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

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 ERK1 and ERK2 genes have been knocked out and ERK function is replaced with a cDNA encoding for a FLAG-ERK2 Q103A mutant and also stably expressing a cRAF-ErT2 expression cassette, were treated with 4OHT for 2hrs to activate ERK signalling via cRAF-ErT2. Activation was performed in the presence of either DMSO, 1uM PD032509 to inhibit Mek acticvity, or 2uM PP1 to inhibit ERK2 Q103A. 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.