Epigenetic modifications, such as DNA methylation and histone modifications, alter DNA accessibility and chromatin structure, thereby regulating patterns of gene expression. DNA methylation mostly occurs in a CpG dinucleotide context and involves the covalent addition of a methyl group at the 5-carbon position of cytosine (5mC), catalyzed by DNA methyltransferases (DNMTs). DNA demethylation is mediated by Ten-eleven translocation (TET) enzymes that successively oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and other derivatives, to eventually derive an unmodified cytosine. Dynamic changes in DNA methylation and demethylation orchestrate the transcriptional network during developmental processes like lineage specification. Alterations in these pathways are implicated in cancer. Cytosine methylation. However, CpG islands (CGIs), characterized by a high CpG density, are often found in promoter regions of genes and are typically hypomethylated. Although methylation of CGIs is canonically associated with transcriptional silencing, recent research has provided evidences refuting this claim. Therefore, molecular mechanisms underlying the association between DNA methylation and regulation of gene expression have proven difficult to decipher.
I will employ quantitative interaction proteomics assay to identify readers of developmentally regulated methylated regions in mouse ESCs. Identified high affinity readers will then be functionally validated using CUT&RUN sequencing. Their potential importance for stem cell identity and lineage commitment will be investigated using perturbation experiments (over-expression and/or CRISPR/Cas9 mediated knockout). This workflow will also be applied to identify and functionally analyze methylation readers in differentiated cells (e.g., neural progenitor cells, intestinal stem cells) which will reveal molecular players pivotal for cellular identity and function. Furthermore, this technique will be employed to highlight methylation readers of cancer-specific methylated promoters to unravel dysregulated pathways in cancer. This technique will be potentially combined with single-locus proteomics (on-going Vermeulen Lab) for studying molecular components involved in dynamic regulation of in vivo activity at a given chromosomal location. Eventually, this protein-DNA binding assay will be optimized to identify readers of oxidized derivatives like hydroxymethylated regions. Thus, the project overall aims to molecularly dissect how ‘readers’ of the epigenome instruct the transcriptional program, thereby coordinating cell-fate dynamics.