Regulation of the RNAPII Pool Is Integral to the DNA Damage Response [RNA-seq]

In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNAPII and genome-wide transcription shutdown. How these responses are connected has remained unclear. Here we show that damage-induced ubiquitylation of RNAPII itself, at a single lysine (RPB1 K1268), is the focal point for DNA damage response coordination. K1268-ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. It is also crucial for transcriptional shutdown, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery – indeed, depletion of the RNAPII pool underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA damage response, and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programmes, in genome instability disorders and even normal cells. To study the role of the RPB1 K1268-ubiquitylation in human cells, we generated switchover model systems (in HEK293 TRex flp In background) whereby an endogenous RPB1 transcript is depleted by highly efficient siRNAs, and simultaneously, expression of a transgenic, siRNA-resistant RPB1 is induced by doxycycline (Dox) - thus "switching over" from endogenous to transgenic RPB1. Transgenic RPB1 is expressed at near-endogenous levels, and is either WT or carries the K1268R mutation to prevent ubiquitylation at this position. Samples were UV irradiated (20 J/m2) 2 days after inducing the switchover (siRNA + Dox treatment), and collected 3h, 8h, 24h and 48 h. Untreated sample was collected at t=0 (just before UV irradiation of other samples).