All Publications


  • ecDNA hubs drive cooperative intermolecular oncogene expression. Nature Hung, K. L., Yost, K. E., Xie, L., Shi, Q., Helmsauer, K., Luebeck, J., Schopflin, R., Lange, J. T., Chamorro Gonzalez, R., Weiser, N. E., Chen, C., Valieva, M. E., Wong, I. T., Wu, S., Dehkordi, S. R., Duffy, C. V., Kraft, K., Tang, J., Belk, J. A., Rose, J. C., Corces, M. R., Granja, J. M., Li, R., Rajkumar, U., Friedlein, J., Bagchi, A., Satpathy, A. T., Tjian, R., Mundlos, S., Bafna, V., Henssen, A. G., Mischel, P. S., Liu, Z., Chang, H. Y. 2021

    Abstract

    Extrachromosomal DNA (ecDNA) is prevalent in human cancers and mediates high expression of oncogenes through gene amplification and altered gene regulation1. Gene induction typically involves cis-regulatory elements that contact and activate genes on the same chromosome2,3. Here we show that ecDNA hubs-clusters of around 10-100 ecDNAs within the nucleus-enable intermolecular enhancer-gene interactions to promote oncogene overexpression. ecDNAs that encode multiple distinct oncogenes form hubs in diverse cancer cell types and primary tumours. Each ecDNA is more likely to transcribe the oncogene when spatially clustered with additional ecDNAs. ecDNA hubs are tethered by the bromodomain and extraterminal domain (BET) protein BRD4 in a MYC-amplified colorectal cancer cell line. The BET inhibitor JQ1 disperses ecDNA hubs and preferentially inhibits ecDNA-derived-oncogene transcription. The BRD4-bound PVT1 promoter is ectopically fused to MYC and duplicated in ecDNA, receiving promiscuous enhancer input to drive potent expression of MYC. Furthermore, the PVT1 promoter on an exogenous episome suffices to mediate gene activation in trans by ecDNA hubs in a JQ1-sensitive manner. Systematic silencing of ecDNA enhancers by CRISPR interference reveals intermolecular enhancer-gene activation among multiple oncogene loci that are amplified on distinct ecDNAs. Thus, protein-tethered ecDNA hubs enable intermolecular transcriptional regulation and may serve as units of oncogene function and cooperative evolution and as potential targets for cancer therapy.

    View details for DOI 10.1038/s41586-021-04116-8

    View details for PubMedID 34819668

  • A human mutation in STAT3 promotes type 1 diabetes through a defect in CD8+ T cell tolerance. The Journal of experimental medicine Warshauer, J. T., Belk, J. A., Chan, A. Y., Wang, J., Gupta, A. R., Shi, Q., Skartsis, N., Peng, Y., Phipps, J. D., Acenas, D., Smith, J. A., Tamaki, S. J., Tang, Q., Gardner, J. M., Satpathy, A. T., Anderson, M. S. 2021; 218 (8)

    Abstract

    Naturally occurring cases of monogenic type 1 diabetes (T1D) help establish direct mechanisms driving this complex autoimmune disease. A recently identified de novo germline gain-of-function (GOF) mutation in the transcriptional regulator STAT3 was found to cause neonatal T1D. We engineered a novel knock-in mouse incorporating this highly diabetogenic human STAT3 mutation (K392R) and found that these mice recapitulated the human autoimmune diabetes phenotype. Paired single-cell TCR and RNA sequencing revealed that STAT3-GOF drives proliferation and clonal expansion of effector CD8+ cells that resist terminal exhaustion. Single-cell ATAC-seq showed that these effector T cells are epigenetically distinct and have differential chromatin architecture induced by STAT3-GOF. Analysis of islet TCR clonotypes revealed a CD8+ cell reacting against known antigen IGRP, and STAT3-GOF in an IGRP-reactive TCR transgenic model demonstrated that STAT3-GOF intrinsic to CD8+ cells is sufficient to accelerate diabetes onset. Altogether, these findings reveal a diabetogenic CD8+ T cell response that is restrained in the presence of normal STAT3 activity and drives diabetes pathogenesis.

    View details for DOI 10.1084/jem.20210759

    View details for PubMedID 34115115

  • Differential usage of transcriptional repressor Zeb2 enhancers distinguishes adult and embryonic hematopoiesis. Immunity Huang, X., Ferris, S. T., Kim, S., Choudhary, M. N., Belk, J. A., Fan, C., Qi, Y., Sudan, R., Xia, Y., Desai, P., Chen, J., Ly, N., Shi, Q., Bagadia, P., Liu, T., Guilliams, M., Egawa, T., Colonna, M., Diamond, M. S., Murphy, T. L., Satpathy, A. T., Wang, T., Murphy, K. M. 2021

    Abstract

    The transcriptional repressor ZEB2 regulates development of many cell fates among somatic, neural, and hematopoietic lineages, but the basis for its requirement in these diverse lineages is unclear. Here, we identified a 400-basepair (bp) region located 165 kilobases (kb) upstream of the Zeb2 transcriptional start site (TSS) that binds the E proteins at several E-box motifs and was active in hematopoietic lineages. Germline deletion of this 400-bp region (Zeb2Delta-165mice) specifically prevented Zeb2 expression in hematopoietic stem cell (HSC)-derived lineages. Zeb2Delta-165 mice lacked development of plasmacytoid dendritic cells (pDCs), monocytes, and B cells. All macrophages in Zeb2Delta-165 mice were exclusively of embryonic origin. Using single-cell chromatin profiling, we identified a second Zeb2 enhancer located at+164-kb that was selectively active in embryonically derived lineages, but not HSC-derived ones. Thus, Zeb2 expression in adult, but not embryonic, hematopoiesis is selectively controlled by the -165-kb Zeb2 enhancer.

    View details for DOI 10.1016/j.immuni.2021.04.015

    View details for PubMedID 34004142

  • Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing. Cell Nunez, J. K., Chen, J., Pommier, G. C., Cogan, J. Z., Replogle, J. M., Adriaens, C., Ramadoss, G. N., Shi, Q., Hung, K. L., Samelson, A. J., Pogson, A. N., Kim, J. Y., Chung, A., Leonetti, M. D., Chang, H. Y., Kampmann, M., Bernstein, B. E., Hovestadt, V., Gilbert, L. A., Weissman, J. S. 2021

    Abstract

    A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.

    View details for DOI 10.1016/j.cell.2021.03.025

    View details for PubMedID 33838111

  • Discovery and functional interrogation of SARS-CoV-2 RNA-host protein interactions. Cell Flynn, R. A., Belk, J. A., Qi, Y., Yasumoto, Y., Wei, J., Alfajaro, M. M., Shi, Q., Mumbach, M. R., Limaye, A., DeWeirdt, P. C., Schmitz, C. O., Parker, K. R., Woo, E., Chang, H. Y., Horvath, T. L., Carette, J. E., Bertozzi, C. R., Wilen, C. B., Satpathy, A. T. 2021

    Abstract

    SARS-CoV-2 is the cause of a pandemic with growing global mortality. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with ChIRP-MS data from three other RNA viruses defined viral specificity of RNA-host protein interactions. Targeted CRISPR screens revealed that the majority of functional RNA-binding proteins protect the host from virus-induced cell death, and comparative CRISPR screens across seven RNA viruses revealed shared and SARS-specific antiviral factors. Finally, by combining the RNA-centric approach and functional CRISPR screens, we demonstrated a physical and functional connection between SARS-CoV-2 and mitochondria, highlighting this organelle as a general platform for antiviral activity. Altogether, these data provide a comprehensive catalog of functional SARS-CoV-2 RNA-host protein interactions, which may inform studies to understand the host-virus interface and nominate host pathways that could be targeted for therapeutic benefit.

    View details for DOI 10.1016/j.cell.2021.03.012

    View details for PubMedID 33743211

  • B cell-specific XIST complex enforces X-inactivation and restrains atypical B cells. Cell Yu, B., Qi, Y., Li, R., Shi, Q., Satpathy, A. T., Chang, H. Y. 2021

    Abstract

    The long non-coding RNA (lncRNA) XIST establishes X chromosome inactivation (XCI) in female cells in early development and thereafter is thought to be largely dispensable. Here, we show XIST is continually required in adult human B cells to silence a subset of X-linked immune genes such as TLR7. XIST-dependent genes lack promoter DNA methylation and require continual XIST-dependent histone deacetylation. XIST RNA-directed proteomics and CRISPRi screen reveal distinctive somatic cell-type-specific XIST complexes and identify TRIM28 that mediates Pol II pausing at promoters of X-linked genes in B cells. Single-cell transcriptome data of female patients with either systemic lupus erythematosus or COVID-19 infection revealed XIST dysregulation, reflected by escape of XIST-dependent genes, in CD11c+ atypical memory B cells (ABCs). XIST inactivation with TLR7 agonism suffices to promote isotype-switched ABCs. These results indicate cell-type-specific diversification and function for lncRNA-protein complexes and suggest expanded roles for XIST in sex-differences in biology and medicine.

    View details for DOI 10.1016/j.cell.2021.02.015

    View details for PubMedID 33735607

  • Concerted localization-resets precede YAP-dependent transcription. Nature communications Franklin, J. M., Ghosh, R. P., Shi, Q., Reddick, M. P., Liphardt, J. T. 2020; 11 (1): 4581

    Abstract

    Yes-associated protein 1 (YAP) is a transcriptional regulator with critical roles in mechanotransduction, organ size control, and regeneration. Here, using advanced tools for real-time visualization of native YAP and target gene transcription dynamics, we show that a cycle of fast exodus of nuclear YAP to the cytoplasm followed by fast reentry to the nucleus ("localization-resets") activates YAP target genes. These "resets" are induced by calcium signaling, modulation of actomyosin contractility, or mitosis. Using nascent-transcription reporter knock-ins of YAP target genes, we show a strict association between these resets and downstream transcription. Oncogenically-transformed cell lines lack localization-resets and instead show dramatically elevated rates of nucleocytoplasmic shuttling of YAP, suggesting an escape from compartmentalization-based control. The single-cell localization and transcription traces suggest that YAP activity is not a simple linear function of nuclear enrichment and point to a model of transcriptional activation based on nucleocytoplasmic exchange properties of YAP.

    View details for DOI 10.1038/s41467-020-18368-x

    View details for PubMedID 32917893

  • Spatial resets modulate YAP-dependent transcription. Franklin, M., Ghosh, R., Shi, Q., Liphardt, J. AMER ASSOC CANCER RESEARCH. 2020: 21
  • Stiff stroma increases breast cancer risk by inducing the oncogene ZNF217. The Journal of clinical investigation Northey, J. J., Barrett, A. S., Acerbi, I., Hayward, M., Talamantes, S., Dean, I. S., Mouw, J. K., Ponik, S. M., Lakins, J. N., Huang, P., Wu, J., Shi, Q., Samson, S., Keely, P. J., Mukhtar, R. A., Liphardt, J. T., Shepherd, J. A., Hwang, E. S., Chen, Y., Hansen, K. C., Littlepage, L. E., Weaver, V. M. 2020

    Abstract

    Women with dense breasts have an increased lifetime risk to malignancy that has been attributed to a higher epithelial density. Quantitative proteomics, collagen analysis and mechanical measurements in normal tissue revealed that stroma in the high density breast contains more oriented, fibrillar collagen, that is stiffer and correlates with higher epithelial cell density. MicroRNA profiling of breast tissue identified microRNA-203 (miR-203) as a matrix stiffness-repressed transcript that is downregulated by collagen density and reduced in the breast epithelium of women with high mammographic density. Culture studies demonstrated that ZNF217 mediates a matrix stiffness and collagen density-induced increase in Akt activity and mammary epithelial cell proliferation. Manipulation of the epithelium in a mouse model of mammographic density supported a causal relationship between stromal stiffness, reduced miR-203, higher levels of the murine homologue Zfp217, and increased Akt activity and mammary epithelial proliferation. ZNF217 was also increased in the normal breast epithelium of women with high mammographic density, correlated positively with epithelial proliferation and density, and inversely with miR-203. The findings identify ZNF217 as a potential target towards which preexisting therapies, such as the Akt inhibitor triciribine, could be used as a chemoprevention agent to reduce cancer risk in women with high mammographic density.

    View details for DOI 10.1172/JCI129249

    View details for PubMedID 32721948

  • Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation. eLife Carter, A. C., Xu, J., Nakamoto, M. Y., Wei, Y., Zarnegar, B. J., Shi, Q., Broughton, J. P., Ransom, R. C., Salhotra, A., Nagaraja, S. D., Li, R., Dou, D. R., Yost, K. E., Cho, S., Mistry, A., Longaker, M. T., Khavari, P. A., Batey, R. T., Wuttke, D. S., Chang, H. Y. 2020; 9

    Abstract

    The Xist lncRNA mediates X chromosome inactivation (XCI)1,2. Here we show that Spen, an Xist-binding repressor protein essential for XCI3-9, binds to ancient retroviral RNA, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen inactivation activates a subset of endogenous retroviral (ERV) elements in mouse embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and ERV RNA transcription. Spen binds directly to ERV RNAs that show structural similarity to the A-repeat of Xist, a region critical for Xist-mediated gene silencing10-11. ERV RNA and Xist A-repeat bind the RRM domains of Spen in a competitive manner. Insertion of an ERV into an A-repeat deficient Xist rescues binding of Xist RNA to Spen and results in strictly local gene silencing in cis. These results suggest that Xist may coopt transposable element RNA-protein interactions to repurpose powerful antiviral chromatin silencing machinery for sex chromosome dosage compensation.

    View details for DOI 10.7554/eLife.54508

    View details for PubMedID 32379046

  • A fluorogenic array for temporally unlimited single-molecule tracking NATURE CHEMICAL BIOLOGY Ghosh, R. P., Franklin, J., Draper, W. E., Shi, Q., Betran, B., Spakowitz, A. J., Liphardt, J. T. 2019; 15 (4): 401-+
  • A fluorogenic array for temporally unlimited single-molecule tracking. Nature chemical biology Ghosh, R. P., Franklin, J. M., Draper, W. E., Shi, Q., Beltran, B., Spakowitz, A. J., Liphardt, J. T. 2019

    Abstract

    We describe three optical tags, ArrayG, ArrayD and ArrayG/N, for intracellular tracking of single molecules over milliseconds to hours. ArrayG is a fluorogenic tag composed of a green fluorescent protein-nanobody array and monomeric wild-type green fluorescent protein binders that are initially dim but brighten ~26-fold on binding with the array. By balancing the rates of binder production, photobleaching and stochastic binder exchange, we achieve temporally unlimited tracking of single molecules. High-speed tracking of ArrayG-tagged kinesins and integrins for thousands of frames reveals novel dynamical features. Tracking of single histones at 0.5Hz for>1hour with the import competent ArrayG/N tag shows that chromosomal loci behave as Rouse polymers with visco-elastic memory and exhibit a non-Gaussian displacement distribution. ArrayD, based on a dihydrofolate reductase nanobody array and dihydrofolate reductase-fluorophore binder, enables dual-color imaging. The arrays combine brightness, fluorogenicity, fluorescence replenishment and extended fluorophore choice, opening new avenues for tracking single molecules in living cells.

    View details for PubMedID 30858596

  • 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

  • Physical confinement induces malignant transformation in mammary epithelial cells. Biomaterials Lu, Y. C., Chu, T. n., Hall, M. S., Fu, D. J., Shi, Q. n., Chiu, A. n., An, D. n., Wang, L. H., Pardo, Y. n., Southard, T. n., Danko, C. G., Liphardt, J. n., Nikitin, A. Y., Wu, M. n., Fischbach, C. n., Coonrod, S. n., Ma, M. n. 2019; 217: 119307

    Abstract

    The physical microenvironment of tumor cells plays an important role in cancer initiation and progression. Here, we present evidence that confinement - a new physical parameter that is apart from matrix stiffness - can also induce malignant transformation in mammary epithelial cells. We discovered that MCF10A cells, a benign mammary cell line that forms growth-arrested polarized acini in Matrigel, transforms into cancer-like cells within the same Matrigel material following confinement in alginate shell hydrogel microcapsules. The confined cells exhibited a range of tumor-like behaviors, including uncontrolled cellular proliferation and invasion. Additionally, 4-6 weeks after transplantation into the mammary fad pads of immunocompromised mice, the confined cells formed large palpable masses that exhibited histological features similar to that of carcinomas. Taken together, our findings suggest that physical confinement represents a previously unrecognized mechanism for malignancy induction in mammary epithelial cells and also provide a new, microcapsule-based, high throughput model system for testing new breast cancer therapeutics.

    View details for DOI 10.1016/j.biomaterials.2019.119307

    View details for PubMedID 31271857