Transcriptional elongation requires DNA break-induced signaling

We have previously shown that RNA polymerase II (Pol II) pause release and transcriptional elongation involve phosphorylation of the factor TRIM28 by the DNA damage response (DDR) kinases ATM and DNA-PK. Here, we report a significant role for DNA breaks and DDR signaling in the mechanisms of transcriptional elongation in stimulus-inducible genes in humans. Our data show the enrichment of TRIM28 and ?H2AX on serum-induced genes and the important function of DNA-PK for Pol II pause release and transcriptional activation-coupled DDR signaling on these genes. ?H2AX accumulation decreases when P-TEFb is inhibited, confirming that DDR signaling results from transcriptional elongation. In addition, transcriptional elongation-coupled DDR signaling involves topoisomerase II because inhibiting this enzyme interferes with Pol II pause release and ?H2AX accumulation. Our findings propose that DDR signaling is required for effective Pol II pause release and transcriptional elongation through a novel mechanism involving TRIM28, DNA-PK, and topoisomerase II Overall design: 44 samples in total. IP targets were gammaH2ax, s2-pol-II, pol-II, pTRIM28, and DNA-pk. Experimental conditions included DMSO treatment (control), pTEFb, topoII-i, dnapk-i. Matched non-specific IP samples used for control in peak calling.