An eRNA Transcription Checkpoint For Diverse Signal-dependent Enhancer Activation Programs [PRO-Seq]

The evidence that several signal and ligand-dependent pathways function by activating regulatory enhancer programs suggests that a “checkpoint” strategy may underline activation of some or even many diversely-regulated enhancers. Here, we report a molecular mechanism common to several acute signal- and ligand-dependent enhancer activation programs based on release of a shared eRNA transcription checkpoint. This requires recruitment of a DNA-PKcs-phosphorylated RING finger repressor, KAP1, functioning as a modulator, inhibiting its association with 7SK and E3 SUMO ligase activity on the CDK9 subunit of P-TEFb, facilitating formation of an activated P-TEFb complex, licensing eRNA elongation. Overcoming this checkpoint for signal-dependent enhancer activation occurs in diverse pathways including estrogen receptor α, NF-κB-regulated proinflammatory, androgen receptor and neuronal depolarization. Therefore, a specific strategy required to convert a basal state enhancer P-TEFb complex to an active state to release a conserved checkpoint is apparently employed by several functionally-important signal/ligand-regulated regulatory enhancers to implement the instructions of endocrine/paracrine system. For hormone treatments, cells were incubated at 37°C and 5% CO2 for at least 3 days in phenol red-free DMEM (GIBCO/Invitrogen) supplemented with 5% charcoal dextran-stripped FBS (GIBCO/Invitrogen). 17-β-Estradiol (E2; Steraloids, Inc.) was added to a final concentration of 100 nM. For knock-down of KAP1 genes, the shRNA were employed.