SNAIL1-mediated downregulation of FOXA proteins facilitates the inactivation of transcriptional enhancer elements at key epithelial genes in colorectal cancer cells.

Sabine Jägle,Monika Schrempp,Melanie Boerries,Andreas Hecht,Sven Beyes,Vivien Freihen,Hauke Busch,Robert A. Weinberg

doi:10.1371/journal.pgen.1007109

Abstract

Phenotypic conversion of tumor cells through epithelial-mesenchymal transition (EMT) requires massive gene expression changes. How these are brought about is 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 anticorrelated in transcriptomes of colorectal tumors and cell lines. In cellular EMT models, ectopically expressed Snail1 directly represses FOXA1 and triggers downregulation of all FOXA family members, suggesting that loss of FOXA expression promotes EMT. Indeed, cells with CRISPR/Cas9-induced FOXA-deficiency acquire mesenchymal characteristics. Furthermore, ChIP-seq data analysis of FOXA chromosomal distribution in relation to chromatin structural features which characterize distinct states of transcriptional activity, revealed preferential localization of FOXA factors 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 enhancers at the CDH1, CDX2 and EPHB3 genes. FOXA-deficiency and 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.

Highlights

The formation of distant organ metastases and acquired therapy resistance represent major challenges for the successful treatment of cancer
In a model of colorectal cancer we found that SNAIL1 represses FOXA factors, and demonstrate that FOXA factors are associated with enhancer elements at epithelial signature genes
Our findings indicate that SNAIL1 induces pervasive repression of epithelial genes through a hierarchical scheme of alterations in transcription factor expression which may be applicable to other instances of cell fate changes and transcriptional reprogramming

Summary

Introduction

The formation of distant organ metastases and acquired therapy resistance represent major challenges for the successful treatment of cancer. The process of epithelial-mesenchymal transition (EMT) is widely believed to promote initial steps in the invasion-metastasis cascade [1]. EMT was linked to reduced cancer cell sensitivity against chemo- and radiotherapy [2,3,4]. Phenotypic changes of tumor cells that undergo EMT include loss of apical-basal cell polarity, alterations in cell-cell and cell-matrix attachment, increased motility, and enhanced invasiveness [1]. The observed adaptations in cellular phenotype are caused by extensive transcriptional reprogramming, during which the expression of epithelial and mesenchymal genes is down- and upregulated, respectively [1,5].

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