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

PLoS Genet. 2017 Nov 20;13(11):e1007109. doi: 10.1371/journal.pgen.1007109. eCollection 2017 Nov.


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.

MeSH terms

  • Binding Sites
  • Cell Line, Tumor / metabolism
  • Colorectal Neoplasms / genetics*
  • Colorectal Neoplasms / metabolism
  • Enhancer Elements, Genetic
  • Epithelial-Mesenchymal Transition
  • Gene Expression Regulation, Neoplastic
  • Genes, Regulator
  • Hepatocyte Nuclear Factor 3-alpha / genetics*
  • Hepatocyte Nuclear Factor 3-alpha / metabolism*
  • Humans
  • Mutagenesis, Site-Directed
  • Promoter Regions, Genetic
  • Regulatory Elements, Transcriptional
  • Snail Family Transcription Factors / genetics
  • Snail Family Transcription Factors / metabolism*


  • FOXA1 protein, human
  • Hepatocyte Nuclear Factor 3-alpha
  • SNAI1 protein, human
  • Snail Family Transcription Factors

Grant support

This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG; to AH (DFG HE2004/11-1; CRC-850 subproject B5) to HB and MB, (CRC-850 subproject Z1), and to HB (EXC-306), and by the German Federal Ministry of Education and Research (BMBF; within the framework of the e:Med research and funding concept (FKZ 01ZX1409B to MB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.