Paul A. Khavari, MD, PhD

All Publications

• Advances in cutaneous squamous cell carcinoma. Nature reviews. Cancer Winge, M. C., Kellman, L. N., Guo, K., Tang, J. Y., Swetter, S. M., Aasi, S. Z., Sarin, K. Y., Chang, A. L., Khavari, P. A. 2023

  Abstract

  Human malignancies arise predominantly in tissues of epithelial origin, where the stepwise transformation from healthy epithelium to premalignant dysplasia to invasive neoplasia involves sequential dysregulation of biological networks that govern essential functions of epithelial homeostasis. Cutaneous squamous cell carcinoma (cSCC) is a prototype epithelial malignancy, often with a high tumour mutational burden. A plethora of risk genes, dominated by UV-induced sun damage, drive disease progression in conjunction with stromal interactions and local immunomodulation, enabling continuous tumour growth. Recent studies have identified subpopulations of SCC cells that specifically interact with the tumour microenvironment. These advances, along with increased knowledge of the impact of germline genetics and somatic mutations on cSCC development, have led to a greater appreciation of the complexity of skin cancer pathogenesis and have enabled progress in neoadjuvant immunotherapy, which has improved pathological complete response rates. Although measures for the prevention and therapeutic management of cSCC are associated with clinical benefit, the prognosis remains poor for advanced disease. Elucidating how the genetic mechanisms that drive cSCC interact with the tumour microenvironment is a current focus in efforts to understand, prevent and treat cSCC.

  View details for DOI 10.1038/s41568-023-00583-5

  View details for PubMedID 37286893

  View details for PubMedCentralID 4833641

• Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation. Cell Miao, W., Porter, D. F., Lopez-Pajares, V., Siprashvili, Z., Meyers, R. M., Bai, Y., Nguyen, D. T., Ko, L. A., Zarnegar, B. J., Ferguson, I. D., Mills, M. M., Jilly-Rehak, C. E., Wu, C., Yang, Y., Meyers, J. M., Hong, A. W., Reynolds, D. L., Ramanathan, M., Tao, S., Jiang, S., Flynn, R. A., Wang, Y., Nolan, G. P., Khavari, P. A. 2023; 186 (1): 80

  Abstract

  Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.

  View details for DOI 10.1016/j.cell.2022.12.004

  View details for PubMedID 36608661

• The dynamic, combinatorial cis-regulatory lexicon of epidermal differentiation. Nature genetics Kim, D. S., Risca, V. I., Reynolds, D. L., Chappell, J., Rubin, A. J., Jung, N., Donohue, L. K., Lopez-Pajares, V., Kathiria, A., Shi, M., Zhao, Z., Deep, H., Sharmin, M., Rao, D., Lin, S., Chang, H. Y., Snyder, M. P., Greenleaf, W. J., Kundaje, A., Khavari, P. A. 2021

  Abstract

  Transcription factors bind DNA sequence motif vocabularies in cis-regulatory elements (CREs) to modulate chromatin state and gene expression during cell state transitions. A quantitative understanding of how motif lexicons influence dynamic regulatory activity has been elusive due to the combinatorial nature of the cis-regulatory code. To address this, we undertook multiomic data profiling of chromatin and expression dynamics across epidermal differentiation to identify 40,103 dynamic CREs associated with 3,609 dynamically expressed genes, then applied an interpretable deep-learning framework to model the cis-regulatory logic of chromatin accessibility. This analysis framework identified cooperative DNA sequence rules in dynamic CREs regulating synchronous gene modules with diverse roles in skin differentiation. Massively parallel reporter assay analysis validated temporal dynamics and cooperative cis-regulatory logic. Variants linked to human polygenic skin disease were enriched in these time-dependent combinatorial motif rules. This integrative approach shows the combinatorial cis-regulatory lexicon of epidermal differentiation and represents a general framework for deciphering the organizational principles of the cis-regulatory code of dynamic gene regulation.

  View details for DOI 10.1038/s41588-021-00947-3

  View details for PubMedID 34650237

• Multimodal Analysis of Composition and Spatial Architecture in Human Squamous Cell Carcinoma. Cell Ji, A. L., Rubin, A. J., Thrane, K., Jiang, S., Reynolds, D. L., Meyers, R. M., Guo, M. G., George, B. M., Mollbrink, A., Bergenstråhle, J., Larsson, L., Bai, Y., Zhu, B., Bhaduri, A., Meyers, J. M., Rovira-Clavé, X., Hollmig, S. T., Aasi, S. Z., Nolan, G. P., Lundeberg, J., Khavari, P. A. 2020; 182 (6): 1661-1662

  View details for DOI 10.1016/j.cell.2020.08.043

  View details for PubMedID 32946785

  View details for PubMedCentralID PMC7505493

• 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. W., Zarnegar, B. J., Greenleaf, W. J., Chang, H. Y., Khavari, P. A. 2018

  Abstract

  Here, we present Perturb-ATAC, a method that combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in 4,300 single cells, encompassing more than 63 genotype-phenotype relationships. Perturb-ATAC in human Blymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning and identified a hierarchy of TFs that govern B cell state, variation, and disease-associated cis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules of cis-elements that specify keratinocyte fate. Combinatorial deletion of all pairs of these TFsuncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful strategy to dissect gene regulatory networks in development and disease.

  View details for PubMedID 30580963

• Repurposing an epithelial sodium channel inhibitor as a therapy for murine and human skin inflammation. Science translational medicine Winge, M. C., Nasrallah, M., Jackrazi, L. V., Guo, K. Q., Fuhriman, J. M., Szafran, R., Ramanathan, M., Gurevich, I., Nguyen, N. T., Siprashvili, Z., Inayathullah, M., Rajadas, J., Porter, D. F., Khavari, P. A., Butte, A. J., Marinkovich, M. P. 2024; 16 (777): eade5915

  Abstract

  Inflammatory skin disease is characterized by a pathologic interplay between skin cells and immunocytes and can result in disfiguring cutaneous lesions and systemic inflammation. Immunosuppression is commonly used to target the inflammatory component; however, these drugs are often expensive and associated with side effects. To identify previously unidentified targets, we carried out a nonbiased informatics screen to identify drug compounds with an inverse transcriptional signature to keratinocyte inflammatory signals. Using psoriasis, a prototypic inflammatory skin disease, as a model, we used pharmacologic, transcriptomic, and proteomic characterization to find that benzamil, the benzyl derivative of the US Food and Drug Administration-approved diuretic amiloride, effectively reversed keratinocyte-driven inflammatory signaling. Through three independent mouse models of skin inflammation (Rac1G12V transgenic mice, topical imiquimod, and human skin xenografts from patients with psoriasis), we found that benzamil disrupted pathogenic interactions between the small GTPase Rac1 and its adaptor NCK1. This reduced STAT3 and NF-κB signaling and downstream cytokine production in keratinocytes. Genetic knockdown of sodium channels or pharmacological inhibition by benzamil prevented excess Rac1-NCK1 binding and limited proinflammatory signaling pathway activation in patient-derived keratinocytes without systemic immunosuppression. Both systemic and topical applications of benzamil were efficacious, suggesting that it may be a potential therapeutic avenue for treating skin inflammation.

  View details for DOI 10.1126/scitranslmed.ade5915

  View details for PubMedID 39661704

• Glucose binds and activates NSUN2 to promote translation and epidermal differentiation. Nucleic acids research Miao, W., Porter, D. F., Li, Y., Meservey, L. M., Yang, Y. Y., Ma, C., Ferguson, I. D., Tien, V. B., Jack, T. M., Ducoli, L., Lopez-Pajares, V., Tao, S., Savage, P. B., Wang, Y., Khavari, P. A. 2024

  Abstract

  Elevations in intracellular glucose concentrations are essential for epithelial cell differentiation by mechanisms that are not fully understood. Glucose has recently been found to directly bind several proteins to alter their functions to enhance differentiation. Among the newly identified glucose-binding proteins is NSUN2, an RNA-binding protein that we identified as indispensable for epidermal differentiation. Glucose was found to bind conserved sequences within NSUN2, enhancing its binding to S-adenosyl-L-methionine and boosting its enzymatic activity. Additionally, glucose enhanced NSUN2's proximity to proteins involved in mRNA translation, with NSUN2 modulating global messenger RNA (mRNA) translation, particularly that of key pro-differentiation mRNAs containing m5C modifications, such as GRHL3. Glucose thus engages diverse molecular mechanisms beyond its energetic roles to facilitate cellular differentiation processes.

  View details for DOI 10.1093/nar/gkae1097

  View details for PubMedID 39565212

• A large-scale TF-DNA interaction map in primary keratinocytes and its relation to skin disease-linked regulatory variants Porter, D. F., Reynolds, D. L., Miao, W., Meyers, R. M., Hong, A. W., Yang, X., Ducoli, L., Mondal, S., Siprashvili, Z., Elfaki, I., Srinivasan, S., Khavari, P. A. ELSEVIER SCIENCE INC. 2024: S71

  View details for Web of Science ID 001276879001174

• p63 cooperates with FOXK1 to mediate oncogenic 3D chromatin dynamics Lopez-Pajares, V., Jung, N., Donohue, L., Guo, M., Khavari, P. A. ELSEVIER SCIENCE INC. 2024: S72

  View details for Web of Science ID 001276879001180

• Mitochondrial Raf1 Regulates Glutamine Catabolism. bioRxiv : the preprint server for biology Shanderson, R. L., Ferguson, I. D., Siprashvili, Z., Ducoli, L., Li, A. M., Miao, W., Srinivasan, S., Velasco, M. G., Li, Y., Ye, J., Khavari, P. A. 2024

  Abstract

  Raf kinases play vital roles in normal mitogenic signaling and cancer, however, the identities of functionally important Raf-proximal proteins throughout the cell are not fully known. Raf1 proximity proteomics/BioID in Raf1-dependent cancer cells unexpectedly identified Raf1-adjacent proteins known to reside in the mitochondrial matrix. Inner-mitochondrial localization of Raf1 was confirmed by mitochondrial purification and super-resolution microscopy. Inside mitochondria, Raf1 associated with glutaminase (GLS) in diverse human cancers and enabled glutaminolysis, an important source of biosynthetic precursors in cancer. These impacts required Raf1 kinase activity and were independent of canonical MAP kinase pathway signaling. Kinase-dead mitochondrial matrix-localized Raf1 impaired glutaminolysis and tumorigenesis in vivo. These data indicate that Raf1 localizes inside mitochondria where it interacts with GLS to engage glutamine catabolism and support tumorigenesis.

  View details for DOI 10.1101/2024.03.08.581297

  View details for PubMedID 38496616

  View details for PubMedCentralID PMC10942467

• Integrative analyses highlight functional regulatory variants associated with neuropsychiatric diseases. Nature genetics Guo, M. G., Reynolds, D. L., Ang, C. E., Liu, Y., Zhao, Y., Donohue, L. K., Siprashvili, Z., Yang, X., Yoo, Y., Mondal, S., Hong, A., Kain, J., Meservey, L., Fabo, T., Elfaki, I., Kellman, L. N., Abell, N. S., Pershad, Y., Bayat, V., Etminani, P., Holodniy, M., Geschwind, D. H., Montgomery, S. B., Duncan, L. E., Urban, A. E., Altman, R. B., Wernig, M., Khavari, P. A. 2023

  Abstract

  Noncoding variants of presumed regulatory function contribute to the heritability of neuropsychiatric disease. A total of 2,221 noncoding variants connected to risk for ten neuropsychiatric disorders, including autism spectrum disorder, attention deficit hyperactivity disorder, bipolar disorder, borderline personality disorder, major depression, generalized anxiety disorder, panic disorder, post-traumatic stress disorder, obsessive-compulsive disorder and schizophrenia, were studied in developing human neural cells. Integrating epigenomic and transcriptomic data with massively parallel reporter assays identified differentially-active single-nucleotide variants (daSNVs) in specific neural cell types. Expression-gene mapping, network analyses and chromatin looping nominated candidate disease-relevant target genes modulated by these daSNVs. Follow-up integration of daSNV gene editing with clinical cohort analyses suggested that magnesium transport dysfunction may increase neuropsychiatric disease risk and indicated that common genetic pathomechanisms may mediate specific symptoms that are shared across multiple neuropsychiatric diseases.

  View details for DOI 10.1038/s41588-023-01533-5

  View details for PubMedID 37857935

  View details for PubMedCentralID 4112379

• A single nucleotide activity map of skin disease variants and transcription factors that modulate them Porter, D., Reynolds, D. L., Meyers, R., Miao, W., Hong, A. W., Yang, X., Ducoli, L., Mondal, S., Siprashvili, Z., Srinivasan, S., Khavari, P. A. ELSEVIER SCIENCE INC. 2023: S148

  View details for Web of Science ID 001037960301113

• Functional characterization of human genomic variation linked to polygenic diseases. Trends in genetics : TIG Fabo, T., Khavari, P. 2023

  Abstract

  The burden of human disease lies predominantly in polygenic diseases. Since the early 2000s, genome-wide association studies (GWAS) have identified genetic variants and loci associated with complex traits. These have ranged from variants in coding sequences to mutations in regulatory regions, such as promoters and enhancers, as well as mutations affecting mediators of mRNA stability and other downstream regulators, such as 5' and 3'-untranslated regions (UTRs), long noncoding RNA (lncRNA), and miRNA. Recent research advances in genetics have utilized a combination of computational techniques, high-throughput in vitro and in vivo screening modalities, and precise genome editing to impute the function of diverse classes of genetic variants identified through GWAS. In this review, we highlight the vastness of genomic variants associated with polygenic disease risk and address recent advances in how genetic tools can be used to functionally characterize them.

  View details for DOI 10.1016/j.tig.2023.02.014

  View details for PubMedID 36997428

• Analyzing RNA-Protein Interactions by Cross-Link Rates and CLIP-seq Libraries. Current protocols Porter, D. F., Garg, R. M., Meyers, R. M., Miao, W., Ducoli, L., Zarnegar, B. J., Khavari, P. A. 2023; 3 (1): e659

  Abstract

  UV cross-linking-based methods are the most common tool to explore in vivo RNA-protein interactions. UV cross-linking enables the freezing of direct interactions in the cell, which can then be mapped by high-throughput sequencing through a family of methods termed CLIP-seq. CLIP-seq measures the distribution of cross-link events by purifying a protein of interest and sequencing the covalently bound RNA fragments. However, there are disagreements and ambiguities as to which proteins are RNA-binding proteins and what interactions are significant as all proteins contact all RNAs at some frequency. Here we describe a protocol for both determining RNA-protein interactions through a combination of RNA library preparation and the measurement of absolute cross-link rates, which helps determine what proteins are RNA-binding proteins and what interactions are significant. This protocol, comprising an updated form of the easyCLIP protocol, describes guidelines for RNA library preparation, oligo and protein standard construction, and the measurement of cross-link rates. These methods are easily visualizable through their fluorescent labels and can be adapted to study RNA-binding properties of both functional, high affinity RNA-binding proteins, and the accidental RNA interactions of non-RNA-binding proteins. © 2023 Wiley Periodicals LLC. Basic Protocol 1: RNA library construction Basic Protocol 2: Determining UV cross-link rates Support Protocol 1: Cross-linking and lysing cells Support Protocol 2: Adapter preparation Support Protocol 3: Preparation of cross-linked RBP standard.

  View details for DOI 10.1002/cpz1.659

  View details for PubMedID 36705610

• A cis-regulatory lexicon of DNA motif combinations mediating cell-type-specific gene regulation. Cell genomics Donohue, L. K., Guo, M. G., Zhao, Y., Jung, N., Bussat, R. T., Kim, D. S., Neela, P. H., Kellman, L. N., Garcia, O. S., Meyers, R. M., Altman, R. B., Khavari, P. A. 2022; 2 (11)

  Abstract

  Gene expression is controlled by transcription factors (TFs) that bind cognate DNA motif sequences in cis-regulatory elements (CREs). The combinations of DNA motifs acting within homeostasis and disease, however, are unclear. Gene expression, chromatin accessibility, TF footprinting, and H3K27ac-dependent DNA looping data were generated and a random-forest-based model was applied to identify 7,531 cell-type-specific cis-regulatory modules (CRMs) across 15 diploid human cell types. A co-enrichment framework within CRMs nominated 838 cell-type-specific, recurrent heterotypic DNA motif combinations (DMCs), which were functionally validated using massively parallel reporter assays. Cancer cells engaged DMCs linked to neoplasia-enabling processes operative in normal cells while also activating new DMCs only seen in the neoplastic state. This integrative approach identifies cell-type-specific cis-regulatory combinatorial DNA motifs in diverse normal and diseased human cells and represents a general framework for deciphering cis-regulatory sequence logic in gene regulation.

  View details for DOI 10.1016/j.xgen.2022.100191

  View details for PubMedID 36742369

• PROBER: overcoming the high noise of DNA-protein interactions studies NATURE METHODS Mondal, S., Khavari, P. 2022; 19 (9): 1044

  View details for DOI 10.1038/s41592-022-01553-9

  View details for Web of Science ID 000857686400013

  View details for PubMedID 35945455

• Glucose controls protein-protein interactions and epidermal differentiation Lopez-Pajares, V., Bhaduri, A., Zhao, Y., Gowrishankar, G., Donohue, L., Guo, M., Guerrero, A., Ji, A., Garcia, O., Gambir, S., Khavari, P. ELSEVIER SCIENCE INC. 2022: S71

  View details for Web of Science ID 000829693000592

• Spatially resolved clonal copy number alterations in benign and malignant tissue. Nature Erickson, A., He, M., Berglund, E., Marklund, M., Mirzazadeh, R., Schultz, N., Kvastad, L., Andersson, A., Bergenstrahle, L., Bergenstrahle, J., Larsson, L., Alonso Galicia, L., Shamikh, A., Basmaci, E., Diaz De Stahl, T., Rajakumar, T., Doultsinos, D., Thrane, K., Ji, A. L., Khavari, P. A., Tarish, F., Tanoglidi, A., Maaskola, J., Colling, R., Mirtti, T., Hamdy, F. C., Woodcock, D. J., Helleday, T., Mills, I. G., Lamb, A. D., Lundeberg, J. 2022; 608 (7922): 360-367

  Abstract

  Defining the transition from benign to malignant tissue is fundamental to improving early diagnosis of cancer1. Here we use a systematic approach to study spatial genome integrity in situ and describe previously unidentified clonal relationships. We used spatially resolved transcriptomics2 to infer spatial copy number variations in >120,000 regions across multiple organs, in benign and malignant tissues. We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue using an organ-wide approach focused on the prostate. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in cancer evolution. We highlight the power of capturing the molecular and spatial continuums in a tissue context and challenge the rationale for treatment paradigms, including focal therapy.

  View details for DOI 10.1038/s41586-022-05023-2

  View details for PubMedID 35948708

• PROBER identifies proteins associated with programmable sequence-specific DNA in living cells. Nature methods Mondal, S., Ramanathan, M., Miao, W., Meyers, R. M., Rao, D., Lopez-Pajares, V., Siprashvili, Z., Reynolds, D. L., Porter, D. F., Ferguson, I., Neela, P., Zhao, Y., Meservey, L. M., Guo, M., Yang, Y., Li, L., Wang, Y., Khavari, P. A. 2022; 19 (8): 959-968

  Abstract

  DNA-protein interactions mediate physiologic gene regulation and may be altered by DNA variants linked to polygenic disease. To enhance the speed and signal-to-noise ratio (SNR) in the identification and quantification of proteins associated with specific DNA sequences in living cells, we developed proximal biotinylation by episomal recruitment (PROBER). PROBER uses high-copy episomes to amplify SNR, and proximity proteomics (BioID) to identify the transcription factors and additional gene regulators associated with short DNA sequences of interest. PROBER quantified both constitutive and inducible association of transcription factors and corresponding chromatin regulators to target DNA sequences and binding quantitative trait loci due to single-nucleotide variants. PROBER identified alterations in regulator associations due to cancer hotspot mutations in the hTERT promoter, indicating that these mutations increase promoter association with specific gene activators. PROBER provides an approach to rapidly identify proteins associated with specific DNA sequences and their variants in living cells.

  View details for DOI 10.1038/s41592-022-01552-w

  View details for PubMedID 35927480

• An essential role for CRL complex signaling in epidermal differentiation Winge, M. C., Reynolds, D. L., Ducoli, L., Meyers, R. M., Khavari, P. ELSEVIER SCIENCE INC. 2022: S123

  View details for Web of Science ID 000829693001265

• Targeted Proteomic Approaches for Proteome-Wide Characterizations of the AMP-Binding Capacities of Kinases. Journal of proteome research Miao, W., Yin, J., Porter, D. F., Jiang, X., Khavari, P. A., Wang, Y. 2022

  Abstract

  Kinases play important roles in cell signaling, and adenosine monophosphate (AMP) is known to modulate cellular energy homeostasis through AMP-activated protein kinase (AMPK). Here, we explored novel AMP-binding kinases by employing a desthiobiotin-conjugated AMP acyl-phosphate probe to enrich efficiently AMP-binding proteins. Together with a parallel-reaction monitoring-based targeted proteomic approach, we uncovered 195 candidate AMP-binding kinases. We also enriched desthiobiotin-labeled peptides from adenine nucleotide-binding sites of kinases and analyzed them using LC-MS/MS in the multiple-reaction monitoring mode, which resulted in the identification of 44 peptides derived from 43 kinases displaying comparable or better binding affinities toward AMP relative to adenosine triphosphate (ATP). Moreover, our proteomic data revealed a potential involvement of AMP in the MAPK pathway through binding directly to the relevant kinases, especially MEK2 and MEK3. Together, we revealed the AMP-binding capacities of a large number of kinases, and our work built a strong foundation for understanding how AMP functions as a second messenger to modulate cell signaling.

  View details for DOI 10.1021/acs.jproteome.2c00225

  View details for PubMedID 35820187

• The spatial landscape of clonal somatic mutations in benign and malignant tissue Erickson, A., Berglund, E., He, M., Marklund, M., Mirzazadeh, R., Schultz, N., Kvastad, L., Andersson, A., Bergenstrahle, L., Bergenstrahle, J., Larsson, L., Shamikh, A., Basmaci, E., De Stahl, T., Rajakumar, T., Thrane, K., Ji, A. L., Khavari, P. A., Tarish, F., Tanoglidi, A., Maaskola, J., Colling, R., Mirtti, T., Hamdy, F., Woodcock, D. J., Helleday, T., Mills, I. G., Lamb, A. D., Lundenberg, J. AMER ASSOC CANCER RESEARCH. 2022

  View details for Web of Science ID 000892509506044

• Super-resolved spatial transcriptomics by deep data fusion. Nature biotechnology Bergenstrahle, L., He, B., Bergenstrahle, J., Abalo, X., Mirzazadeh, R., Thrane, K., Ji, A. L., Andersson, A., Larsson, L., Stakenborg, N., Boeckxstaens, G., Khavari, P., Zou, J., Lundeberg, J., Maaskola, J. 2021

  Abstract

  Current methods for spatial transcriptomics are limited by low spatial resolution. Here we introduce a method that integrates spatial gene expression data with histological image data from the same tissue section to infer higher-resolution expression maps. Using a deep generative model, our method characterizes the transcriptome of micrometer-scale anatomical features and can predict spatial gene expression from histology images alone.

  View details for DOI 10.1038/s41587-021-01075-3

  View details for PubMedID 34845373

• The proximal proteome of 17 SARS-CoV-2 proteins links to disrupted antiviral signaling and host translation. PLoS pathogens Meyers, J. M., Ramanathan, M., Shanderson, R. L., Beck, A., Donohue, L., Ferguson, I., Guo, M. G., Rao, D. S., Miao, W., Reynolds, D., Yang, X., Zhao, Y., Yang, Y., Blish, C., Wang, Y., Khavari, P. A. 2021; 17 (10): e1009412

  Abstract

  Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2.

  View details for DOI 10.1371/journal.ppat.1009412

  View details for PubMedID 34597346

• Mutant collagen COL11A1 enhances cancerous invasion. Oncogene Lee, C. S., Siprashvili, Z., Mah, A., Bencomo, T., Elcavage, L. E., Che, Y., Shenoy, R. M., Aasi, S. Z., Khavari, P. A. 2021

  Abstract

  Collagens are the most abundant proteins in the body and comprise the basement membranes and stroma through which cancerous invasion occurs; however, a pro-neoplastic function for mutant collagens is undefined. Here we identify COL11A1 mutations in 66 of 100 cutaneous squamous cell carcinomas (cSCCs), the second most common U.S. cancer, concentrated in a triple helical region known to produce trans-dominant collagens. Analysis of COL11A1 and other collagen genes found that they are mutated across common epithelial malignancies. Knockout of mutant COL11A1 impairs cSCC tumorigenesis in vivo. Compared to otherwise genetically identical COL11A1 wild-type tissue, gene-edited mutant COL11A1 skin is characterized by induction of beta1 integrin targets and accelerated neoplastic invasion. In mosaic tissue, mutant COL11A1 cells enhanced invasion by neighboring wild-type cells. These results suggest that specific collagens are commonly mutated in cancer and that mutant collagens may accelerate this process.

  View details for DOI 10.1038/s41388-021-02013-y

  View details for PubMedID 34584216

• Integrating single-cell and spatial transcriptomics to elucidate intercellular tissue dynamics. Nature reviews. Genetics Longo, S. K., Guo, M. G., Ji, A. L., Khavari, P. A. 2021

  Abstract

  Single-cell RNA sequencing (scRNA-seq) identifies cell subpopulations within tissue but does not capture their spatial distribution nor reveal local networks of intercellular communication acting in situ. A suite of recently developed techniques that localize RNA within tissue, including multiplexed in situ hybridization and in situ sequencing (here defined as high-plex RNA imaging) and spatial barcoding, can help address this issue. However, no method currently provides as complete a scope of the transcriptome as does scRNA-seq, underscoring the need for approaches to integrate single-cell and spatial data. Here, we review efforts to integrate scRNA-seq with spatial transcriptomics, including emerging integrative computational methods, and propose ways to effectively combine current methodologies.

  View details for DOI 10.1038/s41576-021-00370-8

  View details for PubMedID 34145435

• SARS-CoV-2 B.1.1.7 and B.1.351 spike variants bind human ACE2 with increased affinity. The Lancet. Infectious diseases Ramanathan, M., Ferguson, I. D., Miao, W., Khavari, P. A. 2021

  View details for DOI 10.1016/S1473-3099(21)00262-0

  View details for PubMedID 34022142

• Dissecting intercellular communication in adult human skin with single-cell and spatial transcriptomics Ji, A., Thrane, K., Guo, M., Rubin, A., Kim, D., Hollmig, T., Aasi, S., Lundeberg, J., Khavari, P. ELSEVIER SCIENCE INC. 2021: S17

  View details for Web of Science ID 000641872800093

• Metabolomic identification of an essential glucose-IRF6 axis in differentiation Lopez-Pajares, V., Bhaduri, A., Guerrero, A., Zhao, Y., Donohue, L., Guo, M., Gowrishankar, G., Gambhir, S. S., Khavari, P. ELSEVIER SCIENCE INC. 2021: S24

  View details for Web of Science ID 000641872800132

• easyCLIP analysis of RNA-protein interactions incorporating absolute quantification. Nature communications Porter, D. F., Miao, W., Yang, X., Goda, G. A., Ji, A. L., Donohue, L. K., Aleman, M. M., Dominguez, D., Khavari, P. A. 2021; 12 (1): 1569

  Abstract

  Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP target RNAs. Here, we develop an ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing method, easyCLIP. easyCLIP provides absolute cross-link rates, as well as increased simplicity, efficiency, and capacity to visualize RNA libraries during sequencing library preparation. Measurement of >200 independent cross-link experiments across >35 proteins identifies an RNA cross-link rate threshold that distinguishes RBPs from non-RBPs and defines target RNAs as those with a complex frequency unlikely for a random protein. We apply easyCLIP to the 33 most recurrent cancer mutations across 28 RBPs, finding increased RNA binding per RBP molecule for KHDRBS2 R168C, A1CF E34K and PCBP1 L100P/Q cancer mutations. Quantitating RBP-RNA interactions can thus nominate proteins as RBPs and define the impact of specific disease-associated RBP mutations on RNA association.

  View details for DOI 10.1038/s41467-021-21623-4

  View details for PubMedID 33692367

• The proximal proteome of 17 SARS-CoV-2 proteins links to disrupted antiviral signaling and host translation. bioRxiv : the preprint server for biology Meyers, J. M., Ramanathan, M., Shanderson, R. L., Donohue, L., Ferguson, I., Guo, M. G., Rao, D. S., Miao, W., Reynolds, D., Yang, X., Zhao, Y., Yang, Y. Y., Wang, Y., Khavari, P. A. 2021

  Abstract

  Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2.SARS-CoV-2 is the latest pathogenic coronavirus to emerge as a public health threat. We create a database of proximal host proteins to 17 SARS-CoV-2 viral proteins. We validate that NSP1 is proximal to the EIF3 translation initiation complex and is a potent inhibitor of translation. We also identify ORF6 antagonism of RNA-mediate innate immune signaling. We produce a database of potential host targets of the viral protease NSP5, and create a fluorescence-based assay to screen cleavage of peptide sequences. We believe that this data will be useful for identifying roles for many of the uncharacterized SARS-CoV-2 proteins and provide insights into the pathogenicity of new or emerging coronaviruses.

  View details for DOI 10.1101/2021.02.23.432450

  View details for PubMedID 33655243

  View details for PubMedCentralID PMC7924263

• SARS-CoV-2 B.1.1.7 and B.1.351 Spike variants bind human ACE2 with increased affinity. bioRxiv : the preprint server for biology Ramanathan, M., Ferguson, I. D., Miao, W., Khavari, P. A. 2021

  Abstract

  SARS-CoV2 being highly infectious has been particularly effective in causing widespread infection globally and more variants of SARS-CoV2 are constantly being reported with increased genomic surveillance. In particular, the focus is on mutations of Spike protein, which binds human ACE2 protein enabling SARS-CoV2 entry and infection. Here we present a rapid experimental method leveraging the speed and flexibility of Mircoscale Thermopheresis (MST) to characterize the interaction between Spike Receptor Binding Domain (RBD) and human ACE2 protein. The B.1.351 variant harboring three mutations, (E484K, N501Y, and K417N) binds the ACE2 at nearly five-fold greater affinity than the original SARS-COV-2 RBD. We also find that the B.1.1.7 variant, binds two-fold more tightly to ACE2 than the SARS-COV-2 RBD.

  View details for DOI 10.1101/2021.02.22.432359

  View details for PubMedID 33655251

  View details for PubMedCentralID PMC7924271

• Structural modularity of the XIST ribonucleoprotein complex. Nature communications Lu, Z., Guo, J. K., Wei, Y., Dou, D. R., Zarnegar, B., Ma, Q., Li, R., Zhao, Y., Liu, F., Choudhry, H., Khavari, P. A., Chang, H. Y. 2020; 11 (1): 6163

  Abstract

  Long noncoding RNAs are thought to regulate gene expression by organizing protein complexes through unclear mechanisms. XIST controls the inactivation of an entire X chromosome in female placental mammals. Here we develop and integrate several orthogonal structure-interaction methods to demonstrate that XIST RNA-protein complex folds into an evolutionarily conserved modular architecture. Chimeric RNAs and clustered protein binding in fRIP and eCLIP experiments align with long-range RNA secondary structure, revealing discrete XIST domains that interact with distinct sets of effector proteins. CRISPR-Cas9-mediated permutation of the Xist A-repeat location shows that A-repeat serves as a nucleation center for multiple Xist-associated proteins and m6A modification. Thus modular architecture plays an essential role, in addition to sequence motifs, in determining the specificity of RBP binding and m6A modification. Together, this work builds a comprehensive structure-function model for the XIST RNA-protein complex, and suggests a general strategy for mechanistic studies of large ribonucleoprotein assemblies.

  View details for DOI 10.1038/s41467-020-20040-3

  View details for PubMedID 33268787

• 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

• Genome-wide meta-analysis identifies eight new susceptibility loci for cutaneous squamous cell carcinoma. Nature communications Sarin, K. Y., Lin, Y. n., Daneshjou, R. n., Ziyatdinov, A. n., Thorleifsson, G. n., Rubin, A. n., Pardo, L. M., Wu, W. n., Khavari, P. A., Uitterlinden, A. n., Nijsten, T. n., Toland, A. E., Olafsson, J. H., Sigurgeirsson, B. n., Thorisdottir, K. n., Jorgensen, E. n., Whittemore, A. S., Kraft, P. n., Stacey, S. N., Stefansson, K. n., Asgari, M. M., Han, J. n. 2020; 11 (1): 820

  Abstract

  Cutaneous squamous cell carcinoma (SCC) is one of the most common cancers in the United States. Previous genome-wide association studies (GWAS) have identified 14 single nucleotide polymorphisms (SNPs) associated with cutaneous SCC. Here, we report the largest cutaneous SCC meta-analysis to date, representing six international cohorts and totaling 19,149 SCC cases and 680,049 controls. We discover eight novel loci associated with SCC, confirm all previously associated loci, and perform fine mapping of causal variants. The novel SNPs occur within skin-specific regulatory elements and implicate loci involved in cancer development, immune regulation, and keratinocyte differentiation in SCC susceptibility.

  View details for DOI 10.1038/s41467-020-14594-5

  View details for PubMedID 32041948

• Multimodal Analysis of Composition and Spatial Architecture in Human Squamous Cell Carcinoma. Cell Ji, A. L., Rubin, A. J., Thrane, K. n., Jiang, S. n., Reynolds, D. L., Meyers, R. M., Guo, M. G., George, B. M., Mollbrink, A. n., Bergenstråhle, J. n., Larsson, L. n., Bai, Y. n., Zhu, B. n., Bhaduri, A. n., Meyers, J. M., Rovira-Clavé, X. n., Hollmig, S. T., Aasi, S. Z., Nolan, G. P., Lundeberg, J. n., Khavari, P. A. 2020

  Abstract

  To define the cellular composition and architecture of cutaneous squamous cell carcinoma (cSCC), we combined single-cell RNA sequencing with spatial transcriptomics and multiplexed ion beam imaging from a series of human cSCCs and matched normal skin. cSCC exhibited four tumor subpopulations, three recapitulating normal epidermal states, and a tumor-specific keratinocyte (TSK) population unique to cancer, which localized to a fibrovascular niche. Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing TSK cells as a hub for intercellular communication. Multiple features of potential immunosuppression were observed, including T regulatory cell (Treg) co-localization with CD8 T cells in compartmentalized tumor stroma. Finally, single-cell characterization of human tumor xenografts and in vivo CRISPR screens identified essential roles for specific tumor subpopulation-enriched gene networks in tumorigenesis. These data define cSCC tumor and stromal cell subpopulations, the spatial niches where they interact, and the communicating gene networks that they engage in cancer.

  View details for DOI 10.1016/j.cell.2020.05.039

  View details for PubMedID 32579974

• Genetic and genomic studies of pathogenic EXOSC2 mutations in the newly described disease SHRF implicate the autophagy pathway in disease pathogenesis. Human molecular genetics Yang, X., Bayat, V., DiDonato, N., Zhao, Y., Zarnegar, B., Siprashvili, Z., Lopez-Pajares, V., Sun, T., Tao, S., Li, C., Rump, A., Khavari, P., Lu, B. 2019

  Abstract

  Missense mutations in the RNA exosome component Exosome Component 2 (EXOSC2), also known as Ribosomal RNA-processing Protein 4 (RRP4), were recently identified in two unrelated families with a novel syndrome known as Short stature, Hearing loss, Retinitis pigmentosa, and distinctive Facies (SHRF, #OMIM 617763). Little is known about the mechanism of SHRF pathogenesis. Here we have studied the effect of mutations in EXOSC2/RRP4 in patient-derived lymphoblasts, clustered regularly interspaced short palindromic repeats (CRISPR)-generated mutant fetal keratinocytes, and Drosophila. We determined that human EXOSC2 is an essential gene, and that the pathogenic G198D mutation prevents binding to other RNA exosome components, resulting in protein and complex instability and altered expression and/or activities of critical genes, including those in the autophagy pathway. In parallel, we generated multiple CRISPR knockouts of the fly rrp4 gene. Using these flies, as well as rrp4 mutants with Piggy Bac (PBac) transposon insertion in the 3'UTR and RNAi flies, we determined that fly rrp4 was also essential, that fly rrp4 phenotypes could be rescued by wild-type human EXOSC2 but not the pathogenic form, and that fly rrp4 is critical for eye development and maintenance, muscle ultrastructure, and wing vein development. We found that overexpression of the transcription factor MITF was sufficient to rescue the small eye and adult lethal phenotypes caused by rrp4 inhibition. The autophagy genes ATG1 and ATG17, which are regulated by MITF, had similar effect. Pharmacological stimulation of autophagy with rapamycin also rescued the lethality caused by rrp4 inactivation. Our results implicate defective autophagy in SHRF pathogenesis and suggest therapeutic strategies.

  View details for DOI 10.1093/hmg/ddz251

  View details for PubMedID 31628467

• Ras functional proximity proteomics establishes mTORC2 as new direct ras effector. Oncotarget Kovalski, J. R., Shanderson, R. L., Khavari, P. A. 2019; 10 (50): 5126–35

  Abstract

  Although oncogenic mutations in the three major Ras isoforms, KRAS, HRAS and NRAS, are present in nearly a third of human cancers, therapeutic targeting of Ras remains a challenge due to its structure and complex regulation. However, an in-depth examination of the protein interactome of oncogenic Ras may provide new insights into key regulators, effectors and other mediators of its tumorigenic functions. Previous proteomic analyses have been limited by experimental tools that fail to capture the dynamic, transient nature of Ras cellular interactions. Therefore, in a recent study, we integrated proximity-dependent biotin labeling (BioID) proteomics with CRISPR screening of identified proteins to identify Ras proximal proteins required for Ras-dependent cancer cell growth. Oncogenic Ras was proximal to proteins involved in unexpected biological processes, such as vesicular trafficking and solute transport. Critically, we identified a direct, bona fide interaction between active Ras and the mTOR Complex 2 (mTORC2) that stimulated mTORC2 kinase activity. The oncogenic Ras-mTORC2 interaction resulted in a downstream pro-proliferative transcriptional program and promoted Ras-dependent tumor growth in vivo. Here we provide additional insight into the Ras isoform-specific protein interactomes, highlighting new opportunities for unique tumor-type therapies. Finally, we discuss the active Ras-mTORC2 interaction in detail, providing a more complete understanding of the direct relationship between Ras and mTORC2. Collectively, our findings support a model wherein Ras integrates an expanded array of pro-oncogenic signals to drive tumorigenic processes, including action on mTORC2 as a direct effector of Ras-driven proliferative signals.

  View details for DOI 10.18632/oncotarget.27025

  View details for PubMedID 31497244

• 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

• Impact of a patient-derived hepatitis C viral RNA genome with a mutated microRNA binding site PLOS PATHOGENS Mata, M., Neben, S., Majzoub, K., Carette, J., Ramanathan, M., Khavari, P. A., Sarnow, P. 2019; 15 (5)

  View details for DOI 10.1371/journal.ppat.1007467

  View details for Web of Science ID 000471180400001

• Characterization of novel MAPK interactor with potential in therapeutic development Jiang, T. E., Che, Y., Seelman, A., Guenin, C., Khavari, P. ELSEVIER SCIENCE INC. 2019: S69

  View details for Web of Science ID 000465561503118

• Uncovering causative, noncoding genetic variants in cutaneous diseases Wozniak, G., Shen, Y., Rubin, A., Neela, P., Khavari, P. ELSEVIER SCIENCE INC. 2019: S68

  View details for Web of Science ID 000465561503114

• Dissecting intratumoral heterogeneity and microenvironment interactions in SCC through single-cell RNA-sequencing Ji, A., Rubin, A., Reynolds, D., Guo, M., Bhaduri, A., George, B., Hollmig, S., Aasi, S., Khavari, P. ELSEVIER SCIENCE INC. 2019: S24

  View details for Web of Science ID 000465561502136

• Small non-coding RNA SNORA12 effects MAPK1 signaling Siprashvili, Z., Shenoy, R. M., Elcavage, L., Khavari, P. A. ELSEVIER SCIENCE INC. 2019: S21

  View details for Web of Science ID 000465561502117

• Unraveling keratinocyte gene regulatory networks with single-cell cripsr screening and epigenomic profiling Ramanathan, M., Rubin, A., Parker, K., Satpathy, A., Greenleaf, W., Chang, H., Khavari, P. ELSEVIER SCIENCE INC. 2019: S63

  View details for Web of Science ID 000465561503083

• Profiling of rotavirus 3UTR-binding proteins reveals the ATP synthase subunit ATP5B as a host factor that supports late-stage virus replication JOURNAL OF BIOLOGICAL CHEMISTRY Ren, L., Ding, S., Song, Y., Li, B., Ramanathan, M., Co, J., Amieva, M. R., Khavari, P. A., Greenberg, H. B. 2019; 294 (15): 5993–6006

  View details for DOI 10.1074/jbc.RA118.006004

  View details for Web of Science ID 000465077500030

• Methods to study RNA-protein interactions (vol 16, pg 225, 2019) NATURE METHODS Ramanathan, M., Porter, D. F., Khavari, P. A. 2019; 16 (4): 351

  View details for DOI 10.1038/s41592-019-0366-2

  View details for Web of Science ID 000462620400034

• Author Correction: Methods to study RNA-protein interactions. Nature methods Ramanathan, M., Porter, D. F., Khavari, P. A. 2019

  Abstract

  In the version of this paper originally published, three references were accidentally omitted: Schwartz, J. C. et al. Cell Rep. 5, 918-925 (2013); Tundup, S. et al. FEBS Lett. 580, 1285-1293 (2006); and Itri, F. et al. Biochem. Biophys. Res. Commun. 492, 67-73 (2017). The PDF and HTML versions of the paper now include these as references 58, 59, and 60, respectively, and subsequent references have been renumbered accordingly.

  View details for PubMedID 30850752

• Methods to study RNA-protein interactions. Nature methods Ramanathan, M., Porter, D. F., Khavari, P. A. 2019; 16 (3): 225–34

  Abstract

  Noncoding RNA sequences, including long noncoding RNAs, small nucleolar RNAs, and untranslated mRNA regions, accomplish many of their diverse functions through direct interactions with RNA-binding proteins (RBPs). Recent efforts have identified hundreds of new RBPs that lack known RNA-binding domains, thus underscoring the complexity and diversity of RNA-protein complexes. Recent progress has expanded the number of methods for studying RNA-protein interactions in two general categories: approaches that characterize proteins bound to an RNA of interest (RNA-centric), and those that examine RNAs bound to a protein of interest (protein-centric). Each method has unique strengths and limitations, which makes it important to select optimal approaches for the biological question being addressed. Here we review methods for the study of RNA-protein interactions, with a focus on their suitability for specific applications.

  View details for PubMedID 30804549

• Methods to study RNA-protein interactions NATURE METHODS Ramanathan, M., Porter, D. F., Khavari, P. A. 2019; 16 (3): 225–34

  View details for DOI 10.1038/s41592-019-0330-1

  View details for Web of Science ID 000459804000018

• The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2 MOLECULAR CELL Kovalski, J. R., Bhaduri, A., Zehnder, A. M., Neela, P. H., Che, Y., Wozniak, G. G., Khavari, P. A. 2019; 73 (4): 830-+

  View details for DOI 10.1016/j.molcel.2018.12.001

  View details for Web of Science ID 000459253700017

• Profiling of rotavirus 3'UTR-binding proteins reveals the ATP synthase subunit ATP5B as a host factor that supports late-stage virus replication. The Journal of biological chemistry Ren, L., Ding, S., Song, Y., Li, B., Ramanathan, M., Co, J., Amieva, M. R., Khavari, P. A., Greenberg, H. B. 2019

  Abstract

  Genome replication and virion assembly of segmented RNA viruses are highly coordinated events, tightly regulated by sequence and structural elements in the UTRs of viral RNA. This process is poorly defined and likely requires the participation of host proteins in concert with viral proteins. In this study, we employed a proteomics-based approach, named RNA-protein interaction detection (RaPID), to comprehensively screen for host proteins that bind to a conserved motif within the rotavirus (RV) 3' terminus. Using this assay, we identified ATP5B, a core subunit of the mitochondrial ATP synthase, as having high affinity to the RV 3'UTR consensus sequences. During RV infection, ATP5B bound to the RV 3'UTR and co-localized with viral RNA and viroplasm. Functionally, siRNA-mediated genetic depletion of ATP5B or other ATP synthase subunits such as ATP5A1 and ATP5O reduced the production of infectious viral progeny without significant alteration of intracellular viral RNA levels or RNA translation. Chemical inhibition of ATP synthase diminished RV yield in both conventional cell culture and in human intestinal enteroids, indicating that ATP5B positively regulates late-stage RV maturation in primary intestinal epithelial cells. Collectively, our results shed light on the role of host proteins in RV genome assembly and particle formation and identify ATP5B as a novel pro-RV RNA-binding protein, contributing to our understanding of how host ATP synthases may galvanize virus growth and pathogenesis.

  View details for PubMedID 30770472

• 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

• Impact of a patient-derived hepatitis C viral RNA genome with a mutated microRNA binding site. PLoS pathogens Mata, M. n., Neben, S. n., Majzoub, K. n., Carette, J. n., Ramanathan, M. n., Khavari, P. A., Sarnow, P. n. 2019; 15 (5): e1007467

  Abstract

  Hepatitis C virus (HCV) depends on liver-specific microRNA miR-122 for efficient viral RNA amplification in liver cells. This microRNA interacts with two different conserved sites at the very 5' end of the viral RNA, enhancing miR-122 stability and promoting replication of the viral RNA. Treatment of HCV patients with oligonucleotides that sequester mir-122 resulted in profound loss of viral RNA in phase II clinical trials. However, some patients accumulated in their sera a viral RNA genome that contained a single cytidine to uridine mutation at the third nucleotide from the 5' genomic end. It is shown here that this C3U variant indeed displayed higher rates of replication than that of wild-type HCV when miR-122 abundance is low in liver cells. However, when miR-122 abundance is high, binding of miR-122 to site 1, most proximal to the 5' end in the C3U variant RNA, is impaired without disrupting the binding of miR-122 to site 2. As a result, C3U RNA displays a much lower rate of replication than wild-type mRNA when miR-122 abundance is high in the liver. This phenotype was accompanied by binding of a different set of cellular proteins to the 5' end of the C3U RNA genome. In particular, binding of RNA helicase DDX6 was important for displaying the C3U RNA replication phenotype in liver cells. These findings suggest that sequestration of miR-122 leads to a resistance-associated mutation that has only been observed in treated patients so far, and raises the question about the function of the C3U variant in the peripheral blood.

  View details for PubMedID 31075158

• KRAS regulation by small non-coding RNAs and SNARE proteins. Nature communications Che, Y. n., Siprashvili, Z. n., Kovalski, J. R., Jiang, T. n., Wozniak, G. n., Elcavage, L. n., Khavari, P. A. 2019; 10 (1): 5118

  Abstract

  KRAS receives and relays signals at the plasma membrane (PM) where it transmits extracellular growth factor signals to downstream effectors. SNORD50A/B were recently found to bind KRAS and inhibit its tumorigenic action by unknown mechanisms. KRAS proximity protein labeling was therefore undertaken in SNORD50A/B wild-type and knockout cells, revealing that SNORD50A/B RNAs shape the composition of proteins proximal to KRAS, notably by inhibiting KRAS proximity to the SNARE vesicular transport proteins SNAP23, SNAP29, and VAMP3. To remain enriched on the PM, KRAS undergoes cycles of endocytosis, solubilization, and vesicular transport to the PM. Here we report that SNAREs are essential for the final step of this process, with KRAS localization to the PM facilitated by SNAREs but antagonized by SNORD50A/B. Antagonism between SNORD50A/B RNAs and specific SNARE proteins thus controls KRAS localization, signaling, and tumorigenesis, and disrupting SNARE-enabled KRAS function represents a potential therapeutic opportunity in KRAS-driven cancer.

  View details for DOI 10.1038/s41467-019-13106-4

  View details for PubMedID 31712554

• 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

• 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

• Cancer-Associated Long Noncoding RNA SMRT-2 Controls Epidermal Differentiation JOURNAL OF INVESTIGATIVE DERMATOLOGY Lee, C. S., Mah, A., Aros, C. J., Lopez-Pajares, V., Bhaduri, A., Webster, D. E., Kretz, M., Khavari, P. A. 2018; 138 (6): 1445–49

  View details for PubMedID 29360484

• Single-cell RNA-sequencing reveals SCC intratumoral heterogeneity Ji, A., Rubin, A., Hollmig, S., Aasi, S., Khavari, P. ELSEVIER SCIENCE INC. 2018: S31

  View details for Web of Science ID 000431188500182

• Targeting pathogenic interactions between Rac1 and NCK1 in psoriasis Winge, M. G., Nasrallah, M., Fuhriman, J. M., Ramanathan, M., Azameera, A., Nguyen, N., Inayathullah, M., Rajadas, J., Khavari, P., Butte, A., Marinkovich, M. ELSEVIER SCIENCE INC. 2018: S161

  View details for Web of Science ID 000431498600212

• Phase I / II Clinical Trial for Recessive Dystrophic Epidermolysis Bullosa Using EB-101 (COL7A1 Gene-Corrected Autologous Keratinocytes) Tang, J. Y., Marinkovich, M. P., Siprashvili, Z., Nguyen, N. T., Gorell, E. S., Loutit, K., Dutt-Singkh, Y., Barriga, M., Solis, D., Khuu, P., Furukawa, L., Lorenz, H. P., Leung, T. H., Keene, D. R., Rieger, K. E., Khavari, P. A., Lane, A. T. CELL PRESS. 2018: 158

  View details for Web of Science ID 000435342202011

• KRAS regulation by small non-coding RNAs and SNARE proteins Che, Y., Khavari, P. ELSEVIER SCIENCE INC. 2018: S27

  View details for Web of Science ID 000431188500162

• Dynamic morphogen-p63 chromatin interactions that guide epigenetic changes and p63 activity in surface ectoderm commitment Pattison, J., Melo, S., Piekos, S., Torkelson, J., Mumbach, M. R., Rubin, A., Li, L., Zhen, H., Chang, H., Khavari, P., Oro, A. E. ELSEVIER SCIENCE INC. 2018: S243

  View details for Web of Science ID 000431498600698

• Metabolomic analysis reveals an essential role for glucose in epidermal differentiation Lopez-Pajares, V., Bhaduri, A., Garcia, O., Guerrero, A., Gowrishankar, G., Che, Y., Sanchez, A., Boxer, L., Gambhir, S., Khavari, P. ELSEVIER SCIENCE INC. 2018: S123

  View details for Web of Science ID 000431188500728

• Small non-coding RNAs control the MAPK/ERK pathway Siprashvili, Z., Shenoy, R., Elcavage, L., Khavari, P. ELSEVIER SCIENCE INC. 2018: S27

  View details for Web of Science ID 000431188500159

• Decoding regulatory sequence across skin differentiation with deep learning Kim, D., Risca, V., Chappell, J., Shi, M., Zhao, Z., Jung, N., Chang, H., Snyder, M., Greenleaf, W., Kundaje, A., Khavari, P. ELSEVIER SCIENCE INC. 2018: S135

  View details for Web of Science ID 000431498600057

• Transcript-indexed ATAC-seq for precision immune profiling. Nature medicine Satpathy, A. T., Saligrama, N. n., Buenrostro, J. D., Wei, Y. n., Wu, B. n., Rubin, A. J., Granja, J. M., Lareau, C. A., Li, R. n., Qi, Y. n., Parker, K. R., Mumbach, M. R., Serratelli, W. S., Gennert, D. G., Schep, A. N., Corces, M. R., Khodadoust, M. S., Kim, Y. H., Khavari, P. A., Greenleaf, W. J., Davis, M. M., Chang, H. Y. 2018

  Abstract

  T cells create vast amounts of diversity in the genes that encode their T cell receptors (TCRs), which enables individual clones to recognize specific peptide-major histocompatibility complex (MHC) ligands. Here we combined sequencing of the TCR-encoding genes with assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis at the single-cell level to provide information on the TCR specificity and epigenomic state of individual T cells. By using this approach, termed transcript-indexed ATAC-seq (T-ATAC-seq), we identified epigenomic signatures in immortalized leukemic T cells, primary human T cells from healthy volunteers and primary leukemic T cells from patient samples. In peripheral blood CD4+ T cells from healthy individuals, we identified cis and trans regulators of naive and memory T cell states and found substantial heterogeneity in surface-marker-defined T cell populations. In patients with a leukemic form of cutaneous T cell lymphoma, T-ATAC-seq enabled identification of leukemic and nonleukemic regulatory pathways in T cells from the same individual by allowing separation of the signals that arose from the malignant clone from the background T cell noise. Thus, T-ATAC-seq is a new tool that enables analysis of epigenomic landscapes in clonal T cells and should be valuable for studies of T cell malignancy, immunity and immunotherapy.

  View details for PubMedID 29686426

• RNA-protein interaction detection in living cells. Nature methods Ramanathan, M. n., Majzoub, K. n., Rao, D. S., Neela, P. H., Zarnegar, B. J., Mondal, S. n., Roth, J. G., Gai, H. n., Kovalski, J. R., Siprashvili, Z. n., Palmer, T. D., Carette, J. E., Khavari, P. A. 2018

  Abstract

  RNA-protein interactions play numerous roles in cellular function and disease. Here we describe RNA-protein interaction detection (RaPID), which uses proximity-dependent protein labeling, based on the BirA* biotin ligase, to rapidly identify the proteins that bind RNA sequences of interest in living cells. RaPID displays utility in multiple applications, including in evaluating protein binding to mutant RNA motifs in human genetic disorders, in uncovering potential post-transcriptional networks in breast cancer, and in discovering essential host proteins that interact with Zika virus RNA. To improve the BirA*-labeling component of RaPID, moreover, a new mutant BirA* was engineered from Bacillus subtilis, termed BASU, that enables >1,000-fold faster kinetics and >30-fold increased signal-to-noise ratio over the prior standard Escherichia coli BirA*, thereby enabling direct study of RNA-protein interactions in living cells on a timescale as short as 1 min.

  View details for PubMedID 29400715

• The functions and unique features of long intergenic non-coding RNA. Nature reviews. Molecular cell biology Ransohoff, J. D., Wei, Y. n., Khavari, P. A. 2018; 19 (3): 143–57

  Abstract

  Long intergenic non-coding RNA (lincRNA) genes have diverse features that distinguish them from mRNA-encoding genes and exercise functions such as remodelling chromatin and genome architecture, RNA stabilization and transcription regulation, including enhancer-associated activity. Some genes currently annotated as encoding lincRNAs include small open reading frames (smORFs) and encode functional peptides and thus may be more properly classified as coding RNAs. lincRNAs may broadly serve to fine-tune the expression of neighbouring genes with remarkable tissue specificity through a diversity of mechanisms, highlighting our rapidly evolving understanding of the non-coding genome.

  View details for PubMedID 29138516

• Research Techniques Made Simple: Emerging Methods to Elucidate Protein Interactions through Spatial Proximity JOURNAL OF INVESTIGATIVE DERMATOLOGY Che, Y., Khavari, P. A. 2017; 137 (12): E197–E203

  Abstract

  Interactions between proteins are essential for fundamental cellular processes, and the diversity of such interactions enables the vast variety of functions essential for life. A persistent goal in biological research is to develop assays that can faithfully capture different types of protein interactions to allow their study. A major step forward in this direction came with a family of methods that delineates spatial proximity of proteins as an indirect measure of protein-protein interaction. A variety of enzyme- and DNA ligation-based methods measure protein co-localization in space, capturing novel interactions that were previously too transient or low affinity to be identified. Here we review some of the methods that have been successfully used to measure spatially proximal protein-protein interactions.

  View details for PubMedID 29169465

• Phase I/IIa clinical trial for recessive dystrophic epidermolysis bullosa using EB-101 (COL7A1 gene-corrected autologous keratinocytes) Siprashvili, Z., Nguyen, N. T., Gorell, E. S., Loutit, K., Dutt-Singkh, Y., Nazaroff, J., Khuu, P., Furukawa, L., Lorenz, H. P., Leung, T. H., Keene, D. R., Rieger, K. E., Khavari, P. A., Lane, A. T., Tang, J. Y., Marinkovich, M. P. MARY ANN LIEBERT, INC. 2017: A10

  View details for Web of Science ID 000418410700026

• Phase I/IIa clinical trial for recessive dystrophic epidermolysis bullosa using genetically corrected autologous keratinocytes Siprashvili, Z., Nguyen, N., Gorell, E., Loutit, K., Dutt-Singkh, Y., Nazaroff, J., Khuu, P., Furukawa, L., Lorenz, H., Leung, T., Keene, D., Rieger, K., Khavari, P. A., Lane, A., Tang, J. Y., Marinkovich, M. ELSEVIER SCIENCE INC. 2017: S89

  View details for DOI 10.1016/j.jid.2017.02.539

  View details for Web of Science ID 000406862400513

• K-RAS oncogene activation is regulated by a snoRNA/SNARE protein axis that controls its subcellular transport Che, Y., Siprashvili, Z., Kovalski, J., Khavari, P. A. ELSEVIER SCIENCE INC. 2017: S20

  View details for DOI 10.1016/j.jid.2017.02.131

  View details for Web of Science ID 000406862400117

• RNA-protein interaction detection (RaPID) in living cells uncovers post-transcriptional regulation in carcinogenesis Ramanathan, M., Majzoub, K., Roth, J., Gai, H., Palmer, T., Carette, J., Khavari, P. A. ELSEVIER SCIENCE INC. 2017: S21

  View details for DOI 10.1016/j.jid.2017.02.137

  View details for Web of Science ID 000406862400123

• The SNORD86 small non-coding RNA binds and regulates Rac1 Rios, E., Bhaduri, A., Siprashvili, Z., Zarnegar, B., Khavari, P. A. ELSEVIER SCIENCE INC. 2017: S21

  View details for DOI 10.1016/j.jid.2017.02.135

  View details for Web of Science ID 000406862400121

• Discovery of differential RNA binding and regulation by the APOL4 protein to disease-linked psoriasis CDSN gene variants using RaPID Ransohoff, J. D., Ramanathan, M., Khavari, P. A. ELSEVIER SCIENCE INC. 2017: S71

  View details for DOI 10.1016/j.jid.2017.02.427

  View details for Web of Science ID 000406862400408

• Dynamic and stable enhancer-promoter contacts regulate epidermal terminal differentiation Lopez-Pajares, V., Rubin, A., Barajas, B., Furlan-Magaril, M., Mumbach, M., Greenleaf, W., Kundaje, A., Snyder, M., Chang, H., Fraser, P., Khavari, P. A. ELSEVIER SCIENCE INC. 2017: S80

  View details for DOI 10.1016/j.jid.2017.02.483

  View details for Web of Science ID 000406862400458

• Attenuated netrin-1 receptor mediated regulation of tiam1 is required for rac1 mutant melanoma progression Winge, M. G., Kovalski, J., Nguyen, N. T., Wu, D., Zehnder, A., Khavari, P. A., Marinkovich, M. ELSEVIER SCIENCE INC. 2017: S139

  View details for DOI 10.1016/j.jid.2017.02.836

  View details for Web of Science ID 000406862400805

• 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

• 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

• 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

• CSNK1a1 Regulates PRMT1 to Maintain the Progenitor State in Self-Renewing Somatic Tissue. Developmental cell Bao, X. n., Siprashvili, Z. n., Zarnegar, B. J., Shenoy, R. M., Rios, E. J., Nady, N. n., Qu, K. n., Mah, A. n., Webster, D. E., Rubin, A. J., Wozniak, G. G., Tao, S. n., Wysocka, J. n., Khavari, P. A. 2017; 43 (2): 227–39.e5

  Abstract

  Somatic progenitors sustain tissue self-renewal while suppressing premature differentiation. Protein arginine methyltransferases (PRMTs) affect many processes; however, their role in progenitor function is incompletely understood. PRMT1 was found to be the most highly expressed PRMT in epidermal progenitors and the most downregulated PRMT during differentiation. In targeted mouse knockouts and in long-term regenerated human mosaic epidermis in vivo, epidermal PRMT1 loss abolished progenitor self-renewal and led to premature differentiation. Mass spectrometry of the PRMT1 protein interactome identified the CSNK1a1 kinase, which also proved essential for progenitor maintenance. CSNK1a1 directly bound and phosphorylated PRMT1 to control its genomic targeting to PRMT1-sustained proliferation genes as well as PRMT1-suppressed differentiation genes. Among the latter were GRHL3, whose derepression was required for the premature differentiation seen with PRMT1 and CSNK1a1 loss. Maintenance of the progenitors thus requires cooperation by PRMT1 and CSNK1a1 to sustain proliferation gene expression and suppress premature differentiation driven by GRHL3.

  View details for PubMedID 28943242

• 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

• Safety and Wound Outcomes Following Genetically Corrected Autologous Epidermal Grafts in Patients With Recessive Dystrophic Epidermolysis Bullosa. JAMA Siprashvili, Z., Nguyen, N. T., Gorell, E. S., Loutit, K., Khuu, P., Furukawa, L. K., Lorenz, H. P., Leung, T. H., Keene, D. R., Rieger, K. E., Khavari, P., Lane, A. T., Tang, J. Y., Marinkovich, M. P. 2016; 316 (17): 1808-1817

  Abstract

  Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating, often fatal, inherited blistering disorder caused by mutations in the COL7A1 gene encoding type VII collagen. Support and palliation are the only current therapies.To evaluate the safety and wound outcomes following genetically corrected autologous epidermal grafts in patients with RDEB.Single-center phase 1 clinical trial conducted in the United States of 4 patients with severe RDEB with a measured area of wounds suitable for grafting of at least 100 cm2. Patients with undetectable type VII collagen keratinocyte expression were excluded.Autologous keratinocytes isolated from biopsy samples collected from 4 patients with RDEB were transduced with good manufacturing practice-grade retrovirus carrying full-length human COL7A1 and assembled into epidermal sheet grafts. Type VII collagen gene-corrected grafts (approximately 35 cm2) were transplanted onto 6 wounds in each of the patients (n = 24 grafts).The primary safety outcomes were recombination competent retrovirus, cancer, and autoimmune reaction. Molecular correction was assessed as type VII collagen expression measured by immunofluorescence and immunoelectron microscopy. Wound healing was assessed using serial photographs taken at 3, 6, and 12 months after grafting.The 4 patients (mean age, 23 years [range, 18-32 years]) were all male with an estimated body surface area affected with RDEB of 4% to 30%. All 24 grafts were well tolerated without serious adverse events. Type VII collagen expression at the dermal-epidermal junction was demonstrated on the graft sites by immunofluorescence microscopy in 9 of 10 biopsy samples (90%) at 3 months, in 8 of 12 samples (66%) at 6 months, and in 5 of 12 samples (42%) at 12 months, including correct type VII collagen localization to anchoring fibrils. Wounds with recombinant type VII collagen graft sites displayed 75% or greater healing at 3 months (21 intact graft sites of 24 wound sites; 87%), 6 months (16/24; 67%), and 12 months (12/24; 50%) compared with baseline wound sites.In this preliminary study of 4 patients with RDEB, there was wound healing in some type VII collagen gene-corrected grafts, but the response was variable among patients and among grafted sites and generally declined over 1 year. Long-term follow-up is necessary for these patients, and controlled trials are needed with a broader range of patients to better understand the potential long-term efficacy of genetically corrected autologous epidermal grafts.clinicaltrials.gov Identifier: NCT01263379.

  View details for DOI 10.1001/jama.2016.15588

  View details for PubMedID 27802546

• Factors That May Promote an Effective Local Research Environment JOURNAL OF INVESTIGATIVE DERMATOLOGY Wang, K., Lee, C. S., Marinkovich, M., Chang, H. Y., Oro, A. E., Khavari, P. A. 2016; 136 (8): 1529–31

  View details for PubMedID 27450496

• RAC1 activation drives pathologic interactions between the epidermis and immune cells JOURNAL OF CLINICAL INVESTIGATION Winge, M. C., Ohyama, B., Dey, C. N., Boxer, L. M., Li, W., Ehsani-Chimeh, N., Truong, A. K., Wu, D., Armstrong, A. W., Makino, T., Davidson, M., Starcevic, D., Kislat, A., Nguyen, N. T., Hashimoto, T., Homey, B., Khavari, P. A., Bradley, M., Waterman, E. A., Marinkovich, M. P. 2016; 126 (7): 2661-2677

  Abstract

  Interactions between the epidermis and the immune system govern epidermal tissue homeostasis. These epidermis-immune interactions are altered in the inflammatory disease psoriasis; however, the pathways that underlie this aberrant immune response are not well understood. Here, we determined that Ras-related C3 botulinum toxin substrate 1 (RAC1) is a key mediator of epidermal dysfunction. RAC1 activation was consistently elevated in psoriatic epidermis and primary psoriatic human keratinocytes (PHKCs) exposed to psoriasis-related stimuli, but not in skin from patients with basal or squamous cell carcinoma. Expression of a constitutively active form of RAC1 (RACV12) in mice resulted in the development of lesions similar to those of human psoriasis that required the presence of an intact immune system. RAC1V12-expressing mice and human psoriatic skin showed similar RAC1-dependent signaling as well as transcriptional overlap of differentially expressed epidermal and immune pathways. Coculture of PHKCs with immunocytes resulted in the upregulation of RAC1-dependent proinflammatory cytokines, an effect that was reproduced by overexpressing RAC1 in normal human keratinocytes. In keratinocytes, modulating RAC1 activity altered differentiation, proliferation, and inflammatory pathways, including STAT3, NFκB, and zinc finger protein 750 (ZNF750). Finally, RAC1 inhibition in xenografts composed of human PHKCs and immunocytes abolished psoriasiform hyperplasia and inflammation in vivo. These studies implicate RAC1 as a potential therapeutic target for psoriasis and as a key orchestrator of pathologic epidermis-immune interactions.

  View details for DOI 10.1172/JCI85738

  View details for Web of Science ID 000379094800028

  View details for PubMedID 27294528

  View details for PubMedCentralID PMC4922704

• 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

• Phase I clinical trial for Recessive Dystrophic Epidermolysis Bullosa using genetically corrected autologous keratinocytes Siprashvili, Z., Nguyen, N. T., Gorell, E. S., Loutit, K., Khuu, P., Furukawa, L. K., Lorenz, H. P., Leung, T. H., Keene, D., Rieger, K., Khavari, P. A., Lane, A., Tang, J., Marinkovich, P. ELSEVIER SCIENCE INC. 2016: S65

  View details for DOI 10.1016/j.jid.2016.02.400

  View details for Web of Science ID 000380028800367

• Small non-coding snoRNAs control Ras superfamily GTPases in stem cell differentiation and cancer Zarnegar, B., Rios, E., Siprashvili, Z., Khavari, P. A. ELSEVIER SCIENCE INC. 2016: S17

  View details for DOI 10.1016/j.jid.2016.02.121

  View details for Web of Science ID 000380028800095

• Regulation of epidermal differentiation by MAF and MAFB Lopez-Pajares, V., Barajas, B. C., Rubin, A., Khavari, P. A. ELSEVIER SCIENCE INC. 2016: S59

  View details for DOI 10.1016/j.jid.2016.02.367

  View details for Web of Science ID 000380028800335

• 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

• The noncoding RNAs SNORD50A and SNORD50B bind K-Ras and are recurrently deleted in human cancer. Nature genetics Siprashvili, Z., Webster, D. E., Johnston, D., Shenoy, R. M., Ungewickell, A. J., Bhaduri, A., Flockhart, R., Zarnegar, B. J., Che, Y., Meschi, F., Puglisi, J. D., Khavari, P. A. 2016; 48 (1): 53-58

  Abstract

  Small nucleolar RNAs (snoRNAs) are conserved noncoding RNAs best studied as ribonucleoprotein (RNP) guides in RNA modification. To explore their role in cancer, we compared 5,473 tumor-normal genome pairs to identify snoRNAs with frequent copy number loss. The SNORD50A-SNORD50B snoRNA locus was deleted in 10-40% of 12 common cancers, where its loss was associated with reduced survival. A human protein microarray screen identified direct SNORD50A and SNORD50B RNA binding to K-Ras. Loss of SNORD50A and SNORD50B increased the amount of GTP-bound, active K-Ras and hyperactivated Ras-ERK1/ERK2 signaling. Loss of these snoRNAs also increased binding by farnesyltransferase to K-Ras and increased K-Ras prenylation, suggesting that KRAS mutation might synergize with SNORD50A and SNORD50B loss in cancer. In agreement with this hypothesis, CRISPR-mediated deletion of SNORD50A and SNORD50B in KRAS-mutant tumor cells enhanced tumorigenesis, and SNORD50A and SNORD50B deletion and oncogenic KRAS mutation co-occurred significantly in multiple human tumor types. SNORD50A and SNORD50B snoRNAs thus directly bind and inhibit K-Ras and are recurrently deleted in human cancer.

  View details for DOI 10.1038/ng.3452

  View details for PubMedID 26595770

• Unbiased proteomic-genomic investigation of MHC class I peptide ligands reveals unconventional translation origins Scharf, A. D., Flockhart, R. J., Olsson, N. E., Devabhaktuni, A., Zhang, J., Zhao, X., Khavari, P., Elias, J. E. PERGAMON-ELSEVIER SCIENCE LTD. 2015: 135–36

  View details for Web of Science ID 000366767700026

• Network Analysis Identifies Mitochondria! Regulation of Epidermal Differentiation by MPZL3 and FDXR DEVELOPMENTAL CELL Bhaduri, A., Ungewickell, A., Boxer, L. D., Lopez-Pajares, V., Zarnegar, B. J., Khavari, P. A. 2015; 35 (4): 444-457

  Abstract

  Current gene expression network approaches commonly focus on transcription factors (TFs), biasing network-based discovery efforts away from potentially important non-TF proteins. We developed proximity analysis, a network reconstruction method that uses topological constraints of scale-free, small-world biological networks to reconstruct relationships in eukaryotic systems, independent of subcellular localization. Proximity analysis identified MPZL3 as a highly connected hub that is strongly induced during epidermal differentiation. MPZL3 was essential for normal differentiation, acting downstream of p63, ZNF750, KLF4, and RCOR1, each of which bound near the MPZL3 gene and controlled its expression. MPZL3 protein localized to mitochondria, where it interacted with FDXR, which was itself also found to be essential for differentiation. Together, MPZL3 and FDXR increased reactive oxygen species (ROS) to drive epidermal differentiation. ROS-induced differentiation is dependent upon promotion of FDXR enzymatic activity by MPZL3. ROS induction by the MPZL3 and FDXR mitochondrial proteins is therefore essential for epidermal differentiation.

  View details for DOI 10.1016/j.devcel.2015.10.023

  View details for PubMedID 26609959

• CALML5 is a ZNF750-and TINCR-induced protein that binds stratifin to regulate epidermal differentiation GENES & DEVELOPMENT Sun, B. K., Boxer, L. D., Ransohoff, J. D., Siprashvili, Z., Qu, K., Lopez-Pajares, V., Hollmig, S. T., Khavari, P. A. 2015; 29 (21): 2225-2230

  Abstract

  Outward migration of epidermal progenitors occurs with induction of hundreds of differentiation genes, but the identities of all regulators required for this process are unknown. We used laser capture microdissection followed by RNA sequencing to identify calmodulin-like 5 (CALML5) as the most enriched gene in differentiating outer epidermis. CALML5 mRNA was up-regulated by the ZNF750 transcription factor and then stabilized by the long noncoding RNA TINCR. CALML5 knockout impaired differentiation, abolished keratohyalin granules, and disrupted epidermal barrier function. Mass spectrometry identified SFN (stratifin/14-3-3σ) as a CALML5-binding protein. CALML5 interacts with SFN in suprabasal epidermis, cocontrols 13% of late differentiation genes, and modulates interaction of SFN to some of its binding partners. A ZNF750-TINCR-CALML5-SFN network is thus essential for epidermal differentiation.

  View details for DOI 10.1101/gad.267708.115

  View details for PubMedID 26545810

• DeltaNp63 expression is reduced in aged and photoexposed human epidermis, and down regulated in reconstructed epidermis by aged dermal fibroblasts secretome Boulgana, R., Leblanc-Noblesse, E., Juan, M., Sobilo, L., Bernois, A., Lopez-Pajares, V., Zarnegar, B., Khavari, P., Schnebert, S., Dumas, M. NATURE PUBLISHING GROUP. 2015: S47

  View details for Web of Science ID 000359739400276

• DNMT1 expression is down-regulated in human keratinocytes during differentiation and senescence and by the secretome of aged dermal fibroblasts Boulgana, R., Soyer, M., Jeronimo-Monteiro, V., Leblanc-Noblesse, E., Marteau, C., Lopez-Pajares, V., Zarnegar, B., Khavari, P., Schnebert, S., Dumas, M. NATURE PUBLISHING GROUP. 2015: S47

  View details for Web of Science ID 000359739400274

• ZNF750 expression is reduced in photoexposed sites of the epidermis, by replicative senescence, retinoic acid, and increased by calcium treatment Boulgana, R., Soyer, M., Leblanc-Noblesse, E., Jeronimo-Monteiro, V., Bernois, A., Lopez-Pajares, V., Zarnegar, B., Khavari, P., Schnebert, S., Dumas, M. NATURE PUBLISHING GROUP. 2015: S47

  View details for Web of Science ID 000359739400275

• IQGAP1, an ERK1/2 MAPK scaffold, is an oncogenic target in canine melanoma Zehnder, A., Lee, B., Neela, P., Khavari, P. AMER ASSOC CANCER RESEARCH. 2015

  View details for DOI 10.1158/1538-7445.AM2015-5137

  View details for Web of Science ID 000371597105315

• Phase I clinical trial for recessive dystrophic epidermolysis bullosa using genetically corrected autologous keratinocytes Siprashvili, Z., Nguyen, N. T., Gorell, E., Loutit, K., Khuu, P., Furukawa, L. K., Lorenz, H. P., Leung, T. H., Keene, D. R., Khavari, P., Lane, A., Tang, J. Y., Marinkovich, M. NATURE PUBLISHING GROUP. 2015: S72

  View details for Web of Science ID 000352783200418

• Network analysis identifies MPZL3 as an essential regulator of epidermal differentiation that binds FDXR to induce reactive oxygen species Bhaduri, A., Ungewickell, A., Khavari, P. NATURE PUBLISHING GROUP. 2015: S59

  View details for Web of Science ID 000352783200341

• Calmodulin-like 5 interacts with 14-3-3-sigma/stratifin to regulate late epidermal differentiation Sun, B., Ransohoff, J., Qu, K., Lopez-Pajares, V., Boxer, L. D., Khavari, P. NATURE PUBLISHING GROUP. 2015: S67

  View details for Web of Science ID 000352783200391

• The BAF/SWI/SNF complex controls genome accessibility to p63 during epidermal differentiation Bao, X., Rubin, A., Qu, K., Zhang, J., Giresi, P., Chang, H., Khavari, P. NATURE PUBLISHING GROUP. 2015: S77

  View details for Web of Science ID 000352783200451

• Identification of the KNSTRN proteome by APEX2 targeting Lee, C., Mah, A., Nguyen, C., Khavari, P. NATURE PUBLISHING GROUP. 2015: S25

  View details for Web of Science ID 000352783200146

• A novel therapeutic inhibits Rac1 mediated invasion and metastasis in a newly described in vivo model of human melanoma Winge, M. C., Kovalski, J., Nguyen, N. T., Wu, D., Zehnder, A., Khavari, P., Marinkovich, M. NATURE PUBLISHING GROUP. 2015: S71

  View details for Web of Science ID 000352783200412

• A LncRNA-MAF:MAFB Transcription Factor Network Regulates Epidermal Differentiation DEVELOPMENTAL CELL Lopez-Pajares, V., Qu, K., Zhang, J., Webster, D. E., Barajas, B. C., Siprashvili, Z., Zarnegar, B. J., Boxer, L. D., Rios, E. J., Tao, S., Kretz, M., Khavari, P. A. 2015; 32 (6): 693-706

  Abstract

  Progenitor differentiation requires remodeling of genomic expression; however, in many tissues, such as epidermis, the spectrum of remodeled genes and the transcription factors (TFs) that control them are not fully defined. We performed kinetic transcriptome analysis during regeneration of differentiated epidermis and identified gene sets enriched in progenitors (594 genes), in early (159 genes), and in late differentiation (387 genes). Module mapping of 1,046 TFs identified MAF and MAFB as necessary and sufficient for progenitor differentiation. MAF:MAFB regulated 393 genes altered in this setting. Integrative analysis identified ANCR and TINCR lncRNAs as essential upstream MAF:MAFB regulators. ChIP-seq analysis demonstrated MAF:MAFB binding to known epidermal differentiation TF genes whose expression they controlled, including GRHL3, ZNF750, KLF4, and PRDM1. Each of these TFs rescued expression of specific MAF:MAFB target gene subsets in the setting of MAF:MAFB loss, indicating they act downstream of MAF:MAFB. A lncRNA-TF network is thus essential for epidermal differentiation.

  View details for DOI 10.1016/j.devcel.2015.01.028

  View details for PubMedID 25805135

• 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

• Inhibiting Oncogenic RAS in Multiple Myeloma By Targeting Scaffold-ERK Interactions Inhibiting Oncogenic RAS in Multiple Myeloma By Targeting Scaffold-ERK Interactions Gocke, C. B., Wang, Q., McMillan, R., Penchev, V., Sage, J., Khavari, P. A., Matsui, W. AMER SOC HEMATOLOGY. 2014

  View details for Web of Science ID 000349243501176

• Advances in skin grafting and treatment of cutaneous wounds SCIENCE Sun, B. K., Siprashvili, Z., Khavari, P. A. 2014; 346 (6212): 941-945

  Abstract

  The ability of the skin to repair itself after injury is vital to human survival and is disrupted in a spectrum of disorders. The process of cutaneous wound healing is complex, requiring a coordinated response by immune cells, hematopoietic cells, and resident cells of the skin. We review the classic paradigms of wound healing and evaluate how recent discoveries have enriched our understanding of this process. We evaluate current and experimental approaches to treating cutaneous wounds, with an emphasis on cell-based therapies and skin transplantation.

  View details for DOI 10.1126/science.1253836

  View details for Web of Science ID 000345696000031

• Advances in skin grafting and treatment of cutaneous wounds. Science Sun, B. K., Siprashvili, Z., Khavari, P. A. 2014; 346 (6212): 941-945

  Abstract

  The ability of the skin to repair itself after injury is vital to human survival and is disrupted in a spectrum of disorders. The process of cutaneous wound healing is complex, requiring a coordinated response by immune cells, hematopoietic cells, and resident cells of the skin. We review the classic paradigms of wound healing and evaluate how recent discoveries have enriched our understanding of this process. We evaluate current and experimental approaches to treating cutaneous wounds, with an emphasis on cell-based therapies and skin transplantation.

  View details for DOI 10.1126/science.1253836

  View details for PubMedID 25414301

• Recurrent point mutations in the kinetochore gene KNSTRN in cutaneous squamous cell carcinoma. Nature genetics Lee, C. S., Bhaduri, A., Mah, A., Johnson, W. L., Ungewickell, A., Aros, C. J., Nguyen, C. B., Rios, E. J., Siprashvili, Z., Straight, A., Kim, J., Aasi, S. Z., Khavari, P. A. 2014; 46 (10): 1060-1062

  Abstract

  Here we report the discovery of recurrent mutations concentrated at an ultraviolet signature hotspot in KNSTRN, which encodes a kinetochore protein, in 19% of cutaneous squamous cell carcinomas (SCCs). Cancer-associated KNSTRN mutations, most notably those encoding p.Ser24Phe, disrupt chromatid cohesion in normal cells, occur in SCC precursors, correlate with increased aneuploidy in primary tumors and enhance tumorigenesis in vivo. These findings suggest a role for KNSTRN mutagenesis in SCC development.

  View details for DOI 10.1038/ng.3091

  View details for PubMedID 25194279

• ZNF750 interacts with KLF4 and RCOR1, KDM1A, and CTBP1/2 chromatin regulators to repress epidermal progenitor genes and induce differentiation genes GENES & DEVELOPMENT Boxer, L. D., Barajas, B., Tao, S., Zhang, J., Khavari, P. A. 2014; 28 (18): 2013-2026

  Abstract

  ZNF750 controls epithelial homeostasis by inhibiting progenitor genes while inducing differentiation genes, a role underscored by pathogenic ZNF750 mutations in cancer and psoriasis. How ZNF750 accomplishes these dual gene regulatory impacts is unknown. Here, we characterized ZNF750 as a transcription factor that binds both the progenitor and differentiation genes that it controls at a CCNNAGGC DNA motif. ZNF750 interacts with the pluripotency transcription factor KLF4 and chromatin regulators RCOR1, KDM1A, and CTBP1/2 through conserved PLNLS sequences. ChIP-seq (chromatin immunoprecipitation [ChIP] followed by high-throughput sequencing) and gene depletion revealed that KLF4 colocalizes ∼10 base pairs from ZNF750 at differentiation target genes to facilitate their activation but is unnecessary for ZNF750-mediated progenitor gene repression. In contrast, KDM1A colocalizes with ZNF750 at progenitor genes and facilitates their repression but is unnecessary for ZNF750-driven differentiation. ZNF750 thus controls differentiation in concert with RCOR1 and CTBP1/2 by acting with either KDM1A to repress progenitor genes or KLF4 to induce differentiation genes.

  View details for DOI 10.1101/gad.246579.114

  View details for Web of Science ID 000342277800005

  View details for PubMedCentralID PMC4173152

• ZNF750 interacts with KLF4 and RCOR1, KDM1A, and CTBP1/2 chromatin regulators to repress epidermal progenitor genes and induce differentiation genes. Genes & development Boxer, L. D., Barajas, B., Tao, S., Zhang, J., Khavari, P. A. 2014; 28 (18): 2013-2026

  Abstract

  ZNF750 controls epithelial homeostasis by inhibiting progenitor genes while inducing differentiation genes, a role underscored by pathogenic ZNF750 mutations in cancer and psoriasis. How ZNF750 accomplishes these dual gene regulatory impacts is unknown. Here, we characterized ZNF750 as a transcription factor that binds both the progenitor and differentiation genes that it controls at a CCNNAGGC DNA motif. ZNF750 interacts with the pluripotency transcription factor KLF4 and chromatin regulators RCOR1, KDM1A, and CTBP1/2 through conserved PLNLS sequences. ChIP-seq (chromatin immunoprecipitation [ChIP] followed by high-throughput sequencing) and gene depletion revealed that KLF4 colocalizes ∼10 base pairs from ZNF750 at differentiation target genes to facilitate their activation but is unnecessary for ZNF750-mediated progenitor gene repression. In contrast, KDM1A colocalizes with ZNF750 at progenitor genes and facilitates their repression but is unnecessary for ZNF750-driven differentiation. ZNF750 thus controls differentiation in concert with RCOR1 and CTBP1/2 by acting with either KDM1A to repress progenitor genes or KLF4 to induce differentiation genes.

  View details for DOI 10.1101/gad.246579.114

  View details for PubMedID 25228645

  View details for PubMedCentralID PMC4173152

• Activating HRAS Mutation in Nevus Spilus. journal of investigative dermatology Sarin, K. Y., McNiff, J. M., Kwok, S., Kim, J., Khavari, P. A. 2014; 134 (6): 1766-1768

  View details for DOI 10.1038/jid.2014.6

  View details for PubMedID 24390138

• Enhancer-targeted genome editing selectively blocks innate resistance to oncokinase inhibition GENOME RESEARCH Webster, D. E., Barajas, B., Bussat, R. T., Yan, K. J., Neela, P. H., Flockhart, R. J., Kovalski, J., Zehnder, A., Khavari, P. A. 2014; 24 (5): 751-760

  Abstract

  Thousands of putative enhancers are characterized in the human genome, yet few have been shown to have a functional role in cancer progression. Inhibiting oncokinases, such as EGFR, ALK, ERBB2, and BRAF, is a mainstay of current cancer therapy but is hindered by innate drug resistance mediated by up-regulation of the HGF receptor, MET. The mechanisms mediating such genomic responses to targeted therapy are unknown. Here, we identify lineage-specific enhancers at the MET locus for multiple common tumor types, including a melanoma lineage-specific enhancer 63 kb downstream from the MET TSS. This enhancer displays inducible chromatin looping with the MET promoter to up-regulate MET expression upon BRAF inhibition. Epigenomic analysis demonstrated that the melanocyte-specific transcription factor, MITF, mediates this enhancer function. Targeted genomic deletion (<7 bp) of the MITF motif within the MET enhancer suppressed inducible chromatin looping and innate drug resistance, while maintaining MITF-dependent, inhibitor-induced melanoma cell differentiation. Epigenomic analysis can thus guide functional disruption of regulatory DNA to decouple pro- and anti-oncogenic functions of a dominant transcription factor and block innate resistance to oncokinase therapy.

  View details for DOI 10.1101/gr.166231.113

  View details for PubMedID 24443471

• A IncRNA-MAF:MAFB transcription factor network regulates epidermal differentiation Lopez-Pajares, V., Qu, K., Zhang, J., Webster, D., Barajas, B., Zarnegar, B., Boxer, L., Kretz, M., Khavari, P. NATURE PUBLISHING GROUP. 2014: S44

  View details for Web of Science ID 000334560400255

• The protein arginine methyltransferase PRMT1 acts with the CSNK1a1 kinase to enforce the epidermal progenitor state in vivo Bao, X., Zarnegar, B., Mah, A., Qu, K., Tao, S., Khavari, P. NATURE PUBLISHING GROUP. 2014: S66

  View details for Web of Science ID 000334560400381

• ZNF750 binding to KLF4 and specific epigenetic regulators controls epidermal differentiation Boxer, L. D., Barajas, B., Zhang, J., Zarnegar, B., Khavari, P. NATURE PUBLISHING GROUP. 2014: S68

  View details for Web of Science ID 000334560400391

• Calmodulin-like 5 is a downstream effector of the TINCR long noncoding RNA and is required for late epidermal differentiation Sun, B., Zhang, J., Qu, K., Lopez-Pajares, V., Khavari, P. A. NATURE PUBLISHING GROUP. 2014: S71

  View details for Web of Science ID 000334560400408

• Recurrent point mutations in KNSTRN and disruption of multiple notch signaling genes in cutaneous squamous cell carcinoma Lee, C., Bhaduri, A., Mah, A., Ungewickell, A., Aros, C., Nguyen, C., Siprashvili, Z., Johnson, W., Straight, A., Kim, J., Aasi, S., Khavari, P. NATURE PUBLISHING GROUP. 2014: S35

  View details for Web of Science ID 000334560400201

• Phase 1 clinical trial of genetically corrected autologous epidermal keratinocytes for recessive dystrophic epidermolysis bullosa Siprashvili, Z., Nguyen, N. T., Gorell, E., Khuu, P., Furukawa, L., Lorenz, H. P., Leung, T. H., Keene, D. R., Khavari, P., Marinkovich, M., Lane, A. T. NATURE PUBLISHING GROUP. 2014: S75

  View details for Web of Science ID 000334560400429

• Quantitative analysis of mammalian translation initiation sites by FACS-seq. Molecular systems biology Noderer, W. L., Flockhart, R. J., Bhaduri, A., Diaz de Arce, A. J., Zhang, J., Khavari, P. A., Wang, C. L. 2014; 10: 748-?

  Abstract

  An approach combining fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq) was employed to determine the efficiency of start codon recognition for all possible translation initiation sites (TIS) utilizing AUG start codons. Using FACS-seq, we measured translation from a genetic reporter library representing all 65,536 possible TIS sequences spanning the -6 to +5 positions. We found that the motif RYMRMVAUGGC enhanced start codon recognition and translation efficiency. However, dinucleotide interactions, which cannot be conveyed by a single motif, were also important for modeling TIS efficiency. Our dataset combined with modeling allowed us to predict genome-wide translation initiation efficiency for all mRNA transcripts. Additionally, we screened somatic TIS mutations associated with tumorigenesis to identify candidate driver mutations consistent with known tumor expression patterns. Finally, we implemented a quantitative leaky scanning model to predict alternative initiation sites that produce truncated protein isoforms and compared predictions with ribosome footprint profiling data. The comprehensive analysis of the TIS sequence space enables quantitative predictions of translation initiation based on genome sequence.

  View details for DOI 10.15252/msb.20145136

  View details for PubMedID 25170020

• Activating HRAS Mutation in Agminated Spitz Nevi Arising in a Nevus Spilus. JAMA dermatology Sarin, K. Y., Sun, B. K., Bangs, C. D., Cherry, A., Swetter, S. M., Kim, J., Khavari, P. A. 2013; 149 (9): 1077-1081

  Abstract

  IMPORTANCE Spitz nevi are benign melanocytic proliferations that can sometimes be clinically and histopathologically difficult to distinguish from melanoma. Agminated Spitz nevi have been reported to arise spontaneously, in association with an underlying nevus spilus, or after radiation or chemotherapy. However, to our knowledge, the genetic mechanism for this eruption has not been described. OBSERVATIONS We report a case of agminated Spitz nevi arising in a nevus spilus and use exome sequencing to identify a clonal activating point mutation in HRAS (GenBank 3265) (c.37G→C) in the Spitz nevi and underlying nevus spilus. We also identify a secondary copy number increase involving HRAS on chromosome 11p, which occurs during the development of the Spitz nevi. CONCLUSIONS AND RELEVANCE Our results reveal an activating HRAS mutation in a nevus spilus that predisposes to the formation of Spitz nevi. In addition, we demonstrate a copy number increase in HRAS as a "second hit" during the formation of agminated Spitz nevi, which suggests that both multiple Spitz nevi and solitary Spitz nevi may arise through similar molecular pathways. In addition, we describe a unique investigative approach for the discovery of genetic alterations in Spitz nevi.

  View details for DOI 10.1001/jamadermatol.2013.4745

  View details for PubMedID 23884457

• 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

• Genomic Profiling of a Human Organotypic Model of AEC Syndrome Reveals ZNF750 as an Essential Downstream Target of Mutant TP63 AMERICAN JOURNAL OF HUMAN GENETICS Zarnegar, B. J., Webster, D. E., Lopez-Pajares, V., Hunt, B. V., Qu, K., Yan, K. J., Berk, D. R., Sen, G. L., Khavari, P. A. 2012; 91 (3): 435-443

  Abstract

  The basis for impaired differentiation in TP63 mutant ankyloblepharon-ectodermal dysplasia-clefting (AEC) syndrome is unknown. Human epidermis harboring AEC TP63 mutants recapitulated this impairment, along with downregulation of differentiation activators, including HOPX, GRHL3, KLF4, PRDM1, and ZNF750. Gene-set enrichment analysis indicated that disrupted expression of epidermal differentiation programs under the control of ZNF750 and KLF4 accounted for the majority of disrupted epidermal differentiation resulting from AEC mutant TP63. Chromatin immunoprecipitation (ChIP) analysis and ChIP-sequencing of TP63 binding in differentiated keratinocytes revealed ZNF750 as a direct target of wild-type and AEC mutant TP63. Restoring ZNF750 to AEC model tissue rescued activator expression and differentiation, indicating that AEC TP63-mediated ZNF750 inhibition contributes to differentiation defects in AEC. Incorporating disease-causing mutants into regenerated human tissue can thus dissect pathomechanisms and identify targets that reverse disease features.

  View details for DOI 10.1016/j.ajhg.2012.07.007

  View details for Web of Science ID 000308683100004

  View details for PubMedCentralID PMC3511987

• Transcriptome sequencing in squamous cell carcinoma identifies novel skin malignancy regulated transcripts Lee, C., Ungewickell, A., Bhaduri, A., Qu, K., Webster, D., Aros, C., Mah, A., Kretz, M., Khavari, P. A. NATURE PUBLISHING GROUP. 2012: S29

  View details for Web of Science ID 000302866900171

• BAF53A enforces the epidermal progenitor state by re-targeting the SWI/SNF/BAF chromatin remodeling complex away from differentiation gene promoters 75th Annual Meeting of the Society-for-Investigative-Dermatology Bao, X., Tang, J., Tao, S., Qu, K., Crabtree, G. R., Khavari, P. A. NATURE PUBLISHING GROUP. 2012: S63–S63

  View details for Web of Science ID 000302866900372

• Scaffold-Kinase interaction blockade selectively inhibits Ras-MAPK tumorigenesis 75th Annual Meeting of the Society-for-Investigative-Dermatology Jameson, K. L., Zehnder, A., Khavari, P. A. NATURE PUBLISHING GROUP. 2012: S30–S30

  View details for Web of Science ID 000302866900175

• ZNF750 is a p63 target that induces KLF4 to drive epidermal differentiation 75th Annual Meeting of the Society-for-Investigative-Dermatology Sen, G. L., Boxer, L., Webster, D., Bussat, R., Qu, K., Zarnegar, B., Johnston, D., Siprashvili, Z., Khavari, P. A. NATURE PUBLISHING GROUP. 2012: S74–S74

  View details for Web of Science ID 000302866900437

• Transcriptome sequencing in Sezary Syndrome identifies novel Sezary cell-associated transcripts 75th Annual Meeting of the Society-for-Investigative-Dermatology Lee, C., Ungewickell, A., Bhaduri, A., Qu, K., Webster, D., Armstrong, R., Weng, W., Aros, C., MAH, A., Kretz, M., Kim, Y. H., Khavari, P. A. NATURE PUBLISHING GROUP. 2012: S29–S29

  View details for Web of Science ID 000302866900174

• ZNF750 Is a p63 Target Gene that Induces KLF4 to Drive Terminal Epidermal Differentiation DEVELOPMENTAL CELL Sen, G. L., Boxer, L. D., Webster, D. E., Bussat, R. T., Qu, K., Zarnegar, B. J., Johnston, D., Siprashvili, Z., Khavari, P. A. 2012; 22 (3): 669-677

  Abstract

  Disrupted epidermal differentiation characterizes numerous diseases that impact >25% of the population. In a search for dominant mediators of differentiation, we defined a requirement for ZNF750 in terminal epidermal differentiation. ZNF750 controlled genes mutated in numerous human skin diseases, including FLG, LOR, LCE3B, ALOXE3, and SPINK5. ZNF750 induced progenitor differentiation via an evolutionarily conserved C2H2 zinc finger motif. The epidermal master regulator, p63, bound the ZNF750 promoter and was necessary for its induction. ZNF750 restored differentiation to p63-deficient tissue, suggesting that it acts downstream of p63. A search for functionally important ZNF750 targets via analysis of ZNF750-regulated genes identified KLF4, a transcription factor that activates late epidermal differentiation. ZNF750 binds to KLF4 at multiple sites flanking the transcriptional start site and controls its expression. ZNF750 thus directly links a tissue-specifying factor, p63, to an effector of terminal differentiation, KLF4, and represents a potential future target for disorders of this process.

  View details for DOI 10.1016/j.devcel.2011.12.001

  View details for Web of Science ID 000301701600020

  View details for PubMedID 22364861

  View details for PubMedCentralID PMC3306457

• BAF53A enforces the epidermal progenitor state by re-targeting the SWI/SNF/BAF chromatin remodeling complex away from differentiation gene promoters Bao, X., Tang, J., Lopez-Pajares, V., Tao, S., Qu, K., Crabtree, G. R., Khavari, P. A. AMER SOC CELL BIOLOGY. 2012

  View details for Web of Science ID 000209348604363

• BAF53A enforces the epidermal progenitor state by re-targeting the SWI/SNF/BAF chromatin remodeling complex away from differentiation gene promoters Bao, X., Tang, J., Lopez-Pajares, V., Tao, S., Qu, K., Crabtree, G. R., Khavari, P. A. AMER SOC CELL BIOLOGY. 2012

  View details for Web of Science ID 000209348601053

• 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

• Epidermal tissue differentiation and lipid barrier function requires the long non-coding RNA Linc19A Kretz, M., Webster, D., Siprashvili, Z., Zehnder, A., Lee, C., Flockhart, R., Chow, J., Kim, G., Rinn, J., Chang, H., Khavari, P. NATURE PUBLISHING GROUP. 2011: S66

  View details for Web of Science ID 000289035600396

• Maintenance of the epidermal progenitor state requires the long non-coding RNA Linc8D 71st Annual Meeting of the Society-for-Investigative-Dermatology Lee, C., Kretz, M., Webster, D., Flockhart, R., Qu, K., MAH, A., Aros, C., Chang, H., Rinn, J., Khavari, P. NATURE PUBLISHING GROUP. 2011: S136–S136

  View details for Web of Science ID 000289035600810

• DNMT1 maintains epidermal progenitor function Annual Meeting of the Society-for-Investigative-Dermatology Sen, G. L., Reuter, J. A., Webster, D. E., Zhu, L., Khavari, P. NATURE PUBLISHING GROUP. 2010: S87–S87

  View details for Web of Science ID 000276455100518

• New cancer models assessed via quantitative transcriptome analysis Ridky, T. W., Chow, J. M., Wong, D. J., Khavari, P. A. NATURE PUBLISHING GROUP. 2010: S31

  View details for Web of Science ID 000276455100186

• DNMT1 maintains progenitor function in self-renewing somatic tissue NATURE Sen, G. L., Reuter, J. A., Webster, D. E., Zhu, L., Khavari, P. A. 2010; 463 (7280): 563-U189

  Abstract

  Progenitor cells maintain self-renewing tissues throughout life by sustaining their capacity for proliferation while suppressing cell cycle exit and terminal differentiation. DNA methylation provides a potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through repeated cell divisions. DNA methyltransferase 1 (DNMT1) maintains DNA methylation patterns after cellular replication. Although dispensable for embryonic stem cell maintenance, the role for DNMT1 in maintaining the progenitor state in constantly replenished somatic tissues, such as mammalian epidermis, is unclear. Here we show that DNMT1 is essential for epidermal progenitor cell function. DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation. In tissue, DNMT1 depletion led to exit from the progenitor cell compartment, premature differentiation and eventual tissue loss. Genome-wide analysis showed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation. Furthermore, UHRF1 (refs 9, 10), a component of the DNA methylation machinery that targets DNMT1 to hemi-methylated DNA, is also necessary to suppress premature differentiation and sustain proliferation. In contrast, Gadd45A and B, which promote active DNA demethylation, are required for full epidermal differentiation gene induction. These data demonstrate that proteins involved in the dynamic regulation of DNA methylation patterns are required for progenitor maintenance and self-renewal in mammalian somatic tissue.

  View details for DOI 10.1038/nature08683

  View details for PubMedID 20081831

• Invasive three-dimensional organotypic neoplasia from multiple normal human epithelia Nature Medicine Ridky TW, Khavari PA 2010; 16: 1450-1455
• 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

• Identification of the core gene expression signature and key cell-signaling pathways associated with Ras driven squamous cell carcinoma invasion Ridky, T. W., Wong, D., Khavari, P. A. NATURE PUBLISHING GROUP. 2009: S29

  View details for Web of Science ID 000264994000172

• Ras driven genetically defined invasive human carcinomas generated from primary human epidermal, head and neck, cervical, esophageal, prostate, and breast epithelial cells established within in a three-dimensional organotypic human tissue platform Ridky, T. W., Chow, J., Khavari, P. A. NATURE PUBLISHING GROUP. 2009: S29

  View details for Web of Science ID 000264994000173

• Mammalian epithelial development, homeostasis and Raf-induced hyperplasia rely on Mek1/2 MAPK kinases Scholl, F. A., Dumesic, P. A., Barragan, D. I., Harada, K., Bissonauth, V., Charron, J., Khavari, P. A. NATURE PUBLISHING GROUP. 2007: S76

  View details for Web of Science ID 000245387800448

• Laminin-5 Beta 3 chain promotes epidermal carcinogenesis through type VII collagen binding and pi3k activation Sakai, N., Waterman, E. A., Nguyen, N. E., Horst, B. A., Veitch, D. P., Dey, C. N., Ortiz-Urda, S., Khavari, P. A., Marinkovich, M. NATURE PUBLISHING GROUP. 2006: 24

  View details for Web of Science ID 000242891500143

• Effects of altered calcium ATPase function on epidermal carcinogenesis Kimmel, R. A., Ridky, T. W., Khavari, P. NATURE PUBLISHING GROUP. 2006: 19

  View details for Web of Science ID 000242891500111

• Sustained survival and production of recombinant protein by genetically engineered human fibroblasts after intradermal injection Siprashvili, Z., Ridky, T. W., Khavari, P. NATURE PUBLISHING GROUP. 2006: 85

  View details for Web of Science ID 000242891500507

• TNFR1 signaling to JNK opposes NF-kB to promotes epidermal neoplasia 67th Annual Meeting of the Society-for-Investigative-Dermatology Zhang, J. Y., Tao, S., Ridky, T. W., Khavari, P. NATURE PUBLISHING GROUP. 2006: 92–92

  View details for Web of Science ID 000242891500546

• Invasive human melanocytic neoplasia in a 3-dimensional model using defined genetic elements 35th Annual Meeting of the European-Society-for-Dermatological-Research Adams, A., Chudnovsky, Y., Khavari, P. NATURE PUBLISHING GROUP. 2005: A58–A58

  View details for Web of Science ID 000231862600338

• Use of human tissue to assess the oncogenic activity of melanoma-associated mutations NATURE GENETICS Chudnovsky, Y., Adams, A. E., Robbins, P. B., Lin, Q., Khavari, P. A. 2005; 37 (7): 745-749

  Abstract

  Multiple genetic alterations occur in melanoma, a lethal skin malignancy of increasing incidence. These include mutations that activate Ras and two of its effector cascades, Raf and phosphoinositide 3-kinase (PI3K). Induction of Ras and Raf can be caused by active N-Ras and B-Raf mutants as well as by gene amplification. Activation of PI3K pathway components occurs by PTEN loss and by AKT3 amplification. Melanomas also commonly show impairment of the p16(INK4A)-CDK4-Rb and ARF-HDM2-p53 tumor suppressor pathways. CDKN2A mutations can produce p16(INK4A) and ARF protein loss. Rb bypass can also occur through activating CDK4 mutations as well as by CDK4 amplification. In addition to ARF deletion, p53 pathway disruption can result from dominant negative TP53 mutations. TERT amplification also occurs in melanoma. The extent to which these mutations can induce human melanocytic neoplasia is unknown. Here we characterize pathways sufficient to generate human melanocytic neoplasia and show that genetically altered human tissue facilitates functional analysis of mutations observed in human tumors.

  View details for DOI 10.1038/ng1586

  View details for Web of Science ID 000230196400022

  View details for PubMedID 15951821

  View details for PubMedCentralID PMC3063773

• Type VII collagen is required for cellular invasiveness in epidermal carcinogenesis 66th Annual Meeting of the Society-for-Investigative-Dermatology Ortiz-Urda, S., Garcia, J., Marinkovich, M., Khavari, P. NATURE PUBLISHING GROUP. 2005: A25–A25

  View details for Web of Science ID 000228179900149

• The structural integrity of individual keratin filament networks is dispensable for epidermal tumorigenesis Ridky, T. W., Yang, E., Leachman, S., Khavari, P. BLACKWELL PUBLISHING INC. 2005: A72

  View details for Web of Science ID 000228179900430

• Direct cutaneous plasmid DNA delivery via single intradermal injection of the cis-acting Sleeping Beauty transposon system Siprashvili, Z., Mikkelsen, J. G., Yant, S., Kay, M., Khavari, P. BLACKWELL PUBLISHING INC. 2005: A87

  View details for Web of Science ID 000228179901061

• Disrupting TNFR1-JNK-AP1 action inhibits Ras-driven human epidermal neoplasia 66th Annual Meeting of the Society-for-Investigative-Dermatology Zhang, J. Y., Tao, S., Khavari, P. NATURE PUBLISHING GROUP. 2005: A97–A97

  View details for Web of Science ID 000228179901124

• Functional characterization of class IIPI3-kinase in epidermal differentiation via targeted gene disruption and RNA interference 66th Annual Meeting of the Society-for-Investigative-Dermatology Harada, K., Cai, T., Truong, A., Khavari, P. NATURE PUBLISHING GROUP. 2005: A98–A98

  View details for Web of Science ID 000228179901127

• Two distinct roles for the laminin-5 beta 3 chain in epidermal carcinogenesis and adhesion 66th Annual Meeting of the Society-for-Investigative-Dermatology Waterman, E. A., Ortiz-Urda, S., Nguyen, N. T., Veitch, D. P., Horst, B. A., Dey, C. N., Khavari, P. A., Marinkovich, M. P. NATURE PUBLISHING GROUP. 2005: A22–A22

  View details for Web of Science ID 000228179900128

• Type VII collagen is required for Ras-driven human epidermal tumorigenesis SCIENCE Ortiz-Urda, S., Garcia, J., Green, C. L., Chen, L., Lin, Q., Veitch, D. P., Sakai, L. Y., LEE, H., Marinkovich, M. P., Khavari, P. A. 2005; 307 (5716): 1773-1776

  Abstract

  Type VII collagen defects cause recessive dystrophic epidermolysis bullosa (RDEB), a blistering skin disorder often accompanied by epidermal cancers. To study the role of collagen VII in these cancers, we examined Ras-driven tumorigenesis in RDEB keratinocytes. Cells devoid of collagen VII did not form tumors in mice, whereas those retaining a specific collagen VII fragment (the amino-terminal noncollagenous domain NC1) were tumorigenic. Forced NC1 expression restored tumorigenicity to collagen VII-null epidermis in a non-cell-autonomous fashion. Fibronectin-like sequences within NC1 (FNC1) promoted tumor cell invasion in a laminin 5-dependent manner and were required for tumorigenesis. Tumor-stroma interactions mediated by collagen VII thus promote neoplasia, and retention of NC1 sequences in a subset of RDEB patients may contribute to their increased susceptibility to squamous cell carcinoma.

  View details for DOI 10.1126/science.1106209

  View details for Web of Science ID 000227883900044

  View details for PubMedID 15774758

• The Society's casework in 2004: Review of selected cases (Ancient Monuments Society) TRANSACTIONS OF THE ANCIENT MONUMENTS SOCIETY Lazarov, M., Green, C. L., Zhang, J. Y., Kubo, Y., Dajee, M., Khavari, P. A. 2005; 49: 87–128

  View details for Web of Science ID 000202944300005

• Invasive human melanocytic neoplasia generated using defined genetic elements 65th Annual Meeting of the Society-for-Investigative-Dermatology Chudnovsky, Y., Adams, A. E., Lin, Q., Robbins, P. B., Khavari, P. NATURE PUBLISHING GROUP. 2004: A157–A157

  View details for Web of Science ID 000220660500990

• Class II PI3-kinase mediates cell cycle inhibition and terminal differentiation in epidermis Cai, T., Harada, K., Yang, E., Khavari, P. BLACKWELL PUBLISHING INC. 2004: A77

  View details for Web of Science ID 000220660500508

• Mek1, but not Mek2, alters epidermal growth and differentiation Scholl, F. A., Dumesic, P. A., Charron, J., Khavari, P. BLACKWELL PUBLISHING INC. 2004: A77

  View details for Web of Science ID 000220660500509

• The ATP2C1 golgi ATPase is required for Ras-driven human epidermal neoplasia Kimmel, R., Khavari, P. BLACKWELL PUBLISHING INC. 2004: A89

  View details for Web of Science ID 000220660500579

• NF-kappa B blockade and oncogenic Ras trigger invasive human epidermal neoplasia NATURE Dajee, M., Lazarov, M., Zhang, J. Y., Cai, T., Green, C. L., Russell, A. J., Marinkovich, M. P., Tao, S. Y., Lin, Q., Kubo, Y., Khavari, P. A. 2003; 421 (6923): 639-643

  Abstract

  The nuclear factor NF-kappaB and oncogenic Ras can alter proliferation in epidermis, the most common site of human cancer. These proteins are implicated in epidermal squamous cell carcinoma in mice, however, the potential effects of altering their function are uncertain. Whereas inhibition of NF-kappaB enhances apoptosis in certain tumours, blockade of NF-kappaB predisposes murine skin to squamous cell carcinoma. Because therapeutics inhibiting Ras and NF-kappaB pathways are being developed to treat human cancer, it is essential to assess the effects of altering these regulators. The medical relevance of murine studies is limited, however, by differences between mouse and human skin, and by the greater ease of transforming murine cells. Here we show that in normal human epidermal cells both NF-kappaB and oncogenic Ras trigger cell-cycle arrest. Growth arrest triggered by oncogenic Ras can be bypassed by IkappaBalpha-mediated blockade of NF-kappaB, generating malignant human epidermal tissue resembling squamous cell carcinoma. Human cell tumorigenesis is dependent on laminin 5 and alpha6beta4 integrin. Thus, IkappaBalpha circumvents restraints on growth promotion induced by oncogenic Ras and can act with Ras to induce invasive human tissue neoplasia.

  View details for DOI 10.1038/nature01283

  View details for PubMedID 12571598

• CDK4 coexpression with Ras generates malignant human epidermal tumorigenesis NATURE MEDICINE Lazarov, M., Kubo, Y., Cai, T., Dajee, M., Tarutani, M., Lin, Q., Fang, M., Tao, S. Y., Green, C. L., Khavari, P. A. 2002; 8 (10): 1105-1114

  Abstract

  Ras acts with other proteins to induce neoplasia. By itself, however, strong Ras signaling can suppress proliferation of normal cells. In primary epidermal cells, we found that oncogenic Ras transiently decreases cyclin-dependent kinase (CDK) 4 expression in association with cell cycle arrest in G1 phase. CDK4 co-expression circumvents Ras growth suppression and induces invasive human neoplasia resembling squamous cell carcinoma. Tumorigenesis is dependent on CDK4 kinase function, with cyclin D1 required but not sufficient for this process. In facilitating escape from G1 growth restraints, Ras and CDK4 alter the composition of cyclin D and cyclin E complexes and promote resistance to growth inhibition by INK4 cyclin-dependent kinase inhibitors. These data identify a new role for oncogenic Ras in CDK4 regulation and highlight the functional importance of CDK4 suppression in preventing uncontrolled growth.

  View details for DOI 10.1038/nm779

  View details for PubMedID 12357246

• Stable nonviral genetic correction of inherited human skin disease NATURE MEDICINE Ortiz-Urda, S., Thyagarajan, B., Keene, D. R., Lin, Q., Fang, M., Calos, M. P., Khavari, P. A. 2002; 8 (10): 1166-1170

  Abstract

  Current gene-transfer technologies display limitations in achieving effective gene delivery. Among these limitations are difficulties in stably integrating large corrective sequences into the genomes of long-lived progenitor-cell populations. Current larger-capacity viral vectors suffer from biosafety concerns, whereas plasmid-based approaches have poor efficiency of stable gene transfer. These barriers hinder genetic correction of many severe inherited human diseases, such as the blistering skin disorder recessive dystrophic epidermolysis bullosa (RDEB), caused by mutations in the large COL7A1 gene. To circumvent these barriers, we used the phi C31 bacteriophage integrase, which stably integrates large DNA sequences containing a specific 285-base-pair attB sequence into genomic 'pseudo-attP sites'. phi C31 integrase-based gene transfer stably integrated the COL7A1 cDNA into genomes of primary epidermal progenitor cells from four unrelated RDEB patients. Skin regenerated using these cells displayed stable correction of hallmark RDEB disease features, including Type VII collagen protein expression, anchoring fibril formation and dermal-epidermal cohesion. These findings establish a practical approach to nonviral genetic correction of severe human genetic disorders requiring stable genomic integration of large DNA sequences.

  View details for DOI 10.1038/nm766

  View details for PubMedID 12244305

• Restoration of full-length type VII collagen expression in RDEB via nonviral gene integration using the phiC31 integrase Ortiz-Urda, S., Thyagarajan, B., Calos, M., Khavari, P. BLACKWELL PUBLISHING INC. 2002: 343

  View details for Web of Science ID 000177428100812

• NF-kappa B blockade and oncogenic Ras induce invasive human epidermal neoplasia by circumventing cell cycle arrest Dajee, M., Lazarov, M., Khavari, P. BLACKWELL PUBLISHING INC. 2002: 220

  View details for Web of Science ID 000177428100074

• Bypassing CDK4 suppression by oncogenic Ras leads to invasive human epidermal carcinoma capable of visceral dissemination Lazarov, M., Kubo, Y., Dajee, M., Cai, T., Khavari, P. BLACKWELL PUBLISHING INC. 2002: 219

  View details for Web of Science ID 000177428100073

• Transposase-based stable nonviral laminin 5 gene delivery in junctional epidermolysis bullosa Ortiz-Urda, S., Yant, S., Scholl, F., Siprashvili, Z., Chen, P., Kay, M., Khavari, P. BLACKWELL PUBLISHING INC. 2002: 230

  View details for Web of Science ID 000177428100134

• Laminin 5 integrin beta 4 are required for Ras-driven human epidermal tumorigenesis Dajee, A. M., Lazarov, M., Russell, A., Marinkovich, P., Reuter, J., Khavari, P. BLACKWELL PUBLISHING INC. 2002: 229

  View details for Web of Science ID 000177428100133

• Regulated Ras function in transgenic skin demonstrates potent and reversible Ras effects on epidermal growth and differentiation Tarutani, M., Dajee, M., Cai, T., Khavari, P. BLACKWELL PUBLISHING INC. 2002: 254

  View details for Web of Science ID 000177428100278

• Generation of transgenic skin with regulated NF-kappa B function and epidermis deficient in the Re1A/p65 indicate that NF-kappa B subunits regulate epidermal growth but not differentiation Zhang, Y., Kimmel, R., Tao, S., Khavari, P. NATURE PUBLISHING GROUP. 2002: 267–67

  View details for Web of Science ID 000177428100359

• Potential redundancy of MEK kinase impacts on epidermal growth and differentiation Scholl, F., Dajee, M., Charron, J., Khavari, P. BLACKWELL PUBLISHING INC. 2002: 279

  View details for Web of Science ID 000177428100428

• Upstream regulators of Ras function in epidermal cells Cai, T., Khavari, P. BLACKWELL PUBLISHING INC. 2002: 278

  View details for Web of Science ID 000177428100427

• Impact of Ras effectors RalGDS, Akt and Raf on epidermal growth and differentiation Tarutani, M., Dajee, M., Lin, Q., Khavari, P. BLACKWELL PUBLISHING INC. 2002: 279

  View details for Web of Science ID 000177428100429

• Regulation of CDK4 expression levels by TGF beta, TNF alpha, IL-1, NF-kappa B and Ras determines cellular proliferative responses to these regulators of epidermal growth Lazarov, M., Dajee, M., Kubo, Y., Khavari, P. BLACKWELL PUBLISHING INC. 2002: 279

  View details for Web of Science ID 000177428100433

• Oncogenic Ras and telomerase induce invasive human melanocytic neoplasia Robbins, P., Sheu, S., Chudnovsky, J., Cai, T., Khavari, P. BLACKWELL PUBLISHING INC. 2002: 339

  View details for Web of Science ID 000177428100783

• Regulated lentivectors for controlled and reversible cutaneous gene delivery Siprashvili, Z., Khavari, P. BLACKWELL PUBLISHING INC. 2002: 343

  View details for Web of Science ID 000177428100811

• Stable nonviral genetic correction of inherited human skin disease Nature Medicine Ortiz-Urda S, Thyagarajan B, Keene DR, Lin Q, Fang M, Calos MP, Khavari PA 2002; 8: 1166-1170
• Profiling genomic expression in disorders of epidermal proliferation identifies Egr1 as a potent regulator of epidermal growth. Fang, M., Wee, S. A., Fan, H., Morrisey, G., Tao, S., Lin, Q., Khavari, P. A. CELL PRESS. 2001: 220–20

  View details for Web of Science ID 000171648900226

• Sustainable systemic erythropoietin delivery via a single injection of lentivirus into human skin Baek, S., Sheu, S., Lin, Q., Robbins, P., Khavari, P. BLACKWELL SCIENCE INC. 2001: 549

  View details for Web of Science ID 000170668300986

• I kappa B kinase (IKK) complex function inhibits epidermal growth via NF-kappa B and fails to alter epidermal differentiation Goodnough, J., Robbins, P., Sheu, S., Khavari, P. BLACKWELL SCIENCE INC. 2001: 403

  View details for Web of Science ID 000170668300116

• Spatially localized Ras action inhibits epidermal differentiation and promotes growth capacity via the Raf effector pathway Dajee, M., Deng, H., Tao, S., Khavari, P. NATURE PUBLISHING GROUP. 2001: 403–

  View details for Web of Science ID 000170668300114

• Non-invasive topical immunization by a peptide vaccine against hepatitis B and rubella virus Tao, S., Dajee, M., Wee, S., Khavari, P. NATURE PUBLISHING GROUP. 2001: 435–35

  View details for Web of Science ID 000170668300306

• Induction of invasive human epidermal neoplasia resembling squamous cell carcinoma (SCC) by defined genetic elements Ras and CDK4 Kubo, Y., Dajee, M., Lazarov, M., Tao, S., Khavari, P. NATURE PUBLISHING GROUP. 2001: 472–72

  View details for Web of Science ID 000170668300527

• Impact of specific receptor tyrosine kinases on Ras in epithelial cells Cai, T., Khavari, P. BLACKWELL SCIENCE INC. 2001: 476

  View details for Web of Science ID 000170668300551

• Profiling genomic expression in disorders of epidermal proliferation identifies Egr-1 as a potent regulator of epidermal growth Wee, S., Lin, Q., Tao, S., Fan, H., Khavari, P. NATURE PUBLISHING GROUP. 2001: 477–77

  View details for Web of Science ID 000170668300556

• Induction of invasive human melanocytic neoplasia by defined genetic elements Khavari, P., Sheu, S., Robbins, P., Kubo, Y., Dajee, M., Goodnough, J. BLACKWELL SCIENCE INC. 2001: 483

  View details for Web of Science ID 000170668300594

• Generation of inducible NF-kappa B subunits: Confirmation of a primary growth inhibitory role for NF-kappa B in epidermal cells Zhang, Y., Khavari, P. BLACKWELL SCIENCE INC. 2001: 525

  View details for Web of Science ID 000170668300846

• Regulated induction of Ras and its effectors in epithelial cells Tarutani, M., Dajee, M., Khavari, P. BLACKWELL SCIENCE INC. 2001: 526

  View details for Web of Science ID 000170668300847

• Differential target gene induction mediates opposing growth effects of NF-kappa B in keratinocytes and fibroblasts Hinata, K., Khavari, P. BLACKWELL SCIENCE INC. 2001: 526

  View details for Web of Science ID 000170668300852

• Impact of gene vs. protein replacement on genomic expression patterns in functional epidermolysis bullosa Goodnough, J., Robbins, P., Sheu, S., Khavari, P. BLACKWELL SCIENCE INC. 2001: 541

  View details for Web of Science ID 000170668300939

• Molecular transporters facilitate topical protein transduction into the skin Rothbard, J., Robbins, P., Sheu, S., Oliver, S., Goodnough, J., Wender, P., Khavari, P. BLACKWELL SCIENCE INC. 2001: 549

  View details for Web of Science ID 000170668300985

• Conjugation of arginine oligomers to cyclosporin A facilitates topical delivery and inhibition of inflammation NATURE MEDICINE Rothbard, J. B., Garlington, S., Lin, Q., Kirschberg, T., Kreider, E., McGrane, P. L., Wender, P. A., Khavari, P. A. 2000; 6 (11): 1253-1257

  Abstract

  Many systemically effective drugs such as cyclosporin A are ineffective topically because of their poor penetration into skin. To surmount this problem, we conjugated a heptamer of arginine to cyclosporin A through a pH-sensitive linker to produce R7-CsA. In contrast to unmodified cyclosporin A, which fails to penetrate skin, topically applied R7-CsA was efficiently transported into cells in mouse and human skin. R7-CsA reached dermal T lymphocytes and inhibited cutaneous inflammation. These data establish a general strategy for enhancing delivery of poorly absorbed drugs across tissue barriers and provide a new topical approach to the treatment of inflammatory skin disorders.

  View details for Web of Science ID 000165114800033

  View details for PubMedID 11062537

• Immunization via hair follicles by topical application of naked DNA to normal skin NATURE BIOTECHNOLOGY Fan, H. R., Lin, Q., Morrissey, G. R., Khavari, P. A. 1999; 17 (9): 870-872

  Abstract

  In order to test the immune response generated to small amounts of foreign protein in skin, we applied naked DNA in aqueous solution to untreated normal skin. Topical application of plasmid expression vectors for lacZ and the hepatitis B surface antigen (HBsAg) to intact skin induced antigen-specific immune responses that displayed TH2 features. For HBsAg, specific antibody and cellular responses were induced to the same order of magnitude as those produced by intramuscular injection of the commercially available recombinant HBsAg polypeptide vaccine. Finally, topical gene transfer was dependent on the presence of normal hair follicles.

  View details for Web of Science ID 000082365800027

  View details for PubMedID 10471927

• Analysis of human basal cell carcinoma gene expression at a genomic scale via cDNA microarrays Fan, H., Barry, C. T., Baek, S. C., Lin, Q., Brown, P. O., Khavari, P. A. NATURE PUBLISHING GROUP. 1999: 528–28

  View details for Web of Science ID 000079495800056

• cDNA microarray screening for genes differentially expressed in keratinocyte stem cell enriched populations Freiberg, R., Barry, C., Fang, M., Alvarez-Saavedra, E., Robbins, P., BROWN, P., Khavari, P. NATURE PUBLISHING GROUP. 1999: 560–60

  View details for Web of Science ID 000079495800247

• Genomic analysis of human squamous cell carcinoma gene expression via cDNA microarrays Fan, H., Barry, C., Lin, Q., Baek, S., BROWN, P., Khavari, P. NATURE PUBLISHING GROUP. 1999: 558–58

  View details for Web of Science ID 000079495800234

• Identification of new keratinocyte differentiation candidates genes by a genome-wide screening approach Freiberg, R., Barry, C., BROWN, P., Khavari, P. NATURE PUBLISHING GROUP. 1999: 624–24

  View details for Web of Science ID 000079495800632

• Genome-wide characterization of NF-01 kappa B target genes in human epithelial cells via high density cDNA microarrays Hinata, K., Barry, C., BROWN, P., Khavari, P. NATURE PUBLISHING GROUP. 1999: 637–37

  View details for Web of Science ID 000079495800709

• Sonic hedgehog opposes epithelial cell cycle arrest Fan, H., Wee, S. A., Khavari, P. A. BLACKWELL SCIENCE INC. 1999: 525

  View details for Web of Science ID 000079495800039

• NF-kappa B function determiner localization and features of programmed cell death in epidermis Seitz, C. S., Freiberg, R. A., Khavari, P. A. BLACKWELL SCIENCE INC. 1999: 526

  View details for Web of Science ID 000079495800045

• NF-kappa B-directed transcription is normally localized to non-proliferating cells within epidermis and is strongly induced by ultraviolet injury but not by wounding Gervin, A. M., Memet, S., Freiberg, R. A., Israel, A., Khavari, P. A. BLACKWELL SCIENCE INC. 1999: 530

  View details for Web of Science ID 000079495800068

• Sustainable TGM1 gene delivery in human lamellar ichthyosis patient skin in vivo Lin, Q., Deng, H., Fan, H., Khavari, P. NATURE PUBLISHING GROUP. 1999: 638–38

  View details for Web of Science ID 000079495800713

• Durable laminin 5 01 beta 3 gene delivery vectors for gene therapy of junctional epidermolysis bullosa Robbins, P., Alvarez-Saavedra, E., Lin, Q., Chen, X. J., Khavari, P. BLACKWELL SCIENCE INC. 1999: 639

  View details for Web of Science ID 000079495800722

• Rv inhibition lends to epidermal proliferation in vivo Deng, H., Seitz, C., Khavari, P. NATURE PUBLISHING GROUP. 1999: 641–41

  View details for Web of Science ID 000079495800735

• Genetic immunization by topical application of naked DNA to normal skin Fan, H., Lin, Q., Morrissey, G., Khavari, P. BLACKWELL SCIENCE INC. 1999: 638

  View details for Web of Science ID 000079495800717

• Towards development of an in vitro model of human BCC formation. Oro, A. E., Fan, H., Khavari, P. A., Scott, M. P. ACADEMIC PRESS INC ELSEVIER SCIENCE. 1998: 196–96

  View details for Web of Science ID 000074281900230

• Towards development of an in vitro model of human BC formation. Oro, A. E., Fan, H., Khavari, P. A., Scott, M. P. NATURE PUBLISHING GROUP. 1998: 500–500

  View details for Web of Science ID 000072738200196

• NF-kappa B triggers cell cycle arrest in epidermis via specific induction of the cyclin-dependent kinase inhibitor p21(Cip1). Seitz, C. S., Deng, H., Khavari, P. A. NATURE PUBLISHING GROUP. 1998: 475–75

  View details for Web of Science ID 000072738200047

• Sonic hedgehog regulates keratinocyte differentiation gene expression and cell cycle progression. Fan, H., Khavari, P. A. BLACKWELL SCIENCE INC. 1998: 486

  View details for Web of Science ID 000072738200112

• Durable gene delivery to human epidermis in vivo: Relative contributions of gene regulatory elements and stem cell targeting. Khavari, P. A., Lin, Q., Deng, H. NATURE PUBLISHING GROUP. 1998: 503–

  View details for Web of Science ID 000072738200215

• Sustainable cutaneous gene delivery NATURE BIOTECHNOLOGY Deng, H., Lin, Q., Khavari, P. A. 1997; 15 (13): 1388-1391

  Abstract

  Durable gene delivery to human skin is necessary for lasting correction of human genetic skin disease. Current cutaneous gene-delivery strategies, however, have achieved only transient gene expression, often only within a small percentage of tissue cells. The recent inability to sustain phenotypic correction of human genetic skin disease due to loss of therapeutic gene expression in regenerated epidermal tissue has highlighted this current limitation. In an effort to surmount this problem, we have generated gene delivery vectors that produce more durable gene delivery in human skin tissue in vivo.

  View details for Web of Science ID A1997YK36100031

  View details for PubMedID 9415892

• Induction of basal cell carcinoma features in transgenic human skin expressing Sonic Hedgehog NATURE MEDICINE Fan, H. R., Oro, A. E., Scott, M. P., Khavari, P. A. 1997; 3 (7): 788-792

  Abstract

  Hedgehog (HH) signaling proteins mediate inductive events during animal development. Mutation of the only known HH receptor gene, Patched (PTC), has recently been implicated in inherited and sporadic forms of the most common human cancer, basal cell carcinoma (BCC). In Drosophila, HH acts by inactivating PTC function, raising the possibility that overexpression of Sonic Hedgehog (SHH) in human epidermis might have a tumorigenic effect equivalent to loss of PTC function. We used retroviral transduction of normal human keratinocytes to constitutively express SHH. SHH-expressing cells demonstrated increased expression of both the known HH target, BMP-2B, as well as bcl-2, a protein prominently expressed by keratinocytes in BCCs. These keratinocytes were then used to regenerate human skin transgenic for long terminal repeat-driven SHH (LTR-SHH) on immune-deficient mice. LTR-SHH human skin consistently displays the abnormal specific histologic features seen in BCCs, including downgrowth of epithelial buds into the dermis, basal cell palisading and separation of epidermis from the underlying dermis. In addition, LTR-SHH skin displays the gene expression abnormalities previously described for human BCCs, including decreased BP180/BPAG2 and laminin 5 adhesion proteins and expression of basal epidermal keratins. These data indicate that expression of SHH in human skin recapitulates features of human BCC in vivo, suggest that activation of this conserved signaling pathway contributes to the development of epithelial neoplasia and describe a new transgenic human tissue model of neoplasia.

  View details for Web of Science ID A1997XG76700042

  View details for PubMedID 9212109

• Gene therapy: Progress, problems, prospects. NATURE MEDICINE Blau, H., Khavari, P. 1997; 3 (6): 612-613

  View details for Web of Science ID A1997XB97400028

  View details for PubMedID 9176485

• Corrective gene transfer in the human skin disorder lamellar ichthyosis NATURE MEDICINE Choate, K. A., Medalie, D. A., Morgan, J. R., Khavari, P. A. 1996; 2 (11): 1263-1267

  Abstract

  Lamellar ichthyosis (LI) is a disfiguring skin disease characterized by abnormal epidermal differentiation and defective cutaneous barrier function. LI has been associated with loss of keratinocyte transglutaminase 1 (TGase1), an enzyme believed necessary for normal formation of the cornified epidermal barrier. Using LI as a prototype for therapeutic cutaneous gene delivery, we have used the human skin/immunodeficient mouse xenograft model to correct the molecular, histologic and functional abnormalities of LI patient skin in vivo. We have used TGase1-deficient primary keratinocytes from LI patients combined with high-efficiency transfer of functional TGase1 to regenerate engineered human LI epidermis on immunodeficient mice. Engineered LI epidermis displayed normal TGase1 expression in vivo, unlike unengineered LI epidermis where TGase1 was absent. Epidermal architecture was also normalized by TGase1 restoration, as was expression of the epidermal differentiation marker filaggrin. Engineered LI skin demonstrated restoration of cutaneous barrier function measures to levels seen in epidermis regenerated by keratinocytes from patients with normal skin, indicating functional correction in vivo of the proposed primary pathophysiologic defect in LI. These results confirm a major role for TGase1 in epidermal differentiation and demonstrate a potential future approach to therapeutic gene delivery in human skin.

  View details for Web of Science ID A1996VQ10100046

  View details for PubMedID 8898758

• Fas signal transduction effects in the skin differ by cellular target and magnitude of receptor activation. Khavari, P., Freiberg, R. BLACKWELL SCIENCE INC. 1996: 634

  View details for Web of Science ID A1996UC78700674

• SPECIFIC TRIGGERING OF TNF RECEPTOR SUPERFAMILY SIGNAL-TRANSDUCTION PATHWAYS USING CYTOPLASMIC DOMAINS OF FAS AND TNF-R1 FREIBERG, R. A., DUH, H., SPENCER, D., SCHREIBER, S. L., CRABTREE, G. R., KHAVARI, P. A. BLACKWELL SCIENCE PUBL INC CAMBRIDGE. 1995: 560

  View details for Web of Science ID A1995QR53200055

• SPECIFIC GENE-REGULATION IN KERATINOCYTES AND FIBROBLASTS BY DOSE-DEPENDENT INTRACELLULAR MOLECULAR OLIGOMERIZATION FREIBERG, R. A., HO, S., SPENCER, D. M., WANDLESS, T., SCHREIBER, S. L., CRABTREE, G. R., KHAVARI, P. A. BLACKWELL SCIENCE PUBL INC CAMBRIDGE. 1995: 559

  View details for Web of Science ID A1995QR53200048

• THE RETINOBLASTOMA PROTEIN AND BRG1 FORM A COMPLEX AND COOPERATE TO INDUCE CELL-CYCLE ARREST CELL Dunaief, J. L., Strober, B. E., Guha, S., Khavari, P. A., Alin, K., Luban, J., Begemann, M., Crabtree, G. R., Goff, S. P. 1994; 79 (1): 119-130

  Abstract

  The retinoblastoma tumor suppressor protein (RB) binds several cellular proteins involved in cell cycle progression. Using the yeast two-hybrid system, we found that RB bound specifically to the protein BRG1. BRG1 shares extensive sequence similarity to Drosophila brahma, an activator of homeotic gene expression, and the yeast transcriptional activator SNF2/SW12. BRG1 contains an RB-binding motif found in viral oncoproteins and bound to the A/B pocket and the hypophosphorylated form of RB. BRG1 did not bind RB in viral oncoprotein-transformed cells. Coimmunoprecipitation experiments suggested BRG1 associates with the RB family in vivo. In the human carcinoma cell line SW13, BRG1 exhibited tumor suppressor activity by inducing formation of flat, growth-arrested cells. This activity depended on the ability of BRG1 to cooperate and complex with RB, as both an RB-nonbinding mutant of BRG1 and the sequestration of RB by adenovirus E1A protein abolished flat cell formation.

  View details for Web of Science ID A1994PK58500013

  View details for PubMedID 7923370

• NUCLEOSOME DISRUPTION AND ENHANCEMENT OF ACTIVATOR BINDING BY A HUMAN SW1/SNF COMPLEX NATURE Kwon, H., Imbalzano, A. N., Khavari, P. A., Kingston, R. E., GREEN, M. R. 1994; 370 (6489): 477-481

  Abstract

  CHROMATIN structure can affect the transcriptional activity of eukaryotic structural genes by blocking access of sequence-specific activator proteins (activators) to their promoter-binding sites. For example, the DNA-binding domain of the yeast GAL4 protein interacts very poorly with nucleosome cores compared with naked DNA2 (and see below), and binding of other activators is even more strongly inhibited. The way in which activators bind to nucleosomal DNA is therefore a critical aspect of transcriptional activation. Genetic studies have suggested that the multi-component SWI/SNF complex of Saccharomyces cerevisiae facilitates transcription by altering the structure of the chromatin. Here we identify and partially purify a human homologue of the yeast SWI/SNF complex (hSWI/SNF complex). We show that a partially purified hSWI/SNF complex mediates the ATP-dependent disruption of a nucleosome, thereby enabling the activators, GAL4-VP16 and GAL4-AH, to bind within a nucleosome core. We conclude that the hSWI/SNF complex acts directly to reorganize chromatin structure so as to facilitate binding of transcription factors.

  View details for Web of Science ID A1994PB40700061

  View details for PubMedID 8047169

• INTERACTION BETWEEN THE RETINOBLASTOMA PROTEIN AND THE HUMAN HOMOLOG OF BRAHMA, A REGULATOR OF HOMEOTIC GENES DUNAIEF, J., KHAVARI, P., GUHA, S., ALIN, K., LUBAN, J., CRABTREE, G., GOFF, S. WILEY-LISS. 1994: 175

  View details for Web of Science ID A1994MY17500619

• BRG1 CONTAINS A CONSERVED DOMAIN OF THE SWI2/SNF2 FAMILY NECESSARY FOR NORMAL MITOTIC GROWTH AND TRANSCRIPTION NATURE Khavari, P. A., Peterson, C. L., Tamkun, J. W., Mendel, D. B., Crabtree, G. R. 1993; 366 (6451): 170-174

  Abstract

  Sequence-specific DNA binding activators of gene transcription may be assisted by SWI2 (SNF2), which contains a DNA-dependent ATPase domain. We have isolated a human complementary DNA encoding a 205K nuclear protein, BRG1, that contains extensive homology to SWI2 and Drosophila brahma. We report here that a SWI2/BRG1 chimera with the DNA-dependent ATPase domain replaced by corresponding human sequence restored normal mitotic growth and capacity for transcriptional activation to swi2- yeast cells. Point mutation of the conserved ATP binding site lysine abolished this complementation. This mutation in SWI2 exerted a dominant negative effect on transcription in yeast. A lysine to arginine substitution at the corresponding residue of BRG1 also generated a transcriptional dominant negative in human cells. BRG1 is exclusively nuclear and present in a high M(r) complex of about 2 x 10(6). These results show that the SWI2 family DNA-dependent ATPase domain has functional conservation between yeast and humans and suggest that a SWI/SNF protein complex is required for the activation of selective mammalian genes.

  View details for PubMedID 8232556

• IDENTIFICATION OF A 205-KD KERATINOCYTE-EXPRESSED ACTIVATOR OF GLUCOCORTICOID RECEPTOR (GR)-DRIVEN GENE-TRANSCRIPTION Khavari, P. A., Petersen, C. L. NATURE PUBLISHING GROUP. 1993: 524–24

  View details for Web of Science ID A1993KW39500515

• CHARACTERIZATION OF A COFACTOR THAT REGULATES DIMERIZATION OF A MAMMALIAN HOMEODOMAIN PROTEIN SCIENCE Mendel, D. B., Khavari, P. A., Conley, P. B., Graves, M. K., Hansen, L. P., Admon, A., Crabtree, G. R. 1991; 254 (5039): 1762-1767

  Abstract

  Dimerization among transcription factors has become a recurrent theme in the regulation of eukaryotic gene expression. Hepatocyte nuclear factor-1 alpha (HNF-1 alpha) is a homeodomain-containing protein that functions as a dimer. A dimerization cofactor of HNF-1 alpha (DCoH) was identified that displayed a restricted tissue distribution and did not bind to DNA, but, rather, selectively stabilized HNF-1 alpha dimers. The formation of a stable tetrameric DCoH-HNF-1 alpha complex, which required the dimerization domain of HNF-1 alpha, did not change the DNA binding characteristics of HNF-1 alpha, but enhanced its transcriptional activity. However, DCoH did not confer transcriptional activation to the GAL4 DNA binding domain. These results indicate that DCoH regulates formation of transcriptionally active tetrameric complexes and may contribute to the developmental specificity of the complex.

  View details for PubMedID 1763325