| dc.description.abstract | Current models of enhancer dynamics only weakly describe a role for DNA methylation within the ordered process of cellular differentiation and enhancer activation, both frequently perturbed in human disease. However, DNA methylation is suggested to play a crucial role in cellular differentiation as human embryonic stem cells defective for DNA methylation related proteins (DNMT, TET, TDG) demonstrate impaired or biased cellular differentiation. Past work identified genomic regions with WGBS across discrete hematopoietic intermediate cell types that appear to undergo progressive DNA demethylation depending on the cell fate. Intriguingly, many of these same genomic regions while methylated, exist in nucleosome-depleted regions of the genome. This led to a hypothesis that DNA methylation dynamics and chromatin accessibility are uncoupled molecular events representing distinct stages of enhancer activation during cell differentiation. In pursuing this hypothesis, we developed a methodology (ATAC-Me) to simultaneously probe chromatin accessibility and DNA methylation from a single DNA library by coupling assay for transposase accessible chromatin (ATAC) with subsequent bisulfite conversion. ATAC-Me was applied across a cell fate specification time course of THP1 monocytes differentiated to macrophages with phorbol 12-myristate 13-acetate (PMA) stimulation. ATAC-Me identified multiple waves of chromatin accessibility changes decoupled from DNA methylation changes supporting that concomitant chromatin hyperaccessibility and DNA hypermethylation are a unique stage of enhancer activation. In contrast, we observed that chromatin accessibility dynamics and nearby gene transcriptional responses remain closely coupled regardless of intergenic DNA methylation state. Further, Specific transcription factor motif enrichment and footprints were associated with distinct temporal chromatin accessibility behaviors reflective of gene regulatory activation. In complement to monitoring epigenomic dynamics we also pursued a method development (ATAC-STARR) coupling ATAC with self- transcribing regulatory region sequencing (STARR-seq). Initial tests established ATAC-STARR as a feasible method to monitor open chromatin DNA fragments for enhancer activity. Collectively in this work we have developed the ATAC-Me and ATAC-STARR genomics methods to construct a molecular timeline for enhancer activation events across cellular differentiation with specific focus on epigenomic dynamics. | |
