Project description:Background: The transcription process is controlled by non-coding regulatory elements, more than 70% of which are putative enhancers. These enhancers comprise over 600,000 regions and are marked by histone modifications. However, the mechanisms by which altered enhancers in cancer cooperate within the three-dimensional chromatin architecture to drive oncogenic programs remain poorly understood. Results: By integrating 201 H3K27ac ChIP seq datasets from prostate, we identify 3,216 high confidence prostate cancer-specific putative enhancers. Ultra high resolution chromatin interaction profiling by Region Capture Micro-C at a representative chr6q24.1 locus reveals that these enhancers form cancer specific, highly nested interactions with promoters that coalesce into a multi connected hub absent in normal prostate cells. CRISPR/Cas9 perturbations of these enhancers, examined one by one, distinguish enhancer classes within the hub. Deletion of a central enhancer collapses hub-wide enhancer activities and architecture, leading to the downregulation of target genes, impaired proliferation, and reduced clonogenic growth. In contrast, deletion of a redundant enhancer results in minimal transcriptional changes, as neighboring enhancers rescue cancer signaling through compensatory architectural rewiring that strengthens alternative enhancer-promoter interactions. We also observe that FOXA1, a pioneer transcription factor activated in prostate cancer, directly binds to these enhancers and regulates distinct enhancer classes, leading to varying degrees of chromatin accessibility and gene expression changes. Conclusions: These findings suggest that enhancers function in a coordinated manner, forming multi-connected cancer-specific chromatin interaction hubs, with distinct enhancer classes contributing differently to gene regulation. This study advances our ability to modulate gene expression in a cell type-specific manner, opening new avenues for precision therapies.

Project description:Although technological advances now allow increased tumor profiling, a detailed understanding of the mechanisms leading to the development of different cancers remains elusive. Our approach towards understanding the molecular events that lead to cancer is to characterize changes in transcriptional regulatory networks between normal and tumor tissue. Because enhancer activity is thought to be critical in regulating cell fate decisions, we have focused our studies on distal regulatory elements and transcription factors that bind to these elements. Using DNA methylation data, we identified more than 25,000 enhancers that are differentially activated in breast, prostate, and kidney tumor tissues, as compared to normal tissues. We then developed an analytical approach called TENET (Tracing Enhancer Networks using Epigenetic Traits) that correlates DNA methylation levels at enhancers with gene expression to identify more than 800,000 genome-wide links from enhancers to genes and from genes to enhancers. We found more than 1,200 transcription factors to be involved in these tumor-specific enhancer networks. We further characterized several transcription factors linked to a large number of enhancers in each tumor type, including GATA3 in non-basal breast tumors, HOXC6 and DLX1 in prostate tumors, and ZNF395 in kidney tumors. We showed that HOXC6 and DLX1 are associated with different clusters of prostate tumor-specific enhancers and confer distinct transcriptomic changes upon knockdown in C42B prostate cancer cells. We also discovered de novo motifs enriched in enhancers linked to ZNF395 in kidney tumors. Our studies characterized tumor-specific enhancers and revealed key transcription factors involved in enhancer networks for specific tumor types and subgroups. Our findings, which include a large set of identified enhancers and transcription factors linked to those enhancers in breast, prostate, and kidney cancers, will facilitate understanding of enhancer networks and mechanisms leading to the development of these cancers. Examination of FAIRE and ChIP assays in prostate and breast epithelial cells. RNA-seq assays in C42B, prostate cancer cell line after knocking down of TFs (HOXC6, DLX1).

Project description:FOXA1 is a pioneer factor that is important in hormone dependent cancer cells to stabilise nuclear receptors, such as estrogen receptor (ER) to chromatin. FOXA1 binds to enhancers regions that are enriched in H3K4mono- and dimethylation (H3K4me1, H3K4me2) histone marks and evidence suggests that these marks are requisite events for FOXA1 to associate with enhancers to initate subsequent gene expression events. However, exogenous expression of FOXA1 has been shown to induce H3K4me1 and H3K4me2 signal at enhancer elements and the order of events and the functional importance of these events is not clear. We performed a FOXA1 Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins (RIME) screen in ERα-positive MCF-7 breast cancer cells in order to identify FOXA1 interacting partners and we found histone-lysine N-methyltransferase (MLL3) as the top FOXA1 interacting protein. MLL3 is typically thought to induce H3K4me3 at promoter regions, but recent findings suggest it may contribute to H3K4me1 deposition, in line with our observation that MLL3 associates with an enhancer specific protein. We performed MLL3 ChIP-seq in breast cancer cells and unexpectedly found that MLL3 binds mostly at non-promoter regions enhancers, in contrast to the prevailing hypothesis. MLL3 was shown to occupy regions marked by FOXA1 occupancy and as expected, H3K4me1 and H3K4me2. MLL3 binding was dependent on FOXA1, indicating that FOXA1 recruits MLL3 to chromatin. Motif analysis and subsequent genomic mapping revealed a role for Grainy head like protein-2 (GRHL2) which was shown to co-occupy regions of the chromatin with MLL3. Regions occupied by all three factors, namely FOXA1, MLL3 and GRHL2, were most enriched in H3K4me1. MLL3 silencing decreased H3K4me1 at enhancer elements, but had no appreciable impact on H3K4me3 at enhancer elements. We identify a complex relationship between FOXA1, MLL3 and H3K4me1 at enhancers in breast cancer and propose a mechanism whereby the pioneer factor FOXA1 can interact with a chromatin modifier MLL3, recruiting it to chromatin to facilitate the deposition of H3K4me1 histone marks, subsequently demarcating active enhancer elements.

Project description:Genome-wide association studies (GWAS) have revolutionized the field of cancer genetics, but the causal links between increased genetic risk and onset/progression of disease processes remain to be identified. Here we report the first step in such an endeavor for prostate cancer. We provide a comprehensive annotation of the 77 known risk loci, based upon highly correlated variants in biologically relevant chromatin annotations- we identified 727 such potentially functional SNPs. We also provide a detailed account of possible protein disruption, microRNA target sequence disruption and regulatory response element disruption of all correlated SNPs at r^2≥0.5. Greater than 88% of the 727 SNPs fall within putative enhancers, many of which alter critical residues in the response elements of transcription factors known to be involved in prostate biology. We define as risk enhancers those regions with enhancer chromatin biofeatures in prostate-derived cell lines with prostate-cancer correlated SNPs. To aid in the identification of these enhancers, we performed genomewide ChIP-seq for H3K27-acetylation, a mark of actively engaged enhancer regions, as well as the transcription factor TCF7L2. We analyzed in depth three variants in risk enhancers, two of which show significantly altered androgen sensitivity in LNCaP cells. This includes rs4907792, that is in linkage disequilibrium (r^2=0.91) with an eQTL for NUDT11 (on the X chromosome) in prostate tissue, and rs10486567, the index SNP in intron 3 of the JAZF1 gene on chromosome 7. Rs4907792 is within a critical residue of a strong consensus androgen response element that is interrupted in the protective allele, resulting in a 56% decrease in its androgen sensitivity, whereas rs10486567 affects both NKX3-1 and FOXA-AR motifs where the risk allele results in a 39% increase in basal activity and a 28% fold-increase in androgen stimulated enhancer activity. Identification of such enhancer variants and their potential target genes represents a preliminary step in connecting risk to disease process. ChIP-seq analysis of H3K27Ac in LNCaP charcoal-stripped serum, H3K27Ac in LNCaP charcoal-stripped serum +DHT, TCF7L2 in LNCaP

Dataset Information

Decoding 3D chromatin architecture reveals distinct enhancer classes underlying hierarchical gene regulation in prostate cancer

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets