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SNAIL1-mediated Downregulation of FOXA Proteins Facilitates the Inactivation of Transcriptional Enhancer Elements at Key Epithelial Genes in Colorectal Cancer Cells

ABSTRACT: Converting epithelial into mesenchymal cells through epithelial-mesenchymal transition (EMT) requires massive changes in gene expression. How this is brought about is currently not clear. Here we examined the impact of the EMT master regulator SNAIL1 on the FOXA family of transcription factors which are distinguished by their particular competence to induce chromatin reorganization for the activation of transcriptional enhancer elements. We show that the expression of SNAIL1 and FOXA genes is anti-correlated in transcriptomes of colorectal tumors and cell lines. In two cellular EMT models, ectopically expressed Snail1 downregulates FOXA factors and directly represses FOXA1. To elucidate how FOXA factors contribute to the control of epithelial gene expression, we determined by ChIP-seq data analysis FOXA chromosomal distribution in relation to chromatin structural features characterizing distinct states of transcriptional activity. This revealed a preferential localization of FOXA1 and FOXA2 to transcriptional enhancers at signature genes that distinguish epithelial from mesenchymal colon tumors. To validate the significance of this association, we investigated the impact of FOXA factors on structure and function of transcriptional enhancers at the epithelial genes CDH1, CDX2 and EPHB3. Expression of dominant negative FOXA2 led to chromatin condensation at these enhancer elements. Site- directed mutagenesis of FOXA binding sites in reporter gene constructs and by genome- editing in situ impaired enhancer activity and completely abolished the active chromatin state of the EPHB3 enhancer. Conversely, expression of FOXA factors in cells with inactive CDX2 and EPHB3 enhancers led to chromatin opening and de novo deposition of the H3K4me1 and H3K27ac marks. These findings establish the pioneer function of FOXA factors at enhancer regions of epithelial genes and demonstrate their essential role in maintaining enhancer structure and function. Thus, by repressing FOXA family members, Snail1 targets transcription factors at strategically important positions in gene-regulatory hierarchies which may facilitate transcriptional reprogramming during EMT.

ORGANISM(S): Homo sapiens

PROVIDER: GSE106073 | GEO | 2017/11/10

SECONDARY ACCESSION(S): PRJNA415546

REPOSITORIES: GEO

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Project description:At the molecular level, epithelial-to-mesenchymal transition (EMT) necessitates extensive transcriptional reprogramming which is orchestrated by a small group of gene regulatory proteins. The transcription factor SNAIL1 is a zinc-finger DNA-binding protein and well-known master regulator of EMT. However, knowledge of its immediate target genes is incomplete. Here, we performed ChIP-seq to chart genome-wide SNAIL1 chromosomal binding sites and to identify genes directly regulated by SNAIL1. For this we used the colorectal adenocarcinoma cell line LS174T which was engineered to express hemagglutinin epitope-tagged murine SNAIL1 in a doxycycline inducible fashion. In total, 1501 SNAIL1-HA ChIP-seq peaks were mapped which were linked to 1307 genes based on nearest neighbor relationships. The vast majority of SNAIL1-HA binding events occurred within 1 kb upstream of transcriptional start sites (44%), and in intergenic regions and promoter-distal introns (46%). De novo motif deduction showed that nearly all ChIP-seq peak regions harbored at least one sequence element corresponding to the cognate SNAIL1 DNA-recognition motif 5’-CAGGTG-3’. SNAIL1-HA ChIP-seq peaks included previously determined SNAIL1 binding sites at the FOXA1, EPHB3, ITGB4 and CLDN3 genes, thus validating the ChIP-Seq results. When comparing the genomic distribution of SNAIL1-HA to that of the intestinal stem cell (ISC) transcription factors ASCL2 and TCF7L2, we observed a significant overlap. Furthermore, SNAIL1-HA ChIP-seq peaks are associated with a substantial fraction of ISC signature genes. In two colorectal cancer cell lines, we verified that SNAIL1 decreases ISC marker expression. Likewise, SNAIL1-HA was found to occupy multiple regulatory elements at the proto-oncogene MYB, and the long non-coding RNA (lncRNA) WiNTRLINC1, a recently described regulator of ASCL2. Occupancy of the MYB and WiNTRLINC1 regulatory elements by SNAIL1-HA correlated with the downregulation of the two genes. We propose that SNAIL1-mediated repression of MYB and ISC markers like WiNTRLINC1 contributes to the decrease in proliferation known to be associated with EMT, while simultaneously abrogating stemness features of colorectal cancer cells.

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SNAIL1-mediated Downregulation of FOXA Proteins Facilitates the Inactivation of Transcriptional Enhancer Elements at Key Epithelial Genes in Colorectal Cancer Cells

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