Temporally discordant chromatin accessibility and DNA demethylation define short- and long-term enhancer regulation during cell fate specification.

Chromatin and DNA modifications mediate the transcriptional activity of lineage-specifying enhancers, but recent work challenges the dogma that joint chromatin accessibility and DNA demethylation are prerequisites for transcription. To understand this paradox, we established a highly resolved timeline of their dynamics during neural progenitor cell differentiation. We discovered that, while complete demethylation appears delayed relative to shorter-lived chromatin changes for thousands of enhancers, DNA demethylation actually initiates with 5-hydroxymethylation before appreciable accessibility and transcription factor occupancy is observed. The extended timeline of DNA demethylation creates temporal discordance appearing as heterogeneity in enhancer regulatory states. Few regions ever gain methylation, and resulting enhancer hypomethylation persists long after chromatin activities have dissipated. We demonstrate that the temporal methylation status of CpGs (mC/hmC/C) predicts past, present, and future chromatin accessibility using machine learning models. Thus, chromatin and DNA methylation collaborate on different timescales to shape short- and long-term enhancer regulation during cell fate specification.