Assessment of the impact of DNA methylation on the regulation of enhancer activity – MethylEnh

As mammalian embryonic stem cells (ESCs) prepare for lineage commitment, 5-Cytosine DNA methylation (5mC) global levels shift from low to high. This phenomenon, which is crucial for proper developmental progression, can be recapitulated in cell culture by differentiating mouse ESCs to epiblast-like cells (EpiLCs). Enhancers are key regulatory elements where transcription factors (TF) can bind to establish accurate spatiotemporal expression of target genes, and there is a general anticorrelation between enhancer activity and DNA methylation. The de novo DNA methylation program is largely complete by the epiblast stage, and gives rise to all somatic tissue types.
I have previously implemented a powerful technology in which chromatin features can be simultaneously assayed with the 5mC (EpiMethylTag). This robust technique will allow us to assess enhancer dynamics during cellular transitions from mESCs to EpiLCs to neurons. It has been documented that 5-hydroxymethylcytosine (5hmC, a by-product of DNA demethylation) has a biological function per se, notably for neural system development. Hence we will also assess the 5hmC levels with a modified EpiMethylTag protocol. We will discover the enhancers that are responsive to local and genome-wide perturbations to DNA (de)methylation machinery, and determine their target genes using the high resolution Region Capture MicroC (RCMC) technique.
Finally, using CRISPR/Cas9-based epigenome editing, we will modulate DNA methylation at candidate enhancers to determine the causality between DNAme and enhancer activity. Altogether, this work will utilize powerful methodologies that will generate great insights into enhancer regulation, which has profound implications for our understanding of mammalian development.