Global Regulatory DNA Potentiation by SMARCA4 Propagates to Selective Gene Expression Programs via Domain-Level Remodeling.

The human genome encodes millions of regulatory elements, of which only a small fraction are active within a given cell type. Little is known about the global impact of chromatin remodelers on regulatory DNA landscapes and how this translates to gene expression. We use precision genome engineering to reawaken homozygously inactivated SMARCA4, a central ATPase of the human SWI/SNF chromatin remodeling complex, in lung adenocarcinoma cells. Here, we combine DNase I hypersensitivity, histone modification, and transcriptional profiling to show that SMARCA4 dramatically increases both the number and magnitude of accessible chromatin sites genome-wide, chiefly by unmasking sites of low regulatory factor occupancy. By contrast, transcriptional changes are concentrated within well-demarcated remodeling domains wherein expression of specific genes is gated by both distal element activation and promoter chromatin configuration. Our results provide a perspective on how global chromatin remodeling activity is translated to gene expression via regulatory DNA.

Integrated epigenomic profiling reveals endogenous retrovirus reactivation in renal cell carcinoma.

BACKGROUND: Transcriptional dysregulation drives cancer formation but the underlying mechanisms are still poorly understood. Renal cell carcinoma (RCC) is the most common malignant kidney tumor which canonically activates the hypoxia-inducible transcription factor (HIF) pathway. Despite intensive study, novel therapeutic strategies to target RCC have been difficult to develop. Since the RCC epigenome is relatively understudied, we sought to elucidate key mechanisms underpinning the tumor phenotype and its clinical behavior. METHODS: We performed genome-wide chromatin accessibility (DNase-seq) and transcriptome profiling (RNA-seq) on paired tumor/normal samples from 3 patients undergoing nephrectomy for removal of RCC. We incorporated publicly available data on HIF binding (ChIP-seq) in a RCC cell line. We performed integrated analyses of these high-resolution, genome-scale datasets together with larger transcriptomic data available through The Cancer Genome Atlas (TCGA). FINDINGS: Though HIF transcription factors play a cardinal role in RCC oncogenesis, we found that numerous transcription factors with a RCC-selective expression pattern also demonstrated evidence of HIF binding near their gene body. Examination of chromatin accessibility profiles revealed that some of these transcription factors influenced the tumor's regulatory landscape, notably the stem cell transcription factor POU5F1 (OCT4). Elevated POU5F1 transcript levels were correlated with advanced tumor stage and poorer overall survival in RCC patients. Unexpectedly, we discovered a HIF-pathway-responsive promoter embedded within a endogenous retroviral long terminal repeat (LTR) element at the transcriptional start site of the PSOR1C3 long non-coding RNA gene upstream of POU5F1. RNA transcripts are induced from this promoter and read through PSOR1C3 into POU5F1 producing a novel POU5F1 transcript isoform. Rather than being unique to the POU5F1 locus, we found that HIF binds to several other transcriptionally active LTR elements genome-wide correlating with broad gene expression changes in RCC. INTERPRETATION: Integrated transcriptomic and epigenomic analysis of matched tumor and normal tissues from even a small number of primary patient samples revealed remarkably convergent shared regulatory landscapes. Several transcription factors appear to act downstream of HIF including the potent stem cell transcription factor POU5F1. Dysregulated expression of POU5F1 is part of a larger pattern of gene expression changes in RCC that may be induced by HIF-dependent reactivation of dormant promoters embedded within endogenous retroviral LTRs.

Integrated Functional Genomic Analysis Enables Annotation of Kidney Genome-Wide Association Study Loci.

BACKGROUND: Linking genetic risk loci identified by genome-wide association studies (GWAS) to their causal genes remains a major challenge. Disease-associated genetic variants are concentrated in regions containing regulatory DNA elements, such as promoters and enhancers. Although researchers have previously published DNA maps of these regulatory regions for kidney tubule cells and glomerular endothelial cells, maps for podocytes and mesangial cells have not been available. METHODS: We generated regulatory DNA maps (DNase-seq) and paired gene expression profiles (RNA-seq) from primary outgrowth cultures of human glomeruli that were composed mainly of podocytes and mesangial cells. We generated similar datasets from renal cortex cultures, to compare with those of the glomerular cultures. Because regulatory DNA elements can act on target genes across large genomic distances, we also generated a chromatin conformation map from freshly isolated human glomeruli. RESULTS: We identified thousands of unique regulatory DNA elements, many located close to transcription factor genes, which the glomerular and cortex samples expressed at different levels. We found that genetic variants associated with kidney diseases (GWAS) and kidney expression quantitative trait loci were enriched in regulatory DNA regions. By combining GWAS, epigenomic, and chromatin conformation data, we functionally annotated 46 kidney disease genes. CONCLUSIONS: We demonstrate a powerful approach to functionally connect kidney disease-/trait-associated loci to their target genes by leveraging unique regulatory DNA maps and integrated epigenomic and genetic analysis. This process can be applied to other kidney cell types and will enhance our understanding of genome regulation and its effects on gene expression in kidney disease.