Epithelial–mesenchymal transition (EMT) and its reversal, mesenchymal–epithelial transition (MET) drive tissue reorganization critical for early development. In carcinomas, processing through EMT, MET, or partial states promotes migration, invasion, dormancy, and metastatic colonization. As a reversible process, EMT is inherently regulated at epigenetic and epigenomic levels. To understand the epigenomic nature of reversible EMT and its partial states, we characterized chromatin accessibility dynamics, transcriptomic output, protein expression, and cellular phenotypes during stepwise reversible EMT. We find that the chromatin insulating protein machinery, including CTCF, is suppressed and re-expressed, coincident with broad alterations in chromatin accessibility, during EMT/MET, and is lower in triple-negative breast cancer cell lines with EMT features. Through an analysis of chromatin accessibility using ATAC-seq, we identify that early phases of EMT are characterized by enrichment for AP-1 family member binding motifs, but also by a diminished enrichment for CTCF binding motifs. Through a loss-of-function analysis, we demonstrate that the suppression of CTCF alters cellular plasticity, strengthening the epithelial phenotype via the upregulation of epithelial markers E-cadherin/CDH1 and downregulation of N-cadherin/CDH2. Conversely, the upregulation of CTCF leads to the upregulation of EMT gene expression and an increase in mesenchymal traits. These findings are indicative of a role of CTCF in regulating epithelial–mesenchymal plasticity and gene expression.

Background: Epithelial-mesenchymal transition (EMT) facilitates cellular movements critical for proper development; however, in a carcinoma, EMT promotes metastatic dissemination. Stable intermediate states (partial-EMT) are increasingly implicated in metastatic dissemination while reversal of EMT, termed mesenchymal-epithelial transition (MET), is increasingly implicated in metastatic colonization. To understand the partial and reversible nature of EMT, we characterized chromatin accessibility dynamics, transcriptome changes, protein expression patterns, as well as E-cadherin expression, localization, and gene-level dynamics in mammary epithelial cells undergoing stepwise reversible EMT. Results: While shorter EMT induction induced internalization of E-cadherin protein, surface expression was recovered upon MET without loss of transcript or bulk protein. Conversely, a longer EMT induced stable repression of E-cadherin indicated by loss of chromatin accessibility and induced global expansion of accessible sites across the genome, facilitated by increased engagement of multiple transcription factor families, including AP-1 and SMAD. We observe enrichment for binding sites for the insulator proteins CTCF and BORIS was significantly diminished in both stemness-enriched partial-EMT and partial-MET states and determined that CTCF repression imparts alterations in some histone covalent modifications concomitant with those observed during TGFβ-induced EMT. Conclusions: These findings are indicative of a major role for chromatin looping and reorganization in plasticity, stemness, and partial EMT phenotypes.