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Poly(ADP-ribosylation) of P-TEFb by PARP1 disrupts phase separation to inhibit global transcription after DNA damage

Nature Cell Biology volume 24, pages 513–525 (2022)Cite this article

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Abstract

DNA damage shuts down genome-wide transcription to prevent transcriptional mutagenesis and to initiate repair signalling, but the mechanism to stall elongating RNA polymerase II (Pol II) is not fully understood. Central to the DNA damage response, poly(ADP-ribose) polymerase 1 (PARP1) initiates DNA repair by translocating to the lesions where it catalyses protein poly(ADP-ribosylation). Here we report that PARP1 inhibits Pol II elongation by inactivating the transcription elongation factor P-TEFb, a CDK9–cyclin T1 (CycT1) heterodimer. After sensing damage, the activated PARP1 binds to transcriptionally engaged P-TEFb and modifies CycT1 at multiple positions, including histidine residues that are rarely used as an acceptor site. This prevents CycT1 from undergoing liquid–liquid phase separation that is required for CDK9 to hyperphosphorylate Pol II and to stimulate elongation. Functionally, poly(ADP-ribosylation) of CycT1 promotes DNA repair and cell survival. Thus, the P-TEFb–PARP1 signalling plays a protective role in transcription quality control and genomic stability maintenance after DNA damage.

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Fig. 1: P-TEFb interacts with activated PARP1 after DNA damage.
Fig. 2: Mechanism of PARP1–CycT1 binding.
Fig. 3: PARP1 directly PARylates CycT1 in response to DNA damage.
Fig. 4: PARP1 PARylates CycT1 at multiple sites within and near the HRD.
Fig. 5: PARylation of transcription-engaged CycT1 disrupts phase separation and inhibits the hyperphosphorylation of the Pol II CTD by CDK9.
Fig. 6: PARylation of CycT1 downregulates transcriptional elongation and promotes DNA repair and cell survival after DNA damage.

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Data availability

The raw sequencing data generated in this study have been deposited in the Genome Sequence Archive of The National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (accession number: HRA000946), which are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human. The MS data have been deposited in ProteomeXchange with the dataset identifier PXD031109. All other data supporting the findings of this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank R. Greenberg, E. Song, D. Yin, D. Xu and their colleagues for providing the DSB reporter cell line and other valuable reagents, and L. Li, B. Yang and K. Yuan for excellent suggestions, discussions and technical assistance. This work was supported in part by the National Natural Science Foundation of China (grants 92053114 and 32070632 to H.L. and 81672955 to Y.X.), the Zhejiang Provincial Natural Science Foundation of China (grant LR21C060002 to H.L.), the Xiamen Southern Oceanographic Center (grant 17GYY002NF02 to X.G.) and the National Institutes of Health (grant R01AI41757 to Q.Z.).

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  1. These authors contributed equally: Huanyi Fu, Rongdiao Liu, Zixuan Jia.

Authors and Affiliations

  1. Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China

    Huanyi Fu, Zixuan Jia, Ran Li, Wenxuan Zhu, Yangqing Shao, Jun Huang & Huasong Lu

  2. State Key Laboratory of Cellular Stress Biology and Fujian Provincial Key Laboratory of Innovative Drug Target Research, School of Pharmaceutical Sciences, Xiamen University, Xiamen, China

    Rongdiao Liu, Feifeng Zhu, Yuhua Xue & Xiang Gao

  3. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA

    Rongdiao Liu, Yiyang Jin, Kunxin Luo, Huasong Lu & Qiang Zhou

  4. The MOE Key Laboratory of Biosystems Homeostasis & Protection, Life Sciences Institute, Zhejiang University, Hangzhou, China

    Jun Huang

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Contributions

H.F., R. Liu, X.G., H.L. and Q.Z. designed the research and analysed the data. H.F., R. Liu, Z.J., R. Li, F.Z., W.Z., Y.S., Y.J. and H.L. performed the experiments. Y.X., J.H. and K.L. provided valuable discussion. H.F., H.L. and Q.Z. wrote the manuscript. H.L. and Q.Z. conceived and directed the project. All the authors discussed the results and commented on the manuscript.

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Correspondence to Xiang Gao, Huasong Lu or Qiang Zhou.

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Extended data

Extended Data Fig. 1 Kinetics of CycT1 recruitment to laser-induced DNA lesions.

a, HeLa cells expressing GFP-CycT1 were subjected to laser microirradiation and examined with live-cell imaging at the indicated time points. The entry and exit points of the laser beam are indicated by arrowheads. Scale bar = 10 μM. Bottom: The relative intensities of GFP-CycT1 signals at the irradiation sites were measured at the indicated time points. b&c, HeLa cells were untreated or treated with MNNG or/and the inhibitor of ATM (ATMi) (b) or DNA-PK (DNA-PKi) (c) as indicated. Whole cell extracts were analyzed by Western blotting for the indicated proteins. d, Unmodified or in vitro PARylated mCherry-PARP1 (0.1 mg/ml) was subjected to droplet formation assay and examined by fluorescence microscopy for mCherry fluorescence. Scale bar = 10 μM. Experiments in a and d were repeated independently twice with similar results. All Western blots are representative of three independent experiments. Gel source data are available online.

Source data

Extended Data Fig. 2 CycT1 but not CDK9 in P-TEFb is PARylated in response to treatment with MNNG.

a, Schematic diagram depicting different regions on the PAR polymer that are recognized by pan-ADPr, αpADPr (10H) and pADPr, respectively. b, CycT1 was affinity-purified from HeLa cells either untreated or treated with MNNG and analyzed by Western blotting (WB) with pan-ADPr and αpADPr (10H). c, CDK9 was purified from HeLa cells, which were untreated or treated with MNNG, and analyzed by WB. d, HeLa or F1C2 cells stably expressing CDK9-F were treated with MNNG. Anti-Flag immunoprecipitates (IP) were analyzed by WB for the indicated proteins. All Western blots are representative of three independent experiments. Gel source data are available online.

Source data

Extended Data Fig. 3 PARylation of CycT1 depends on PARP1 activation and is dynamically modulated by enzymes controlling PAR synthesis and degradation in response to certain types of DNA damage.

a, CycT1 was affinity-purified from HeLa cells either untreated or treated with H2O2 and analyzed by Western blotting (WB) with pan-ADPr and pADPr. b, HeLa or F1C2 cells stably expressing CDK9-F were either untreated or treated with UV. Nuclear extracts (NE; input) and anti-Flag immunoprecipitates (IP) were analyzed by WB for the indicated proteins. c, Whole cell extracts (WCE) of cells either untreated or treated with UV were analyzed by WB for the indicated proteins. d, WCE of cells treated with the indicated agents were examined by WB with the antibodies marked on the left. e, HeLa cells were untreated or treated with H2O2 for the indicated periods of time. CycT1 immunoprecipitated from WCE were analyzed by WB. f, WCE of cells treated with MNNG for the indicated periods of time were analyzed by WB for the indicated proteins. Right: Quantification of WB signals for the indicated proteins. PAR-CycT1 was quantified based on the signals in Fig. 3c,g, The PARG protein levels in control and PARG knockdown cells were analyzed by WB. h, & i, CycT1 purified from NE of HeLa cells treated with the indicated chemicals were analyzed by WB. j, HeLa cells expressing the indicated CycT1-F proteins were untreated or treated with MNNG. The anti-Flag IP from WCE were analyzed by WB. All Western blots are representative of three independent experiments. Gel source data are available online.

Source data

Extended Data Fig. 4 Mutations of D and E residues in CycT1 IDR into A does not diminish MNNG-induced CycT1 PARylation; PARG treatment generates a homogenous pool of MARylated CycT1.

a, HeLa cells expressing the indicated CycT1-F proteins were untreated or treated with MNNG. The anti-Flag immunoprecipitates (IP) from whole cell extracts (WCE) were analyzed by Western blotting (WB). b, The immobilized af1521-Strep was incubated with WCE of HeLa cells untreated or treated with MNNG or/and PARG as indicated. The input and bound proteins were analyzed by WB. c, A schematic representation displaying PARG’s hydrolysis activity toward the PAR chain to leave behind a mono-ADP-ribose unit of 541.06 Dalton (Da). d, The Af1521-enriched CycT1-IDRL17A was untreated or treated with PARG in vitro and then analyzed by WB. All Western blots are representative of three independent experiments. Gel source data are available online.

Source data

Extended Data Fig. 5 Identification of CycT1 PARylation sites by ETD LC-MS/MS and mutation of CycT1 PARylation sites doesn’t affect the binding to PAR and P-TEFb partners.

a, The electron transfer dissociation (ETD) tandem mass spectra of the identified ADP-ribosylated peptides of CycT1, with c-ions and z-ions marked in red and blue, respectively. b, The sequence of the CycT1 IDRL region (aa 401-650) with the 12 identified PARylation sites highlighted in red and the remaining serine residues labeled in blue. c, Purified and immobilized WT or mutant CycT1-F was incubated with autoPARylated PARP1. The input and pull-down proteins were analyzed by Dot blotting (DB) or Western blotting (WB). d, Nuclear extracts (NE) of HeLa cells expressing WT or mutant CycT1-F proteins and anti-Flag immunoprecipitates (IP) from NE were analyzed by WB for the indicated proteins. All Western blots are representative of three independent experiments. Gel source data are available online.

Source data

Extended Data Fig. 6 Transcriptionally engaged CycT1 is preferentially targeted for PARylation upon MNNG-induced DNA damage, which inhibits CycT1’s phase separation.

a, & b, CycT1 affinity-purified from whole cell extracts (WCE) of HeLa cells, which were untreated or treated with the indicated chemicals, were analyzed by Western blotting (WB). c, Nuclear extracts (NE) of control or MNNG-treated HeLa cells were subjected to immunoprecipitation with anti-HEXIM1 antibody. The immunoprecipitates (IP) were analyzed by WB. d, The LARP7 protein levels in NE of HeLa cells expressing a LARP7-specific shRNA only in the presence of doxycycline (Dox) were analyzed by WB. e, HeLa cells expressing GFP-CycT1 were untreated (control) or treated with the indicated chemical(s). The distribution patterns of GFP-CycT1 were analyzed by fluorescence microscopy. Scale bar = 10 μM. f, WT and mutant GFP-CycT1-IDR were subjected to in vitro ADP-ribosylation reactions containing NAD+ or not and then analyzed by WB. g, Solutions containing 2 mg/ml of WT or mutant GFP-CycT1-IDR were subjected to droplet formation assay and examined by fluorescence microscopy for GFP signals. Scale bar = 20 μM. Right: Quantification of the sizes of droplets in each group. The box plots show the minimum, first quartile, median, third quartile and maximum with n represents the number of droplets: GFP-CycT1-IDR-WT (n = 849), GFP-CycT1-IDR-Mut2 (n = 621). h, Solutions containing GFP-CycT1-IDR Mut2 proteins that were in vitro PARylated or not were subjected to droplet formation assay and analyzed as in g. Scale bar = 25 μM. Right: Quantification of the sizes of droplets in each group. The box plots are as described in g with n represents the number of droplets: GFP-CycT1-IDR-Mut2-Control (n = 1346), GFP-CycT1-IDR-Mut2-PARylation (n = 1371). i, Liquid droplets containing GFP-CycT1-IDR (0.1 mg/ml) and mCherry-PAR-PARP1 (0.025 mg/ml) were incubated with or without NAD+ and then analyzed by WB. j, Samples analyzed in i were further examined by fluorescence microscopy for GFP and mCherry fluorescence signals. Scale bar = 25 μM. Right: Quantification of the sizes of droplets in each group. The box plots are as described in g with n represents the number of droplets: GFP-CycT1-IDR − NAD+ (n = 1410), GFP-CycT1-IDR + NAD+ (n = 2483); statistical analysis was performed using two-tailed unpaired t-tests; ****P < 0.0001. Experiment in e was repeated independently three times with similar results. g, h, j, Data are representative of three independent experiments with similar results. All Western blots are representative of three independent experiments. Gel source data are available online.

Source data

Extended Data Fig. 7 MNNG/H2O2-induced CycT1 PARylation inhibits P-TEFb’s transcriptional elongation activity.

a, A procedure for isolating newly synthesized, 4-thiouridine (4sU)-labeled RNA for subsequent analysis by real-time RT-PCR. b, Transcription from the cellular FOS and JUN gene promoters in untreated or MNNG-treated cells was investigated by 4sU-labeling followed by qRT-PCR. The mRNA levels in untreated cells were artificially set to 1.0. The error bars indicate mean ± s.d. with n = 3 biologically independent samples; statistical analysis was performed using two-tailed unpaired t-tests; ***P < 0.001. c, Transcription from the promoters of HIV-1, FOS and JUN in untreated or H2O2-treated cells was analyzed as in b. The error bars indicate mean ± s.d. with n = 3 biologically independent samples; statistical analysis was performed using two-tailed unpaired t-tests; ***P < 0.001; **P = 0.0085. d, A diagram of double strand break (DSB) induction in U2OS-265 cells. ER-mCherry-LacI-FokI-DD is stabilized and directed to LacO repeats to introduce DSBs by the addition of Shield-1 and 4-OH. Transcription of the reporter gene is induced with Doxycycline after DSB induction. e, Transcription from the reporter and GAPDH gene promoters in untreated or DSB-induced cells was measured by qRT-PCR (mean ± s.d., n = 3 biologically independent samples) and the ratios of “Reporter gene vs. GAPDH” are shown. The signals were normalized to non-DSB conditions, which were set to 1.0. f, A diagram illustrating the experimental design involving the incubation with MNNG for the indicated periods of time with the last 20 min of the incubation performed also in the presence of 5-EU to label the newly synthesized. g, HeLa cells were untreated or treated with MNNG for the indicated time periods. Newly synthesized RNA was labeled with 5-EU following the scheme in d and then detected using Alexa fluor 488. Scale bar = 50 μM. Right: Quantification of the 5-EU fluorescence intensity per cell. Red lines indicate the mean 5-EU intensity in each group. n represents the number of cells examined in a representative assay out of 3 independent experiments: MNNG 0 min (n = 181), MNNG 20 min (n = 181), MNNG 40 min (n = 180), MNNG 60 min (n = 183); statistical analysis was performed using two-tailed unpaired t-tests; ****P < 0.0001. h, HeLa cells expressing GFP-CycT1 were treated with MNNG for the indicated time periods and examined by fluorescence microscopy. Scale bar = 10 μM. Bottom: Quantification of the enrichment of GFP-CycT1, which is calculated as the ratio of mean fluorescence intensity per nuclear condensate versus the mean fluorescence intensity of the entire cell nucleus. The box plots show the minimum, first quartile, median, third quartile and maximum with n represents the number of cells examined in a representative assay out of 3 independent experiments: MNNG 0 min (n = 71), MNNG 20 min (n = 84), MNNG 40 min (n = 67), MNNG 60 min (n = 65); statistical analysis was performed using two-tailed unpaired t-tests; ****P < 0.0001. i, Volcano plot showing differential gene expression based on genome-wide sequencing of 4sU-labeled RNA in DMSO- or MNNG-treated cells. The negative log10 p-values test the significance of enrichment (y axis) and are plotted against the average log2 fold changes in expression (x axis). Genes not differentially expressed are displayed as black dots. Colored dots are genes showing at least 2-fold changes (red indicates increase and green decrease) in expression with p-values less than 0.01. p-values were determined using Fisher’s exact test. j, Scatterplot of log10 fold changes in the expression of genes in MNNG- versus i-CDK9-treated cells. The r value denotes the Pearson correlation coefficient; and the p value (< 2.2e-16) displays statistical significance of the correlation as calculated by two-tailed test. k, & l, HeLa cells were depleted of CycT1 and reconstituted with WT or Mut2 CycT1. Transcription from the JUN (k) and FOS (l) gene promoters in untreated or MNNG-treated cells was investigated by 4sU-labeling followed by qRT-PCR analysis. The mRNA levels in untreated cells were artificially set to 1.0. The error bars indicate mean ± s.d. with n = 3 biologically independent samples; statistical analysis was performed using two-tailed unpaired t-tests; ***P < 0.001.

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Extended Data Fig. 8 NELF-E, NuRD, ATM, and DNA-PK are not involved in transcriptional silencing induced by MNNG.

a, & c, HeLa cells were transfected with the siRNA targeting NELF-E (a) or MTA1 (a NuRD subunit, c) and harvested at 72 hr post-transfection to examine the mRNA levels of NELF-E (a) and MTA1 (c) by qRT-PCR analysis. The error bars indicate mean ± s.d. with n = 3 biologically independent samples; statistical analysis was performed using two-tailed unpaired t-tests; ***P < 0.001. b, & d, HeLa cells were transfected with control siRNA (siCon) or a specific siRNA targeting NELF-E (b) or MTA1 (d) and then treated with DMSO or MNNG as indicated. Newly synthesized RNA was labeled with 5-EU and detected using Alexa fluor 488. Scale bar = 50 μM. Right: Quantification of the 5-EU fluorescence intensity per cell. Red lines indicate the mean 5-EU intensity in each group. n represents the number of cells examined in a representative assay out of 3 independent experiments: DMSO siCon in b (n = 189), DMSO siNELF-E (n = 170), MNNG siCon in b (n = 157), MNNG siNELF-E (n = 190), DMSO siCon in d (n = 203), DMSO siMTA1 (n = 205), MNNG siCon in d (n = 198), MNNG siMTA1 (n = 193). e, HeLa cells were untreated or pre-treated with ATMi or DNA-PKi and then treated with DMSO or MNNG as indicated. Newly synthesized RNA was detected and quantified as in b. Scale bar = 50 μM. Right: Quantification of the 5-EU fluorescence intensity per cell. Red lines indicate the mean 5-EU intensity in each group. n represents the number of cells examined in a representative assay out of 3 independent experiments: DMSO-Control (n = 158), DMSO-ATMi (n = 160), DMSO-DNAPKi (n = 133), MNNG-Control (n = 152), MNNG-ATMi (n = 144), MNNG- DNAPKi (n = 149).

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Extended Data Fig. 9 PARP1 activity is required to promote DNA repair and cell survival upon DNA damage.

a, HeLa cells were depleted of CycT1 and reconstituted with WT or Mut2 CycT1. Cell viability was measured before or after the exposure to the indicated doses of UV. The error bars indicate mean ± s.d. with n = 3 biologically independent samples. b, Control or AZD2281-treated HeLa cells depleted of CycT1 and reconstituted with WT or Mut2 CycT1 were either mock treated or treated with IR (5 Gy) and allowed to recover for the indicated time periods. WCE was examined by WB to detect γH2AX and actin. c, Control or AZD2281-treated HeLa cells depleted of CycT1 and reconstituted with WT or Mut2 CycT1 were analyzed by immunofluorescence staining at 12 hr after IR (5 Gy) treatment with the anti-γH2AX antibody. DNA was counterstained by DAPI. Scale bar = 10 μM. Right: Quantification of the number of γH2AX foci per cell. Red lines indicate the mean γH2AX foci in each cell populations. n represents the number of cells examined in a representative assay out of 3 independent experiments: CycT1-WT-DMSO (n = 148), CycT1-WT-AZD2281 (n = 110), CycT1-Mut2-DMSO (n = 138), CycT1-Mut2-AZD2281 (n = 110). All Western blots are representative of three independent experiments. Gel source data are available online.

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Extended Data Fig. 10 A working model.

A model illustrating how the PARP1-CycT1 signaling pathway senses base lesions or strand breaks caused by certain DNA damaging agents and induces CycT1 PARylation to inhibit P-TEFb phase separation and P-TEFb-dependent Pol II elongation.

Supplementary information

Supplementary Information

Flow cytometry gating strategy associated with Fig. 6k.

Reporting Summary

Peer Review File

Supplementary Table 1

List of MS-identified proteins.

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Fu, H., Liu, R., Jia, Z. et al. Poly(ADP-ribosylation) of P-TEFb by PARP1 disrupts phase separation to inhibit global transcription after DNA damage. Nat Cell Biol 24, 513–525 (2022). https://doi.org/10.1038/s41556-022-00872-5

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