Transcriptional elongation requires DNA break-induced signalling

Abstract

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 signalling in the mechanisms of transcriptional elongation in stimulus-inducible genes in humans. Our data show the enrichment of TRIM28 and γH2AX on serum-induced genes and the important function of DNA-PK for Pol II pause release and transcriptional activation-coupled DDR signalling on these genes. γH2AX accumulation decreases when P-TEFb is inhibited, confirming that DDR signalling results from transcriptional elongation. In addition, transcriptional elongation-coupled DDR signalling involves topoisomerase II because inhibiting this enzyme interferes with Pol II pause release and γH2AX accumulation. Our findings propose that DDR signalling is required for effective Pol II pause release and transcriptional elongation through a novel mechanism involving TRIM28, DNA-PK and topoisomerase II.

Creative damage unleashes transcription

“…2 It has been shown that some of the components of the NHEJ pathway such as DNA-dependent kinase (DNA-PK), Ku70 and Ku80 become associated with activated genes, including estrogen-induced genes, heat shock genes and serum-inducible immediate early (IE) genes. 4,6 The NHEJ intermediates were shown to be associated with ERinduced genes in a complex with Poly (ADP-Ribose) Polymerase 1 (PARP1), Topoisomerase IIb and transcription appeared to require Topoisomerase IIb-mediated nicking of sequences within the promoter of the presenilin 2 (pS2) gene 4 (Fig. 1).…”

“…Inducible gene transcription has been shown to be accompanied by the generation of DNA double strand breaks (DSB) in cells activated by agents as diverse as heat shock, growth serum, androgens, neuronal activation or estrogens. 1,[4][5][6][7] Increased DSB have been detected directly in transcribing cells, as well as indirectly by the Comet assay and by incorporation into chromatin of the histone gH2Ax, a surrogate marker of DNA damage. [5][6][7] Indeed direct generation of DSB in the promoters of the EGR1 and NPAS4 genes, using a CRISPR-CAS approach, was sufficient to drive transcription even in the absence of external stimuli.…”

“…1,[4][5][6][7] Increased DSB have been detected directly in transcribing cells, as well as indirectly by the Comet assay and by incorporation into chromatin of the histone gH2Ax, a surrogate marker of DNA damage. [5][6][7] Indeed direct generation of DSB in the promoters of the EGR1 and NPAS4 genes, using a CRISPR-CAS approach, was sufficient to drive transcription even in the absence of external stimuli. 7 Repair of DSBs requires DNA recombination repair by mechanisms including homologous recombination (HR) and non-homologous end joining repair (NHEJ).…”

“…In fact a role for Topoisomerase IIb in transcriptional activation appeared to be a general finding in a number of the studies. 4,6,7 It is not clear which exact stimulus provokes Topoisomerase IIb to bind and induce DSBs in transcribing genes, although direct recruitment of the enzyme by the androgen receptor has been observed. 1 The roles of Topoisomerase IIb nicking and recruitment of the NHEJ complex in transcription are still to be fully determined.…”

“…7 In addition, DNA-PK and Topoisomerase II may play roles in the regulation of transcription by the relief of posttranscriptional Pol II pausing in HSP and IE genes. 6 A mechanism involving phosphorylation by DNA-PK of the Pol II pausing regulator Kap1/TRIM28 appeared to be required for transcriptional activation. 6 It is notable that in the latter studies, elevated levels of activated phospho-DNA-PK and gH2AX were detected throughout the ORFs of transcribing IE genes.…”