AFF4 globally affects the release of paused RNA polymerase II in HEL cells.

P-TEFb, a heterodimer of the kinase CDK9 and Cyclin T1, is a critical regulator of promoter-proximal pause release of Pol II in metazoans. It is capable of forming three larger complexes, including the super elongation complex (SEC), the BRD4/P-TEFb complex and the 7SK snRNP. In the SEC or the BRD4/P-TEFb complex, P-TEFb is enzymatically active, while in the 7SK snRNP, its activity is inhibited. The SEC consists of AFF1 or 4, ENL or AF9, ELL1, 2 or 3 and EAF1 or 2 in addition to P-TEFb, the only subunit with catalytic activity, and the noncatalytic subunits have been found to be able to regulate pause release through P-TEFb. We and others recently found that AFF1, ENL and AF9 are capable of regulating transcriptional initiation, but it is unknown yet whether AFF4 is also capable of doing so. With respect to the gene regulation selectivity of the SEC and the BRD4/P-TEFb complex, one recent study showed that in human DLD-1 cells, the SEC only regulates pause release of heat shock (HS) genes, whereas the BRD4/P-TEFb complex regulates pause release of the rest of the genes. However, it is unclear whether those mechanisms are general. In this study for the purpose of further understanding the role of AFF4 in transcriptional regulation, we found that AFF4 knockdown by RNA interference in human HEL cells decreased not only cellular level but also global chromatin occupancy of CTD serine 2 phosphorylated Pol II. Direct target genes of AFF4 were identified by RNA-seq and CUT&Tag. Notably, we found by ChIP-seq and PRO-seq that AFF4 loss also increased promoter-proximal pause of Pol II on several hundred HS and thousands of non-HS genes. Mechanistically, AFF4 promotes pause release likely by facilitating the binding of P-TEFb to Pol II. These results suggest that extent of the impact of AFF4 on pause release is likely to be context-dependent or cell-type dependent.