Abstract
Epidermal barrier function requires differentiation initiation from a sustainable pool of progenitors. The mechanisms underlying the earliest events initiating differentiation remain unclear. Here we show that RNA Polymerase II (Pol II) pause release, controlled by the CDK9 kinase activity, plays a crucial role in the rapid induction of differentiation. Using RNA-seq in combination with Pol II ChIP-seq, we identified a cluster of upregulated genes featuring robust Pol II pause release in keratinocyte differentiation. A subset of these genes showed rapid induction within 3 hours by Peptidomimetic inhibitors targeting the super elongation complex (SEC), in keratinocytes cultured in the undifferentiation condition. A longer-term (24 hour) treatment led to significant changes (fold change >2, p<0.05) of 1286 keratinocyte-differentiation-signature genes. Mechanistically, we found that the SEC scaffold protein AFF1, but not AFF4, is essential for repressing differentiation in the progenitor state. AFF1 knockdown induced epidermal hypoplasia and impaired progenitor regenerative capacity. We further identified that AFF1 represses differentiation in progenitors through sequestering CDK9 in the inactive state via its interaction with HEXIM1. AFF1 and HEXIM1 directly bind near the transcription start sites of 92 rapid-response genes, sustaining Pol II pausing in the progenitor state. These rapid response genes include ATF3, whose overexpression is sufficient to drive the activation of other differentiation-activating transcription factors, including ZNF750, PRDM1, OVOL1, and GRHL3. In addition, we found that the AFF1 and CDK9 activity are both involved in the immediate of response of PKC-signaling activation. Taken together, our findings suggest a model that AFF1 and HEXIM1 mediated CDK9 activity switch underlies the initial steps of epidermal progenitor differentiation, in response to cellular signaling such as PKC activation.
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