CTCF-mediated regulation of partial epithelial-mesenchymal transition states

About This Grant

Project Summary Cellular plasticity, including epithelial and mesenchymal transition (EMT), whether partial, complete or reversed, as mesenchymal-epithelial transition-(MET), is critical throughout development and implicated in wound healing, cancer metastasis, and fibrotic disorders. The reversibility or irreversibility of EMT is critical to successful implementation of these cellular programs yet is not well understood. We have shown that EMT results in diminished expression and chromatin interaction of the DNA-binding chromatin looping factor, CTCF. CTCF has been well characterized as a transcription factor and insulator protein capable of regulating formation of topologically associating domains (TADs) when bound to CTCF binding sites (CTCF-BSs). TADs define semi-stable looping structures which can incorporate multiple genes and which impact gene expression. We have shown that CTCF loss accompanies EMT and yet, paradoxically, causes a more epithelial phenotype as measured by E-cadherin and N-cadherin expression. Moreover, cells with low CTCF expression respond differently to TGF-β induced EMT by retaining E-cadherin while still elevating N-cadherin, thus eliciting a distinct partial EMT state. How CTCF and TADs effect these outcomes is unknown. To achieve our long-term goal which is to understand the epigenomic impacts on epithelial-mesenchymal plasticity and elucidate associated mechanistic underpinnings, we propose to test the hypothesis plasticity or irreversibility between epithelial-mesenchymal states, including distinct partial states, is founded upon dynamic chromatin looping patterns, driven by withdrawal and re-engagement of CTCF from CTCF-BSs at key EMT genes. In Aim 1, we will map the EMT-associated redistribution of chromatin loops and TADs, in combination with gene expression changes, during reversible EMT. We will utilize a model of reversible EMT to detect and quantify changes in CTCF binding, chromatin looping, and enhancer-promoter interactions using ChIP-seq and HiC-seq at discrete timepoints. Furthermore, we have uncovered key CTCF engagement sites at CDH1, CDH2, and ZEB1 with potential regulatory roles for EMT. In Aim 2A: we will determine the functional outcome of reduced CTCF expression to the phenotype of partial EMT. We will interrogate this hybrid EMT phenotype by phenotypic analysis including an examination of the relevance of the dual-cadherin state towards collective and individual cell migration. In Aim 2B: we will delineate the contribution of specific CTCF-BSs within these loci to chromatin looping and epithelial-mesenchymal plasticity. We will elucidate the mechanism through a focus on the epigenetic state of these loci by performing 3C-qPCR and ChIP-qPCR at the relevant loci in timecourse and knockdown models. Overall, we will uncover fundamental mechanisms used by cells to that have undergone reversible EMT at the level of chromatin looping and TAD distribution. Furthermore, this work provides opportunities for undergraduate students to perform interdisciplinary research in cellular and computational biology, while addressing fundamental questions in epigenetics and cellular plasticity.