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Stabilization of AFF1 by PARylation ensures transcriptional restart after DNA damage

Nature Chemical Biology (2025)Cite this article

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Abstract

Precise control of transcription is essential for cell survival under stress conditions, including DNA damage. While mechanisms of DNA damage-induced transcriptional silencing are well characterized, how transcription resumes remains less understood. Here we identify a new role for poly(ADP-ribose) polymerase 1 (PARP1) in transcriptional restart during the DNA damage response (DDR) through a mechanism termed poly(ADP-ribose)-mediated stabilization (PARSTA) of AFF1. Upon DNA damage, PARP1 binds to and PARylates AFF1 in a region targeted by the E3 ligase Siah1, preventing AFF1 ubiquitination and promoting its stability. This stabilization supports efficient transcriptional recovery after DNA damage. Notably, cells resistant to genotoxic stress exhibit elevated PARP1 activity and AFF1 levels, while AFF1 depletion impairs DNA repair and survival. Together, these findings expand PARP1’s role to the transcriptional recovery phase in DDR and suggest that targeting the PARSTA pathway may offer therapeutic potential in diseases characterized by hyperactive PARP1 and elevated levels of AFF1.

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Fig. 1: AFF1 responds to DNA damage in a PARP1-dependent manner.
Fig. 2: DNA damage induces PARP1–AFF1 interaction.
Fig. 3: AFF1 is PARylated by PARP1 following DNA damage.
Fig. 4: PARylation of AFF1 enhances protein stability by disrupting its interaction with Siah1.
Fig. 5: AFF1 is required for transcriptional recovery after DNA damage.
Fig. 6: The PARP1–AFF1 axis is essential for cell survival in response to DNA damage.

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Data availability

All data that were generated and/or analyzed in this study are included in the article and the Supplementary Information. The raw sequencing data generated in this study were deposited to the Genome Sequence Archive of the National Genomics Data Center (HRA010143). MS data were uploaded to ProteomeXchange (PXD049977). Source data are provided with this paper.

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Acknowledgements

We thank the core facility of the Life Sciences Institute for technical assistance. This work was supported in part by the National Key R&D Program of China (2020YFA0908100), the National Natural Science Foundation of China (32570646, 32370591 and 32470588), the Zhejiang Provincial Natural Science Foundation of China (LRG25C060001), the Natural Science Foundation of Xiamen (3502Z202473020), the Scientific Research Foundation of State Key Laboratory of Vaccines for Infectious Diseases, the Xiang An Biomedicine Laboratory (2023XAKJ0102052) and the Fundamental Research Funds for the Central Universities (226-2025-00028).

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Author notes

  1. These authors contributed equally: Feifeng Zhu, Huanyi Fu, Wenxuan Zhu, Chengyu Li.

Authors and Affiliations

  1. Fujian Provincial Key Laboratory of Innovative Drug Target Research, School of Pharmaceutical Sciences, Affiliated Xiamen Eye Center & Xiang’an Hospital, Xiamen University, Xiamen, China

    Feifeng Zhu, Yafei Guo, Mengting Huang, Yaohui He & Yuhua Xue

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

    Feifeng Zhu, Huanyi Fu, Wenxuan Zhu, Chengyu Li, Min Yang, Zeming Feng, Yangqing Shao, Zhuo Li, Yitong Zhang, Huipeng Jiao, Jun Huang, Long Zhang & Huasong Lu

  3. State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Xiamen University, Xiamen, China

    Feifeng Zhu, Yafei Guo, Mengting Huang, Yuhua Xue & Huasong Lu

  4. School of Biological Sciences, Faculty of Science, The University of Hong Kong, Hong Kong, China

    Huanyi Fu & Qiang Zhou

  5. Tongji University Cancer Center, Shanghai Tenth People’s Hospital, School of Medicine, Tongji University, Shanghai, China

    Yi Lu

  6. Department of Oncology, Affiliated Zhejiang Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Zhibing Wu

  7. The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory of Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China

    Jun Huang & Long Zhang

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  1. Feifeng Zhu
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Contributions

F.Z., H.F., W.Z. and C.L. performed the majority of the molecular biology and biochemistry experiments. M.Y., Z.F., Y.S., Z.L., Y.G., M.H., Y.Z. and Y.H. helped with the imaging and MS experiments. F.Z., H.F. and W.Z. analyzed the data. Y.L., Z.W., Q.Z., H.J., J.H. and L.Z. provided valuable discussion. F.Z., Y.X. and H.L. designed and supervised the project and wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Yuhua Xue or Huasong Lu.

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Nature Chemical Biology thanks Xiaohua Shen and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Proteomic analysis of chromatin-bound factions in response to DNA damage.

a, Correlation analysis depicting the TMT labeling quantification of chromatin-bound fractions derived from cells untreated or treated with H2O2. Quantitative mass spectrometry (MS) analysis for each group was performed in triplicate as indicated by 1-3. b, c, Gene Ontology analysis of up-regulated proteins (b) or down-regulated proteins (c) bound to chromatin after H2O2 treatment. GO enrichment analysis was conducted using Metascape, with statistical significance calculated by cumulative hypergeometric test (default setting). Only terms with Min Enrichment ≥ 1.5, Min Overlap ≥ 3 and P < 0.01 were retained. Bar plots display –log₁₀ (P value) of enriched terms for up-regulated (b) and down-regulated (c) proteins after H2O2 treatment. d, Functional interaction network of proteins showed increased binding to PAR-PARP1 and chromatin retention after H2O2. e, f, Lysates from HeLa control, or PARP1 KD (e) or PARP2 KD (f) cells were analyzed by WB. g, h, Control or PARP1 KD (g) or PARP2 KD (h) cells were untreated or treated with 2 mM H2O2 for 30 min. After fractionation, soluble and chromatin-bound fractions 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. 2 AFF1 binds to PARP1.

a, Confocal microscopy images of cells expressing different GFP-AFF1 variants. b, Lysates from HeLa cells expressing different GFP-AFF1 variants were analyzed by WB. c, HeLa cells expressing different GFP-AFF1 variants were subjected to co-immunoprecipitation assays. Input and anti-Flag IPs were analyzed by WB. d, Immobilized PARP1-Strep or PAR-PARP1-Strep was incubated with purified F-AFF1 for pull-down (PD) assays. Input and PD samples were analyzed by WB. e, g, Quantification of PLA assay shown in Fig. 2g (e) and Fig. 2h (g). Red lines indicate the mean intensity in each group. n = 20. f, HeLa cells expressing GFP or GFP-AFF1 were subjected to laser microirradiation and then analyzed by immunofluorescence staining with anti-PARP1. Bottom: line plot of fluorescence intensity along the dashed line. Scale bars, 20 μM in a and 10 μM in f. Experiment in a was representative of three independent experiments with similar results. Experiment in f was repeated twice, yielding similar results. All Western blots are representative of three independent experiments. Gel source data are available online.

Source Data

Extended Data Fig. 3 AFF1 is PARylated by PARP1.

a, f, g, h, WB of affinity-purified AFF1-F from cells exposed to increasing IR doses (a), or H2O2 for indicated times (f), or H2O2 ± SK575 (g), or H2O2 ± DNA-PK/ATM inhibitors (h). b, WB of Input and affinity-purified AFF1-F from cells ± H2O2 using pan-ADPr, poly-ADPr, mono-ADPr antibodies as indicated. c, Purified and immobilized af1521-Strep was incubated with lysates from AFF1-F-expressing cells ± H2O2. Input and PD samples were analyzed by WB. d, WB of affinity-purified AFF1-F from soluble / chromatin fractions of cells ± H2O2. e, WB of affinity-purified various SEC subunits from cells ± H2O2. i, j, WB of affinity-purified AFF1-F from control or PARP2 KD (i) or PARG KD (j) cells ± H2O2. k, m, Lysates from HeLa control or PARG KD (k) or HPF1 KD (m) cells were analyzed by WB. l, Affinity-purified PARylated AFF1-F were treated with PARG or NH2OH, followed by WB analysis. n, Chromatin retention of GFP-AFF1 in H2O2-exposed cells pretreated ± ActD / DRB following pre-extraction. Bottom: percentage of cells showing nuclear signal (n = 3 biologically independent replicates). Error bars indicate mean ± s.d. o, WB of soluble / chromatin fractions from cells pretreated with DRB ± H2O2. p, Quantification of GFP-AFF1 enrichment at damage sites following treatment with DMSO, ActD or AZD2281 (n = 19). Box plots show minimum, quartiles, and maximum that are representative of three independent experiments. q, WB of affinity-purified AFF1-F from cells pretreated with ActD or DRB ± H2O2, and then detected via Odyssey imaging. Scale bars, 10 μM. All Western blots are representative of three independent experiments. Gel source data are available online.

Source Data

Extended Data Fig. 4 Identification of AFF1 PARylation sites by MS.

a, WB of affinity-purified AFF1-601-900-F from control or HPF1 KD cells ± H2O2. b, Confocal microscopy images of cells expressing different AFF1 variants. Scale bar, 20 μM. c, WB of affinity-purified AFF1-F variants as indicated from cells ± H2O2. d, WB of in vitro PARylation of AFF1-601-900-F using recombinant PARP1 and indicated components. e, f, g, Fully annotated ETD tandem mass spectra of representative peptides containing an ADP-ribosylated residue, with both c-ions (red) and z-ions (blue) pinpointing the modification site. h, The sequence of the AFF1 aa 601-900 region with the 16 identified PARylation sites highlighted in red and the remaining serine residues in blue. i, HeLa cells expressing Flag-tagged AFF1-WT, -M1 or -M2 were subjected to co-immunoprecipitation assays. Input and anti-Flag IPs were analyzed by WB. j, Purified and immobilized Flag-tagged AFF1-WT, -M1 or -M2 proteins were incubated with PAR for in vitro binding analysis. Input and PD samples were analyzed by dot blotting or WB. All Western blots are representative of three independent experiments. Gel source data are available online.

Source Data

Extended Data Fig. 5 AFF1 becomes stabilized after PARylation.

a, b, HeLa cells expressing Flag-tagged AFF1-WT (a) or AFF1-M2 (b) or not (-) were untreated or treated with 2 mM H2O2 for 30 min. Input and anti-Flag IPs were analyzed by WB. c, HeLa cells expressing AFF1-M2 and/or CycT1-Mut2 as indicated were subjected to co-IP assays. Input and anti-Flag IPs were analyzed by WB. d, e, HeLa cells expressing AFF1-F were pretreated with DMSO or AZD2281 (e), or left untreated (d), and then exposed to 0.25 mM H2O2 or 1 μM Doxorubicin for the indicated times. Affinity-purified AFF1-F proteins were analyzed by WB. f, HeLa cells expressing Flag-tagged AFF1-WT or AFF1-M2 were exposed to 0.25 mM H2O2 or 1 μM Doxorubicin for the indicated times and then analyzed as in e. g, h, WB of affinity-purified AFF1-WT or -2R from cells untreated or treated with 1 μM Doxorubicin (g) or 2 mM H2O2 (h). i, j, k, l, b, HeLa cells were treated with CHX to block protein synthesis and harvested at indicated times after no treatment or treatment with MG132 (i) or MNNG (j) or MMS (k) or UV (l). m, Control or PARP1 KO cells expressing PARP1-E988A were treated ana analyzed as in j. n, HeLa cells expressing Flag-tagged AFF1-WT or AFF1-M2 were treated ana analyzed as in j. o, The mRNA levels of Siah1 in control or Siah1 KD cells were measured by qRT-PCR. Error bars indicate mean ± s.d., n = 3 biologically independent samples. p, HeLa cells expressing AFF1-F and/or His-Ub were treated with 2 mM H2O2 for 30 min and then followed by MG132 incubation for 8 hrs. Cells were harvested for Ni-NTA pull-down assays and analyzed by WB. All Western blots are representative of three independent experiments. Gel source data are available online.

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Extended Data Fig. 6 Depletion of AFF1 attenuates transcriptional recovery after DNA damage.

a, b, Metaplots of average TT-seq signals for genes (10-25 kb, 25-50 kb, 50-100 kb; a) or AFF1 targets (b) in control/AFF1 KD cells at different times after H2O2 treatment. c, Box plot of log2 fold change in TT-seq signals of AFF1 targets at 2.5 h post-H2O2 vs untreated. Box plots show minimum, quartiles, and maximum, n = 10229. d, GO analysis of 2311 AFF1 targets with impaired recovery in KD cells. e, Metaplots of spike-in normalized TT-seq signals in control or AFF1 KD cells at different time points post-H2O2. f, h, Heatmaps of pSer2 occupancy across gene bodies in control (f) or AFF1 KD (h) cells post-H2O2. g, i, Metaplots of pSer2 ChIP-seq signals in control (g) or AFF1 KD (i) cells at different time points post-H2O2. j, k, Metaplots showing the average level of nascent transcription of genes in control (j) or AFF1 KD (k) cells. Wavefront positions are defined as the distance from the TSS where the reads density drops below an arbitrary threshold (dashed line). l, WB of lysates from AFF1 KD cells reconstituted with AFF1-WT or M2 ± 0.25 mM H2O2 for 5 min, followed by recovery for the indicated times. m, HeLa cells pre-treated with DMSO or AZD2281 were treated ± H2O2 for 5 min and allowed to recover for the indicated times. Nascent RNA was labeled with 5-EU for 20 min and quantified by fluorescence. Quantification: relative fluorescence intensity per cell normalized to mock (set to 100). Red lines indicate mean intensity. n = 97,100,109,74,95,108, left to right. Scale bars = 20 μM. Statistical analysis was performed using two-tailed unpaired t-tests. ****P < 0.0001. All Western blots are representative of three independent experiments. Gel source data are available online.

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Extended Data Fig. 7 Depletion of AFF1 enhances the sensitivity of cancer cells to genotoxic stress.

a, Representative colony formation assay images (as Fig.7a). b, k, WB of lysates from control or AFF1 KD HeLa (b) or CR (k) cells. c, Colony formation of control or AFF1 KD cells with increasing IR doses (n = 3). d, o, Control or AFF1 KD HeLa (d) or CR (o) cells were exposed to 2 Gy IR and then harvested at the indicated time points for WB analysis. e, γH2AX immunofluorescence in control or AFF1 KD cells after 5 Gy IR. Right: quantification of γH2AX foci per cell. Red lines indicate mean, n = 100. f, Alkaline comet assays of control, PARP1 KD, or AFF1 KD cells at the indicated time points after treatment with 0.25 mM H2O2 for 5 min. Quantification showing the tail moment per cell. Red lines indicate the mean in each group. n = 98. Statistical analysis was performed using two-tailed unpaired t-tests. ****P < 0.0001, ***P < 0.001. g, m, Colony formation of CNE2 or CR cells (g, n = 3) or CR control or AFF1 KD cells (m, n = 3) with increasing IR doses. h, Crystal violet staining of CNE2 or CR cells ± AZD2281 1 week after 10 Gy IR. i, LFQ intensities of indicated proteins in CNE2 or CR cells. j, qRT-PCR analysis of AFF1 mRNA levels. l, Proliferation curves of control, AFF1 KD, and AFF4 KD CR cells (n = 3). n, SA-β-gal staining of control or AFF1 KD CR cells 3 days after 6 Gy IR. Right: quantification of the number of SA-β-gal positive cells per field. p, q, Graphs of isolated tumors in Fig.7k (p) or Fig.7 m (q). All error bars indicate mean ± s.d. with n = 3 biologically independent samples. All Western blots are representative of three independent experiments. Gel source data are available online.

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Zhu, F., Fu, H., Zhu, W. et al. Stabilization of AFF1 by PARylation ensures transcriptional restart after DNA damage. Nat Chem Biol (2025). https://doi.org/10.1038/s41589-025-02045-5

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