Substained DNA demethylation and discordant chromatin accessibility dynamics define enhancer regulation during neural progenitor cell differentiation [ATACme]

Epigenetic mechanisms govern the transcriptional activity of lineage-specifying enhancers; but recent work challenges the dogma that both chromatin accessibility and DNA hypomethylation are prerequisites for transcription, highlighting a need to understand their coordinated dynamics. We established a highly-resolved timeline of DNA demethylation, chromatin accessibility, and transcription factor (TF) occupancy during early human cell differentiation. We show >30,000 lineage-specifying enhancers undergo rapid and transient accessibility changes associated with distinct periods of TF expression. By contrast, enhancer DNA methylation changes are prolonged, unidirectional and delayed relative to chromatin dynamics, creating discordant epigenetic states. Using 6-base sequencing to detect methyl-intermediate, 5hmC, revealed that, for a subset of enhancers, TET-mediated, active demethylation begins prior to, and is maintained independently of, TF binding. In fact, machine learning models trained on 5-hydroxymethylation can predict of future chromatin states. Complete demethylation persists long after TF binding and accessibility have dissipated, suggesting that long-lasting hypomethylation of certain enhancers is a historical record of previous activity.