A single RNA polymerase II ubiquitylation site coordinates transcription with the DNA damage response
Description
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.
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Cells were lysed in 9 M urea, 20mM HEPES (pH=7.8), supplemented with 100 units/ml of benzonase and sonicated to reduce viscosity (3 mm probe, 50 % amplitude, 3 x 15 sec bursts, on ice). Total of 10 mg of protein per sample were used as estimated by Bradford protein assay. Lysates were reduced with 10 mM dithiothreitol (DTT) for 30 min at room temperature, followed by alkylation with 20 mM chloroacetamide for 30 min at room temperature in the dark. Lysates were digested initially with LysC (Promega) for 2 hours at 37⁰C. The lysates were then diluted with 20 mM HEPES, 5 % acetonitrile to a final urea concentration of less than 2 M. The samples were digested 1:100 enzyme to protein ratio (w/w) with trypsin (Promega) overnight at 37⁰C. The next day, two additional aliquots of trypsin were added and incubated at 37 ⁰C four hours each. After the digestion the samples were acidified with TFA (Thermo Fisher Scientific) to final concentration of 1 % (v/v). All insoluble material was removed by centrifugation and the supernatant was desalted with C18 SepPak solid-phase extraction cartridges (SPE) (Waters) and lyophilized for 2 days. Peptides containing the diGly remnant were enriched using K-ϵ-GG affinity resin (Cell Signaling Technology) according to the manufacturer's instructions. Samples enriched for peptides containing the diGly remnant were resuspended in 20 μl of 20 mM HEPES (pH 8.5). Isobaric labeling of the peptides was performed using the 10- plex tandem mass tag (TMT) reagents. For fractionation the high pH reversed-phase peptide fractionation kit (Thermo Fisher Scientific) was used. Peptide mixtures from TMT 10-plex labelled samples were chromatographically resolved on an EASY-spray PepMap RSLC C18 column (2μm, 100Å, 75μm X 50cm ID) using an Ultimate 3000 RSLCnano system over a 180 min gradient at 40°C. The peptides were separated using linear gradient of 2-35 % solvent B over 153 min. All spectra wereacquired using an Orbitrap Fusion Lumos Tribrid mass spectrometer. Xcalibur 2.0 software was used to control data acquisition. The instrument was operated in data dependent acquisition mode with top scan speed set at 3 s. MS1 spectra were acquired in the Orbitrap at a resolution of 120 000 and an ion target of 4E5. The MS2 precursors were isolated using the quadrupole and analyzed in the Orbitrap at 60 000 resolution, with an AGC target of 1E5 and a max injection time of 105 ms. Precursors were fragmented by HCD at a normalized collision energy of 38 %. For the SPS-MS3 method precursors were fragmented by CID at a normalized collision energy (NCE) of 35 %. Following acquisition of each MS2 spectrum, SPS MS3 scan was collected on the top 10 most intense ions in the MS2 spectrum when the diagnostic 344.2137 ion was detected. SPS-MS3 precursors were fragmented by high energy collision-induced dissociation and analyzed using the Orbitrap. All raw data files were analysed using the MaxQuant computational platform.