Dynamic Lineage Priming by ERK is Driven by Transcription Factor-Independent Enhancer Regulation [Med24_KO]

Extensive analysis if the means by which ERK affects transcription via enhancer regulation in mouse embryonic stem cells. This study combines analysis of mRNA, and nascent RNA dynamics and correlates this with changes in transcription factor, co-factor, and RNAPII binding and activity. Overall design: Mouse embryonic stem cells, where both Med24 has been knocked out and Med24 function is replaced with a TET regulated cDNA encoding for a FLAG-Med24 and also stably expressing a cRAF-ErT2-T2A-RTTA expression cassette. Cells were maintained for several passages in then presence (WT) or absence (KO) of Doxycycline (500nM) prior to stimulation with 4OHT to activate ERK. Cells were treated with 4OHT for 2hrs or 8hrs to activate ERK signalling via cRAF-ErT2. 15 minutes prior to RNA isolation, cells were treated with 5mM ethynyl uridine to label newly synthesized RNA . Subsequent to total RNA isolation, labelled RNA was purified by standard chemistry, libraries prepared, and sequenced.