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Expression levels of retinoic acid receptor gamma (NR1B3/RARG, encodes RARγ), are commonly reduced in prostate cancer (PCa). Therefore we sought to establish the cellular and gene regulatory consequences of reduced RARγ expression, and determine RARγ regulatory mechanisms.RARGshRNA approaches in non-malignant (RWPE-1 and HPr1-AR) and malignant (LNCaP) prostate models revealed that reducing RARγ levels, rather than adding exogenous retinoid ligand, had the greatest impact on prostate cell viability and gene expression. ChIP-Seq defined the RARγ cistrome which was significantly enriched at active enhancers associated with AR binding sites. Reflecting a significant genomic role for RARγ to regulate androgen signaling, RARγ knockdown in HPr1-AR cells significantly regulated the magnitude of the AR transcriptome. RARγ down-regulation was explained by increased miR-96 in PCa cell and mouse models, and TCGA PCa cohorts. Biochemical approaches confirmed that miR-96 directly regulated RARγ expression and function Capture of the miR-96 targetome by biotin-miR96 identified that RARγ and a number of RARγ interacting co-factors includingTACC1were all targeted by miR-96, and expression of these genes were prominently altered, positively and negatively, in the TCGA-PRAD cohort. Differential gene expression analyses between tumors in the TCGA-PRAD cohort with lower quartile expression levels ofRARGandTACC1and upper quartile miR-96, compared to the reverse, identified a gene network including several RARγ target genes (e.g.SOX15) that significantly associated with worse disease free survival (hazard ratio 2.23, 95% CI 1.58 to 2.88, p=0.015). In summary, miR-96 targets a RARγ network to govern AR signaling, PCa progression and disease outcome.Conflict of interest:The authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.FUNDINGLESCacknowledges support, in part, of Roswell Park Comprehensive Cancer Center-University of Pittsburg Cancer Institute Ovarian Cancer Specialized Program of Research Excellence National Institutes of Health [P50CA159981-01A1].MDLacknowledges support of Molecular Pharmacology and Experimental Therapeutics NRSA T32 program [T32CA009072] held at Roswell Park Comprehensive Cancer Center.MJCandDJSacknowledges support in part from the Prostate program of the Department of Defense Congressionally Directed Medical Research Programs [W81XWH-14-1-0608, W81XWH-11-2-0033] and the National Cancer Institute (NCI) grant P30CA016056 involving the use of Roswell Park Comprehensive Cancer Center’s Genomic Shared Resource.MJC, GL, AR, HWandPvdBacknowledges support from the European Union-United States Atlantis Program [P116J090011].MJCandLESCacknowledge support from the National Cancer Institute (NCI) grant P30CA016056 involving the use of OSUCCC The James, CCSG P30CA016058

SUMMARYStem-cell derived in vitro systems, such as organoids or embryoids, hold great potential for modeling in vivo biology and engineering living systems with novel functions. To unlock that potential, we need new ways to elicit higher-level organization. Here we show that adding extraembryonic endoderm (XEN) cells to mouse gastruloids leads to the formation of neural epithelia. By single-cell RNA-seq, imaging and differentiation experiments, we demonstrate the neural characteristics and spatial patterning of the epithelial tissue. We further show that the XEN cells differentiate reciprocally to a visceral endoderm-like state. Finally, we demonstrate that local inhibition of WNT signaling and production of a basement membrane by the XEN cells underlie the formation of the neuroepithelial tissue. In summary, we establish “XEN Enhanced Gastruloids” (XEGs) to explore heterotypic cellular interactions as a means to achieve complex, tissue-level organization in vitro.

Song et al. used an allelic reporter approach to show super-enhancers allelic DNA methylation dynamics underlies locus-specific heterogeneity, which functionally impacts transcription and cellular states of mouse embryonic stem cells., NIH (Grants HD045022, 1U19AI131135, 5R01MH104610, and 1R01GM114864)

The current understanding of mammalian kidney development is largely based on mouse models. Recent landmark studies revealed pervasive differences in renal embryogenesis between mouse and human. The scarcity of detailed gene expression data in humans therefore hampers a thorough understanding of human kidney development and the possible developmental origin of kidney diseases. In this paper, we present a single-cell transcriptomics study of the human fetal kidney. We identified 22 cell types and a host of marker genes. Comparison of samples from different developmental ages revealed continuous gene expression changes in podocytes. To demonstrate the usefulness of our data set, we explored the heterogeneity of the nephrogenic niche, localized podocyte precursors, and confirmed disease-associated marker genes. With close to 18,000 renal cells from five different developmental ages, this study provides a rich resource for the elucidation of human kidney development, easily accessible through an interactive web application., A detailed transcriptomic characterization of the human fetal kidney at the single-cell level allows the identification of 22 cell types and a host of novel marker genes, revealing heterogeneity in the nephrogenic niche and localized podocyte precursors., Author summary Regenerative medicine offers an exciting avenue to potential cures of kidney disease. However, a detailed knowledge of the structure and embryonic development of the kidney is crucial, both for stimulating regeneration in the body and for growing healthy kidney tissue in a dish. Most of such knowledge has been obtained from mice, whose development differs in crucial ways from that of humans. We therefore studied the composition of human fetal kidney tissue from five developmental ages, using a technique that can measure gene expression in individual cells. Our measurements revealed 22 distinguishable cell types, some of which we localized in the tissue by fluorescence microscopy. We found several subpopulations of nephron progenitors—cells that give rise to the nephron, the functional unit of the kidney. Our study also focused on the development of podocytes, a cell type that is crucial for the filtration function of the kidney, and our results might inform attempts to recreate these cells in a dish. We hope that our data set, made conveniently accessible through a web application, will help scientists develop new regenerative medicine approaches to kidney disease.

To define the functions of NCOR1 we developed an integrative analysis that combined ENCODE and NCI-60 data, followed by in vitro validation. NCOR1 and H3K9me3 ChIP-Seq, FAIRE-seq and DNA CpG methylation interactions were related to gene expression using bootstrapping approaches. Most NCOR1 combinations (24/44) were associated with significantly elevated level expression of protein coding genes and only very few combinations related to gene repression. DAVID's biological process annotation revealed that elevated gene expression was uniquely associated with acetylation and ETS binding. A matrix of gene and drug interactions built on NCI-60 data identified that Imatinib significantly targeted the NCOR1 governed transcriptome. Stable knockdown of NCOR1 in K562 cells slowed growth and significantly repressed genes associated with NCOR1 cistrome, again, with the GO terms acetylation and ETS binding, and significantly dampened sensitivity to Imatinib-induced erythroid differentiation. Mining public microarray data revealed that NCOR1-targeted genes were significantly enriched in Imatinib response gene signatures in cell lines and chronic myelogenous leukemia (CML) patients. These approaches integrated cistrome, transcriptome and drug sensitivity relationships to reveal that NCOR1 function is surprisingly most associated with elevated gene expression, and that these targets, both in CML cell lines and patients, associate with sensitivity to Imatinib.

The transcription factor Sox2 controls the fate of pluripotent stem cells and neural stem cells. This gatekeeper function requires well-regulated Sox2 levels. We postulated that Sox2 regulation is partially controlled by the Sox2 overlapping long non-coding RNA (lncRNA) gene Sox2ot. Here we show that the RNA levels of Sox2ot and Sox2 are inversely correlated during neural differentiation of mouse embryonic stem cells (ESCs). Through allele-specific enhanced transcription of Sox2ot in mouse Sox2eGFP knockin ESCs we demonstrate that increased Sox2ot transcriptional activity reduces Sox2 RNA levels in an allele-specific manner. Enhanced Sox2ot transcription, yielding lower Sox2 RNA levels, correlates with a decreased chromatin interaction of the upstream regulatory sequence of Sox2 and the ESC-specific Sox2 super enhancer. Our study indicates that, in addition to previously reported in trans mechanisms, Sox2ot can regulate Sox2 by an allele-specific mechanism, in particular during development.

SummaryDuring in vitro differentiation, pluripotent stem cells undergo extensive remodeling of their gene expression profile. While studied extensively at the transcriptome level, much less is known about protein dynamics. Here, we measured mRNA and protein levels of 7459 genes during differentiation of embryonic stem cells (ESCs). This comprehensive data set revealed pervasive discordance between mRNA and protein. The high temporal resolution of the data made it possible to determine protein turnover rates genome-wide by fitting a kinetic model. This model further enabled us to systematically identify dynamic post-transcriptional regulation. Moreover, we linked different modes of regulation to the function of specific gene sets. Finally, we showed that the kinetic model can be applied to singlecell transcriptomics data to predict protein levels in differentiated cell types. In conclusion, our comprehensive data set, easily accessible through a web application, is a valuable resource for the discovery of post-transcriptional regulation in ESC differentiation.

NCOR1 is a co-regulator of multiple transcription factors (TFs) and is mutated in major cancers. We mined publically available data-sets to reveal the diversity, function and druggability of the NCOR1 cistrome. From ENCODE, in K562 chronic myelogenous leukemia cells, NCOR1 ChIP-Seq data were integrated with H3K9me3 (a functional consequence), CpG methylation levels and open and closed chromatin states in a matrix of 45 interactions that were combined with RNA-Seq data. Boot-strapping approaches then revealed significantly affected gene networks. Surprisingly, only the combination of NCOR1, H3K9me3 and an intermediate level of CpG methylation, in open chromatin, significantly associated with a network of 212 repressed genes (lo_gene). By contrast, 15 different NCOR1 combinatorial binding patterns associated with significantly elevated gene expression. For example, NCOR1 and low CpG methylation associated with a network of 908 significantly elevated genes (hi_gene). A simple linear regression model was built on the correlation of NCOR1 and 11 different histone modifications with RNA-seq values, across 81 bins spanning gene loci, to reveal the optimal bin predictor of gene expression. NCOR1 binding at the TSS significantly and positively correlated with gene expression, and mirrored the patterns of the activating histone mark H3K4me3. Transcription factor and GO enrichment in the lo_gene and hi_gene networks revealed these significant associations with different TFs and GO terms. In the lo_genes network the most significantly associated TF was regulatory factor X (RFX1), (FDR=1e-4), whereas in the hi_gene network it was Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2), (FDR=2.7e-23). GO analyses revealed these cohorts were most associated with skeletal development (FDR, 2e-3) and transcription regulation (FDR 1.8e-2) terms respectively, underscoring their diverse functions. Mining the NCI60 database, combined with bootstrap approaches, identified that the NCOR1 hi_gene (upregulated) network was most significantly enriched for genes associated with sensitivity towards HDAC inhibitors, including vorinostat, compared to other drug classes including RTKi. Finally, mining a GEO microarray data set of 59 CML patients revealed that significantly up-regulated genes in Imatinib resistant patients were significantly enriched for targets associated with vorinostat sensitivity in the NCI60. Together this integration pipeline in R, from ENCODE, GEO and NCI60, forms a highly flexible pipeline that can be applied to any DNA binding protein or epigenetic state as a prelude to drug redeployment and/or precision medicine approaches. Specifically, these data suggest that cancer related functions of NCOR1 are surprisingly most associated with sustained gene expression, and these targets, both in CML cell lines and patients, associate with sensitivity to clinically approved HDAC inhibitors. Citation Format: Mark D. Long, Patrick van den Berg, Moray J. Campbell, Prashant K. Singh, Sebastiano Battaglia. An integrative genomic pipeline to target the NCOR1 cistrome with precision. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4186. doi:10.1158/1538-7445.AM2014-4186

Article, Background The nuclear hormone receptor superfamily acts as a genomic sensor of diverse signals. Their actions are often intertwined with other transcription factors. Nuclear hormone receptors are targets for many therapeutic drugs, and include the vitamin D receptor (VDR). VDR signaling is pleotropic, being implicated in calcaemic function, antibacterial actions, growth control, immunomodulation and anti-cancer actions. Specifically, we hypothesized that the biologically significant relationships between the VDR transcriptome and phenotype-associated biology could be discovered by integrating the known VDR transcription factor binding sites and all published trait- and disease-associated SNPs. By integrating VDR genome-wide binding data (ChIP-seq) with the National Human Genome Research Institute (NHGRI) GWAS catalog of SNPs we would see where and which target gene interactions and pathways are impacted by inherited genetic variation in VDR binding sites, indicating which of VDR’s multiple functions are most biologically significant. Results To examine how genetic variation impacts VDR function we overlapped 23,409 VDR genomic binding peaks from six VDR ChIP-seq datasets with 191,482 SNPs, derived from GWAS-significant SNPs (Lead SNPs) and their correlated variants (r 2 > 0.8) from HapMap3 and the 1000 genomes project. In total, 574 SNPs (71 Lead and 503 SNPs in linkage disequilibrium with Lead SNPs) were present at VDR binding loci and associated with 211 phenotypes. For each phenotype a hypergeometric test was used to determine if SNPs were enriched at VDR binding sites. Bonferroni correction for multiple testing across the 211 phenotypes yielded 42 SNPs that were either disease- or phenotype-associated with seven predominately immune related including self-reported allergy; esophageal cancer was the only cancer phenotype. Motif analyses revealed that only two of these 42 SNPs reside within a canonical VDR binding site (DR3 motif), and that 1/3 of the 42 SNPs significantly impacted binding and gene regulation by other transcription factors, including NF-κB. This suggests a plausible link for the potential cross-talk between VDR and NF-κB. Conclusions These analyses showed that VDR peaks are enriched for SNPs associated with immune phenotypes suggesting that VDR immunomodulatory functions are amongst its most important actions. The enrichment of genetic variation in non-DR3 motifs suggests a significant role for the VDR to bind in multimeric complexes containing other transcription factors that are the primary DNA binding component. Our work provides a framework for the combination of ChIP-seq and GWAS findings to provide insight into the underlying phenotype-associated biology of a given transcription factor., published version, http://purl.org/eprint/status/PeerReviewed