlo, M., Wesche, D. J., Martin, L., Ware, C. B., Blish, C. A., Chang, H. Y., Pera, R. A., Wysocka, J. 2015; 522 (7555): 221-?

  Abstract

  Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections, and comprise nearly 8% of the human genome. The most recently acquired human ERV is HERVK(HML-2), which repeatedly infected the primate lineage both before and after the divergence of the human and chimpanzee common ancestor. Unlike most other human ERVs, HERVK retained multiple copies of intact open reading frames encoding retroviral proteins. However, HERVK is transcriptionally silenced by the host, with the exception of in certain pathological contexts such as germ-cell tumours, melanoma or human immunodeficiency virus (HIV) infection. Here we demonstrate that DNA hypomethylation at long terminal repeat elements representing the most recent genomic integrations, together with transactivation by OCT4 (also known as POU5F1), synergistically facilitate HERVK expression. Consequently, HERVK is transcribed during normal human embryogenesis, beginning with embryonic genome activation at the eight-cell stage, continuing through the emergence of epiblast cells in preimplantation blastocysts, and ceasing during human embryonic stem cell derivation from blastocyst outgrowths. Remarkably, we detected HERVK viral-like particles and Gag proteins in human blastocysts, indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product, the HERVK accessory protein Rec, in a pluripotent cell line is sufficient to increase IFITM1 levels on the cell surface and inhibit viral infection, suggesting at least one mechanism through which HERVK can induce viral restriction pathways in early embryonic cells. Moreover, Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy, indicating that complex interactions between retroviral proteins and host factors can fine-tune pathways of early human development.

  View details for DOI 10.1038/nature14308

  View details for Web of Science ID 000356016700040

  View details for PubMedID 25896322

  View details for PubMedCentralID PMC4503379

• Long Noncoding RNA in Hematopoiesis and Immunity IMMUNITY Satpathy, A. T., Chang, H. Y. 2015; 42 (5): 792-804

  Abstract

  Dynamic gene expression during cellular differentiation is tightly coordinated by transcriptional and post-transcriptional mechanisms. An emerging theme is the central role of long noncoding RNAs (lncRNAs) in the regulation of this specificity. Recent advances demonstrate that lncRNAs are expressed in a lineage-specific manner and control the development of several cell types in the hematopoietic system. Moreover, specific lncRNAs are induced to modulate innate and adaptive immune responses. lncRNAs can function via RNA-DNA, RNA-RNA, and RNA-protein target interactions. As a result, they affect several stages of gene regulation, including chromatin modification, mRNA biogenesis, and protein signaling. We discuss recent advances, future prospects, and challenges in understanding the roles of lncRNAs in immunity and immune-mediated diseases.

  View details for DOI 10.1016/j.immuni.2015.05.004

  View details for Web of Science ID 000354827400007

• Systematic Discovery of Xist RNA Binding Proteins CELL Chu, C., Zhang, Q. C., da Rocha, S. T., Flynn, R. A., Bharadwaj, M., Calabrese, J. M., Magnuson, T., Heard, E., Chang, H. Y. 2015; 161 (2): 404-416

  Abstract

  Noncoding RNAs (ncRNAs) function with associated proteins to effect complex structural and regulatory outcomes. To reveal the composition and dynamics of specific noncoding RNA-protein complexes (RNPs) in vivo, we developed comprehensive identification of RNA binding proteins by mass spectrometry (ChIRP-MS). ChIRP-MS analysis of four ncRNAs captures key protein interactors, including a U1-specific link to the 3' RNA processing machinery. Xist, an essential lncRNA for X chromosome inactivation (XCI), interacts with 81 proteins from chromatin modification, nuclear matrix, and RNA remodeling pathways. The Xist RNA-protein particle assembles in two steps coupled with the transition from pluripotency to differentiation. Specific interactors include HnrnpK, which participates in Xist-mediated gene silencing and histone modifications but not Xist localization, and Drosophila Split ends homolog Spen, which interacts via the A-repeat domain of Xist and is required for gene silencing. Thus, Xist lncRNA engages with proteins in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing.

  View details for DOI 10.1016/j.cell.2015.03.025

  View details for PubMedID 25843628

• Structural imprints in vivo decode RNA regulatory mechanisms. Nature Spitale, R. C., Flynn, R. A., Zhang, Q. C., Crisalli, P., Lee, B., Jung, J., Kuchelmeister, H. Y., Batista, P. J., Torre, E. A., Kool, E. T., Chang, H. Y. 2015; 519 (7544): 486-490

  Abstract

  Visualizing the physical basis for molecular behaviour inside living cells is a great challenge for biology. RNAs are central to biological regulation, and the ability of RNA to adopt specific structures intimately controls every step of the gene expression program. However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles include only two of the four nucleotides that make up RNA. Here we present a novel biochemical approach, in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE), which enables the first global view, to our knowledge, of RNA secondary structures in living cells for all four bases. icSHAPE of the mouse embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguish different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved from in vitro conditions, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA-protein interactions and N(6)-methyladenosine (m(6)A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.

  View details for DOI 10.1038/nature14263

  View details for PubMedID 25799993

• Structural imprints in vivo decode RNA regulatory mechanisms NATURE Spitale, R. C., Flynn, R. A., Zhang, Q. C., Crisalli, P., Lee, B., Jung, J., Kuchelmeister, H. Y., Batista, P. J., Torre, E. A., Kool, E. T., Chang, H. Y. 2015; 519 (7544): 486-?

  Abstract

  Visualizing the physical basis for molecular behaviour inside living cells is a great challenge for biology. RNAs are central to biological regulation, and the ability of RNA to adopt specific structures intimately controls every step of the gene expression program. However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles include only two of the four nucleotides that make up RNA. Here we present a novel biochemical approach, in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE), which enables the first global view, to our knowledge, of RNA secondary structures in living cells for all four bases. icSHAPE of the mouse embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguish different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved from in vitro conditions, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA-protein interactions and N(6)-methyladenosine (m(6)A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.

  View details for DOI 10.1038/nature14263

  View details for Web of Science ID 000351602800059

  View details for PubMedID 25799993

  View details for PubMedCentralID PMC4376618

• Detecting riboSNitches with RNA folding algorithms: a genome-wide benchmark NUCLEIC ACIDS RESEARCH Corley, M., Solem, A., Qu, K., Chang, H. Y., Laederach, A. 2015; 43 (3): 1859-1868

  Abstract

  Ribonucleic acid (RNA) secondary structure prediction continues to be a significant challenge, in particular when attempting to model sequences with less rigidly defined structures, such as messenger and non-coding RNAs. Crucial to interpreting RNA structures as they pertain to individual phenotypes is the ability to detect RNAs with large structural disparities caused by a single nucleotide variant (SNV) or riboSNitches. A recently published human genome-wide parallel analysis of RNA structure (PARS) study identified a large number of riboSNitches as well as non-riboSNitches, providing an unprecedented set of RNA sequences against which to benchmark structure prediction algorithms. Here we evaluate 11 different RNA folding algorithms' riboSNitch prediction performance on these data. We find that recent algorithms designed specifically to predict the effects of SNVs on RNA structure, in particular remuRNA, RNAsnp and SNPfold, perform best on the most rigorously validated subsets of the benchmark data. In addition, our benchmark indicates that general structure prediction algorithms (e.g. RNAfold and RNAstructure) have overall better performance if base pairing probabilities are considered rather than minimum free energy calculations. Although overall aggregate algorithmic performance on the full set of riboSNitches is relatively low, significant improvement is possible if the highest confidence predictions are evaluated independently.

  View details for DOI 10.1093/nar/gkv010

  View details for PubMedID 25618847

• RNA helicase DDX21 coordinates transcription and ribosomal RNA processing. Nature Calo, E., Flynn, R. A., Martin, L., Spitale, R. C., Chang, H. Y., Wysocka, J. 2015; 518 (7538): 249-253

  Abstract

  DEAD-box RNA helicases are vital for the regulation of various aspects of the RNA life cycle, but the molecular underpinnings of their involvement, particularly in mammalian cells, remain poorly understood. Here we show that the DEAD-box RNA helicase DDX21 can sense the transcriptional status of both RNA polymerase (Pol) I and II to control multiple steps of ribosome biogenesis in human cells. We demonstrate that DDX21 widely associates with Pol I- and Pol II-transcribed genes and with diverse species of RNA, most prominently with non-coding RNAs involved in the formation of ribonucleoprotein complexes, including ribosomal RNA, small nucleolar RNAs (snoRNAs) and 7SK RNA. Although broad, these molecular interactions, both at the chromatin and RNA level, exhibit remarkable specificity for the regulation of ribosomal genes. In the nucleolus, DDX21 occupies the transcribed rDNA locus, directly contacts both rRNA and snoRNAs, and promotes rRNA transcription, processing and modification. In the nucleoplasm, DDX21 binds 7SK RNA and, as a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, is recruited to the promoters of Pol II-transcribed genes encoding ribosomal proteins and snoRNAs. Promoter-bound DDX21 facilitates the release of the positive transcription elongation factor b (P-TEFb) from the 7SK snRNP in a manner that is dependent on its helicase activity, thereby promoting transcription of its target genes. Our results uncover the multifaceted role of DDX21 in multiple steps of ribosome biogenesis, and provide evidence implicating a mammalian RNA helicase in RNA modification and Pol II elongation control.

  View details for DOI 10.1038/nature13923

  View details for PubMedID 25470060

• RNA helicase DDX21 coordinates transcription and ribosomal RNA processing. Nature Calo, E., Flynn, R. A., Martin, L., Spitale, R. C., Chang, H. Y., Wysocka, J. 2015; 518 (7538): 249-253

  View details for DOI 10.1038/nature13923

  View details for PubMedID 25470060

• Structure and Thermodynamics of N-6-Methyladenosine in RNA: A Spring-Loaded Base Modification JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Roost, C., Lynch, S. R., Batista, P. J., Qu, K., Chang, H. Y., Kool, E. T. 2015; 137 (5): 2107-2115

  Abstract

  N(6)-Methyladenosine (m(6)A) modification is hypothesized to control processes such as RNA degradation, localization, and splicing. However, the molecular mechanisms by which this occurs are unclear. Here, we measured structures of an RNA duplex containing m(6)A in the GGACU consensus, along with an unmodified RNA control, by 2D NMR. The data show that m(6)A-U pairing in the double-stranded context is accompanied by the methylamino group rotating from its energetically preferred syn geometry on the Watson-Crick face to the higher-energy anti conformation, positioning the methyl group in the major groove. Thermodynamic measurements of m(6)A in duplexes reveal that it is destabilizing by 0.5-1.7 kcal/mol. In contrast, we show that m(6)A in unpaired positions base stacks considerably more strongly than the unmodified base, adding substantial stabilization in single-stranded locations. Transcriptome-wide nuclease mapping of methylated RNA secondary structure from human cells reveals a structural transition at methylated adenosines, with a tendency to single-stranded structure adjacent to the modified base.

  View details for DOI 10.1021/ja513080v

  View details for PubMedID 25611135

• Dissecting noncoding and pathogen RNA-protein interactomes RNA-A PUBLICATION OF THE RNA SOCIETY Flynn, R. A., Martin, L., Spitale, R. C., Do, B. T., Sagan, S. M., Zarnegar, B., Qu, K., Khavari, P. A., Quake, S. R., Sarnow, P., Chang, H. Y. 2015; 21 (1): 135-143

  Abstract

  RNA-protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA-protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and nonhuman RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and nonhuman transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2-bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA-protein recognition across the diversity of human and pathogen RNAs.

  View details for DOI 10.1261/rna.047803.114

  View details for PubMedID 25411354

• A novel ATAC-seq approach reveals lineage-specific reinforcement of the open chromatin landscape via cooperation between BAF and p63. Genome biology Bao, X., Rubin, A. J., Qu, K., Zhang, J., Giresi, P. G., Chang, H. Y., Khavari, P. A. 2015; 16 (1): 284-?

  Abstract

  Open chromatin regions are correlated with active regulatory elements in development and are dysregulated in diseases. The BAF (SWI/SNF) complex is essential for development, and has been demonstrated to remodel reconstituted chromatin in vitro and to control the accessibility of a few individual regions in vivo. However, it remains unclear where and how BAF controls the open chromatin landscape to regulate developmental processes, such as human epidermal differentiation.Using a novel "on-plate" ATAC-sequencing approach for profiling open chromatin landscapes with a low number of adherent cells, we demonstrate that the BAF complex is essential for maintaining 11.6 % of open chromatin regions in epidermal differentiation. These BAF-dependent open chromatin regions are highly cell-type-specific and are strongly enriched for binding sites for p63, a master epidermal transcription factor. The DNA sequences of p63 binding sites intrinsically favor nucleosome formation and are inaccessible in other cell types without p63 to prevent ectopic activation. In epidermal cells, BAF and p63 mutually recruit each other to maintain 14,853 open chromatin regions. We further demonstrate that BAF and p63 cooperatively position nucleosomes away from p63 binding sites and recruit transcriptional machinery to control tissue differentiation.BAF displays high specificity in controlling the open chromatin landscape during epidermal differentiation by cooperating with the master transcription factor p63 to maintain lineage-specific open chromatin regions.

  View details for DOI 10.1186/s13059-015-0840-9

  View details for PubMedID 26683334

• Technologies to probe functions and mechanisms of long noncoding RNAs NATURE STRUCTURAL & MOLECULAR BIOLOGY Chu, C., Spitale, R. C., Chang, H. Y. 2015; 22 (1): 29-35

  Abstract

  Thousands of long noncoding RNAs (lncRNAs) have been discovered, but their functional characterization has been slowed by a limited set of research tools. Here we review emerging RNA-centric methods to interrogate the intrinsic structure of lncRNAs as well as their genomic localization and biochemical partners. Understanding these technologies, including their advantages and caveats, and developing them in the future will be essential to progress from description to comprehension of the myriad roles of lncRNAs.

  View details for DOI 10.1038/nsmb.2921

  View details for PubMedID 25565030

• m(6)A RNA Modification Controls Cell Fate Transition in Mammalian Embryonic Stem Cells. Cell stem cell Batista, P. J., Molinie, B., Wang, J., Qu, K., Zhang, J., Li, L., Bouley, D. M., Lujan, E., Haddad, B., Daneshvar, K., Carter, A. C., Flynn, R. A., Zhou, C., Lim, K., Dedon, P., Wernig, M., Mullen, A. C., Xing, Y., Giallourakis, C. C., Chang, H. Y. 2014; 15 (6): 707-719

  Abstract

  N6-methyl-adenosine (m(6)A) is the most abundant modification on messenger RNAs and is linked to human diseases, but its functions in mammalian development are poorly understood. Here we reveal the evolutionary conservation and function of m(6)A by mapping the m(6)A methylome in mouse and human embryonic stem cells. Thousands of messenger and long noncoding RNAs show conserved m(6)A modification, including transcripts encoding core pluripotency transcription factors. m(6)A is enriched over 3' untranslated regions at defined sequence motifs and marks unstable transcripts, including transcripts turned over upon differentiation. Genetic inactivation or depletion of mouse and human Mettl3, one of the m(6)A methylases, led to m(6)A erasure on select target genes, prolonged Nanog expression upon differentiation, and impaired ESC exit from self-renewal toward differentiation into several lineages in vitro and in vivo. Thus, m(6)A is a mark of transcriptome flexibility required for stem cells to differentiate to specific lineages.

  View details for DOI 10.1016/j.stem.2014.09.019

  View details for PubMedID 25456834

• RNA structural analysis by evolving SHAPE chemistry WILEY INTERDISCIPLINARY REVIEWS-RNA Spitale, R. C., Flynn, R. A., Torre, E. A., Kool, E. T., Chang, H. Y. 2014; 5 (6): 867-881

  Abstract

  RNA is central to the flow of biological information. From transcription to splicing, RNA localization, translation, and decay, RNA is intimately involved in regulating every step of the gene expression program, and is thus essential for health and understanding disease. RNA has the unique ability to base-pair with itself and other nucleic acids to form complex structures. Hence the information content in RNA is not simply its linear sequence of bases, but is also encoded in complex folding of RNA molecules. A general chemical functionality that all RNAs have is a 2'-hydroxyl group in the ribose ring, and the reactivity of the 2'-hydroxyl in RNA is gated by local nucleotide flexibility. In other words, the 2'-hydroxyl is reactive at single-stranded and conformationally flexible positions but is unreactive at nucleotides constrained by base-pairing. Recent efforts have been focused on developing reagents that modify RNA as a function of RNA 2' hydroxyl group reactivity. Such RNA structure probing techniques can be read out by primer extension in experiments termed RNA SHAPE (selective 2'- hydroxyl acylation and primer extension). Herein, we describe the efforts devoted to the design and utilization of SHAPE probes for characterizing RNA structure. We also describe current technological advances that are being applied to utilize SHAPE chemistry with deep sequencing to probe many RNAs in parallel. The merging of chemistry with genomics is sure to open the door to genome-wide exploration of RNA structure and function.

  View details for DOI 10.1002/wrna.1253

  View details for Web of Science ID 000344453000009

• Physiological roles of long noncoding RNAs: insight from knockout mice TRENDS IN CELL BIOLOGY Li, L., Chang, H. Y. 2014; 24 (10): 594-602

  Abstract

  Long noncoding RNAs (lncRNAs) are a pervasive and recently recognized class of genes. lncRNAs have been proposed to modulate gene expression and nuclear architecture, but their physiological functions are still largely unclear. Several recent efforts to inactivate lncRNA genes in mouse models have shed light on their functions. Different genetic strategies have yielded specific lessons about the roles of lncRNA transcription, the lncRNA transcript itself, and underlying sequence elements. Current results indicate important functions for lncRNAs in organ development, immunity, organismal viability, and in human diseases.

  View details for DOI 10.1016/j.tcb.2014.06.003

  View details for Web of Science ID 000343630000006

  View details for PubMedCentralID PMC4177945

• An Integrated Cell Purification and Genomics Strategy Reveals Multiple Regulators of Pancreas Development PLOS GENETICS Benitez, C. M., Qu, K., Sugiyama, T., Pauerstein, P. T., Liu, Y., Tsai, J., Gu, X., Ghodasara, A., Arda, H. E., Zhang, J., Dekker, J. D., Tucker, H. O., Chang, H. Y., Kim, S. K. 2014; 10 (10)

  Abstract

  The regulatory logic underlying global transcriptional programs controlling development of visceral organs like the pancreas remains undiscovered. Here, we profiled gene expression in 12 purified populations of fetal and adult pancreatic epithelial cells representing crucial progenitor cell subsets, and their endocrine or exocrine progeny. Using probabilistic models to decode the general programs organizing gene expression, we identified co-expressed gene sets in cell subsets that revealed patterns and processes governing progenitor cell development, lineage specification, and endocrine cell maturation. Purification of Neurog3 mutant cells and module network analysis linked established regulators such as Neurog3 to unrecognized gene targets and roles in pancreas development. Iterative module network analysis nominated and prioritized transcriptional regulators, including diabetes risk genes. Functional validation of a subset of candidate regulators with corresponding mutant mice revealed that the transcription factors Etv1, Prdm16, Runx1t1 and Bcl11a are essential for pancreas development. Our integrated approach provides a unique framework for identifying regulatory genes and functional gene sets underlying pancreas development and associated diseases such as diabetes mellitus.

  View details for DOI 10.1371/journal.pgen.1004645

  View details for Web of Science ID 000344650700023

  View details for PubMedCentralID PMC4199491

• Revealing long noncoding RNA architecture and functions using domain-specific chromatin isolation by RNA purification. Nature biotechnology Quinn, J. J., Ilik, I. A., Qu, K., Georgiev, P., Chu, C., Akhtar, A., Chang, H. Y. 2014; 32 (9): 933-40

  Abstract

  Little is known about the functional domain architecture of long noncoding RNAs (lncRNAs) because of a relative paucity of suitable methods to analyze RNA function at a domain level. Here we describe domain-specific chromatin isolation by RNA purification (dChIRP), a scalable technique to dissect pairwise RNA-RNA, RNA-protein and RNA-chromatin interactions at the level of individual RNA domains in living cells. dChIRP of roX1, a lncRNA essential for Drosophila melanogaster X-chromosome dosage compensation, reveals a 'three-fingered hand' ribonucleoprotein topology. Each RNA finger binds chromatin and the male-specific lethal (MSL) protein complex and can individually rescue male lethality in roX-null flies, thus defining a minimal RNA domain for chromosome-wide dosage compensation. dChIRP improves the RNA genomic localization signal by >20-fold relative to previous techniques, and these binding sites are correlated with chromosome conformation data, indicating that most roX-bound loci cluster in a nuclear territory. These results suggest dChIRP can reveal lncRNA architecture and function with high precision and sensitivity.

  View details for DOI 10.1038/nbt.2943

  View details for PubMedID 24997788

• Revealing long noncoding RNA architecture and functions using domain-specific chromatin isolation by RNA purification NATURE BIOTECHNOLOGY Quinn, J. J., Ilik, I. A., Qu, K., Georgiev, P., Chu, C., Alchtar, A., Chang, H. Y. 2014; 32 (9): 933-940

  Abstract

  Little is known about the functional domain architecture of long noncoding RNAs (lncRNAs) because of a relative paucity of suitable methods to analyze RNA function at a domain level. Here we describe domain-specific chromatin isolation by RNA purification (dChIRP), a scalable technique to dissect pairwise RNA-RNA, RNA-protein and RNA-chromatin interactions at the level of individual RNA domains in living cells. dChIRP of roX1, a lncRNA essential for Drosophila melanogaster X-chromosome dosage compensation, reveals a 'three-fingered hand' ribonucleoprotein topology. Each RNA finger binds chromatin and the male-specific lethal (MSL) protein complex and can individually rescue male lethality in roX-null flies, thus defining a minimal RNA domain for chromosome-wide dosage compensation. dChIRP improves the RNA genomic localization signal by >20-fold relative to previous techniques, and these binding sites are correlated with chromosome conformation data, indicating that most roX-bound loci cluster in a nuclear territory. These results suggest dChIRP can reveal lncRNA architecture and function with high precision and sensitivity.

  View details for DOI 10.1038/nbt.2943

  View details for Web of Science ID 000342600300033

• RNA switch at enhancers. Nature genetics Quinn, J. J., Chang, H. Y. 2014; 46 (9): 929-931

  Abstract

  Polycomb/Trithorax response elements (PRE/TREs) are genetic elements that can stably silence or activate genes. A new study describes how long noncoding RNAs (lncRNAs) transcribed from opposite strands of the Drosophila melanogaster vestigial PRE/TRE throw the switch between these two opposing epigenetic states.

  View details for DOI 10.1038/ng.3074

  View details for PubMedID 25162802

• Dicer-microRNA-Myc circuit promotes transcription of hundreds of long noncoding RNAs NATURE STRUCTURAL & MOLECULAR BIOLOGY Zheng, G. X., Do, B. T., Webster, D. E., Khavari, P. A., Chang, H. Y. 2014; 21 (7): 585-590

  Abstract

  Long noncoding RNAs (lncRNAs) are important regulators of cell fate, yet little is known about mechanisms controlling lncRNA expression. Here we show that transcription is quantitatively different for lncRNAs and mRNAs--as revealed by deficiency of Dicer (Dcr), a key RNase that generates microRNAs (miRNAs). Dcr loss in mouse embryonic stem cells led unexpectedly to decreased levels of hundreds of lncRNAs. The canonical Dgcr8-Dcr-miRNA pathway is required for robust lncRNA transcriptional initiation and elongation. Computational and genetic epistasis analyses demonstrated that Dcr activation of the oncogenic transcription factor cMyc is partly responsible for lncRNA expression. A quantitative metric of mRNA-lncRNA decoupling revealed that Dcr and cMyc differentially regulate lncRNAs versus mRNAs in diverse cell types and in vivo. Thus, numerous lncRNAs may be modulated as a class in development and disease, notably where Dcr and cMyc act.

  View details for DOI 10.1038/nsmb.2842

  View details for Web of Science ID 000338689800005

• Dicer-microRNA-Myc circuit promotes transcription of hundreds of long noncoding RNAs. Nature structural & molecular biology Zheng, G. X., Do, B. T., Webster, D. E., Khavari, P. A., Chang, H. Y. 2014; 21 (7): 585-590

  Abstract

  Long noncoding RNAs (lncRNAs) are important regulators of cell fate, yet little is known about mechanisms controlling lncRNA expression. Here we show that transcription is quantitatively different for lncRNAs and mRNAs--as revealed by deficiency of Dicer (Dcr), a key RNase that generates microRNAs (miRNAs). Dcr loss in mouse embryonic stem cells led unexpectedly to decreased levels of hundreds of lncRNAs. The canonical Dgcr8-Dcr-miRNA pathway is required for robust lncRNA transcriptional initiation and elongation. Computational and genetic epistasis analyses demonstrated that Dcr activation of the oncogenic transcription factor cMyc is partly responsible for lncRNA expression. A quantitative metric of mRNA-lncRNA decoupling revealed that Dcr and cMyc differentially regulate lncRNAs versus mRNAs in diverse cell types and in vivo. Thus, numerous lncRNAs may be modulated as a class in development and disease, notably where Dcr and cMyc act.

  View details for DOI 10.1038/nsmb.2842

  View details for PubMedID 24929436

• Long noncoding RNAs in cell-fate programming and reprogramming. Cell stem cell Flynn, R. A., Chang, H. Y. 2014; 14 (6): 752-761

  Abstract

  In recent years, long noncoding RNAs (lncRNAs) have emerged as an important class of regulators of gene expression. lncRNAs exhibit several distinctive features that confer unique regulatory functions, including exquisite cell- and tissue-specific expression and the capacity to transduce higher-order spatial information. Here we review evidence showing that lncRNAs exert critical functions in adult tissue stem cells, including skin, brain, and muscle, as well as in developmental patterning and pluripotency. We highlight new approaches for ascribing lncRNA functions and discuss mammalian dosage compensation as a classic example of an lncRNA network coupled to stem cell differentiation.

  View details for DOI 10.1016/j.stem.2014.05.014

  View details for PubMedID 24905165

• Quantitative analysis of RNA-protein interactions on a massively parallel array reveals biophysical and evolutionary landscapes. Nature biotechnology Buenrostro, J. D., Araya, C. L., Chircus, L. M., Layton, C. J., Chang, H. Y., Snyder, M. P., Greenleaf, W. J. 2014; 32 (6): 562-568

  Abstract

  RNA-protein interactions drive fundamental biological processes and are targets for molecular engineering, yet quantitative and comprehensive understanding of the sequence determinants of affinity remains limited. Here we repurpose a high-throughput sequencing instrument to quantitatively measure binding and dissociation of a fluorescently labeled protein to >10(7) RNA targets generated on a flow cell surface by in situ transcription and intermolecular tethering of RNA to DNA. Studying the MS2 coat protein, we decompose the binding energy contributions from primary and secondary RNA structure, and observe that differences in affinity are often driven by sequence-specific changes in both association and dissociation rates. By analyzing the biophysical constraints and modeling mutational paths describing the molecular evolution of MS2 from low- to high-affinity hairpins, we quantify widespread molecular epistasis and a long-hypothesized, structure-dependent preference for G:U base pairs over C:A intermediates in evolutionary trajectories. Our results suggest that quantitative analysis of RNA on a massively parallel array (RNA-MaP) provides generalizable insight into the biophysical basis and evolutionary consequences of sequence-function relationships.

  View details for DOI 10.1038/nbt.2880

  View details for PubMedID 24727714

• Quantitative analysis of RNA-protein interactions on a massively parallel array reveals biophysical and evolutionary landscapes. Nature biotechnology Buenrostro, J. D., Araya, C. L., Chircus, L. M., Layton, C. J., Chang, H. Y., Snyder, M. P., Greenleaf, W. J. 2014; 32 (6): 562-568

  Abstract

  RNA-protein interactions drive fundamental biological processes and are targets for molecular engineering, yet quantitative and comprehensive understanding of the sequence determinants of affinity remains limited. Here we repurpose a high-throughput sequencing instrument to quantitatively measure binding and dissociation of a fluorescently labeled protein to >10(7) RNA targets generated on a flow cell surface by in situ transcription and intermolecular tethering of RNA to DNA. Studying the MS2 coat protein, we decompose the binding energy contributions from primary and secondary RNA structure, and observe that differences in affinity are often driven by sequence-specific changes in both association and dissociation rates. By analyzing the biophysical constraints and modeling mutational paths describing the molecular evolution of MS2 from low- to high-affinity hairpins, we quantify widespread molecular epistasis and a long-hypothesized, structure-dependent preference for G:U base pairs over C:A intermediates in evolutionary trajectories. Our results suggest that quantitative analysis of RNA on a massively parallel array (RNA-MaP) provides generalizable insight into the biophysical basis and evolutionary consequences of sequence-function relationships.

  View details for DOI 10.1038/nbt.2880

  View details for PubMedID 24727714

• Identification of genes promoting skin youthfulness by genome-wide association study. journal of investigative dermatology Chang, A. L., Atzmon, G., Bergman, A., Brugmann, S., Atwood, S. X., Chang, H. Y., Barzilai, N. 2014; 134 (3): 651-657

  Abstract

  To identify genes that promote facial skin youthfulness (SY), a genome-wide association study on an Ashkenazi Jewish discovery group (n=428) was performed using Affymetrix 6.0 Single-Nucleotide Polymorphism (SNP) Array. After SNP quality controls, 901,470 SNPs remained for analysis. The eigenstrat method showed no stratification. Cases and controls were identified by global facial skin aging severity including intrinsic and extrinsic parameters. Linear regression adjusted for age and gender, with no significant differences in smoking history, body mass index, menopausal status, or personal or family history of centenarians. Six SNPs met the Bonferroni threshold with Pallele<10(-8); two of these six had Pgenotype<10(-8). Quantitative trait loci mapping confirmed linkage disequilibrium. The six SNPs were interrogated by MassARRAY in a replication group (n=436) with confirmation of rs6975107, an intronic region of KCND2 (potassium voltage-gated channel, Shal-related family member 2) (Pgenotype=0.023). A second replication group (n=371) confirmed rs318125, downstream of DIAPH2 (diaphanous homolog 2 (Drosophila)) (Pallele=0.010, Pgenotype=0.002) and rs7616661, downstream of EDEM1 (ER degradation enhancer, mannosidase α-like 1) (Pgenotype=0.042). DIAPH2 has been associated with premature ovarian insufficiency, an aging phenotype in humans. EDEM1 associates with lifespan in animal models, although not humans. KCND2 is expressed in human skin, but has not been associated with aging. These genes represent new candidate genes to study the molecular basis of healthy skin aging.

  View details for DOI 10.1038/jid.2013.381

  View details for PubMedID 24037343

• Landscape and variation of RNA secondary structure across the human transcriptome. Nature Wan, Y., Qu, K., Zhang, Q. C., Flynn, R. A., Manor, O., Ouyang, Z., Zhang, J., Spitale, R. C., Snyder, M. P., Segal, E., Chang, H. Y. 2014; 505 (7485): 706-709

  Abstract

  In parallel to the genetic code for protein synthesis, a second layer of information is embedded in all RNA transcripts in the form of RNA structure. RNA structure influences practically every step in the gene expression program. However, the nature of most RNA structures or effects of sequence variation on structure are not known. Here we report the initial landscape and variation of RNA secondary structures (RSSs) in a human family trio (mother, father and their child). This provides a comprehensive RSS map of human coding and non-coding RNAs. We identify unique RSS signatures that demarcate open reading frames and splicing junctions, and define authentic microRNA-binding sites. Comparison of native deproteinized RNA isolated from cells versus refolded purified RNA suggests that the majority of the RSS information is encoded within RNA sequence. Over 1,900 transcribed single nucleotide variants (approximately 15% of all transcribed single nucleotide variants) alter local RNA structure. We discover simple sequence and spacing rules that determine the ability of point mutations to impact RSSs. Selective depletion of 'riboSNitches' versus structurally synonymous variants at precise locations suggests selection for specific RNA shapes at thousands of sites, including 3' untranslated regions, binding sites of microRNAs and RNA-binding proteins genome-wide. These results highlight the potentially broad contribution of RNA structure and its variation to gene regulation.

  View details for DOI 10.1038/nature12946

  View details for PubMedID 24476892

• Essential role of lncRNA binding for WDR5 maintenance of active chromatin and embryonic stem cell pluripotency. eLife Yang, Y. W., Flynn, R. A., Chen, Y., Qu, K., Wan, B., Wang, K. C., Lei, M., Chang, H. Y. 2014; 3

  Abstract

  The WDR5 subunit of the MLL complex enforces active chromatin and can bind RNA; the relationship between these two activities is unclear. Here we identify a RNA binding pocket on WDR5, and discover a WDR5 mutant (F266A) that selectively abrogates RNA binding without affecting MLL complex assembly or catalytic activity. Complementation in ESCs shows that WDR5 F266A mutant is unable to accumulate on chromatin, and is defective in gene activation, maintenance of histone H3 lysine 4 trimethylation, and ESC self renewal. We identify a family of ESC messenger and lncRNAs that interact with wild type WDR5 but not F266A mutant, including several lncRNAs known to be important for ESC gene expression. These results suggest that specific RNAs are integral inputs into the WDR5-MLL complex for maintenance of the active chromatin state and embryonic stem cell fates. DOI: http://dx.doi.org/10.7554/eLife.02046.001.

  View details for DOI 10.7554/eLife.02046

  View details for PubMedID 24521543

• Essential role of lncRNA binding for WDR5 maintenance of active chromatin and embryonic stem cell pluripotency. eLife Yang, Y. W., Flynn, R. A., Chen, Y., Qu, K., Wan, B., Wang, K. C., Lei, M., Chang, H. Y. 2014; 3

  View details for DOI 10.7554/eLife.02046

  View details for PubMedID 24521543

• Identification of a Selective Polymerase Enables Detection of N-6-Methyladenosine in RNA JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Harcourt, E. M., Ehrenschwender, T., Batista, P. J., Chang, H. Y., Kool, E. T. 2013; 135 (51): 19079-19082

  Abstract

  N(6)-methyladenosine (m(6)A) is the most abundant mRNA modification and has important links to human health. While recent studies have successfully identified thousands of mammalian RNA transcripts containing the modification, it is extremely difficult to identify the exact location of any specific m(6)A. Here we have identified a polymerase with reverse transcriptase activity (from Thermus thermophilus) that is selective by up to 18-fold for incorporation of thymidine opposite unmodified A over m(6)A. We show that the enzyme can be used to locate and quantify m(6)A in synthetic RNAs by analysis of pausing bands, and have used the enzyme in tandem with a nonselective polymerase to locate the presence and position of m(6)A in high-abundance cellular RNAs. By this approach we demonstrate that the long-undetermined position of m(6)A in mammalian 28S rRNA is nucleotide 4190.

  View details for DOI 10.1021/ja4105792

  View details for PubMedID 24328136

• Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nature methods Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y., Greenleaf, W. J. 2013; 10 (12): 1213-1218

  Abstract

  We describe an assay for transposase-accessible chromatin using sequencing (ATAC-seq), based on direct in vitro transposition of sequencing adaptors into native chromatin, as a rapid and sensitive method for integrative epigenomic analysis. ATAC-seq captures open chromatin sites using a simple two-step protocol with 500-50,000 cells and reveals the interplay between genomic locations of open chromatin, DNA-binding proteins, individual nucleosomes and chromatin compaction at nucleotide resolution. We discovered classes of DNA-binding factors that strictly avoided, could tolerate or tended to overlap with nucleosomes. Using ATAC-seq maps of human CD4(+) T cells from a proband obtained on consecutive days, we demonstrated the feasibility of analyzing an individual's epigenome on a timescale compatible with clinical decision-making.

  View details for DOI 10.1038/nmeth.2688

  View details for PubMedID 24097267

• Hierarchical Mechanisms for Direct Reprogramming of Fibroblasts to Neurons CELL Wapinski, O. L., Vierbuchen, T., Qu, K., Lee, Q. Y., Chanda, S., Fuentes, D. R., Giresi, P. G., Ng, Y. H., Marro, S., Neff, N. F., Drechsel, D., Martynoga, B., Castro, D. S., Webb, A. E., Suedhof, T. C., Brunet, A., Guillemot, F., Chang, H. Y., Wernig, M. 2013; 155 (3): 621-635

  Abstract

  Direct lineage reprogramming is a promising approach for human disease modeling and regenerative medicine, with poorly understood mechanisms. Here, we reveal a hierarchical mechanism in the direct conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an "on-target" pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myt1l do not access fibroblast chromatin productively on their own; instead, Ascl1 recruits Brn2 to Ascl1 sites genome wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, we identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, a precise match between pioneer factors and the chromatin context at key target genes is determinative for transdifferentiation to neurons and likely other cell types.

  View details for DOI 10.1016/j.cell.2013.09.028

  View details for PubMedID 24243019

• Augmenting Endogenous Wnt Signaling Improves Skin Wound Healing PLOS ONE Whyte, J. L., Smith, A. A., Liu, B., Manzano, W. R., Evans, N. D., Dhamdhere, G. R., Fang, M. Y., Chang, H. Y., Oro, A. E., Helms, J. A. 2013; 8 (10)

  Abstract

  Wnt signaling is required for both the development and homeostasis of the skin, yet its contribution to skin wound repair remains controversial. By employing Axin2(LacZ/+) reporter mice we evaluated the spatial and temporal distribution patterns of Wnt responsive cells, and found that the pattern of Wnt responsiveness varies with the hair cycle, and correlates with wound healing potential. Using Axin2(LacZ/LacZ) mice and an ear wound model, we demonstrate that amplified Wnt signaling leads to improved healing. Utilizing a biochemical approach that mimics the amplified Wnt response of Axin2(LacZ/LacZ) mice, we show that topical application of liposomal Wnt3a to a non-healing wound enhances endogenous Wnt signaling, and results in better skin wound healing. Given the importance of Wnt signaling in the maintenance and repair of skin, liposomal Wnt3a may have widespread application in clinical practice.

  View details for DOI 10.1371/journal.pone.0076883

  View details for Web of Science ID 000326029300061

  View details for PubMedID 24204695

  View details for PubMedCentralID PMC3799989

• Targeted disruption of hotair leads to homeotic transformation and gene derepression. Cell reports Li, L., Liu, B., Wapinski, O. L., Tsai, M., Qu, K., Zhang, J., Carlson, J. C., Lin, M., Fang, F., Gupta, R. A., Helms, J. A., Chang, H. Y. 2013; 5 (1): 3-12

  Abstract

  Long noncoding RNAs (lncRNAs) are thought to be prevalent regulators of gene expression, but the consequences of lncRNA inactivation in vivo are mostly unknown. Here, we show that targeted deletion of mouse Hotair lncRNA leads to derepression of hundreds of genes, resulting in homeotic transformation of the spine and malformation of metacarpal-carpal bones. RNA sequencing and conditional inactivation reveal an ongoing requirement of Hotair to repress HoxD genes and several imprinted loci such as Dlk1-Meg3 and Igf2-H19 without affecting imprinting choice. Hotair binds to both Polycomb repressive complex 2, which methylates histone H3 at lysine 27 (H3K27), and Lsd1 complex, which demethylates histone H3 at lysine 4 (H3K4) in vivo. Hotair inactivation causes H3K4me3 gain and, to a lesser extent, H3K27me3 loss at target genes. These results reveal the function and mechanisms of Hotair lncRNA in enforcing a silent chromatin state at Hox and additional genes.

  View details for DOI 10.1016/j.celrep.2013.09.003

  View details for PubMedID 24075995

• HOXB13 Mediates Tamoxifen Resistance and Invasiveness in Human Breast Cancer by Suppressing ERa and Inducing IL-6 Expression. Cancer research Shah, N., Jin, K., Cruz, L., Park, S., Sadik, H., Cho, S., Goswami, C. P., Nakshatri, H., Gupta, R., Chang, H. Y., Zhang, Z., Cimino-Mathews, A., Cope, L., Umbricht, C., Sukumar, S. 2013; 73 (17): 5449-5458

  Abstract

  Most breast cancers expressing the estrogen receptor α (ERα) are treated successfully with the receptor antagonist tamoxifen (TAM), but many of these tumors recur. Elevated expression of the homeodomain transcription factor HOXB13 correlates with TAM-resistance in ERα-positive (ER+) breast cancer, but little is known regarding the underlying mechanism. Our comprehensive evaluation of HOX gene expression using tiling microarrays, with validation, showed that distant metastases from TAM-resistant patients also displayed high HOXB13 expression, suggesting a role for HOXB13 in tumor dissemination and survival. Here we show that HOXB13 confers TAM resistance by directly downregulating ERα transcription and protein expression. HOXB13 elevation promoted cell proliferation in vitro and growth of tumor xenografts in vivo. Mechanistic investigations showed that HOXB13 transcriptionally upregulated interleukin (IL)-6, activating the mTOR pathway via STAT3 phosphorylation to promote cell proliferation and fibroblast recruitment. Accordingly, mTOR inhibition suppressed fibroblast recruitment and proliferation of HOXB13-expressing ER+ breast cancer cells and tumor xenografts, alone or in combination with TAM. Taken together, our results establish a function for HOXB13 in TAM resistance through direct suppression of ERα and they identify the IL-6 pathways as mediator of disease progression and recurrence. Cancer Res; 73(17); 5449-58. ©2013 AACR.

  View details for DOI 10.1158/0008-5472.CAN-13-1178

  View details for PubMedID 23832664

• Tandem Stem-Loops in roX RNAs Act Together to Mediate X Chromosome Dosage Compensation in Drosophila MOLECULAR CELL Ilik, I. A., Quinn, J. J., Georgiev, P., Tavares-Cadete, F., Maticzka, D., Toscano, S., Wan, Y., Spitale, R. C., Luscombe, N., Backofen, R., Chang, H. Y., Akhtar, A. 2013; 51 (2): 156-173

  Abstract

  Dosage compensation in Drosophila is an epigenetic phenomenon utilizing proteins and long noncoding RNAs (lncRNAs) for transcriptional upregulation of the male X chromosome. Here, by using UV crosslinking followed by deep sequencing, we show that two enzymes in the Male-Specific Lethal complex, MLE RNA helicase and MSL2 ubiquitin ligase, bind evolutionarily conserved domains containing tandem stem-loops in roX1 and roX2 RNAs in vivo. These domains constitute the minimal RNA unit present in multiple copies in diverse arrangements for nucleation of the MSL complex. MLE binds to these domains with distinct ATP-independent and ATP-dependent behavior. Importantly, we show that different roX RNA domains have overlapping function, since only combinatorial mutations in the tandem stem-loops result in severe loss of dosage compensation and consequently male-specific lethality. We propose that repetitive structural motifs in lncRNAs could provide plasticity during multiprotein complex assemblies to ensure efficient targeting in cis or in trans along chromosomes.

  View details for DOI 10.1016/j.molcel.2013.07.001

  View details for Web of Science ID 000322424200005

  View details for PubMedID 23870142

• Genome-wide mapping of RNA structure using nuclease digestion and high-throughput sequencing. Nature protocols Wan, Y., Qu, K., Ouyang, Z., Chang, H. Y. 2013; 8 (5): 849-869

  Abstract

  RNA structure is important for RNA function and regulation, and there is growing interest in determining the RNA structure of many transcripts. Here we provide a detailed protocol for the parallel analysis of RNA structure (PARS) for probing RNA secondary structures genome-wide. In this method, enzymatic footprinting is coupled to high-throughput sequencing to provide secondary structure data for thousands of RNAs simultaneously. The entire experimental protocol takes ∼5 d to complete, and sequencing and data analysis take an additional 6-8 d. PARS was developed using the yeast genome as proof of principle, but its approach should be applicable to probing RNA structures from different transcriptomes and structural dynamics under diverse solution conditions.

  View details for DOI 10.1038/nprot.2013.045

  View details for PubMedID 23558785

• Cytotopic localization by long noncoding RNAs CURRENT OPINION IN CELL BIOLOGY Batista, P. J., Chang, H. Y. 2013; 25 (2): 195-199

  Abstract

  Cells are highly organized structures. In addition to membrane delimited organelles, proteins and RNAs can organize themselves into specific domains. Some examples include stress granules and subnuclear bodies. This level of organization is essential for the correct execution of multiple processes in the cell, ranging from cell signaling to assembly of structures such as the ribosomes. Here we will review evidence that noncoding RNAs play a critical role in the establishment and regulation of these domains. The unique abilities of RNA to mark the genome in a gene-specific and condition-specific manner and to serve as tethers nominate them as ideal molecular address codes.

  View details for DOI 10.1016/j.ceb.2012.12.001

  View details for Web of Science ID 000317886100008

  View details for PubMedID 23279909

  View details for PubMedCentralID PMC3633454

• Long Noncoding RNAs: Cellular Address Codes in Development and Disease CELL Batista, P. J., Chang, H. Y. 2013; 152 (6): 1298-1307

  Abstract

  In biology as in real estate, location is a cardinal organizational principle that dictates the accessibility and flow of informational traffic. An essential question in nuclear organization is the nature of the address code--how objects are placed and later searched for and retrieved. Long noncoding RNAs (lncRNAs) have emerged as key components of the address code, allowing protein complexes, genes, and chromosomes to be trafficked to appropriate locations and subject to proper activation and deactivation. lncRNA-based mechanisms control cell fates during development, and their dysregulation underlies some human disorders caused by chromosomal deletions and translocations.

  View details for DOI 10.1016/j.cell.2013.02.012

  View details for PubMedID 23498938

• The NeST Long ncRNA Controls Microbial Susceptibility and Epigenetic Activation of the Interferon-gamma Locus CELL Gomez, J. A., Wapinski, O. L., Yang, Y. W., Bureau, J., Gopinath, S., Monack, D. M., Chang, H. Y., Brahic, M., Kirkegaard, K. 2013; 152 (4): 743-754

  Abstract

  Long noncoding RNAs (lncRNAs) are increasingly appreciated as regulators of cell-specific gene expression. Here, an enhancer-like lncRNA termed NeST (nettoie Salmonella pas Theiler's [cleanup Salmonella not Theiler's]) is shown to be causal for all phenotypes conferred by murine viral susceptibility locus Tmevp3. This locus was defined by crosses between SJL/J and B10.S mice and contains several candidate genes, including NeST. The SJL/J-derived locus confers higher lncRNA expression, increased interferon-γ (IFN-γ) abundance in activated CD8(+) T cells, increased Theiler's virus persistence, and decreased Salmonella enterica pathogenesis. Transgenic expression of NeST lncRNA alone was sufficient to confer all phenotypes of the SJL/J locus. NeST RNA was found to bind WDR5, a component of the histone H3 lysine 4 methyltransferase complex, and to alter histone 3 methylation at the IFN-γ locus. Thus, this lncRNA regulates epigenetic marking of IFN-γ-encoding chromatin, expression of IFN-γ, and susceptibility to a viral and a bacterial pathogen.

  View details for DOI 10.1016/j.cell.2013.01.015

  View details for PubMedID 23415224

• Rejuvenation of Gene Expression Pattern of Aged Human Skin by Broadband Light Treatment: A Pilot Study JOURNAL OF INVESTIGATIVE DERMATOLOGY Chang, A. L., Bitter, P. H., Qu, K., Lin, M., Rapicavoli, N. A., Chang, H. Y. 2013; 133 (2): 394-402

  Abstract

  Studies in model organisms suggest that aged cells can be functionally rejuvenated, but whether this concept applies to human skin is unclear. Here we apply 3'-end sequencing for expression quantification ("3-seq") to discover the gene expression program associated with human photoaging and intrinsic skin aging (collectively termed "skin aging"), and the impact of broadband light (BBL) treatment. We find that skin aging was associated with a significantly altered expression level of 2,265 coding and noncoding RNAs, of which 1,293 became "rejuvenated" after BBL treatment; i.e., they became more similar to their expression level in youthful skin. Rejuvenated genes (RGs) included several known key regulators of organismal longevity and their proximal long noncoding RNAs. Skin aging is not associated with systematic changes in 3'-end mRNA processing. Hence, BBL treatment can restore gene expression pattern of photoaged and intrinsically aged human skin to resemble young skin. In addition, our data reveal, to our knowledge, a previously unreported set of targets that may lead to new insights into the human skin aging process.

  View details for DOI 10.1038/jid.2012.287

  View details for PubMedID 22931923

• SeqFold: Genome-scale reconstruction of RNA secondary structure integrating high-throughput sequencing data GENOME RESEARCH Ouyang, Z., Snyder, M. P., Chang, H. Y. 2013; 23 (2): 377-387

  Abstract

  We present an integrative approach, SeqFold, that combines high-throughput RNA structure profiling data with computational prediction for genome-scale reconstruction of RNA secondary structures. SeqFold transforms experimental RNA structure information into a structure preference profile (SPP) and uses it to select stable RNA structure candidates representing the structure ensemble. Under a high-dimensional classification framework, SeqFold efficiently matches a given SPP to the most likely cluster of structures sampled from the Boltzmann-weighted ensemble. SeqFold is able to incorporate diverse types of RNA structure profiling data, including parallel analysis of RNA structure (PARS), selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), fragmentation sequencing (FragSeq) data generated by deep sequencing, and conventional SHAPE data. Using the known structures of a wide range of mRNAs and noncoding RNAs as benchmarks, we demonstrate that SeqFold outperforms or matches existing approaches in accuracy and is more robust to noise in experimental data. Application of SeqFold to reconstruct the secondary structures of the yeast transcriptome reveals the diverse impact of RNA secondary structure on gene regulation, including translation efficiency, transcription initiation, and protein-RNA interactions. SeqFold can be easily adapted to incorporate any new types of high-throughput RNA structure profiling data and is widely applicable to analyze RNA structures in any transcriptome.

  View details for DOI 10.1101/gr.138545.112

  View details for PubMedID 23064747

• Control of somatic tissue differentiation by the long non-coding RNA TINCR. Nature Kretz, M., Siprashvili, Z., Chu, C., Webster, D. E., Zehnder, A., Qu, K., Lee, C. S., Flockhart, R. J., Groff, A. F., Chow, J., Johnston, D., Kim, G. E., Spitale, R. C., Flynn, R. A., Zheng, G. X., Aiyer, S., Raj, A., Rinn, J. L., Chang, H. Y., Khavari, P. A. 2013; 493 (7431): 231-235

  Abstract

  Several of the thousands of human long non-coding RNAs (lncRNAs) have been functionally characterized; however, potential roles for lncRNAs in somatic tissue differentiation remain poorly understood. Here we show that a 3.7-kilobase lncRNA, terminal differentiation-induced ncRNA (TINCR), controls human epidermal differentiation by a post-transcriptional mechanism. TINCR is required for high messenger RNA abundance of key differentiation genes, many of which are mutated in human skin diseases, including FLG, LOR, ALOXE3, ALOX12B, ABCA12, CASP14 and ELOVL3. TINCR-deficient epidermis lacked terminal differentiation ultrastructure, including keratohyalin granules and intact lamellar bodies. Genome-scale RNA interactome analysis revealed that TINCR interacts with a range of differentiation mRNAs. TINCR-mRNA interaction occurs through a 25-nucleotide 'TINCR box' motif that is strongly enriched in interacting mRNAs and required for TINCR binding. A high-throughput screen to analyse TINCR binding capacity to approximately 9,400 human recombinant proteins revealed direct binding of TINCR RNA to the staufen1 (STAU1) protein. STAU1-deficient tissue recapitulated the impaired differentiation seen with TINCR depletion. Loss of UPF1 and UPF2, both of which are required for STAU1-mediated RNA decay, however, did not have differentiation effects. Instead, the TINCR-STAU1 complex seems to mediate stabilization of differentiation mRNAs, such as KRT80. These data identify TINCR as a key lncRNA required for somatic tissue differentiation, which occurs through lncRNA binding to differentiation mRNAs to ensure their expression.

  View details for DOI 10.1038/nature11661

  View details for PubMedID 23201690

• Control of somatic tissue differentiation by the long non-coding RNA TINCR NATURE Kretz, M., Siprashvili, Z., Chu, C., Webster, D. E., Zehnder, A., Qu, K., Lee, C. S., Flockhart, R. J., Groff, A. F., Chow, J., Johnston, D., Kim, G. E., Spitale, R. C., Flynn, R. A., Zheng, G. X., Aiyer, S., Raj, A., Rinn, J. L., Chang, H. Y., Khavari, P. A. 2013; 493 (7431): 231-U245

  Abstract

  Several of the thousands of human long non-coding RNAs (lncRNAs) have been functionally characterized; however, potential roles for lncRNAs in somatic tissue differentiation remain poorly understood. Here we show that a 3.7-kilobase lncRNA, terminal differentiation-induced ncRNA (TINCR), controls human epidermal differentiation by a post-transcriptional mechanism. TINCR is required for high messenger RNA abundance of key differentiation genes, many of which are mutated in human skin diseases, including FLG, LOR, ALOXE3, ALOX12B, ABCA12, CASP14 and ELOVL3. TINCR-deficient epidermis lacked terminal differentiation ultrastructure, including keratohyalin granules and intact lamellar bodies. Genome-scale RNA interactome analysis revealed that TINCR interacts with a range of differentiation mRNAs. TINCR-mRNA interaction occurs through a 25-nucleotide 'TINCR box' motif that is strongly enriched in interacting mRNAs and required for TINCR binding. A high-throughput screen to analyse TINCR binding capacity to approximately 9,400 human recombinant proteins revealed direct binding of TINCR RNA to the staufen1 (STAU1) protein. STAU1-deficient tissue recapitulated the impaired differentiation seen with TINCR depletion. Loss of UPF1 and UPF2, both of which are required for STAU1-mediated RNA decay, however, did not have differentiation effects. Instead, the TINCR-STAU1 complex seems to mediate stabilization of differentiation mRNAs, such as KRT80. These data identify TINCR as a key lncRNA required for somatic tissue differentiation, which occurs through lncRNA binding to differentiation mRNAs to ensure their expression.

  View details for DOI 10.1038/nature11661

  View details for Web of Science ID 000313259600041

  View details for PubMedID 23201690

  View details for PubMedCentralID PMC3674581

• RNA SHAPE analysis in living cells. Nature chemical biology Spitale, R. C., Crisalli, P., Flynn, R. A., Torre, E. A., Kool, E. T., Chang, H. Y. 2013; 9 (1): 18-20

  Abstract

  RNA structure has important roles in practically every facet of gene regulation, but the paucity of in vivo structural probes limits current understanding. Here we design, synthesize and demonstrate two new chemical probes that enable selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) in living cells. RNA structures in human, mouse, fly, yeast and bacterial cells are read out at single-nucleotide resolution, revealing tertiary contacts and RNA-protein interactions.

  View details for DOI 10.1038/nchembio.1131

  View details for PubMedID 23178934

• A mammalian pseudogene lncRNA at the interface of inflammation and anti-inflammatory therapeutics. eLife Rapicavoli, N. A., Qu, K., Zhang, J., Mikhail, M., Laberge, R., Chang, H. Y. 2013; 2

  Abstract

  Pseudogenes are thought to be inactive gene sequences, but recent evidence of extensive pseudogene transcription raised the question of potential function. Here we discover and characterize the sets of mouse lncRNAs induced by inflammatory signaling via TNFα. TNFα regulates hundreds of lncRNAs, including 54 pseudogene lncRNAs, several of which show exquisitely selective expression in response to specific cytokines and microbial components in a NF-κB-dependent manner. Lethe, a pseudogene lncRNA, is selectively induced by proinflammatory cytokines via NF-κB or glucocorticoid receptor agonist, and functions in negative feedback signaling to NF-κB. Lethe interacts with NF-κB subunit RelA to inhibit RelA DNA binding and target gene activation. Lethe level decreases with organismal age, a physiological state associated with increased NF-κB activity. These findings suggest that expression of pseudogenes lncRNAs are actively regulated and constitute functional regulators of inflammatory signaling. DOI:http://dx.doi.org/10.7554/eLife.00762.001.

  View details for DOI 10.7554/eLife.00762

  View details for PubMedID 23898399

• Systematic reconstruction of RNA functional motifs with high-throughput microfluidics NATURE METHODS Martin, L., Meier, M., Lyons, S. M., Sit, R. V., Marzluff, W. F., Quake, S. R., Chang, H. Y. 2012; 9 (12): 1192-U85

  Abstract

  We present RNA-mechanically induced trapping of molecular interactions (RNA-MITOMI), a microfluidic platform that allows integrated synthesis and functional assays for programmable RNA libraries. The interaction of a comprehensive library of RNA mutants with stem-loop-binding protein precisely defined the RNA structural and sequence features that govern affinity. The functional motif reconstructed in a single experiment on our platform uncovers new binding specificities and enriches interpretation of phylogenetic data.

  View details for DOI 10.1038/NMETH.2225

  View details for PubMedID 23142872

• Identification of proteins binding coding and non-coding human RNAs using protein microarrays BMC GENOMICS Siprashvili, Z., Webster, D. E., Kretz, M., Johnston, D., Rinn, J. L., Chang, H. Y., Khavari, P. A. 2012; 13

  Abstract

  The regulation and function of mammalian RNAs has been increasingly appreciated to operate via RNA-protein interactions. With the recent discovery of thousands of novel human RNA molecules by high-throughput RNA sequencing, efficient methods to uncover RNA-protein interactions are urgently required. Existing methods to study proteins associated with a given RNA are laborious and require substantial amounts of cell-derived starting material. To overcome these limitations, we have developed a rapid and large-scale approach to characterize binding of in vitro transcribed labeled RNA to ~9,400 human recombinant proteins spotted on protein microarrays.We have optimized methodology to probe human protein microarrays with full-length RNA molecules and have identified 137 RNA-protein interactions specific for 10 coding and non-coding RNAs. Those proteins showed strong enrichment for common human RNA binding domains such as RRM, RBD, as well as K homology and CCCH type zinc finger motifs. Previously unknown RNA-protein interactions were discovered using this technique, and these interactions were biochemically verified between TP53 mRNA and Staufen1 protein as well as between HRAS mRNA and CNBP protein. Functional characterization of the interaction between Staufen 1 protein and TP53 mRNA revealed a novel role for Staufen 1 in preserving TP53 RNA stability.Our approach demonstrates a scalable methodology, allowing rapid and efficient identification of novel human RNA-protein interactions using RNA hybridization to human protein microarrays. Biochemical validation of newly identified interactions between TP53-Stau1 and HRAS-CNBP using reciprocal pull-down experiments, both in vitro and in vivo, demonstrates the utility of this approach to study uncharacterized RNA-protein interactions.

  View details for DOI 10.1186/1471-2164-13-633

  View details for PubMedID 23157412

• Genome-wide Measurement of RNA Folding Energies MOLECULAR CELL Wan, Y., Qu, K., Ouyang, Z., Kertesz, M., Li, J., Tibshirani, R., Makino, D. L., Nutter, R. C., Segal, E., Chang, H. Y. 2012; 48 (2): 169-181

  Abstract

  RNA structural transitions are important in the function and regulation of RNAs. Here, we reveal a layer of transcriptome organization in the form of RNA folding energies. By probing yeast RNA structures at different temperatures, we obtained relative melting temperatures (Tm) for RNA structures in over 4000 transcripts. Specific signatures of RNA Tm demarcated the polarity of mRNA open reading frames and highlighted numerous candidate regulatory RNA motifs in 3' untranslated regions. RNA Tm distinguished noncoding versus coding RNAs and identified mRNAs with distinct cellular functions. We identified thousands of putative RNA thermometers, and their presence is predictive of the pattern of RNA decay in vivo during heat shock. The exosome complex recognizes unpaired bases during heat shock to degrade these RNAs, coupling intrinsic structural stabilities to gene regulation. Thus, genome-wide structural dynamics of RNA can parse functional elements of the transcriptome and reveal diverse biological insights.

  View details for DOI 10.1016/j.molcel.2012.08.008

  View details for PubMedID 22981864

• Detection of Long Non-Coding RNA in Archival Tissue: Correlation with Polycomb Protein Expression in Primary and Metastatic Breast Carcinoma PLOS ONE Chisholm, K. M., Wan, Y., Li, R., Montgomery, K. D., Chang, H. Y., West, R. B. 2012; 7 (10)

  Abstract

  A major function of long non-coding RNAs (lncRNAs) is regulating gene expression through changes in chromatin state. Experimental evidence suggests that in cancer, they can influence Polycomb Repressive Complexes (PRC) to retarget to an occupancy pattern resembling that of the embryonic state. We have previously demonstrated that the expression level of lncRNA in the HOX locus, including HOTAIR, is a predictor of breast cancer metastasis. In this current project, RNA in situ hybridization of probes to three different lncRNAs (HOTAIR, ncHoxA1, and ncHoxD4), as well a immunohistochemical staining of EZH2, is undertaken in formalin-fixed paraffin-embedded breast cancer tissues in a high throughput tissue microarray format to correlate expression with clinicopathologic features. Though overall EZH2 and HOTAIR expression levels were highly correlated, the subset of cases with strong HOTAIR expression correlated with ER and PR positivity, while the subset of cases with strong EZH2 expression correlated with an increased proliferation rate, ER and PR negativity, HER2 underexpression, and triple negativity. Co-expression of HOTAIR and EZH2 trended with a worse outcome. In matched primary and metastatic cancers, both HOTAIR and EZH2 had increased expression in the metastatic carcinomas. This is the first study to show that RNA in situ hybridization of formalin fixed paraffin-embedded clinical material can be used to measure levels of long non-coding RNAs. This approach offers a method to make observations on lncRNAs that may influence the cancer epigenome in a tissue-based technique.

  View details for DOI 10.1371/journal.pone.0047998

  View details for PubMedID 23133536

• Transcriptome sequencing in Sezary syndrome identifies Sezary cell and mycosis fungoides-associated lncRNAs and novel transcripts BLOOD Lee, C. S., Ungewickell, A., Bhaduri, A., Qu, K., Webster, D. E., Armstrong, R., Weng, W., Aros, C. J., Mah, A., Chen, R. O., Lin, M., Sundram, U., Chang, H. Y., Kretz, M., Kim, Y. H., Khavari, P. A. 2012; 120 (16): 3288-3297

  Abstract

  Sézary syndrome (SS) is an aggressive cutaneous T-cell lymphoma (CTCL) of unknown etiology in which malignant cells circulate in the peripheral blood. To identify viral elements, gene fusions, and gene expression patterns associated with this lymphoma, flow cytometry was used to obtain matched pure populations of malignant Sézary cells (SCs) versus nonmalignant CD4(+) T cells from 3 patients for whole transcriptome, paired-end sequencing with an average depth of 112 million reads per sample. Pathway analysis of differentially expressed genes identified mis-regulation of PI3K/Akt, TGFβ, and NF-κB pathways as well as T-cell receptor signaling. Bioinformatic analysis did not detect either nonhuman transcripts to support a viral etiology of SS or recurrently expressed gene fusions, but it did identify 21 SC-associated annotated long noncoding RNAs (lncRNAs). Transcriptome assembly by multiple algorithms identified 13 differentially expressed unannotated transcripts termed Sézary cell-associated transcripts (SeCATs) that include 12 predicted lncRNAs and a novel transcript with coding potential. High-throughput sequencing targeting the 3' end of polyadenylated transcripts in archived tumors from 24 additional patients with tumor-stage CTCL confirmed the differential expression of SC-associated lncRNAs and SeCATs in CTCL. Our findings characterize the SS transcriptome and support recent reports that implicate lncRNA dysregulation in human malignancies.

  View details for DOI 10.1182/blood-2012-04-423061

  View details for PubMedID 22936659

• A Molecular Signature for Purified Definitive Endoderm Guides Differentiation and Isolation of Endoderm from Mouse and Human Embryonic Stem Cells STEM CELLS AND DEVELOPMENT Wang, P., McKnight, K. D., Wong, D. J., Rodriguez, R. T., Sugiyama, T., Gu, X., Ghodasara, A., Qu, K., Chang, H. Y., Kim, S. K. 2012; 21 (12): 2273-2287

  Abstract

  Embryonic definitive endoderm (DE) generates the epithelial compartment of vital organs such as liver, pancreas, and intestine. However, purification of DE in mammals has not been achieved, limiting the molecular "definition" of endoderm, and hindering our understanding of DE development and attempts to produce endoderm from sources such as embryonic stem (ES) cells. Here, we describe purification of mouse DE using fluorescence-activated cell sorting (FACS) and mice harboring a transgene encoding enhanced green fluorescent protein (eGFP) inserted into the Sox17 locus, which is expressed in the embryonic endoderm. Comparison of patterns of signaling pathway activation in native mouse DE and endoderm-like cells generated from ES cells produced novel culture modifications that generated Sox17-eGFP⁺ progeny whose gene expression resembled DE more closely than achieved with standard methods. These studies also produced new FACS methods for purifying DE from nontransgenic mice and mouse ES cell cultures. Parallel studies of a new human SOX17-eGFP ES cell line allowed analysis of endoderm differentiation in vitro, leading to culture modifications that enhanced expression of an endoderm-like signature. This work should accelerate our understanding of mechanisms regulating DE development in mice and humans, and guide further use of ES cells for tissue replacement.

  View details for DOI 10.1089/scd.2011.0416

  View details for PubMedID 22236333

• The Transcription Factor ZNF217 Is a Prognostic Biomarker and Therapeutic Target during Breast Cancer Progression CANCER DISCOVERY Littlepage, L. E., Adler, A. S., Kouros-Mehr, H., Huang, G., Chou, J., Krig, S. R., Griffith, O. L., Korkola, J. E., Qu, K., Lawson, D. A., Xue, Q., Sternlicht, M. D., Dijkgraaf, G. J., Yaswen, P., Rugo, H. S., Sweeney, C. A., Collins, C. C., Gray, J. W., Chang, H. Y., Werb, Z. 2012; 2 (7): 638-651

  Abstract

  The transcription factor ZNF217 is a candidate oncogene in the amplicon on chromosome 20q13 that occurs in 20% to 30% of primary human breast cancers and that correlates with poor prognosis. We show that Znf217 overexpression drives aberrant differentiation and signaling events, promotes increased self-renewal capacity, mesenchymal marker expression, motility, and metastasis, and represses an adult tissue stem cell gene signature downregulated in cancers. By in silico screening, we identified candidate therapeutics that at low concentrations inhibit growth of cancer cells expressing high ZNF217. We show that the nucleoside analogue triciribine inhibits ZNF217-induced tumor growth and chemotherapy resistance and inhibits signaling events [e.g., phospho-AKT, phospho-mitogen-activated protein kinase (MAPK)] in vivo. Our data suggest that ZNF217 is a biomarker of poor prognosis and a therapeutic target in patients with breast cancer and that triciribine may be part of a personalized treatment strategy in patients overexpressing ZNF217. Because ZNF217 is amplified in numerous cancers, these results have implications for other cancers.This study finds that ZNF217 is a poor prognostic indicator and therapeutic target in patients with breast cancer and may be a strong biomarker of triciribine treatment efficacy in patients. Because previous clinical trials for triciribine did not include biomarkers of treatment efficacy, this study provides a rationale for revisiting triciribine in the clinical setting as a therapy for patients with breast cancer who overexpress ZNF217.

  View details for DOI 10.1158/2159-8290.CD-12-0093

  View details for Web of Science ID 000306365800042

  View details for PubMedID 22728437

  View details for PubMedCentralID PMC3546490

• Identification of genes promoting exceptional skin youthfulness 75th Annual Meeting of the Society-for-Investigative-Dermatology Chang, A. S., Atzmon, G., Bergman, A., Chang, H. Y., Barzilai, N. NATURE PUBLISHING GROUP. 2012: S65–S65

  View details for Web of Science ID 000302866900385

• Uncovering the role of genomic "dark matter" in human disease JOURNAL OF CLINICAL INVESTIGATION Martin, L., Chang, H. Y. 2012; 122 (5): 1589-1595

  Abstract

  The human genome encodes thousands of long noncoding RNAs (lncRNAs). Although most remain functionally uncharacterized biological "dark matter," lncRNAs have garnered considerable attention for their diverse roles in human biology, including developmental programs and tumor suppressor gene networks. As the number of lncRNAs associated with human disease grows, ongoing research efforts are focusing on their regulatory mechanisms. New technologies that enable enumeration of lncRNA interaction partners and determination of lncRNA structure are well positioned to drive deeper understanding of their functions and involvement in pathogenesis. In turn, lncRNAs may become targets for therapeutic intervention or new tools for biotechnology.

  View details for DOI 10.1172/JCI60020

  View details for PubMedID 22546862

• Active chromatin and noncoding RNAs: an intimate relationship CURRENT OPINION IN GENETICS & DEVELOPMENT Flynn, R. A., Chang, H. Y. 2012; 22 (2): 172-178

  Abstract

  Eukaryotic genomes are packaged into chromatin, where diverse histone modifications can demarcate chromatin domains that facilitate or block gene expression. While silent chromatin has been associated with long noncoding RNAs (lncRNAs) for some time, new studies suggest that noncoding RNAs also modulate the active chromatin state. Divergent, antisense, and enhancer-like intergenic noncoding RNAs can either activate or repress gene expression by altering histone H3 lysine 4 methylation. An emerging class of enhancer-like lncRNAs may link chromosome structure to chromatin state and establish active chromatin domains. The confluence of several new technologies promises to rapidly expand this fascinating topic of investigation.

  View details for DOI 10.1016/j.gde.2011.11.002

  View details for PubMedID 22154525

• Chromatin Isolation by RNA Purification (ChIRP) JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Chu, C., Quinn, J., Chang, H. Y. 2012

  View details for DOI 10.3791/3912

  View details for Web of Science ID 000209222800051

• Suppression of progenitor differentiation requires the long noncoding RNA ANCR GENES & DEVELOPMENT Kretz, M., Webster, D. E., Flockhart, R. J., Lee, C. S., Zehnder, A., Lopez-Pajares, V., Qu, K., Zheng, G. X., Chow, J., Kim, G. E., Rinn, J. L., Chang, H. Y., Siprashvili, Z., Khavari, P. A. 2012; 26 (4): 338-343

  Abstract

  Long noncoding RNAs (lncRNAs) regulate diverse processes, yet a potential role for lncRNAs in maintaining the undifferentiated state in somatic tissue progenitor cells remains uncharacterized. We used transcriptome sequencing and tiling arrays to compare lncRNA expression in epidermal progenitor populations versus differentiating cells. We identified ANCR (anti-differentiation ncRNA) as an 855-base-pair lncRNA down-regulated during differentiation. Depleting ANCR in progenitor-containing populations, without any other stimuli, led to rapid differentiation gene induction. In epidermis, ANCR loss abolished the normal exclusion of differentiation from the progenitor-containing compartment. The ANCR lncRNA is thus required to enforce the undifferentiated cell state within epidermis.

  View details for DOI 10.1101/gad.182121.111

  View details for Web of Science ID 000300626800004

  View details for PubMedID 22302877

  View details for PubMedCentralID PMC3289881

• Aging, Rejuvenation, and Epigenetic Reprogramming: Resetting the Aging Clock CELL Rando, T. A., Chang, H. Y. 2012; 148 (1-2): 46-57

  Abstract

  The underlying cause of aging remains one of the central mysteries of biology. Recent studies in several different systems suggest that not only may the rate of aging be modified by environmental and genetic factors, but also that the aging clock can be reversed, restoring characteristics of youthfulness to aged cells and tissues. This Review focuses on the emerging biology of rejuvenation through the lens of epigenetic reprogramming. By defining youthfulness and senescence as epigenetic states, a framework for asking new questions about the aging process emerges.

  View details for DOI 10.1016/j.cell.2012.01.003

  View details for PubMedID 22265401

• Chromatin isolation by RNA purification (ChIRP). Journal of visualized experiments : JoVE Chu, C., Quinn, J., Chang, H. Y. 2012

  Abstract

  Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression (1,2,3,4,5,6,7). The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion (8,9). While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation (10,11). However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide( 3,12,13), but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex (14); HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex (13). Prior studies mapping RNA occupancy at chromatin have revealed substantial insights (15,16), but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution (17). This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

  View details for DOI 10.3791/3912

  View details for PubMedID 22472705

• Genome Regulation by Long Noncoding RNAs ANNUAL REVIEW OF BIOCHEMISTRY, VOL 81 Rinn, J. L., Chang, H. Y. 2012; 81: 145-166

  Abstract

  The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. The discovery of extensive transcription of large RNA transcripts that do not code for proteins, termed long noncoding RNAs (lncRNAs), provides an important new perspective on the centrality of RNA in gene regulation. Here, we discuss genome-scale strategies to discover and characterize lncRNAs. An emerging theme from multiple model systems is that lncRNAs form extensive networks of ribonucleoprotein (RNP) complexes with numerous chromatin regulators and then target these enzymatic activities to appropriate locations in the genome. Consistent with this notion, lncRNAs can function as modular scaffolds to specify higher-order organization in RNP complexes and in chromatin states. The importance of these modes of regulation is underscored by the newly recognized roles of long RNAs for proper gene control across all kingdoms of life.

  View details for DOI 10.1146/annurev-biochem-051410-092902

  View details for Web of Science ID 000305765500008

  View details for PubMedID 22663078

• Transcriptional profiling of long non-coding RNAs and novel transcribed regions across a diverse panel of archived human cancers GENOME BIOLOGY Brunner, A. L., Beck, A. H., Edris, B., Sweeney, R. T., Zhu, S. X., Li, R., Montgomery, K., Varma, S., Gilks, T., Guo, X., Foley, J. W., Witten, D. M., Giacomini, C. P., Flynn, R. A., Pollack, J. R., Tibshirani, R., Chang, H. Y., van de Rijn, M., West, R. B. 2012; 13 (8)

  Abstract

  BACKGROUND: Molecular characterization of tumors has been critical for identifying important genes in cancer biology and for improving tumor classification and diagnosis. Long non-coding RNAs, as a new, relatively unstudied class of transcripts, provide a rich opportunity to identify both functional drivers and cancer-type-specific biomarkers. However, despite the potential importance of long non-coding RNAs to the cancer field, no comprehensive survey of long non-coding RNA expression across various cancers has been reported. RESULTS: We performed a sequencing-based transcriptional survey of both known long non-coding RNAs and novel intergenic transcripts across a panel of 64 archival tumor samples comprising 17 diagnostic subtypes of adenocarcinomas, squamous cell carcinomas and sarcomas. We identified hundreds of transcripts from among the known 1,065 long non-coding RNAs surveyed that showed variability in transcript levels between the tumor types and are therefore potential biomarker candidates. We discovered 1,071 novel intergenic transcribed regions and demonstrate that these show similar patterns of variability between tumor types. We found that many of these differentially expressed cancer transcripts are also expressed in normal tissues. One such novel transcript specifically expressed in breast tissue was further evaluated using RNA in situ hybridization on a panel of breast tumors. It was shown to correlate with low tumor grade and estrogen receptor expression, thereby representing a potentially important new breast cancer biomarker. CONCLUSIONS: This study provides the first large survey of long non-coding RNA expression within a panel of solid cancers and also identifies a number of novel transcribed regions differentially expressed across distinct cancer types that represent candidate biomarkers for future research.

  View details for Web of Science ID 000315867500009

• High throughput automated chromatin immunoprecipitation as a platform for drug screening and antibody validation LAB ON A CHIP Wu, A. R., Kawahara, T. L., Rapicavoli, N. A., van Riggelen, J., Shroff, E. H., Xu, L., Felsher, D. W., Chang, H. Y., Quake, S. R. 2012; 12 (12): 2190-2198

  Abstract

  Chromatin immunoprecipitation (ChIP) is an assay for interrogating protein-DNA interactions that is increasingly being used for drug target discovery and screening applications. Currently the complexity of the protocol and the amount of hands-on time required for this assay limits its use to low throughput applications; furthermore, variability in antibody quality poses an additional obstacle in scaling up ChIP for large scale screening purposes. To address these challenges, we report HTChIP, an automated microfluidic-based platform for performing high-throughput ChIP screening measurements of 16 different targets simultaneously, with potential for further scale-up. From chromatin to analyzable PCR results only takes one day using HTChIP, as compared to several days up to one week for conventional protocols. HTChIP can also be used to test multiple antibodies and select the best performer for downstream ChIP applications, saving time and reagent costs of unsuccessful ChIP assays as a result of poor antibody quality. We performed a series of characterization assays to demonstrate that HTChIP can rapidly and accurately evaluate the epigenetic states of a cell, and that it is sensitive enough to detect the changes in the epigenetic state induced by a cytokine stimulant over a fine temporal resolution. With these results, we believe that HTChIP can introduce large improvements in routine ChIP, antibody screening, and drug screening efficiency, and further facilitate the use of ChIP as a valuable tool for research and discovery.

  View details for DOI 10.1039/c2lc21290k

  View details for PubMedID 22566096

• Genomic Maps of Long Noncoding RNA Occupancy Reveal Principles of RNA-Chromatin Interactions MOLECULAR CELL Chu, C., Qu, K., Zhong, F. L., Artandi, S. E., Chang, H. Y. 2011; 44 (4): 667-678

  Abstract

  Long noncoding RNAs (lncRNAs) are key regulators of chromatin state, yet the nature and sites of RNA-chromatin interaction are mostly unknown. Here we introduce Chromatin Isolation by RNA Purification (ChIRP), where tiling oligonucleotides retrieve specific lncRNAs with bound protein and DNA sequences, which are enumerated by deep sequencing. ChIRP-seq of three lncRNAs reveal that RNA occupancy sites in the genome are focal, sequence-specific, and numerous. Drosophila roX2 RNA occupies male X-linked gene bodies with increasing tendency toward the 3' end, peaking at CES sites. Human telomerase RNA TERC occupies telomeres and Wnt pathway genes. HOTAIR lncRNA preferentially occupies a GA-rich DNA motif to nucleate broad domains of Polycomb occupancy and histone H3 lysine 27 trimethylation. HOTAIR occupancy occurs independently of EZH2, suggesting the order of RNA guidance of Polycomb occupancy. ChIRP-seq is generally applicable to illuminate the intersection of RNA and chromatin with newfound precision genome wide.

  View details for DOI 10.1016/j.molcel.2011.08.027

  View details for PubMedID 21963238

• Direct Lineage Conversion of Terminally Differentiated Hepatocytes to Functional Neurons CELL STEM CELL Marro, S., Pang, Z. P., Yang, N., Tsai, M., Qu, K., Chang, H. Y., Suedhof, T. C., Wernig, M. 2011; 9 (4): 374-382

  Abstract

  Several recent studies have showed that mouse and human fibroblasts can be directly reprogrammed into induced neuronal (iN) cells, bypassing a pluripotent intermediate state. However, fibroblasts represent heterogeneous mesenchymal progenitor cells that potentially contain neural crest lineages, and the cell of origin remained undefined. This raises the fundamental question of whether lineage reprogramming is possible between cell types derived from different germ layers. Here, we demonstrate that terminally differentiated hepatocytes can be directly converted into functional iN cells. Importantly, single-cell and genome-wide expression analyses showed that fibroblast- and hepatocyte-derived iN cells not only induced a neuronal transcriptional program, but also silenced their donor transcriptome. The remaining donor signature decreased over time and could not support functional hepatocyte properties. Thus, the reprogramming factors lead to a binary lineage switch decision rather than an induction of hybrid phenotypes, but iN cells retain a small but detectable epigenetic memory of their donor cells.

  View details for DOI 10.1016/j.stem.2011.09.002

  View details for PubMedID 21962918

• Molecular Mechanisms of Long Noncoding RNAs MOLECULAR CELL Wang, K. C., Chang, H. Y. 2011; 43 (6): 904-914

  Abstract

  Long noncoding RNAs (lncRNAs) are an important class of pervasive genes involved in a variety of biological functions. Here we discuss the emerging archetypes of molecular functions that lncRNAs execute-as signals, decoys, guides, and scaffolds. For each archetype, examples from several disparate biological contexts illustrate the commonality of the molecular mechanisms, and these mechanistic views provide useful explanations and predictions of biological outcomes. These archetypes of lncRNA function may be a useful framework to consider how lncRNAs acquire properties as biological signal transducers and hint at their possible origins in evolution. As new lncRNAs are being discovered at a rapid pace, the molecular mechanisms of lncRNAs are likely to be enriched and diversified.

  View details for DOI 10.1016/j.molcel.2011.08.018

  View details for PubMedID 21925379

• Disruption of PPAR gamma/beta-catenin-mediated regulation of apelin impairs BMP-induced mouse and human pulmonary arterial EC survival JOURNAL OF CLINICAL INVESTIGATION Alastalo, T., Li, M., Perez, V. D., Pham, D., Sawada, H., Wang, J. K., Koskenvuo, M., Wang, L., Freeman, B. A., Chang, H. Y., Rabinovitch, M. 2011; 121 (9): 3735-3746

  Abstract

  Reduced bone morphogenetic protein receptor 2 (BMPR2) expression in patients with pulmonary arterial hypertension (PAH) can impair pulmonary arterial EC (PAEC) function. This can adversely affect EC survival and promote SMC proliferation. We hypothesized that interventions to normalize expression of genes that are targets of BMPR2 signaling could restore PAEC function and prevent or reverse PAH. Here we have characterized, in human PAECs, a BMPR2-mediated transcriptional complex between PPARγ and β-catenin and shown that disruption of this complex impaired BMP-mediated PAEC survival. Using whole genome-wide ChIP-Chip promoter analysis and gene expression microarrays, we delineated PPARγ/β-catenin-dependent transcription of target genes including APLN, which encodes apelin. We documented reduced PAEC expression of apelin in PAH patients versus controls. In cell culture experiments, we showed that apelin-deficient PAECs were prone to apoptosis and promoted pulmonary arterial SMC (PASMC) proliferation. Conversely, we established that apelin, like BMPR2 ligands, suppressed proliferation and induced apoptosis of PASMCs. Consistent with these functions, administration of apelin reversed PAH in mice with reduced production of apelin resulting from deletion of PPARγ in ECs. Taken together, our findings suggest that apelin could be effective in treating PAH by rescuing BMPR2 and PAEC dysfunction.

  View details for DOI 10.1172/JCI43382

  View details for PubMedID 21821917

• Understanding the transcriptome through RNA structure NATURE REVIEWS GENETICS Wan, Y., Kertesz, M., Spitale, R. C., Segal, E., Chang, H. Y. 2011; 12 (9): 641-655

  Abstract

  RNA structure is crucial for gene regulation and function. In the past, transcriptomes have largely been parsed by primary sequences and expression levels, but it is now becoming feasible to annotate and compare transcriptomes based on RNA structure. In addition to computational prediction methods, the recent advent of experimental techniques to probe RNA structure by high-throughput sequencing has enabled genome-wide measurements of RNA structure and has provided the first picture of the structural organization of a eukaryotic transcriptome - the 'RNA structurome'. With additional advances in method refinement and interpretation, structural views of the transcriptome should help to identify and validate regulatory RNA motifs that are involved in diverse cellular processes and thereby increase understanding of RNA function.

  View details for DOI 10.1038/nrg3049

  View details for PubMedID 21850044

• Crystal structure of the N-terminal region of human Ash2L shows a winged-helix motif involved in DNA binding EMBO REPORTS Chen, Y., Wan, B., Wang, K. C., Cao, F., Yang, Y., Protacio, A., Dou, Y., Chang, H. Y., Lei, M. 2011; 12 (8): 797-803

  Abstract

  Ash2L is a core component of the MLL family histone methyltransferases and has an important role in regulating the methylation of histone H3 on lysine 4. Here, we report the crystal structure of the N-terminal domain of Ash2L and reveal a new function of Ash2L. The structure shows that Ash2L contains an atypical PHD finger that does not have histone tail-binding activity. Unexpectedly, the structure shows a previously unrecognized winged-helix motif that directly binds to DNA. The DNA-binding-deficient mutants of Ash2L reduced Ash2L localization to the HOX locus. Strikingly, a single mutation in Ash2L(WH) (K131A) breaks the chromatin domain boundary, suggesting that Ash2L also has a role in chromosome demarcation.

  View details for DOI 10.1038/embor.2011.101

  View details for Web of Science ID 000293326500014

  View details for PubMedID 21660059

  View details for PubMedCentralID PMC3147254

• Extensive and coordinated transcription of noncoding RNAs within cell-cycle promoters NATURE GENETICS Hung, T., Wang, Y., Lin, M. F., Koegel, A. K., Kotake, Y., Grant, G. D., Horlings, H. M., Shah, N., Umbricht, C., Wang, P., Wang, Y., Kong, B., Langerod, A., Borresen-Dale, A., Kim, S. K., van de Vijver, M., Sukumar, S., Whitfield, M. L., Kellis, M., Xiong, Y., Wong, D. J., Chang, H. Y. 2011; 43 (7): 621-U196

  Abstract

  Transcription of long noncoding RNAs (lncRNAs) within gene regulatory elements can modulate gene activity in response to external stimuli, but the scope and functions of such activity are not known. Here we use an ultrahigh-density array that tiles the promoters of 56 cell-cycle genes to interrogate 108 samples representing diverse perturbations. We identify 216 transcribed regions that encode putative lncRNAs, many with RT-PCR-validated periodic expression during the cell cycle, show altered expression in human cancers and are regulated in expression by specific oncogenic stimuli, stem cell differentiation or DNA damage. DNA damage induces five lncRNAs from the CDKN1A promoter, and one such lncRNA, named PANDA, is induced in a p53-dependent manner. PANDA interacts with the transcription factor NF-YA to limit expression of pro-apoptotic genes; PANDA depletion markedly sensitized human fibroblasts to apoptosis by doxorubicin. These findings suggest potentially widespread roles for promoter lncRNAs in cell-growth control.

  View details for DOI 10.1038/ng.848

  View details for PubMedID 21642992

• Long noncoding RNAs and human disease TRENDS IN CELL BIOLOGY Wapinski, O., Chang, H. Y. 2011; 21 (6): 354-361

  Abstract

  A new class of transcripts, long noncoding RNAs (lncRNAs), has been recently found to be pervasively transcribed in the genome. Multiple lines of evidence increasingly link mutations and dysregulations of lncRNAs to diverse human diseases. Alterations in the primary structure, secondary structure, and expression levels of lncRNAs as well as their cognate RNA-binding proteins underlie diseases ranging from neurodegeneration to cancer. Recent progress suggests that the involvement of lncRNAs in human diseases could be far more prevalent than previously appreciated. We review the evidence linking lncRNAs to diverse human diseases and highlight fundamental concepts in lncRNA biology that still need to be clarified to provide a robust framework for lncRNA genetics.

  View details for DOI 10.1016/j.tcb.2011.04.001

  View details for Web of Science ID 000292238200005

  View details for PubMedID 21550244

• Dynamic Chromatin Localization of Sirt6 Shapes Stress- and Aging-Related Transcriptional Networks PLOS GENETICS Kawahara, T. L., Rapicavoli, N. A., Wu, A. R., Qu, K., Quake, S. R., Chang, H. Y. 2011; 7 (6)

  Abstract

  The sirtuin Sirt6 is a NAD-dependent histone deacetylase that is implicated in gene regulation and lifespan control. Sirt6 can interact with the stress-responsive transcription factor NF-κB and regulate some NF-κB target genes, but the full scope of Sirt6 target genes as well as dynamics of Sirt6 occupancy on chromatin are not known. Here we map Sirt6 occupancy on mouse promoters genome-wide and show that Sirt6 occupancy is highly dynamic in response to TNF-α. More than half of Sirt6 target genes are only revealed upon stress-signaling. The majority of genes bound by NF-κB subunit RelA recruit Sirt6, and dynamic Sirt6 relocalization is largely driven in a RelA-dependent manner. Integrative analysis with global gene expression patterns in wild-type, Sirt6-/-, and double Sirt6-/- RelA-/- cells reveals the epistatic relationships between Sirt6 and RelA in shaping diverse temporal patterns of gene expression. Genes under the direct joint control of Sirt6 and RelA include several with prominent roles in cell senescence and organismal aging. These data suggest dynamic chromatin relocalization of Sirt6 as a key output of NF-κB signaling in stress response and aging.

  View details for DOI 10.1371/journal.pgen.1002153

  View details for PubMedID 21738489

• RNA templating the epigenome Long noncoding RNAs as molecular scaffolds EPIGENETICS Spitale, R. C., Tsai, M., Chang, H. Y. 2011; 6 (5): 539-543

  Abstract

  Cellular pathways must be synergized, controlled and organized to manage homeostasis. To achieve high selectivity within the crowded cellular milieu the cell utilizes scaffolding complexes whose role is to bring molecules in proximity thereby controlling and enhancing intermolecular interactions and signaling events. To date, scaffolds have been shown to be composed of proteinaceous units; however, recent evidence has supported the idea that non-coding RNAs may also play a similar role. In this point of view article we discuss recent data on ncRNA scaffolds, with particular focus on ncRNA HOTAIR. Using our current knowledge of signaling networks we discuss the role that RNA may play in writing and regulating histone modifications and the information needed for correct gene expression. Further, we speculate on additional, yet undiscovered roles that ncRNAs may be playing as molecular scaffolds.

  View details for DOI 10.4161/epi.6.5.15221

  View details for PubMedID 21393997

• A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression NATURE Wang, K. C., Yang, Y. W., Liu, B., Sanyal, A., Corces-Zimmerman, R., Chen, Y., Lajoie, B. R., Protacio, A., Flynn, R. A., Gupta, R. A., Wysocka, J., Lei, M., Dekker, J., Helms, J. A., Chang, H. Y. 2011; 472 (7341): 120-U158

  Abstract

  The genome is extensively transcribed into long intergenic noncoding RNAs (lincRNAs), many of which are implicated in gene silencing. Potential roles of lincRNAs in gene activation are much less understood. Development and homeostasis require coordinate regulation of neighbouring genes through a process termed locus control. Some locus control elements and enhancers transcribe lincRNAs, hinting at possible roles in long-range control. In vertebrates, 39 Hox genes, encoding homeodomain transcription factors critical for positional identity, are clustered in four chromosomal loci; the Hox genes are expressed in nested anterior-posterior and proximal-distal patterns colinear with their genomic position from 3' to 5'of the cluster. Here we identify HOTTIP, a lincRNA transcribed from the 5' tip of the HOXA locus that coordinates the activation of several 5' HOXA genes in vivo. Chromosomal looping brings HOTTIP into close proximity to its target genes. HOTTIP RNA binds the adaptor protein WDR5 directly and targets WDR5/MLL complexes across HOXA, driving histone H3 lysine 4 trimethylation and gene transcription. Induced proximity is necessary and sufficient for HOTTIP RNA activation of its target genes. Thus, by serving as key intermediates that transmit information from higher order chromosomal looping into chromatin modifications, lincRNAs may organize chromatin domains to coordinate long-range gene activation.

  View details for DOI 10.1038/nature09819

  View details for PubMedID 21423168

• High throughput RNA sequencing of Sezary syndrome and cutaneous squamous cell carcinoma 71st Annual Meeting of the Society-for-Investigative-Dermatology Lee, C., Qu, K., Webster, D., Kretz, M., MAH, A., Ungewickell, A., Armstrong, R., Weng, W., Kim, Y., Chang, H., Khavari, P. NATURE PUBLISHING GROUP. 2011: S22–S22

  View details for Web of Science ID 000289035600128

• Long noncoding RNA programs active chromatin domain to coordinate homeotic gene expression 71st Annual Meeting of the Society-for-Investigative-Dermatology Wang, K. C., Yang, Y. W., Liu, B., Sanyal, A., Corces-Zimmerman, R., Chen, Y., Lajoie, B. R., Protacio, A., Flynn, R. A., Gupta, R. A., Wysocka, J., Lei, M., Dekker, J., Helms, J. A., Chang, H. Y. NATURE PUBLISHING GROUP. 2011: S63–S63

  View details for Web of Science ID 000289035600373

• Long Intergenic Noncoding RNAs: New Links in Cancer Progression CANCER RESEARCH Tsai, M., Spitale, R. C., Chang, H. Y. 2011; 71 (1): 3-7

  Abstract

  The process of cancer metastasis involves a series of sequential and complex steps. Here we give a perspective on recent results regarding noncoding transcription in cancer progression, focusing on the emerging role of long intergenic noncoding RNAs (lincRNAs). LincRNAs target chromatin modification complexes or RNA-binding proteins to alter gene expression programs. Similarly to miRNAs, lincRNAs exhibit distinct gene expression patterns in primary tumors and metastases. We discuss how lincRNAs can be used for cancer diagnosis and prognosis and serve as potential therapeutic targets.

  View details for DOI 10.1158/0008-5472.CAN-10-2483

  View details for PubMedID 21199792

• Noncoding RNA Landmarks of Pluripotency and Reprogramming CELL STEM CELL Ouyang, Z., Zheng, G. X., Chang, H. Y. 2010; 7 (6): 649-650

  View details for DOI 10.1016/j.stem.2010.11.018

  View details for PubMedID 21112559

• G1 arrest and differentiation can occur independently of Rb family function JOURNAL OF CELL BIOLOGY Wirt, S. E., Adler, A. S., Gebala, V., Weimann, J. M., Schaffer, B. E., Saddic, L. A., Viatour, P., Vogel, H., Chang, H. Y., Meissner, A., Sage, J. 2010; 191 (4): 809-825

  Abstract

  The ability of progenitor cells to exit the cell cycle is essential for proper embryonic development and homeostasis, but the mechanisms governing cell cycle exit are still not fully understood. Here, we tested the requirement for the retinoblastoma (Rb) protein and its family members p107 and p130 in G0/G1 arrest and differentiation in mammalian cells. We found that Rb family triple knockout (TKO) mouse embryos survive until days 9-11 of gestation. Strikingly, some TKO cells, including in epithelial and neural lineages, are able to exit the cell cycle in G0/G1 and differentiate in teratomas and in culture. This ability of TKO cells to arrest in G0/G1 is associated with the repression of key E2F target genes. Thus, G1 arrest is not always dependent on Rb family members, which illustrates the robustness of cell cycle regulatory networks during differentiation and allows for the identification of candidate pathways to inhibit the expansion of cancer cells with mutations in the Rb pathway.

  View details for DOI 10.1083/jcb.201003048

  View details for Web of Science ID 000284737200014

  View details for PubMedID 21059851

  View details for PubMedCentralID PMC2983066

• Genome-wide measurement of RNA secondary structure in yeast NATURE Kertesz, M., Wan, Y., Mazor, E., Rinn, J. L., Nutter, R. C., Chang, H. Y., Segal, E. 2010; 467 (7311): 103-107

  Abstract

  The structures of RNA molecules are often important for their function and regulation, yet there are no experimental techniques for genome-scale measurement of RNA structure. Here we describe a novel strategy termed parallel analysis of RNA structure (PARS), which is based on deep sequencing fragments of RNAs that were treated with structure-specific enzymes, thus providing simultaneous in vitro profiling of the secondary structure of thousands of RNA species at single nucleotide resolution. We apply PARS to profile the secondary structure of the messenger RNAs (mRNAs) of the budding yeast Saccharomyces cerevisiae and obtain structural profiles for over 3,000 distinct transcripts. Analysis of these profiles reveals several RNA structural properties of yeast transcripts, including the existence of more secondary structure over coding regions compared with untranslated regions, a three-nucleotide periodicity of secondary structure across coding regions and an anti-correlation between the efficiency with which an mRNA is translated and the structure over its translation start site. PARS is readily applicable to other organisms and to profiling RNA structure in diverse conditions, thus enabling studies of the dynamics of secondary structure at a genomic scale.

  View details for DOI 10.1038/nature09322

  View details for PubMedID 20811459

• HOTAIR Flight of noncoding RNAs in cancer metastasis CELL CYCLE Wan, Y., Chang, H. Y. 2010; 9 (17): 3391-3392

  View details for DOI 10.4161/cc.9.17.13122

  View details for Web of Science ID 000281621700004

  View details for PubMedID 20864820

  View details for PubMedCentralID PMC3066151

• Long noncoding RNA in genome regulation Prospects and mechanisms RNA BIOLOGY Hung, T., Chang, H. Y. 2010; 7 (5): 582-585

  Abstract

  Long noncoding RNAs (lncRNAs) are pervasively transcribed and critical regulators of the epigenome[1, 2]. These long, polyadenylated RNAs do not code for proteins, but function directly as RNAs, recruiting chromatin modifiers to mediate transcriptional changes in processes ranging from X-inactivation (XIST) to imprinting (H19)[3]. The recent discovery that lncRNA HOTAIR can link chromatin changes to cancer metastasis[4] furthers the relevance of lncRNAs to human disease. Here, we discuss lncRNAs as regulatory modules and explore the implications for disease pathogenesis. Although large-scale analyses of mammalian transcriptomes have revealed that more than 50% of transcripts have no protein coding potential[2, 5, 6], the functions of these putative transcripts are largely unknown. A subset of these noncoding transcripts are termed long noncoding RNAs (lncRNAs), based on an arbitrary minimum length of 200 nucleotides. LncRNAs are roughly classified based on their position relative to protein-coding genes: intergenic (between genes), intragenic/intronic (within genes), and antisense[2]. Initial efforts to characterize these molecules demonstrated that they function in cis, regulating their immediate genomic neighbors. Examples include AIR, XIST, and Kcnq1ot (reviewed in [1, 7, 8]), which recruit chromatin modifying complexes to silence adjacent sites. The scope of lncRNAs in gene regulation was advanced with the finding that lncRNA HOTAIR exhibited trans regulatory capacities. HOTAIR is transcribed at the intersection of opposing chromatin domains in the HOXC locus, but targets Polycomb Repressive Complex 2 (PRC2) to silence 40 kilobases of HOXD[9], a locus involved in developmental patterning. A subsequent study revealed that HOTAIR is overexpressed in approximately one quarter of human breast cancers, directing PRC2 to approximately 800 ectopic sites in the genome, which leads to histone H3 lysine 27 trimethylation and changes in gene expression[4]. The impacts of lncRNA-mediated chromatin changes are noteworthy: not only did HOTAIR drive metastasis in a mouse model, but HOTAIR expression in human breast cancer was found to be an independent prognostic marker for death and metastasis[4]. The fact that HOTAIR drives chromatin reprogramming genome-wide suggests that long-range regulation by lncRNAs may be a widespread mechanism. This is supported by a study showing that > 20% of tested lncRNAs are bound by PRC2 and other chromatin modifiers[10]. Furthermore, this is an underestimate of the total RNAs involved in chromatin modification, as PRC2 target genes also transcribe smaller 50-200 nt RNAs that interact with SUZ12 to mediate gene repression[11]. These findings provoke questions regarding the initial triggers for HOTAIR overexpression and whether understanding of lncRNA mechanics may have clinical relevance.

  View details for PubMedID 20930520

• Long Noncoding RNA as Modular Scaffold of Histone Modification Complexes SCIENCE Tsai, M., Manor, O., Wan, Y., Mosammaparast, N., Wang, J. K., Lan, F., Shi, Y., Segal, E., Chang, H. Y. 2010; 329 (5992): 689-693

  Abstract

  Long intergenic noncoding RNAs (lincRNAs) regulate chromatin states and epigenetic inheritance. Here, we show that the lincRNA HOTAIR serves as a scaffold for at least two distinct histone modification complexes. A 5' domain of HOTAIR binds polycomb repressive complex 2 (PRC2), whereas a 3' domain of HOTAIR binds the LSD1/CoREST/REST complex. The ability to tether two distinct complexes enables RNA-mediated assembly of PRC2 and LSD1 and coordinates targeting of PRC2 and LSD1 to chromatin for coupled histone H3 lysine 27 methylation and lysine 4 demethylation. Our results suggest that lincRNAs may serve as scaffolds by providing binding surfaces to assemble select histone modification enzymes, thereby specifying the pattern of histone modifications on target genes.

  View details for DOI 10.1126/science.1192002

  View details for PubMedID 20616235

• Tumor suppression by the histone demethylase UTX CELL CYCLE Tsai, M., Wang, J. K., Chang, H. Y. 2010; 9 (11): 2043-2044

  View details for Web of Science ID 000279148400001

  View details for PubMedID 20505331

  View details for PubMedCentralID PMC3311649

• Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis NATURE Gupta, R. A., Shah, N., Wang, K. C., Kim, J., Horlings, H. M., Wong, D. J., Tsai, M., Hung, T., Argani, P., Rinn, J. L., Wang, Y., Brzoska, P., Kong, B., Li, R., West, R. B., van de Vijver, M. J., Sukumar, S., Chang, H. Y. 2010; 464 (7291): 1071-U148

  Abstract

  Large intervening non-coding RNAs (lincRNAs) are pervasively transcribed in the genome yet their potential involvement in human disease is not well understood. Recent studies of dosage compensation, imprinting, and homeotic gene expression suggest that individual lincRNAs can function as the interface between DNA and specific chromatin remodelling activities. Here we show that lincRNAs in the HOX loci become systematically dysregulated during breast cancer progression. The lincRNA termed HOTAIR is increased in expression in primary breast tumours and metastases, and HOTAIR expression level in primary tumours is a powerful predictor of eventual metastasis and death. Enforced expression of HOTAIR in epithelial cancer cells induced genome-wide re-targeting of Polycomb repressive complex 2 (PRC2) to an occupancy pattern more resembling embryonic fibroblasts, leading to altered histone H3 lysine 27 methylation, gene expression, and increased cancer invasiveness and metastasis in a manner dependent on PRC2. Conversely, loss of HOTAIR can inhibit cancer invasiveness, particularly in cells that possess excessive PRC2 activity. These findings indicate that lincRNAs have active roles in modulating the cancer epigenome and may be important targets for cancer diagnosis and therapy.

  View details for DOI 10.1038/nature08975

  View details for Web of Science ID 000276635000045

  View details for PubMedID 20393566

  View details for PubMedCentralID PMC3049919

• A TGF beta-Responsive Gene Signature Is Associated with a Subset of Diffuse Scleroderma with Increased Disease Severity JOURNAL OF INVESTIGATIVE DERMATOLOGY Sargent, J. L., Milano, A., Bhattacharyya, S., Varga, J., Connolly, M. K., Chang, H. Y., Whitfield, M. L. 2010; 130 (3): 694-705

  Abstract

  Systemic sclerosis is a complex disease with widespread skin fibrosis and variable visceral organ involvement. Since transforming growth factor-beta (TGFbeta) has been implicated in driving fibrosis in systemic sclerosis, a mechanism-derived gene expression signature was used to assay TGFbeta-responsive gene expression in the skin of patients with systemic sclerosis (SSc). Primary dermal fibroblasts from patients with diffuse SSc (dSSc) and healthy controls were treated with TGFbeta, and the genome-wide gene expression was measured on DNA microarrays over a time course of 24 hours. Eight hundred and ninety-four probes representing 674 uniquely annotated genes were identified as TGFbeta responsive. Expression of the TGFbeta-responsive signature was examined in skin biopsies from 17 dSSc, seven limited SSc (lSSc), three morphea patients, and six healthy controls. The TGFbeta-responsive signature was expressed in 10 out of 17 dSSc skin biopsies, but was not found in lSSc, morphea, or healthy control biopsies. Expression of dSSC the TGFbeta-responsive signature stratifies patients into two major groups, one of which corresponds to the "diffuse-proliferation" intrinsic subset that showed higher modified Rodnan skin score and a higher likelihood of scleroderma lung disease. The TGFbeta-responsive signature is found in only a subset of dSSc patients who could be targeted by specific therapies.

  View details for DOI 10.1038/jid.2009.318

  View details for Web of Science ID 000275017600013

  View details for PubMedID 19812599

• The histone demethylase UTX enables RB-dependent cell fate control GENES & DEVELOPMENT Wang, J. K., Tsai, M., Poulin, G., Adler, A. S., Chen, S., Liu, H., Shi, Y., Chang, H. Y. 2010; 24 (4): 327-332

  Abstract

  Trimethylation of histone H3 on Lys 27 (H3K27me3) is key for cell fate regulation. The H3K27me3 demethylase UTX functions in development and tumor suppression with undefined mechanisms. Here, genome-wide chromatin occupancy analysis of UTX and associated histone modifications reveals distinct classes of UTX target genes, including genes encoding Retinoblastoma (RB)-binding proteins. UTX removes H3K27me3 and maintains expression of several RB-binding proteins, enabling cell cycle arrest. Genetic interactions in mammalian cells and Caenorhabditis elegans show that UTX regulates cell fates via RB-dependent pathways. Thus, UTX defines an evolutionarily conserved mechanism to enable coordinate transcription of a RB network in cell fate control.

  View details for DOI 10.1101/gad.1882610

  View details for PubMedID 20123895

• Anatomic Demarcation of Cells: Genes to Patterns SCIENCE Chang, H. Y. 2009; 326 (5957): 1206-1207

  Abstract

  An organizing principle of the diverse cell types in multicellular organisms is their anatomic location. In turn, anatomic location is patterned by the positional identities of cells along developmental axes. Recent progress in functional genomics and chromatin biology illustrates how cells use specific gene expression programs to encode location. Dynamic chromatin states of key genes, notably the Hox loci, serve as the internal representation in cells of their positional identity within the animal.

  View details for DOI 10.1126/science.1175686

  View details for PubMedID 19965461

• Identification, molecular characterization, clinical prognosis, and therapeutic targeting of human bladder tumor-initiating cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chan, K. S., Espinosa, I., Chao, M., Wong, D., Ailles, L., Diehn, M., Gill, H., Presti, J., Chang, H. Y., van de Rijn, M., Shortliffe, L., Weissman, I. L. 2009; 106 (33): 14016-14021

  Abstract

  Major clinical issues in bladder cancer include the identification of prediction markers and novel therapeutic targets for invasive bladder cancer. In the current study, we describe the isolation and characterization of a tumor-initiating cell (T-IC) subpopulation in primary human bladder cancer, based on the expression of markers similar to that of normal bladder basal cells (Lineage-CD44(+)CK5(+)CK20(-)). The bladder T-IC subpopulation was defined functionally by its enriched ability to induce xenograft tumors in vivo that recapitulated the heterogeneity of the original tumor. Further, molecular analysis of more than 300 bladder cancer specimens revealed heterogeneity among activated oncogenic pathways in T-IC (e.g., 80% Gli1, 45% Stat3, 10% Bmi-1, and 5% beta-catenin). Despite this molecular heterogeneity, we identified a unique bladder T-IC gene signature by gene chip analysis. This T-IC gene signature, which effectively distinguishes muscle-invasive bladder cancer with worse clinical prognosis from non-muscle-invasive (superficial) cancer, has significant clinical value. It also can predict the progression of a subset of recurring non-muscle-invasive cancers. Finally, we found that CD47, a protein that provides an inhibitory signal for macrophage phagocytosis, is highly expressed in bladder T-ICs compared with the rest of the tumor. Blockade of CD47 by a mAb resulted in macrophage engulfment of bladder cancer cells in vitro. In summary, we have identified a T-IC subpopulation with potential prognostic and therapeutic value for invasive bladder cancer.

  View details for DOI 10.1073/pnas.0906549106

  View details for PubMedID 19666525

• Gene dates, parties and galas Symposium on Chromatin Dynamics and Higher Order Organization EMBO REPORTS Chang, H. Y., Cuvier, O., Dekker, J. 2009; 10 (7): 689-693

  View details for DOI 10.1038/embor.2009.136

  View details for PubMedID 19525922

• Tumor Vasculature Is Regulated by PHD2-Mediated Angiogenesis and Bone Marrow-Derived Cell Recruitment CANCER CELL Chan, D. A., Kawahara, T. L., Sutphin, P. D., Chang, H. Y., Chi, J., Giaccia, A. J. 2009; 15 (6): 527-538

  Abstract

  Sustained angiogenesis, through either local sprouting (angiogenesis) or the recruitment of bone marrow-derived cells (BMDCs) (vasculogenesis), is essential to the development of a tumor. How BMDCs are recruited to the tumor and their contribution to the tumor vasculature is poorly understood. Here, we demonstrate that both IL-8 and angiogenin contribute to the complementary pathways of angiogenesis and BMDC mobilization to increase tumor growth. These two factors are regulated by PHD2 in a HIF-independent but NF-kappaB-dependent manner. PHD2 levels are decreased in human cancers, compared with corresponding normal tissue, and correlate with an increase in mature blood vessels. Thus, PHD2 plays a critical role in regulating tumor angiogenesis.

  View details for DOI 10.1016/j.ccr.2009.04.010

  View details for PubMedID 19477431

• Modeling Inducible Human Tissue Neoplasia Identifies an Extracellular Matrix Interaction Network Involved in Cancer Progression CANCER CELL Reuter, J. A., Ortiz-Urda, S., Kretz, M., Garcia, J., Scholl, F. A., Pasmooij, A. M., Cassarino, D., Chang, H. Y., Khavari, P. A. 2009; 15 (6): 477-488

  Abstract

  To elucidate mechanisms of cancer progression, we generated inducible human neoplasia in three-dimensionally intact epithelial tissue. Gene expression profiling of both epithelia and stroma at specific time points during tumor progression revealed sequential enrichment of genes mediating discrete biologic functions in each tissue compartment. A core cancer progression signature was distilled using the increased signaling specificity of downstream oncogene effectors and subjected to network modeling. Network topology predicted that tumor development depends on specific extracellular matrix-interacting network hubs. Blockade of one such hub, the beta1 integrin subunit, disrupted network gene expression and attenuated tumorigenesis in vivo. Thus, integrating network modeling and temporal gene expression analysis of inducible human neoplasia provides an approach to prioritize and characterize genes functioning in cancer progression.

  View details for DOI 10.1016/j.ccr.2009.04.002

  View details for Web of Science ID 000266686500006

  View details for PubMedID 19477427

  View details for PubMedCentralID PMC3050547

• Regeneration, repair and remembering identity: the three Rs of Hox gene expression TRENDS IN CELL BIOLOGY Wang, K. C., Helms, J. A., Chang, H. Y. 2009; 19 (6): 268-275

  Abstract

  Hox genes encode transcription factors that specify embryonic positional identity in cells and guide tissue differentiation. Recent advances have greatly increased our understanding of the epigenetic mechanisms that ensure the faithful expression of Hox genes in adult cells and which involve the interplay of histone methylation, demethylation and intergenic transcription of long non-coding RNAs. The transcriptional memory of Hox genes poses both an opportunity and a challenge for regenerative medicine. Matching the positional identity of transplanted stem cells with that of the host environment, as reflected by their respective Hox profiles, is likely to be required to achieve regenerative healing. Strategies to manipulate the plasticity of Hox gene expression will probably become a major focus in regenerative medicine.

  View details for DOI 10.1016/j.tcb.2009.03.007

  View details for PubMedID 19428253

• Hierarchical Maintenance of MLL Myeloid Leukemia Stem Cells Employs a Transcriptional Program Shared with Embryonic Rather Than Adult Stem Cells CELL STEM CELL Somervaille, T. C., Matheny, C. J., Spencer, G. J., Iwasaki, M., Rinn, J. L., Witten, D. M., Chang, H. Y., Shurtleff, S. A., Downing, J. R., Cleary, M. L. 2009; 4 (2): 129-140

  Abstract

  The genetic programs that promote retention of self-renewing leukemia stem cells (LSCs) at the apex of cellular hierarchies in acute myeloid leukemia (AML) are not known. In a mouse model of human AML, LSCs exhibit variable frequencies that correlate with the initiating MLL oncogene and are maintained in a self-renewing state by a transcriptional subprogram more akin to that of embryonic stem cells (ESCs) than to that of adult stem cells. The transcription/chromatin regulatory factors Myb, Hmgb3, and Cbx5 are critical components of the program and suffice for Hoxa/Meis-independent immortalization of myeloid progenitors when coexpressed, establishing the cooperative and essential role of an ESC-like LSC maintenance program ancillary to the leukemia-initiating MLL/Hox/Meis program. Enriched expression of LSC maintenance and ESC-like program genes in normal myeloid progenitors and poor-prognosis human malignancies links the frequency of aberrantly self-renewing progenitor-like cancer stem cells (CSCs) to prognosis in human cancer.

  View details for DOI 10.1016/j.stem.2008.11.015

  View details for PubMedID 19200802

• Molecular Framework for Response to Imatinib Mesylate in Systemic Sclerosis ARTHRITIS AND RHEUMATISM Chung, L., Fiorentino, D. F., Benbarak, M. J., Adler, A. S., Mariano, M. M., Paniagua, R. T., Milano, A., Connolly, M. K., Ratiner, B. D., Wiskocil, R. L., Whitfield, M. L., Chang, H. Y., Robinson, W. H. 2009; 60 (2): 584-591

  Abstract

  Systemic sclerosis (SSc) is an autoimmune disease in which the tyrosine kinases platelet-derived growth factor receptor (PDGFR) and Abl are hypothesized to contribute to the fibrosis and vasculopathy of the skin and internal organs. Herein we describe 2 patients with early diffuse cutaneous SSc (dcSSc) who experienced reductions in cutaneous sclerosis in response to therapy with the tyrosine kinase inhibitor imatinib mesylate. Immunohistochemical analyses of skin biopsy specimens demonstrated reductions of phosphorylated PDGFRbeta and Abl with imatinib therapy. By gene expression profiling, an imatinib-responsive signature specific to dcSSc was identified (P < 10(-8)). The response of these patients and the findings of the analyses suggest that PDGFRbeta and Abl play critical, synergistic roles in the pathogenesis of SSc, and that imatinib targets a gene expression program that is frequently dysregulated in dcSSc.

  View details for DOI 10.1002/art.24221

  View details for PubMedID 19180499

• ING4 Mediates Crosstalk between Histone H3 K4 Trimethylation and H3 Acetylation to Attenuate Cellular Transformation MOLECULAR CELL Hung, T., Binda, O., Champagne, K. S., Kuo, A. J., Johnson, K., Chang, H. Y., Simon, M. D., Kutateladze, T. G., Gozani, O. 2009; 33 (2): 248-256

  Abstract

  Aberrations in chromatin dynamics play a fundamental role in tumorigenesis, yet relatively little is known of the molecular mechanisms linking histone lysine methylation to neoplastic disease. ING4 (Inhibitor of Growth 4) is a native subunit of an HBO1 histone acetyltransferase (HAT) complex and a tumor suppressor protein. Here we show a critical role for specific recognition of histone H3 trimethylated at lysine 4 (H3K4me3) by the ING4 PHD finger in mediating ING4 gene expression and tumor suppressor functions. The interaction between ING4 and H3K4me3 augments HBO1 acetylation activity on H3 tails and drives H3 acetylation at ING4 target promoters. Further, ING4 facilitates apoptosis in response to genotoxic stress and inhibits anchorage-independent cell growth, and these functions depend on ING4 interactions with H3K4me3. Together, our results demonstrate a mechanism for brokering crosstalk between H3K4 methylation and H3 acetylation and reveal a molecular link between chromatin modulation and tumor suppressor mechanisms.

  View details for DOI 10.1016/j.molcel.2008.12.016

  View details for PubMedID 19187765

• SIRT6 Links Histone H3 Lysine 9 Deacetylation to NF-kappa B-Dependent Gene Expression and Organismal Life Span CELL Kawahara, T. L., Michishita, E., Adler, A. S., Damian, M., Berber, E., Lin, M., McCord, R. A., Ongaigui, K. C., Boxer, L. D., Chang, H. Y., Chua, K. F. 2009; 136 (1): 62-74

  Abstract

  Members of the sirtuin (SIRT) family of NAD-dependent deacetylases promote longevity in multiple organisms. Deficiency of mammalian SIRT6 leads to shortened life span and an aging-like phenotype in mice, but the underlying molecular mechanisms are unclear. Here we show that SIRT6 functions at chromatin to attenuate NF-kappaB signaling. SIRT6 interacts with the NF-kappaB RELA subunit and deacetylates histone H3 lysine 9 (H3K9) at NF-kappaB target gene promoters. In SIRT6-deficient cells, hyperacetylation of H3K9 at these target promoters is associated with increased RELA promoter occupancy and enhanced NF-kappaB-dependent modulation of gene expression, apoptosis, and cellular senescence. Computational genomics analyses revealed increased activity of NF-kappaB-driven gene expression programs in multiple Sirt6-deficient tissues in vivo. Moreover, haploinsufficiency of RelA rescues the early lethality and degenerative syndrome of Sirt6-deficient mice. We propose that SIRT6 attenuates NF-kappaB signaling via H3K9 deacetylation at chromatin, and hyperactive NF-kappaB signaling may contribute to premature and normal aging.

  View details for DOI 10.1016/j.cell.2008.10.052

  View details for PubMedID 19135889

• Genome-Wide Views of Chromatin Structure ANNUAL REVIEW OF BIOCHEMISTRY Rando, O. J., Chang, H. Y. 2009; 78: 245-271

  Abstract

  Eukaryotic genomes are packaged into a nucleoprotein complex known as chromatin, which affects most processes that occur on DNA. Along with genetic and biochemical studies of resident chromatin proteins and their modifying enzymes, mapping of chromatin structure in vivo is one of the main pillars in our understanding of how chromatin relates to cellular processes. In this review, we discuss the use of genomic technologies to characterize chromatin structure in vivo, with a focus on data from budding yeast and humans. The picture emerging from these studies is the detailed chromatin structure of a typical gene, where the typical behavior gives insight into the mechanisms and deep rules that establish chromatin structure. Important deviation from the archetype is also observed, usually as a consequence of unique regulatory mechanisms at special genomic loci. Chromatin structure shows substantial conservation from yeast to humans, but mammalian chromatin has additional layers of complexity that likely relate to the requirements of multicellularity such as the need to establish faithful gene regulatory mechanisms for cell differentiation.

  View details for DOI 10.1146/annurev.biochem.78.071107.134639

  View details for Web of Science ID 000268069200011

  View details for PubMedID 19317649

  View details for PubMedCentralID PMC2811691

• Global Expression Profiling in Atopic Eczema Reveals Reciprocal Expression of Inflammatory and Lipid Genes PLOS ONE Saaf, A. M., Tengvall-Linder, M., Chang, H. Y., Adler, A. S., Wahlgren, C., Scheynius, A., Nordenskjold, M., Bradley, M. 2008; 3 (12)

  Abstract

  Atopic eczema (AE) is a common chronic inflammatory skin disorder. In order to dissect the genetic background several linkage and genetic association studies have been performed. Yet very little is known about specific genes involved in this complex skin disease, and the underlying molecular mechanisms are not fully understood.We used human DNA microarrays to identify a molecular picture of the programmed responses of the human genome to AE. The transcriptional program was analyzed in skin biopsy samples from lesional and patch-tested skin from AE patients sensitized to Malassezia sympodialis (M. sympodialis), and corresponding biopsies from healthy individuals. The most notable feature of the global gene-expression pattern observed in AE skin was a reciprocal expression of induced inflammatory genes and repressed lipid metabolism genes. The overall transcriptional response in M. sympodialis patch-tested AE skin was similar to the gene-expression signature identified in lesional AE skin. In the constellation of genes differentially expressed in AE skin compared to healthy control skin, we have identified several potential susceptibility genes that may play a critical role in the pathological condition of AE. Many of these genes, including genes with a role in immune responses, lipid homeostasis, and epidermal differentiation, are localized on chromosomal regions previously linked to AE.Through genome-wide expression profiling, we were able to discover a distinct reciprocal expression pattern of induced inflammatory genes and repressed lipid metabolism genes in skin from AE patients. We found a significant enrichment of differentially expressed genes in AE with cytobands associated to the disease, and furthermore new chromosomal regions were found that could potentially guide future region-specific linkage mapping in AE. The full data set is available at http://microarray-pubs.stanford.edu/eczema.

  View details for DOI 10.1371/journal.pone.0004017

  View details for Web of Science ID 000265463000012

  View details for PubMedID 19107207

  View details for PubMedCentralID PMC2603322

• Stemness, cancer and cancer stem cells CELL CYCLE Wong, D. J., Segal, E., Chang, H. Y. 2008; 7 (23): 3622-3624

  Abstract

  The ability of cancers to grow indefinitely has fueled the idea that cancer and stem cells may have common underlying mechanisms. Detailed gene expression maps have now shown the diversity and distinctiveness in gene expression programs associated with stemness in embryonic and adult stem cells. These maps have further revealed a shared transcriptional program in embryonic stem cells (ESC) and cancer stem cells. Surprisingly, forced activation of an ESC-like gene expression program in adult epithelial cells can reprogram them into human cancer stem cells and achieve pathologic self-renewal. The ability to create induced cancer stem cells (iCSC) may provide opportunities to better define the biology of cancer stem cells in order to trace or eliminate them in human patients.

  View details for PubMedID 19029796

• Combining biological gene expression signatures in predicting outcome in breast cancer: An alternative to supervised classification EUROPEAN JOURNAL OF CANCER Nuyten, D. S., Hastie, T., Chi, J. A., Chang, H. Y., van de Vijver, M. J. 2008; 44 (15): 2319-2329

  Abstract

  Gene expression profiling has been extensively used to predict outcome in breast cancer patients. We have previously reported on biological hypothesis-driven analysis of gene expression profiling data and we wished to extend this approach through the combinations of various gene signatures to improve the prediction of outcome in breast cancer.We have used gene expression data (25.000 gene probes) from a previously published study of tumours from 295 early stage breast cancer patients from the Netherlands Cancer Institute using updated follow-up. Tumours were assigned to three prognostic groups using the previously reported Wound-response and hypoxia-response signatures, and the outcome in each of these subgroups was evaluated.We have assigned invasive breast carcinomas from 295 stages I and II breast cancer patients to three groups based on gene expression profiles subdivided by the wound-response signature (WS) and hypoxia-response signature (HS). These three groups are (1) quiescent WS/non-hypoxic HS; (2) activated WS/non-hypoxic HS or quiescent WS/hypoxic tumours and (3) activated WS/hypoxic HS. The overall survival at 15 years for patients with tumours in groups 1, 2 and 3 are 79%, 59% and 27%, respectively. In multivariate analysis, this signature is not only independent of clinical and pathological risk factors; it is also the strongest predictor of outcome. Compared to a previously identified 70-gene prognosis profile, obtained with supervised classification, the combination of signatures performs roughly equally well and might have additional value in the ER-negative subgroup. In the subgroup of lymph node positive patients, the combination signature outperforms the 70-gene signature in multivariate analysis. In addition, in multivariate analysis, the WS/HS combination is a stronger predictor of outcome compared to the recently reported invasiveness gene signature combined with the WS.A combination of biological gene expression signatures can be used to identify a powerful and independent predictor for outcome in breast cancer patients.

  View details for DOI 10.1016/j.ejca.2008.07.015

  View details for Web of Science ID 000261020800031

  View details for PubMedID 18715778

  View details for PubMedCentralID PMC3756930

• Deletional tolerance mediated by extrathymic Aire-expressing cells SCIENCE Gardner, J. M., DeVoss, J. J., Friedman, R. S., Wong, D. J., Tan, Y. X., Zhou, X., Johannes, K. P., Su, M. A., Chang, H. Y., Krummel, M. F., Anderson, M. S. 2008; 321 (5890): 843-847

  Abstract

  The prevention of autoimmunity requires the elimination of self-reactive T cells during their development and maturation. The expression of diverse self-antigens by stromal cells in the thymus is essential to this process and depends, in part, on the activity of the autoimmune regulator (Aire) gene. Here we report the identification of extrathymic Aire-expressing cells (eTACs) resident within the secondary lymphoid organs. These stromally derived eTACs express a diverse array of distinct self-antigens and are capable of interacting with and deleting naïve autoreactive T cells. Using two-photon microscopy, we observed stable antigen-specific interactions between eTACs and autoreactive T cells. We propose that such a secondary network of self-antigen-expressing stromal cells may help reinforce immune tolerance by preventing the maturation of autoreactive T cells that escape thymic negative selection.

  View details for DOI 10.1126/science.1159407

  View details for Web of Science ID 000258261000048

  View details for PubMedID 18687966

  View details for PubMedCentralID PMC2532844

• Control of differentiation in a self-renewing mammalian tissue by the histone demethylase JMJD3 GENES & DEVELOPMENT Sen, G. L., Webster, D. E., Barragan, D. I., Chang, H. Y., Khavari, P. A. 2008; 22 (14): 1865-1870

  Abstract

  The recent discovery of H3K27me3 demethylases suggests that H3K27me3 may dynamically regulate gene expression, but this potential role in mammalian tissue homeostasis remains uncharacterized. In the epidermis, a tissue that balances stem cell self-renewal with differentiation, H3K27me3, occupies the promoters of many differentiation genes. During calcium-induced differentiation, H3K27me3 was erased at these promoters in concert with loss of PcG protein occupancy and increased binding by the H3K27me3 demethylase, JMJD3. Within epidermal tissue, JMJD3 depletion blocked differentiation, while active JMJD3 dominantly induced it. These results indicate that epigenetic derepression by JMJD3 controls mammalian epidermal differentiation.

  View details for DOI 10.1101/gad.1673508

  View details for Web of Science ID 000257643400003

  View details for PubMedID 18628393

  View details for PubMedCentralID PMC2492733

• Mechanisms of an autoimmunity syndrome in mice caused by a dominant mutation in Aire JOURNAL OF CLINICAL INVESTIGATION Su, M. A., Giang, K., Zumer, K., Jiang, H., Oven, I., Rinn, J. L., DeVoss, J. J., Johannes, K. P., Lu, W., Gardner, J., Chang, A., Bubulya, P., Chang, H. Y., Peterlin, B. M., Anderson, M. S. 2008; 118 (5): 1712-1726

  Abstract

  Homozygous loss-of-function mutations in AIRE cause autoimmune polyglandular syndrome type 1 (APS 1), which manifests in a classic triad of hypoparathyroidism, adrenal insufficiency, and candidiasis. Interestingly, a kindred with a specific G228W AIRE variant presented with an autosomal dominant autoimmune phenotype distinct from APS 1. We utilized a novel G228W-knockin mouse model to show that this variant acted in a dominant-negative manner to cause a unique autoimmunity syndrome. In addition, the expression of a large number of Aire-regulated thymic antigens was partially inhibited in these animals, demonstrating the importance of quantitative changes in thymic antigen expression in determining organ-specific autoimmunity. Furthermore, the dominant-negative effect of the G228W variant was exerted through recruitment of WT Aire away from active sites of transcription in the nucleus of medullary thymic epithelial cells in vivo. Together, these results may demonstrate a mechanism by which autoimmune predisposition to phenotypes distinct from APS 1 can be mediated in a dominant-negative fashion by Aire.

  View details for DOI 10.1172/JCI34523

  View details for Web of Science ID 000255490100016

  View details for PubMedID 18414681

  View details for PubMedCentralID PMC2293336

• A systems biology approach to anatomic diversity of skin JOURNAL OF INVESTIGATIVE DERMATOLOGY Rinn, J. L., Wang, J. K., Liu, H., Montgomery, K., van de Rijn, M., Chang, H. Y. 2008; 128 (4): 776-782

  Abstract

  Human skin exhibits exquisite site-specific morphologies and functions. How are these site-specific differences specified during development, maintained in adult homeostasis, and potentially perturbed by disease processes? Here, we review progress in understanding the anatomic patterning of fibroblasts, a major constituent cell type of the dermis and key participant in epithelial-mesenchymal interactions. The gene expression programs of human fibroblasts largely reflect the superimposition of three gene expression profiles that demarcate the fibroblast's position relative to three developmental axes. The HOX family of homeodomain transcription factors is implicated in specifying site-specific transcriptional programs. The use of gene, tiling, and tissue microarrays together gives a comprehensive view of the gene regulation involved in patterning the skin.

  View details for DOI 10.1038/sj.jid.5700986

  View details for PubMedID 18337710

• Module map of stem cell genes guides creation of epithelial cancer stem cells CELL STEM CELL Wong, D. J., Liu, H., Ridky, T. W., Cassarino, D., Segal, E., Chang, H. Y. 2008; 2 (4): 333-344

  Abstract

  Self-renewal is a hallmark of stem cells and cancer, but existence of a shared stemness program remains controversial. Here, we construct a gene module map to systematically relate transcriptional programs in embryonic stem cells (ESCs), adult tissue stem cells, and human cancers. This map reveals two predominant gene modules that distinguish ESCs and adult tissue stem cells. The ESC-like transcriptional program is activated in diverse human epithelial cancers and strongly predicts metastasis and death. c-Myc, but not other oncogenes, is sufficient to reactivate the ESC-like program in normal and cancer cells. In primary human keratinocytes transformed by Ras and I kappa B alpha, c-Myc increases the fraction of tumor-initiating cells by 150-fold, enabling tumor formation and serial propagation with as few as 500 cells. c-Myc-enhanced tumor initiation is cell-autonomous and independent of genomic instability. Thus, activation of an ESC-like transcriptional program in differentiated adult cells may induce pathologic self-renewal characteristic of cancer stem cells.

  View details for DOI 10.1016/j.stem.2008.02.009

  View details for PubMedID 18397753

• SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin NATURE Michishita, E., McCord, R. A., Berber, E., Kioi, M., Padilla-Nash, H., Damian, M., Cheung, P., Kusumoto, R., Kawahara, T. L., Barrett, J. C., Chang, H. Y., Bohr, V. A., Ried, T., Gozani, O., Chua, K. F. 2008; 452 (7186): 492-U16

  Abstract

  The Sir2 deacetylase regulates chromatin silencing and lifespan in Saccharomyces cerevisiae. In mice, deficiency for the Sir2 family member SIRT6 leads to a shortened lifespan and a premature ageing-like phenotype. However, the molecular mechanisms of SIRT6 function are unclear. SIRT6 is a chromatin-associated protein, but no enzymatic activity of SIRT6 at chromatin has yet been detected, and the identity of physiological SIRT6 substrates is unknown. Here we show that the human SIRT6 protein is an NAD+-dependent, histone H3 lysine 9 (H3K9) deacetylase that modulates telomeric chromatin. SIRT6 associates specifically with telomeres, and SIRT6 depletion leads to telomere dysfunction with end-to-end chromosomal fusions and premature cellular senescence. Moreover, SIRT6-depleted cells exhibit abnormal telomere structures that resemble defects observed in Werner syndrome, a premature ageing disorder. At telomeric chromatin, SIRT6 deacetylates H3K9 and is required for the stable association of WRN, the factor that is mutated in Werner syndrome. We propose that SIRT6 contributes to the propagation of a specialized chromatin state at mammalian telomeres, which in turn is required for proper telomere metabolism and function. Our findings constitute the first identification of a physiological enzymatic activity of SIRT6, and link chromatin regulation by SIRT6 to telomere maintenance and a human premature ageing syndrome.

  View details for DOI 10.1038/nature06736

  View details for PubMedID 18337721

• Systematic functional characterization of cis-regulatory motifs in human core promoters GENOME RESEARCH Sinha, S., Adler, A. S., Field, Y., Chang, H. Y., Segal, E. 2008; 18 (3): 477-488

  Abstract

  A large number of cis-regulatory motifs involved in transcriptional control have been identified, but the regulatory context and biological processes in which many of them function are unknown. Here, we computationally identify the sets of human core promoters targeted by motifs, and systematically characterize their function by using a robust gene-set-based approach and diverse sources of biological data. We find that the target sets of most motifs contain both genes with similar function and genes that are coregulated in vivo, thereby suggesting both the biological process regulated by the motifs and the conditions in which this regulation may occur. Our analysis also identifies many motifs whose target sets are predicted to be regulated by a common microRNA, suggesting a connection between transcriptional and post-transcriptional control processes. Finally, we predict novel roles for uncharacterized motifs in the regulation of specific biological processes and certain types of human cancer, and experimentally validate four such predictions, suggesting regulatory roles for four uncharacterized motifs in cell cycle progression. Our analysis thus provides a concrete framework for uncovering the biological function of cis-regulatory motifs genome wide.

  View details for DOI 10.1101/gr.6828808

  View details for Web of Science ID 000253766700014

  View details for PubMedID 18256240

  View details for PubMedCentralID PMC2259112

• Reversal of aging by NF kappa B blockade CELL CYCLE Adler, A. S., Kawahara, T. L., Segal, E., Chang, H. Y. 2008; 7 (5): 556-559

  Abstract

  Genetic studies in model organisms such as yeast, worms, flies, and mice leading to lifespan extension suggest that longevity is subject to regulation. In addition, various system-wide interventions in old animals can reverse features of aging. To better understand these processes, much effort has been put into the study of aging on a molecular level. In particular, genome-wide microarray analysis of differently aged individual organisms or tissues has been used to track the global expression changes that occur during normal aging. Although these studies consistently implicate specific pathways in aging processes, there is little conservation between the individual genes that change. To circumvent this problem, we have recently developed a novel computational approach to discover transcription factors that may be responsible for driving global expression changes with age. We identified the transcription factor NFkappaB as a candidate activator of aging-related transcriptional changes in multiple human and mouse tissues. Genetic blockade of NFkappaB in the skin of chronologically aged mice reversed the global gene expression program and tissue characteristics to those of young mice, demonstrating for the first time that disruption of a single gene is sufficient to reverse features of aging, at least for the short-term.

  View details for PubMedID 18256548

• A dermal HOX transcriptional program regulates site-specific epidermal fate GENES & DEVELOPMENT Rinn, J. L., Wang, J. K., Allen, N., Brugmann, S. A., Mikels, A. J., Liu, H., Ridky, T. W., Stadler, H. S., Nusse, R., Helms, J. A., Chang, H. Y. 2008; 22 (3): 303-307

  Abstract

  Reciprocal epithelial-mesenchymal interactions shape site-specific development of skin. Here we show that site-specific HOX expression in fibroblasts is cell-autonomous and epigenetically maintained. The distal-specific gene HOXA13 is continually required to maintain the distal-specific transcriptional program in adult fibroblasts, including expression of WNT5A, a morphogen required for distal development. The ability of distal fibroblasts to induce epidermal keratin 9, a distal-specific gene, is abrogated by depletion of HOXA13, but rescued by addition of WNT5A. Thus, maintenance of appropriate HOX transcriptional program in adult fibroblasts may serve as a source of positional memory to differentially pattern the epithelia during homeostasis and regeneration.

  View details for DOI 10.1101/gad.1610508

  View details for PubMedID 18245445

• CSN5 isopeptidase activity links COP9 signalosome activation to breast cancer progression CANCER RESEARCH Adler, A. S., Littlepage, L. E., Lin, M., Kawahara, T. L., Wong, D. J., Werb, Z., Chang, H. Y. 2008; 68 (2): 506-515

  Abstract

  CSN5 has been implicated as a candidate oncogene in human breast cancers by genetic linkage with activation of the poor-prognosis, wound response gene expression signature. CSN5 is a subunit of the eight-protein COP9 signalosome, a signaling complex with multiple biochemical activities; the mechanism of CSN5 action in cancer development remains poorly understood. Here, we show that CSN5 isopeptidase activity is essential for breast epithelial transformation and progression. Amplification of CSN5 is required for transformation of primary human breast epithelial cells by defined oncogenes. The transforming effects of CSN5 require CSN subunits for assembly of the full COP9 signalosome and the isopeptidase activity of CSN5, which potentiates the transcriptional activity of MYC. Transgenic inhibition of CSN5 isopeptidase activity blocks breast cancer progression evoked by MYC and RAS in vivo. These results highlight CSN5 isopeptidase activity in breast cancer progression, suggesting it as a therapeutic target in aggressive human breast cancers.

  View details for DOI 10.1158/0008-5472.CAN-07-3060

  View details for PubMedID 18199546

• Revealing targeted therapy for human cancer by gene module maps CANCER RESEARCH Wong, D. J., Nuyten, D. S., Regev, A., Lin, M., Adler, A. S., Segal, E., van de Vijver, M. J., Chang, H. Y. 2008; 68 (2): 369-378

  Abstract

  A major goal of cancer research is to match specific therapies to molecular targets in cancer. Genome-scale expression profiling has identified new subtypes of cancer based on consistent patterns of variation in gene expression, leading to improved prognostic predictions. However, how these new genetic subtypes of cancers should be treated is unknown. Here, we show that a gene module map can guide the prospective identification of targeted therapies for genetic subtypes of cancer. By visualizing genome-scale gene expression in cancer as combinations of activated and deactivated functional modules, gene module maps can reveal specific functional pathways associated with each subtype that might be susceptible to targeted therapies. We show that in human breast cancers, activation of a poor-prognosis "wound signature" is strongly associated with induction of both a mitochondria gene module and a proteasome gene module. We found that 3-bromopyruvic acid, which inhibits glycolysis, selectively killed breast cells expressing the mitochondria and wound signatures. In addition, inhibition of proteasome activity by bortezomib, a drug approved for human use in multiple myeloma, abrogated wound signature expression and selectively killed breast cells expressing the wound signature. Thus, gene module maps may enable rapid translation of complex genomic signatures in human disease to targeted therapeutic strategies.

  View details for DOI 10.1158/0008-5472.CAN-07-0382

  View details for PubMedID 18199530

• Motif module map reveals enforcement of aging by continual NF-kappa B activity GENES & DEVELOPMENT Adler, A. S., Sinha, S., Kawahara, T. L., Zhang, J. Y., Segal, E., Chang, H. Y. 2007; 21 (24): 3244-3257

  Abstract

  Aging is characterized by specific alterations in gene expression, but their underlying mechanisms and functional consequences are not well understood. Here we develop a systematic approach to identify combinatorial cis-regulatory motifs that drive age-dependent gene expression across different tissues and organisms. Integrated analysis of 365 microarrays spanning nine tissue types predicted fourteen motifs as major regulators of age-dependent gene expression in human and mouse. The motif most strongly associated with aging was that of the transcription factor NF-kappaB. Inducible genetic blockade of NF-kappaB for 2 wk in the epidermis of chronologically aged mice reverted the tissue characteristics and global gene expression programs to those of young mice. Age-specific NF-kappaB blockade and orthogonal cell cycle interventions revealed that NF-kappaB controls cell cycle exit and gene expression signature of aging in parallel but not sequential pathways. These results identify a conserved network of regulatory pathways underlying mammalian aging and show that NF-kappaB is continually required to enforce many features of aging in a tissue-specific manner.

  View details for DOI 10.1101/gad.1588507

  View details for PubMedID 18055696

• A histone H3 lysine 27 demethylase regulates animal posterior development NATURE Lan, F., Bayliss, P. E., Rinn, J. L., Whetstine, J. R., Wang, J. K., Chen, S., Iwase, S., Alpatov, R., Issaeva, I., Canaani, E., Roberts, T. M., Chang, H. Y., Shi, Y. 2007; 449 (7163): 689-U3

  Abstract

  The recent discovery of a large number of histone demethylases suggests a central role for these enzymes in regulating histone methylation dynamics. Histone H3K27 trimethylation (H3K27me3) has been linked to polycomb-group-protein-mediated suppression of Hox genes and animal body patterning, X-chromosome inactivation and possibly maintenance of embryonic stem cell (ESC) identity. An imbalance of H3K27 methylation owing to overexpression of the methylase EZH2 has been implicated in metastatic prostate and aggressive breast cancers. Here we show that the JmjC-domain-containing related proteins UTX and JMJD3 catalyse demethylation of H3K27me3/2. UTX is enriched around the transcription start sites of many HOX genes in primary human fibroblasts, in which HOX genes are differentially expressed, but is selectively excluded from the HOX loci in ESCs, in which HOX genes are largely silent. Consistently, RNA interference inhibition of UTX led to increased H3K27me3 levels at some HOX gene promoters. Importantly, morpholino oligonucleotide inhibition of a zebrafish UTX homologue resulted in mis-regulation of hox genes and a striking posterior developmental defect, which was partially rescued by wild-type, but not by catalytically inactive, human UTX. Taken together, these findings identify a small family of H3K27 demethylases with important, evolutionarily conserved roles in H3K27 methylation regulation and in animal anterior-posterior development.

  View details for DOI 10.1038/nature06192

  View details for Web of Science ID 000250045000036

  View details for PubMedID 17851529

• Turning skin into embryonic stem cells NATURE MEDICINE Chang, H. Y., Cotsarelis, G. 2007; 13 (7): 783-784

  View details for DOI 10.1038/nm0707-783

  View details for Web of Science ID 000247902800016

  View details for PubMedID 17618266

• Functional demarcation of active and silent chromatin domains in human HOX loci by Noncoding RNAs CELL Rinn, J. L., Kertesz, M., Wang, J. K., Squazzo, S. L., Xu, X., Brugmann, S. A., Goodnough, L. H., Helms, J. A., Farnham, P. J., Segal, E., Chang, H. Y. 2007; 129 (7): 1311-1323

  Abstract

  Noncoding RNAs (ncRNA) participate in epigenetic regulation but are poorly understood. Here we characterize the transcriptional landscape of the four human HOX loci at five base pair resolution in 11 anatomic sites and identify 231 HOX ncRNAs that extend known transcribed regions by more than 30 kilobases. HOX ncRNAs are spatially expressed along developmental axes and possess unique sequence motifs, and their expression demarcates broad chromosomal domains of differential histone methylation and RNA polymerase accessibility. We identified a 2.2 kilobase ncRNA residing in the HOXC locus, termed HOTAIR, which represses transcription in trans across 40 kilobases of the HOXD locus. HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and is required for PRC2 occupancy and histone H3 lysine-27 trimethylation of HOXD locus. Thus, transcription of ncRNA may demarcate chromosomal domains of gene silencing at a distance; these results have broad implications for gene regulation in development and disease states.

  View details for DOI 10.1016/j.cell.2007.05.022

  View details for PubMedID 17604720

• Decoding global gene expression programs in liver cancer by noninvasive imaging NATURE BIOTECHNOLOGY Segal, E., Sirlin, C. B., Ooi, C., Adler, A. S., Gollub, J., Chen, X., Chan, B. K., Matcuk, G. R., Barry, C. T., Chang, H. Y., Kuo, M. D. 2007; 25 (6): 675-680

  Abstract

  Paralleling the diversity of genetic and protein activities, pathologic human tissues also exhibit diverse radiographic features. Here we show that dynamic imaging traits in non-invasive computed tomography (CT) systematically correlate with the global gene expression programs of primary human liver cancer. Combinations of twenty-eight imaging traits can reconstruct 78% of the global gene expression profiles, revealing cell proliferation, liver synthetic function, and patient prognosis. Thus, genomic activity of human liver cancers can be decoded by noninvasive imaging, thereby enabling noninvasive, serial and frequent molecular profiling for personalized medicine.

  View details for DOI 10.1038/nbt1306

  View details for Web of Science ID 000247077500025

  View details for PubMedID 17515910

• A transcriptional program mediating entry into cellular quiescence PLOS GENETICS Liu, H., Adler, A. S., Segal, E., Chang, H. Y. 2007; 3 (6): 996-1008

  Abstract

  The balance of quiescence and cell division is critical for tissue homeostasis and organismal health. Serum stimulation of fibroblasts is well studied as a classic model of entry into the cell division cycle, but the induction of cellular quiescence, such as by serum deprivation (SD), is much less understood. Here we show that SS and SD activate distinct early transcriptional responses genome-wide that converge on a late symmetric transcriptional program. Several serum deprivation early response genes (SDERGs), including the putative tumor suppressor genes SALL2 and MXI1, are required for cessation of DNA synthesis in response to SD and induction of additional SD genes. SDERGs are coordinately repressed in many types of human cancers compared to their normal counterparts, and repression of SDERGs predicts increased risk of cancer progression and death in human breast cancers. These results identify a gene expression program uniquely responsive to loss of growth factor signaling; members of SDERGs may constitute novel growth inhibitors that prevent cancer.

  View details for DOI 10.1371/journal.pgen.0030091

  View details for PubMedID 17559306

• Genome-wide analysis of KAP1 binding suggests autoregulation of KRAB-ZNFs PLOS GENETICS O'Geen, H., Squazzo, S. L., Iyengar, S., Blahnik, K., Rinn, J. L., Chang, H. Y., Green, R., Farnham, P. J. 2007; 3 (6): 916-926

  Abstract

  We performed a genome-scale chromatin immunoprecipitation (ChIP)-chip comparison of two modifications (trimethylation of lysine 9 [H3me3K9] and trimethylation of lysine 27 [H3me3K27]) of histone H3 in Ntera2 testicular carcinoma cells and in three different anatomical sources of primary human fibroblasts. We found that in each of the cell types the two modifications were differentially enriched at the promoters of the two largest classes of transcription factors. Specifically, zinc finger (ZNF) genes were bound by H3me3K9 and homeobox genes were bound by H3me3K27. We have previously shown that the Polycomb repressive complex 2 is responsible for mediating trimethylation of lysine 27 of histone H3 in human cancer cells. In contrast, there is little overlap between H3me3K9 targets and components of the Polycomb repressive complex 2, suggesting that a different histone methyltransferase is responsible for the H3me3K9 modification. Previous studies have shown that SETDB1 can trimethylate H3 on lysine 9, using in vitro or artificial tethering assays. SETDB1 is thought to be recruited to chromatin by complexes containing the KAP1 corepressor. To determine if a KAP1-containing complex mediates trimethylation of the identified H3me3K9 targets, we performed ChIP-chip assays and identified KAP1 target genes using human 5-kb promoter arrays. We found that a large number of genes of ZNF transcription factors were bound by both KAP1 and H3me3K9 in normal and cancer cells. To expand our studies of KAP1, we next performed a complete genomic analysis of KAP1 binding using a 38-array tiling set, identifying ~7,000 KAP1 binding sites. The identified KAP1 targets were highly enriched for C2H2 ZNFs, especially those containing Krüppel-associated box (KRAB) domains. Interestingly, although most KAP1 binding sites were within core promoter regions, the binding sites near ZNF genes were greatly enriched within transcribed regions of the target genes. Because KAP1 is recruited to the DNA via interaction with KRAB-ZNF proteins, we suggest that expression of KRAB-ZNF genes may be controlled via an auto-regulatory mechanism involving KAP1.

  View details for DOI 10.1371/journal.pgen.0030089

  View details for Web of Science ID 000248349300006

  View details for PubMedID 17542650

  View details for PubMedCentralID PMC1885280

• Patterning skin pigmentation via dickkopf JOURNAL OF INVESTIGATIVE DERMATOLOGY Chang, H. Y. 2007; 127 (5): 994-995

  Abstract

  One of the striking regional variations in skin is its pigmentation. Yamaguchi et al. further dissect the mechanism of regional pigmentation by assessing the effects of dickkopf 1 (DKK1), an antagonist of the Wnt pathway produced in lightly pigmented skin, on melanocyte gene expression. The results provide a plethora of candidate genes that may mediate DKK1's inhibitory effects on melanocyte function.

  View details for DOI 10.1038/sj.jid.5700636

  View details for PubMedID 17435783

• GSK3 beta mediates suppression of cyclin D2 expression by tumor suppressor PTEN ONCOGENE Huang, W., Chang, H. Y., Fei, T., Wu, H., Chen, Y. 2007; 26 (17): 2471-2482

  Abstract

  PTEN, encoding a lipid phosphatase, is a tumor suppressor gene and is mutated in various types of cancers. It is reported to regulate G1 to S phase transition of the cell cycle by influencing the expression, protein stability and subcellular location of cyclin D1. Here, we provide evidence that PTEN modulates the transcription and protein stability of cyclin D2. Targeted deletion of Pten in mouse embryonic fibroblasts (MEFs) endowed cells with greater potential to overcome G1 arrest than wild-type MEFs and led to the elevated expression of cyclin D2, which was suppressed by the introduction of PTEN. We further defined a pathway involving GSK3beta and beta-catenin/TCF in PTEN-mediated suppression of cyclin D2 transcription. LiCl, an inhibitor of GSK3beta, abolished inhibitory effect of PTEN on cyclin D2 expression, and TCF members could directly bind to the promoter of cyclin D2 and regulate its transcription in a CREB-dependent manner. Our results indicate that the downregulation of cyclin D2 expression by PTEN is mediated by the GSK3beta/beta-catenin/TCF pathway in cooperation with CREB, and suggest a convergence from the PI-3 kinase/PTEN pathway and the Wnt pathway in modulation of cyclin D2 expression.

  View details for DOI 10.1038/sj.onc.1210033

  View details for Web of Science ID 000245831000007

  View details for PubMedID 17043650

• Spontaneous autoimmunity prevented by thymic expression of a single self-antigen JOURNAL OF EXPERIMENTAL MEDICINE DeVoss, J., Hou, Y., Johannes, K., Lu, W., Liou, G. I., Rinn, J., Chang, H., Caspi, R., Fong, L., Anderson, M. S. 2006; 203 (12): 2727-2735

  Abstract

  The expression of self-antigen in the thymus is believed to be responsible for the deletion of autoreactive T lymphocytes, a critical process in the maintenance of unresponsiveness to self. The Autoimmune regulator (Aire) gene, which is defective in the disorder autoimmune polyglandular syndrome type 1, has been shown to promote the thymic expression of self-antigens. A clear link, however, between specific thymic self-antigens and a single autoimmune phenotype in this model has been lacking. We show that autoimmune eye disease in aire-deficient mice develops as a result of loss of thymic expression of a single eye antigen, interphotoreceptor retinoid-binding protein (IRBP). In addition, lack of IRBP expression solely in the thymus, even in the presence of aire expression, is sufficient to trigger spontaneous eye-specific autoimmunity. These results suggest that failure of thymic expression of selective single self-antigens can be sufficient to cause organ-specific autoimmune disease, even in otherwise self-tolerant individuals.

  View details for DOI 10.1084/jem.20061864

  View details for Web of Science ID 000242339700015

  View details for PubMedID 17116738

  View details for PubMedCentralID PMC2118158

• Bone morphogenetic protein antagonist gremlin 1 is widely expressed by cancer-associated stromal cells and can promote tumor cell proliferation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sneddon, J. B., Zhen, H. H., Montgomery, K., van de Rijn, M., Tward, A. D., West, R., Gladstone, H., Chang, H. Y., Morganroth, G. S., Oro, A. E., Brown, P. O. 2006; 103 (40): 14842-14847

  Abstract

  Although tissue microenvironments play critical roles in epithelial development and tumorigenesis, the factors mediating these effects are poorly understood. In this work, we used a genomic approach to identify factors produced by cells in the microenvironment of basal cell carcinoma (BCC) of the skin, one of the most common human cancers. The global gene expression programs of stromal cell cultures derived from human BCCs showed consistent, systematic differences from those derived from nontumor skin. The gene most consistently expressed at a higher level in BCC tumor stromal cells compared with those from nontumor skin was GREMLIN 1, which encodes a secreted antagonist of the bone morphogenetic protein (BMP) pathway. BMPs and their antagonists are known to play a crucial role in stem and progenitor cell biology as regulators of the balance between expansion and differentiation. Consistent with the hypothesis that BMP antagonists might have a similar role in cancer, we found GREMLIN 1 expression in the stroma of human BCC tumors but not in normal skin in vivo. Furthermore, BMP 2 and 4 are expressed by BCC cells. Ex vivo, BMP inhibits, and Gremlin 1 promotes, proliferation of cultured BCC cells. We further found that GREMLIN 1 is expressed by stromal cells in many carcinomas but not in the corresponding normal tissue counterparts that we examined. Our data suggest that BMP antagonists may be important constituents of tumor stroma, providing a favorable microenvironment for cancer cell survival and expansion in many cancers.

  View details for DOI 10.1073/pnas.0606857103

  View details for PubMedID 17003113

• MYC can induce DNA breaks in vivo and in vitro independent of reactive oxygen species CANCER RESEARCH Ray, S., Atkuri, K. R., Deb-Basu, D., Adler, A. S., Chang, H. Y., Herzenberg, L. A., Felsher, D. W. 2006; 66 (13): 6598-6605

  Abstract

  MYC overexpression is thought to initiate tumorigenesis by inducing cellular proliferation and growth and to be restrained from causing tumorigenesis by inducing cell cycle arrest, cellular senescence, and/or apoptosis. Here we show that MYC can induce DNA breaks both in vitro and in vivo independent of increased production of reactive oxygen species (ROS). We provide an insight into the specific circumstances under which MYC generates ROS in vitro and propose a possible mechanism. We found that MYC induces DNA double-strand breaks (DSBs) independent of ROS production in murine lymphocytes in vivo as well as in normal human foreskin fibroblasts (NHFs) in vitro in normal (10%) serum, as measured by gammaH2AX staining. However, NHFs cultured in vitro in low serum (0.05%) and/or ambient oxygen saturation resulted in ROS-associated oxidative damage and DNA single-strand breaks (SSBs), as measured by Ape-1 staining. In NHFs cultured in low versus normal serum, MYC induced increased expression of CYP2C9, a gene product well known to be associated with ROS production. Specific inhibition of CYP2C9 by small interfering RNA was shown to partially inhibit MYC-induced ROS production. Hence, MYC overexpression can induce ROS and SSBs under some conditions, but generally induces widespread DSBs in vivo and in vitro independent of ROS production.

  View details for DOI 10.1158/0008-5472.CAN-05-3115

  View details for PubMedID 16818632

• Anatomic demarcation by positional variation in fibroblast gene expression programs PLOS GENETICS Rinn, J. L., Bondre, C., Gladstone, H. B., Brown, P. O., Chang, H. Y. 2006; 2 (7): 1084-1096

  Abstract

  Fibroblasts are ubiquitous mesenchymal cells with many vital functions during development, tissue repair, and disease. Fibroblasts from different anatomic sites have distinct and characteristic gene expression patterns, but the principles that govern their molecular specialization are poorly understood. Spatial organization of cellular differentiation may be achieved by unique specification of each cell type; alternatively, organization may arise by cells interpreting their position along a coordinate system. Here we test these models by analyzing the genome-wide gene expression profiles of primary fibroblast populations from 43 unique anatomical sites spanning the human body. Large-scale differences in the gene expression programs were related to three anatomic divisions: anterior-posterior (rostral-caudal), proximal-distal, and dermal versus nondermal. A set of 337 genes that varied according to these positional divisions was able to group all 47 samples by their anatomic sites of origin. Genes involved in pattern formation, cell-cell signaling, and matrix remodeling were enriched among this minimal set of positional identifier genes. Many important features of the embryonic pattern of HOX gene expression were retained in fibroblasts and were confirmed both in vitro and in vivo. Together, these findings suggest that site-specific variations in fibroblast gene expression programs are not idiosyncratic but rather are systematically related to their positional identities relative to major anatomic axes.

  View details for DOI 10.1371/journal.pgen.0020119

  View details for PubMedID 16895450

• From description to causality - Mechanisms of gene expression signatures in cancer CELL CYCLE Adler, A. S., Chang, H. Y. 2006; 5 (11): 1148-1151

  Abstract

  Global gene expression profiles of thousands of cancer samples have been completed, giving rise to hundreds of gene expression signatures (GES). Although many expression signatures show promise in predicting patient prognosis or response to therapies, the usefulness of the signatures in understanding the underlying mechanisms of cancer has not been fully exploited. While "reverse genomic" methods can test specific hypotheses of gene regulation, they fare less well in deciphering novel or combinatorial mechanisms of gene regulation. Recently we described SLAMS (stepwise linkage analysis of microarray signatures), a novel method that can prospectively identify genetic regulators of gene expression signatures in cancer. Applying SLAMS on a poor-prognosis wound signature in human breast cancer, we identified CSN5-mediated ubiquitination of MYC as a novel mechanism to activate a biological program favoring metastasis.

  View details for PubMedID 16721055

• Genetic regulators of large-scale transcriptional signatures in cancer NATURE GENETICS Adler, A. S., Lin, M. H., Horlings, H., Nuyten, D. S., Van de Vijver, M. J., Chang, H. Y. 2006; 38 (4): 421-430

  Abstract

  Gene expression signatures encompassing dozens to hundreds of genes have been associated with many important parameters of cancer, but mechanisms of their control are largely unknown. Here we present a method based on genetic linkage that can prospectively identify functional regulators driving large-scale transcriptional signatures in cancer. Using this method we show that the wound response signature, a poor-prognosis expression pattern of 512 genes in breast cancer, is induced by coordinate amplifications of MYC and CSN5 (also known as JAB1 or COPS5). This information enabled experimental recapitulation, functional assessment and mechanistic elucidation of the wound signature in breast epithelial cells.

  View details for DOI 10.1038/ng1752

  View details for PubMedID 16518402

• Predicting a local recurrence after breast-conserving therapy by gene expression profiling BREAST CANCER RESEARCH Nuyten, D. S., Kreike, B., Hart, A. A., Chi, J. A., Sneddon, J. B., Wessels, L. F., Peterse, H. J., Bartelink, H., Brown, P. O., Chang, H. Y., van de Vijver, M. J. 2006; 8 (5)

  Abstract

  To tailor local treatment in breast cancer patients there is a need for predicting ipsilateral recurrences after breast-conserving therapy. After adequate treatment (excision with free margins and radiotherapy), young age and incompletely excised extensive intraductal component are predictors for local recurrence, but many local recurrences can still not be predicted. Here we have used gene expression profiling by microarray analysis to identify gene expression profiles that can help to predict local recurrence in individual patients.By using previously established gene expression profiles with proven value in predicting metastasis-free and overall survival (wound-response signature, 70-gene prognosis profile and hypoxia-induced profile) and training towards an optimal prediction of local recurrences in a training series, we establish a classifier for local recurrence after breast-conserving therapy.Validation of the different gene lists shows that the wound-response signature is able to separate patients with a high (29%) or low (5%) risk of a local recurrence at 10 years (sensitivity 87.5%, specificity 75%). In multivariable analysis the classifier is an independent predictor for local recurrence.Our findings indicate that gene expression profiling can identify subgroups of patients at increased risk of developing a local recurrence after breast-conserving therapy.

  View details for DOI 10.1186/bcr1614

  View details for Web of Science ID 000243169100015

  View details for PubMedID 17069664

  View details for PubMedCentralID PMC1779489

• Microarray analysis of stem cells and differentiation STEM CELL TOOLS AND OTHER EXPERIMENTAL PROTOCOLS Chang, H. Y., Thomson, J. A., Chen, X. 2006; 420: 225-254

  Abstract

  Microarrays have revolutionized molecular biology and enabled biologists to perform global analysis on the expression of tens of thousands of genes simultaneously. They have been widely used in gene discovery, biomarker determination, disease classification, and studies of gene regulation. Microarrays have been applied in stem cell research to identify major features or expression signatures that define stem cells and characterize their differentiation programs toward specific lineages. Here we provide a review of the microarray technology, including the introduction of array platforms, experimental designs, RNA isolation and amplification, array hybridization, and data analysis. We also detail examples that apply microarray technology to address several of the main questions in stem cell biology.

  View details for DOI 10.1016/S0076-6879(06)20010-7

  View details for PubMedID 17161699

• Learning more from microarrays: Insights from modules and networks JOURNAL OF INVESTIGATIVE DERMATOLOGY Wong, D. J., Chang, H. Y. 2005; 125 (2): 175-182

  Abstract

  Global gene expression patterns can provide comprehensive molecular portraits of biologic diversity and complex disease states, but understanding the physiologic meaning and genetic basis of the myriad gene expression changes have been a challenge. Several new analytic strategies have now been developed to improve the interpretation of microarray data. Because genes work together in groups to carry out specific functions, defining the unit of analysis by coherent changes in biologically meaningful sets of genes, termed modules, improves our understanding of the biological processes underlying the gene expression changes. The gene module approach has been used in exploratory discovery of defective oxidative phosphorylation in diabetes mellitus and also has allowed definitive hypothesis testing on a genomic scale for the relationship between wound healing and cancer and for the oncogenic mechanism of cyclin D. To understand the genetic basis of global gene expression patterns, computational modeling of regulatory networks can highlight key regulators of the gene expression changes, and many of these predictions can now be experimentally validated using global chromatin-immunoprecipitation analysis.

  View details for DOI 10.1111/j.0022-202X.2005.23827.x

  View details for PubMedID 16098025

• Robustness, scalability, and integration of a wound-response gene expression signature in predicting breast cancer survival PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chang, H. Y., Nuyten, D. S., Sneddon, J. B., Hastie, T., Tibshirani, R., Sorlie, T., Dai, H. Y., He, Y. D., Van't Veer, L. J., Bartelink, H., van de Rijn, M., Brown, P. O., van de Vijver, M. J. 2005; 102 (10): 3738-3743

  Abstract

  Based on the hypothesis that features of the molecular program of normal wound healing might play an important role in cancer metastasis, we previously identified consistent features in the transcriptional response of normal fibroblasts to serum, and used this "wound-response signature" to reveal links between wound healing and cancer progression in a variety of common epithelial tumors. Here, in a consecutive series of 295 early breast cancer patients, we show that both overall survival and distant metastasis-free survival are markedly diminished in patients whose tumors expressed this wound-response signature compared to tumors that did not express this signature. A gene expression centroid of the wound-response signature provides a basis for prospectively assigning a prognostic score that can be scaled to suit different clinical purposes. The wound-response signature improves risk stratification independently of known clinico-pathologic risk factors and previously established prognostic signatures based on unsupervised hierarchical clustering ("molecular subtypes") or supervised predictors of metastasis ("70-gene prognosis signature").

  View details for DOI 10.1073/pnas.0409462102

  View details for PubMedID 15701700

• Gene expression signature of fibroblast serum response predicts human cancer progression: similarities between tumors and wounds. PLoS biology Chang, H. Y., Sneddon, J. B., Alizadeh, A. A., Sood, R., West, R. B., Montgomery, K., Chi, J., van de Rijn, M., Botstein, D., Brown, P. O. 2004; 2 (2): E7-?

  Abstract

  Cancer invasion and metastasis have been likened to wound healing gone awry. Despite parallels in cellular behavior between cancer progression and wound healing, the molecular relationships between these two processes and their prognostic implications are unclear. In this study, based on gene expression profiles of fibroblasts from ten anatomic sites, we identify a stereotyped gene expression program in response to serum exposure that appears to reflect the multifaceted role of fibroblasts in wound healing. The genes comprising this fibroblast common serum response are coordinately regulated in many human tumors, allowing us to identify tumors with gene expression signatures suggestive of active wounds. Genes induced in the fibroblast serum-response program are expressed in tumors by the tumor cells themselves, by tumor-associated fibroblasts, or both. The molecular features that define this wound-like phenotype are evident at an early clinical stage, persist during treatment, and predict increased risk of metastasis and death in breast, lung, and gastric carcinomas. Thus, the transcriptional signature of the response of fibroblasts to serum provides a possible link between cancer progression and wound healing, as well as a powerful predictor of the clinical course in several common carcinomas.

  View details for PubMedID 14737219

• Gene expression signature of fibroblast serum response predicts human cancer progression: Similarities between tumors and wounds PLOS BIOLOGY Chang, H. Y., Sneddon, J. B., Alizadeh, A. A., Sood, R., West, R. B., Montgomery, K., Chi, J. T., van de Rijn, M., Botstein, D., Brown, P. O. 2004; 2 (2): 206-214

  Abstract

  Cancer invasion and metastasis have been likened to wound healing gone awry. Despite parallels in cellular behavior between cancer progression and wound healing, the molecular relationships between these two processes and their prognostic implications are unclear. In this study, based on gene expression profiles of fibroblasts from ten anatomic sites, we identify a stereotyped gene expression program in response to serum exposure that appears to reflect the multifaceted role of fibroblasts in wound healing. The genes comprising this fibroblast common serum response are coordinately regulated in many human tumors, allowing us to identify tumors with gene expression signatures suggestive of active wounds. Genes induced in the fibroblast serum-response program are expressed in tumors by the tumor cells themselves, by tumor-associated fibroblasts, or both. The molecular features that define this wound-like phenotype are evident at an early clinical stage, persist during treatment, and predict increased risk of metastasis and death in breast, lung, and gastric carcinomas. Thus, the transcriptional signature of the response of fibroblasts to serum provides a possible link between cancer progression and wound healing, as well as a powerful predictor of the clinical course in several common carcinomas.

  View details for DOI 10.1371/journal.pbio.0020007

  View details for Web of Science ID 000189314400013

  View details for PubMedCentralID PMC314300

• Kinetics and specificity of Fas ligand induction in toxic epidermal necrolysis ARCHIVES OF DERMATOLOGY Chang, H. Y., Cooper, Z. A., Swetter, S. A., Marinkovich, M. P. 2004; 140 (2): 242-244

  View details for Web of Science ID 000188877100024

  View details for PubMedID 14967808

• Endothelial cell diversity revealed by global expression profiling PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chi, J. T., Chang, H. Y., Haraldsen, G., Jahnsen, F. L., Troyanskaya, O. G., Chang, D. S., Wang, Z., Rockson, S. G., van de Rijn, M., Botstein, D., Brown, P. O. 2003; 100 (19): 10623-10628

  Abstract

  The vascular system is locally specialized to accommodate widely varying blood flow and pressure and the distinct needs of individual tissues. The endothelial cells (ECs) that line the lumens of blood and lymphatic vessels play an integral role in the regional specialization of vascular structure and physiology. However, our understanding of EC diversity is limited. To explore EC specialization on a global scale, we used DNA microarrays to determine the expression profile of 53 cultured ECs. We found that ECs from different blood vessels and microvascular ECs from different tissues have distinct and characteristic gene expression profiles. Pervasive differences in gene expression patterns distinguish the ECs of large vessels from microvascular ECs. We identified groups of genes characteristic of arterial and venous endothelium. Hey2, the human homologue of the zebrafish gene gridlock, was selectively expressed in arterial ECs and induced the expression of several arterial-specific genes. Several genes critical in the establishment of left/right asymmetry were expressed preferentially in venous ECs, suggesting coordination between vascular differentiation and body plan development. Tissue-specific expression patterns in different tissue microvascular ECs suggest they are distinct differentiated cell types that play roles in the local physiology of their respective organs and tissues.

  View details for DOI 10.1073/pnas.1434429100

  View details for PubMedID 12963823

• Eruptive xanthomas associated with olanzapine use ARCHIVES OF DERMATOLOGY Chang, H. Y., Ridky, T. W., Kimball, A. B., Hughes, E., Oro, A. E. 2003; 139 (8): 1045-1048

  Abstract

  Since their introduction to the US market, atypical antipsychotic drugs, such as olanzapine, have been widely prescribed for the management of psychosis and have increasingly been used in dermatologic settings for the treatment of psychogenic dermatoses. Mild hyperglycemia and hypertriglyceridemia have been documented from the use of these medications, but the range of effects on metabolism and the effects on skin are poorly characterized. OBSERVETION: We describe 3 patients who developed eruptive xanthomas, 1 of whom had relative insulin insufficiency, after starting olanzapine therapy. These cases further support the association of severe dyslipidemia with olanzapine use in selected patients.With the increasing use of atypical antipsychotic agents in the dermatologic setting, the dyslipidemia that develops in association with olanzapine use emphasizes the need for periodic metabolic studies in high-risk patients.

  View details for PubMedID 12925394

• Myelogenous leukemia cutis resembling stasis dermatitis JOURNAL OF THE AMERICAN ACADEMY OF DERMATOLOGY Chang, H. Y., Wong, K. M., Bosenberg, M., McKee, P. H., Haynes, H. A. 2003; 49 (1): 128-129

  Abstract

  Leukemia cutis may clinically mimic many inflammatory dermatoses. A patient with myelodysplastic syndrome presented with an acute eruption of bilateral, lower-extremity, tender, indurated, brown plaques that clinically resembled chronic stasis dermatitis. Histologic study revealed a dermal myeloblastic leukemic infiltrate.

  View details for DOI 10.1067/mjd.2003.233

  View details for Web of Science ID 000184135000021

  View details for PubMedID 12833025

• Genomewide view of gene silencing by small interfering RNAs PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chi, J. T., Chang, H. Y., Wang, N. N., Chang, D. S., Dunphy, N., Brown, P. O. 2003; 100 (11): 6343-6346

  Abstract

  RNA interference (RNAi) is an evolutionarily conserved mechanism in plant and animal cells that directs the degradation of messenger RNAs homologous to short double-stranded RNAs termed small interfering RNA (siRNA). The ability of siRNA to direct gene silencing in mammalian cells has raised the possibility that siRNA might be used to investigate gene function in a high throughput fashion or to modulate gene expression in human diseases. The specificity of siRNA-mediated silencing, a critical consideration in these applications, has not been addressed on a genomewide scale. Here we show that siRNA-induced gene silencing of transient or stably expressed mRNA is highly gene-specific and does not produce secondary effects detectable by genomewide expression profiling. A test for transitive RNAi, extension of the RNAi effect to sequences 5' of the target region that has been observed in Caenorhabditis elegans, was unable to detect this phenomenon in human cells.

  View details for DOI 10.1073/pnas.1037853100

  View details for PubMedID 12730368

• Diversity, topographic differentiation, and positional memory in human fibroblasts PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chang, H. Y., Chi, J. T., Dudoit, S., Bondre, C., van de Rijn, M., Botstein, D., Brown, P. O. 2002; 99 (20): 12877-12882

  Abstract

  A fundamental feature of the architecture and functional design of vertebrate animals is a stroma, composed of extracellular matrix and mesenchymal cells, which provides a structural scaffold and conduit for blood and lymphatic vessels, nerves, and leukocytes. Reciprocal interactions between mesenchymal and epithelial cells are known to play a critical role in orchestrating the development and morphogenesis of tissues and organs, but the roles played by specific stromal cells in controlling the design and function of tissues remain poorly understood. The principal cells of stromal tissue are called fibroblasts, a catch-all designation that belies their diversity. We characterized genome-wide patterns of gene expression in cultured fetal and adult human fibroblasts derived from skin at different anatomical sites. Fibroblasts from each site displayed distinct and characteristic transcriptional patterns, suggesting that fibroblasts at different locations in the body should be considered distinct differentiated cell types. Notable groups of differentially expressed genes included some implicated in extracellular matrix synthesis, lipid metabolism, and cell signaling pathways that control proliferation, cell migration, and fate determination. Several genes implicated in genetic diseases were found to be expressed in fibroblasts in an anatomic pattern that paralleled the phenotypic defects. Finally, adult fibroblasts maintained key features of HOX gene expression patterns established during embryogenesis, suggesting that HOX genes may direct topographic differentiation and underlie the detailed positional memory in fibroblasts.

  View details for DOI 10.1073/pnas.162488599

  View details for PubMedID 12297622

• Proteases for cell suicide: Functions and regulation of caspases MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS Chang, H. Y., Yang, X. L. 2000; 64 (4): 821-?

  Abstract

  Caspases are a large family of evolutionarily conserved proteases found from Caenorhabditis elegans to humans. Although the first caspase was identified as a processing enzyme for interleukin-1beta, genetic and biochemical data have converged to reveal that many caspases are key mediators of apoptosis, the intrinsic cell suicide program essential for development and tissue homeostasis. Each caspase is a cysteine aspartase; it employs a nucleophilic cysteine in its active site to cleave aspartic acid peptide bonds within proteins. Caspases are synthesized as inactive precursors termed procaspases; proteolytic processing of procaspase generates the tetrameric active caspase enzyme, composed of two repeating heterotypic subunits. Based on kinetic data, substrate specificity, and procaspase structure, caspases have been conceptually divided into initiators and effectors. Initiator caspases activate effector caspases in response to specific cell death signals, and effector caspases cleave various cellular proteins to trigger apoptosis. Adapter protein-mediated oligomerization of procaspases is now recognized as a universal mechanism of initiator caspase activation and underlies the control of both cell surface death receptor and mitochondrial cytochrome c-Apaf-1 apoptosis pathways. Caspase substrates have bene identified that induce each of the classic features of apoptosis, including membrane blebbing, cell body shrinkage, and DNA fragmentation. Mice deficient for caspase genes have highlighted tissue- and signal-specific pathways for apoptosis and demonstrated an independent function for caspase-1 and -11 in cytokine processing. Dysregulation of caspases features prominently in many human diseases, including cancer, autoimmunity, and neurodegenerative disorders, and increasing evidence shows that altering caspase activity can confer therapeutic benefits.

  View details for Web of Science ID 000167056400008

  View details for PubMedID 11104820

• Dissecting Fas signaling with an altered-specificity death-domain mutant: Requirement of FADD binding for apoptosis but not Jun N-terminal kinase activation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chang, H. Y., Yang, X. L., Baltimore, D. 1999; 96 (4): 1252-1256

  Abstract

  Fas is a cell surface death receptor that regulates peripheral tolerance and lymphoid homeostasis. In many pathologic conditions, ectopic Fas activation mediates tissue destruction. Several proteins that can bind to the cytoplasmic death domain of Fas have been implicated in Fas signal transduction. Here we show that FADD, which couples Fas to pro-caspase-8, and, Daxx, which couples Fas to the Jun N-terminal kinase pathway, bind independently to the Fas death domain. We have isolated a death domain mutant, termed FasDelta, that selectively binds Daxx but not FADD. In tranfected tissue culture cells, FasDelta activated Jun N-terminal kinase normally but was impaired in cell death induction. These results suggest that FADD and Daxx activate two independent pathways downstream of Fas and confirm the essential role of FADD binding in apoptosis induction.

  View details for Web of Science ID 000078698400018

  View details for PubMedID 9990010

• Activation of apoptosis signal regulating kinase 1 (ASK1) by the adapter protein Daxx SCIENCE Chang, H. Y., Nishitoh, H., Yang, X. L., ICHIJO, H., Baltimore, D. 1998; 281 (5384): 1860-1863

  Abstract

  The Fas death receptor can activate the Jun NH2-terminal kinase (JNK) pathway through the receptor-associated protein Daxx. Daxx was found to activate the JNK kinase kinase ASK1, and overexpression of a kinase-deficient ASK1 mutant inhibited Fas- and Daxx-induced apoptosis and JNK activation. Fas activation induced Daxx to interact with ASK1, which consequently relieved an inhibitory intramolecular interaction between the amino- and carboxyl-termini of ASK1, activating its kinase activity. The Daxx-ASK1 connection completes a signaling pathway from a cell surface death receptor to kinase cascades that modulate nuclear transcription factors.

  View details for Web of Science ID 000076007100056

  View details for PubMedID 9743501

• Essential role of CED-4 oligomerization in CED-3 activation and apoptosis SCIENCE Yang, X. L., Chang, H. Y., Baltimore, D. 1998; 281 (5381): 1355-1357

  Abstract

  Control of the activation of apoptosis is important both in development and in protection against cancer. In the classic genetic model Caenorhabditis elegans, the pro-apoptotic protein CED-4 activates the CED-3 caspase and is inhibited by the Bcl-2-like protein CED-9. Both processes are mediated by protein-protein interaction. Facilitating the proximity of CED-3 zymogen molecules was found to induce caspase activation and cell death. CED-4 protein oligomerized in cells and in vitro. This oligomerization induced CED-3 proximity and competed with CED-4:CED-9 interaction. Mutations that abolished CED-4 oligomerization inactivated its ability to activate CED-3. Thus, the mechanism of control is that CED-3 in CED-3:CED-4 complexes is activated by CED-4 oligomerization, which is inhibited by binding of CED-9 to CED-4.

  View details for Web of Science ID 000075666800051

  View details for PubMedID 9721101

• Autoproteolytic activation of pro-caspases by oligomerization MOLECULAR CELL Yang, X. L., Chang, H. Y., Baltimore, D. 1998; 1 (2): 319-325

  Abstract

  Initiation of apopotosis requires the conversion of procaspases to mature caspases. Here we show that oligomerization of pro-caspases is sufficient to induce proteolytic generation of mature caspase subunits and activation of their cell death activity. Deletion of the protein interaction motif DED from pro-caspase-8 greatly suppresses its apoptotic activity. Cell death activity can be restored by oligomerization of pro-caspase-8 protease domains by two heterologous inducible oligomerization systems. Induced oligomerization also activates the apoptotic activity of pro-caspase-1 but not pro-caspase-3. In vitro, oligomerization leads to pro-caspase processing to from the mature caspase subunits; this processing requires the intrinsic caspase activity of zymogens and proceeds via a novel order of cleavage events.

  View details for Web of Science ID 000072970500016

  View details for PubMedID 9659928

• Daxx, a novel Fas-binding protein that activates JNK and apoptosis CELL Yang, X. L., KHOSRAVIFAR, R., Chang, H. Y., Baltimore, D. 1997; 89 (7): 1067-1076

  Abstract

  The Fas cell surface receptor induces apoptosis upon receptor oligomerization. We have identified a novel signaling protein, termed Daxx, that binds specifically to the Fas death domain. Overexpression of Daxx enhances Fas-mediated apoptosis and activates the Jun N-terminal kinase (JNK) pathway. A C-terminal portion of Daxx interacts with the Fas death domain, while a different region activates both JNK and apoptosis. The Fas-binding domain of Daxx is a dominant-negative inhibitor of both Fas-induced apoptosis and JNK activation, while the FADD death domain partially inhibits death but not JNK activation. The Daxx apoptotic pathway is sensitive to both Bcl-2 and dominant-negative JNK pathway components and acts cooperatively with the FADD pathway. Thus, Daxx and FADD define two distinct apoptotic pathways downstream of Fas.

  View details for Web of Science ID A1997XG83000011

  View details for PubMedID 9215629

• ASYMMETRIC RETRACTION OF GROWTH CONE FILOPODIA FOLLOWING FOCAL INACTIVATION OF CALCINEURIN NATURE Chang, H. Y., Takei, K., SYDOR, A. M., Born, T., Rusnak, F., Jay, D. G. 1995; 376 (6542): 686-690

  Abstract

  The neuronal growth cone is thought to be the site of decision making in nerve growth and guidance. One likely mechanism of how the growth cone translates various extracellular cues into directed motility involves rises in intracellular calcium. A variety of physiological cues, such as adhesion molecules and neurotransmitters, increases intracellular calcium, and artificial manipulations of growth cone calcium levels affect growth cone morphology and neurite outgrowth. The molecular events downstream of calcium fluxes are incompletely understood. Here we show that calcineurin, a protein phosphatase enriched in growth cones that is dependent on calcium ions and calmodulin, functions in neurite outgrowth and directed filopodial motility in cultured chick dorsal root ganglia neurons. Cyclosporin A and FK506, inhibitors of calcineurin, delayed neuritogenesis and inhibited neurite extension. Chromophore-assisted laser inactivation of calcineurin in regions of growth cones causes localized filopodial and lamellipodial retraction and influences the direction of subsequent outgrowth. We suggest that a spatial distribution of calcineurin activity within the growth cone can regulate motility and direct outgrowth.

  View details for Web of Science ID A1995RQ67200060

  View details for PubMedID 7544441

• ACTIVATION OF CLN1 AND CLN2 G(1) CYCLIN GENE-EXPRESSION BY BCK2 MOLECULAR AND CELLULAR BIOLOGY DICOMO, C. J., Chang, H., Arndt, K. T. 1995; 15 (4): 1835-1846

  Abstract

  The Saccharomyces cerevisiae CLN3 protein, a G1 cyclin, positively regulates the expression of CLN1 and CLN2, two additional G1 cyclins whose expression during late G1 is activated, in part, by the transcription factors SWI4 and SWI6. We isolated 12 complementation groups of mutants that require CLN3. The members of one of these complementation groups have mutations in the BCK2 gene. In a wild-type CLN3 genetic background, bck2 mutants have a normal growth rate but have a larger cell size, are more sensitive to alpha-factor, and have a modest defect in the accumulation of CLN1 and CLN2 RNA. In the absence of CLN3, bck2 mutations cause an extremely slow growth rate: the cells accumulate in late G1 with very low levels of CLN1 and CLN2 RNA. The slow growth rate and long G1 delay of bck2 cln3 mutants are cured by heterologous expression of CLN2. Moreover, overexpression of BCK2 induces very high levels of CLN1, CLN2, and HCS26 RNAs. The results suggest that BCK2 and CLN3 provide parallel activation pathways for the expression of CLN1 and CLN2 during late G1.

  View details for Web of Science ID A1995QM49200001

  View details for PubMedID 7891677

• ACTIVE-SITE MUTANTS OF HUMAN CYCLOPHILIN-A SEPARATE PEPTIDYL-PROLYL ISOMERASE ACTIVITY FROM CYCLOSPORINE-A BINDING AND CALCINEURIN INHIBITION PROTEIN SCIENCE ZYDOWSKY, L. D., Etzkorn, F. A., Chang, H. Y., Ferguson, S. B., STOLZ, L. A., Ho, S. I., Walsh, C. T. 1992; 1 (9): 1092-1099

  Abstract

  Based on recent X-ray structural information, six site-directed mutants of human cyclophilin A (hCyPA) involving residues in the putative active site--H54, R55, F60, Q111, F113, and H126--have been constructed, overexpressed, and purified from Escherichia coli to homogeneity. The proteins W121A (Liu, J., Chen, C.-M., & Walsh, C.T., 1991a, Biochemistry 30, 2306-2310), H54Q, R55A, F60A, Q111A, F113A, and H126Q were assayed for cis-trans peptidyl-prolyl isomerase (PPIase) activity, their ability to bind the immunosuppressive drug cyclosporin A (CsA), and protein phosphatase 2B (calcineurin) inhibition in the presence of CsA. Results indicate that H54Q, Q111A, F113A, and W121A retain 3-15% of the catalytic efficiency (kcat/Km) of wild-type recombinant hCyPA. The remaining three mutants (R55A, F60A, and H126Q) each retain less than 1% of the wild-type catalytic efficiency, indicating participation by these residues in PPIase catalysis. Each of the mutants bound to a CsA affinity matrix. The mutants R55A, F60A, F113A, and H126Q inhibited calcineurin in the presence of CsA, whereas W121A did not. Although CsA is a competitive inhibitor of PPIase activity, it can complex with enzymatically inactive cyclophilins and inhibit the phosphatase activity of calcineurin.

  View details for Web of Science ID A1992JR69300003

  View details for PubMedID 1338979

• CYCLOSPORINE-MEDIATED INHIBITION OF BOVINE CALCINEURIN BY CYCLOPHILIN-A AND CYCLOPHILIN-B PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA SWANSON, S. K., Born, T., ZYDOWSKY, L. D., Cho, H. J., Chang, H. Y., Walsh, C. T., Rusnak, F. 1992; 89 (9): 3741-3745

  Abstract

  The Ca(2+)- and calmodulin-dependent protein phosphatase calcineurin is inhibited by the immunosuppressant drug cyclosporin A in the presence of cyclophilin A or B. Of the two isoforms, cyclophilin B is more potent by a factor of 2-5 when either the phosphoprotein [32P]casein or the [32P]phosphoserine [Ser(32P)] form of the 19-residue bovine cardiac cAMP-dependent protein kinase regulatory subunit peptide RII, [Ser(32P)15]RII, is used as substrate. With [Ser(32P15]RII as substrate, the concentrations of the cyclosporin A.cyclophilin A and cyclosporin A.cyclophilin B complexes, which cause 50% inhibition of calcineurin activity, are 120 and 50 nM, respectively. Lowering the concentration of calcineurin 80% with [32P]casein as substrate lowered the apparent inhibition constant for each complex even further; 50% inhibition of calcineurin was observed at 40 nM for cyclosporin A.cyclophilin A, whereas it was less than 10 nM for cyclosporin A.cyclophilin B. In all inhibition assays with [32P]casein or [Ser(32P)15]RII, the concentration of calcineurin required for measurable phosphatase activity is such that these complexes behave as tight-binding inhibitors of calcineurin, and steady-state kinetics cannot be used to assess inhibition patterns or Ki values. Limited trypsinization of calcineurin produces a fragment that is still inhibited, indicating that the interaction of cyclosporin.cyclophilin with calcineurin does not require either calmodulin or Ca2+.

  View details for Web of Science ID A1992HR85300015

  View details for PubMedID 1315036