AFF1-HEXIM1 cooperation suppresses rapid-response differentiation initiators to enforce epidermal progenitor maintenance

In this study we identified an essential role of the SEC, a crucial regulator of Pol II dynamics, in repressing differentiation for epidermal progenitor maintenance. This repression of differentiation is specifically mediated by the scaffold protein AFF1, but not AFF4. Mechanistically, we find that AFF1-SEC associates with the HEXIM1-containing inactive-PTEFb to directly repress a group of rapid-response genes, which feature robust Pol II pausing near their promoters in the progenitor state. De-repression of these genes occurs within 3 hours of SEC-PTEFb disruption by the peptidomimetic inhibitors. These rapid-response genes include the transcription factor ATF3. Increased ATF3 level is sufficient to promote the expression of key differentiation activators, such as PRDM1, OVOL1, GRHL3 and ZNF750. Furthermore, we find that the dissociation of inactivate P-TEFb from SEC mediates the earliest events of PKC signaling, in triggering epidermal differentiation. Overall design: We performed ChIP-seq for Pol II in progenitors and in differentiated keratinocytes. We additionally performed ChIP-seq for AFF1 and HEXIM1 in progenitors. We completed RNA-seq for AFF1 knockdown (KD), AFF4 KD, and HEXIM1 KD. We also performed RNA-seq after TPA and KL drug treatments.