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A SETD2–CDK1–lamin axis maintains nuclear morphology and genome stability

Nature Cell Biology volume 27, pages 1327–1341 (2025)Cite this article

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Abstract

Histone methyltransferases regulate chromatin organization and are frequently mutated in human diseases, including cancer. One such often mutated methyltransferase, SETD2, associates with transcribing RNA polymerase II and catalyses H3K36me3—a modification that contributes to gene transcription, splicing and DNA repair. Although its catalytic function is well-characterized, its non-catalytic roles remain unclear. Here we reveal a catalysis-independent function of SETD2 in nuclear lamina stability and genome integrity. Through its intrinsically disordered amino terminus, SETD2 associates with lamina-associated proteins, including lamin A/C, lamin B1 and emerin. Loss of SETD2 or its N terminus leads to severe nuclear morphology defects and genome instability, mirroring lamina dysfunction. Mechanistically, the N terminus of SETD2 serves as a scaffold for the mitotic kinase CDK1 and lamins, facilitating lamin phosphorylation and depolymerization during mitosis. Restoration of the N-terminal regions required for interaction with CDK1 and lamins rescues nuclear morphology and suppresses tumorigenic growth in a clear cell renal cell carcinoma model with SETD2 haploinsufficiency. These findings reveal a previously unrecognized role of SETD2 in nuclear lamina organization and genome maintenance that probably extends to its role as a tumour suppressor.

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Fig. 1: SETD2 interacts with nuclear lamina proteins.
Fig. 2: SETD2 loss disrupts nuclear lamina architecture.
Fig. 3: SETD2 absence impairs lamin phosphorylation during G2/M.
Fig. 4: Non-enzymatic function of SETD2 in the maintenance of nuclear morphology.
Fig. 5: The SETD2 N terminus associates with the mitotic lamin kinase CDK1.
Fig. 6: The SETD2 N terminus enhances CDK1-mediated lamin phosphorylation and is required to suppress ccRCC tumour growth.
Scheme 1: Modified synthesis of EPZ-719.

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Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD040771. Source data are provided with this paper.

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Acknowledgements

We thank all members of the Strahl and Davis laboratories for their valuable input. We thank S. Prasanth and M. Emanuele for critical comments on the manuscript, R. Berlow and M. Begley for advice related to protein expression, W. Salmon for advice and helpful suggestions related to microscopy, K. Ikegami for sharing LMNA mutant cell lines, W. Legant and F. Rahman for help with microscopy, J. Cook for sharing cell lines and reagents and Z. Mayo for help with the initial experiments. We thank the UNC Proteomics Core Facility and the UNC Hooker Imaging Core Facility, which are supported in part by a NCI Cancer Center Core Support Grant (grant number 2P30CA016086-45) to the UNC Lineberger Comprehensive Cancer Center. This work was supported by an NIH grant to B.D.S. (grant number GM126900), a DOD grant (grant number W81XWH2110786) to F.M.M., an NIH grant (grant number R01CA275082) to F.M.M. and W.K.R., and a CPRIT award (grant number RR190058) to Q.Z. L.V. is supported by NIH 5T32CA009592.

Author information

Author notes

  1. These authors contributed equally: Cheng Zhang, Phu G. Nguyen, James M. Metts.

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Abid Khan, Phu G. Nguyen, James M. Metts, Lucas C. Collins, Kanishk Jain, Amanda L. Brademeyer & Brian D. Strahl

  2. Department of Pathology, UT Southwestern Medical Center, Dallas, TX, USA

    Cheng Zhang & Qing Zhang

  3. Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Twin Cities, Minneapolis, MN, USA

    Kanishk Jain

  4. UNC Proteomics Core Facility, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    C. Allie Mills & Laura E. Herring

  5. The James Cancer Hospital, Ohio State University Comprehensive Cancer Center, Columbus, OH, USA

    Logan Vlach, W. Kimryn Rathmell & Frank M. Mason

  6. Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Kelin Li & Jeffrey Aubé

  7. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Brittany M. Bowman, Michael B. Major, Ian J. Davis & Brian D. Strahl

  8. Department of Cell Biology & Physiology, Washington University in St. Louis, St. Louis, MO, USA

    Brittany M. Bowman & Michael B. Major

  9. Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Ian J. Davis

  10. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center, Dallas, TX, USA

    Qing Zhang

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Contributions

A.K. and B.D.S. conceived the study. A.K., P.G.N. and J.M.M. performed all the experiments with help from L.C.C. and A.L.B. C.Z. performed the 3D colony growth assays and mice experiments. K.J. purified proteins. W.K.R., F.M.M. and L.V. shared dTAG-related reagents. B.M.B. and M.B.M. helped with initial mass spectrometry experiments. C.A.M. and L.E.H. performed mass spectrometry analyses. K.L. and J.A. synthesized EPZ-719. I.J.D. and Q.Z. provided technical resources and conceptual ideas. A.K. and B.D.S. analysed data and wrote the paper with input from all authors.

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Correspondence to Brian D. Strahl.

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B.D.S. is a co-founder and board member of EpiCypher, Inc. and holds equity in the company. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 SETD2 interacts with nuclear lamina proteins.

a, Schematic of SETD2 with its indicated domains. b, Fluorescence intensity profiles of SETD2, lamin A/C and DAPI in interphase or mitotic cells. Scale bar, 10 µm. c, Proximity ligation assay (PLA) on SETD2-KD cells expressing Halo-Flag-tagged SETD2 using anti-SETD2 and anti-lamin A/C antibodies. PLA pairs are in green; SETD2 (red) visualized using fluorescent Halo ligand JFX-549. DNA was counterstained with DAPI. Scale bar, 10 µm. d, Quantitative analysis of PLAs between SETD2 and lamin A/C shown in c. Two-tailed unpaired Student’s t-test was performed from two independent biological replicates, with n (left to right) = 55, 41, 7 cells; mean ± s.e.m.

Extended Data Fig. 2 SETD2 loss during G2/M leads to nuclear lamina defects.

a, Representative confocal microscopy images of control and SETD2-KD HKC cells immunostained with anti-lamin A/C and lamin B1 antibodies. DNA counterstained with DAPI. b, Representative confocal microscopy images of control and Setd2-KO MEF cells immunostained with anti-lamin B1 antibody. DNA counterstained with DAPI. c, Schematic of cell synchronization by double thymidine block and release method used in d–f. d, Immunoblot analysis of indicated proteins in untreated and dTAG-47 treated (at 6 h post release) cells. e, Representative images of either untreated or dTAG-47 treated SETD2–FKBP cells, fixed at the indicated timepoints post release from thymidine block and immunostained with anti-lamin A/C antibody. DNA counterstained with DAPI. f, Quantification of nuclear defects observed in e. Ordinary one-way ANOVA with Sidak’s multiple comparison test was performed on data from two independent biological replicates, with n (left to right) = 614, 558, 735, 654, 754, 693 cells; mean ± s.e.m. g, Schematic of cell synchronization by serum starvation of Setd2 (w/flox) MEFs. h, Immunoblot analysis of indicated proteins in untreated and tamoxifen treated (Setd2-KO) MEF cells upon release from G0/G1 block. i, Representative images of either control or tamoxifen-treated MEF cells at the indicated timepoints post release from serum starvation, immunostained with anti-lamin A/C antibody. j, Quantification of nuclear defects from data in i. Two-way ANOVA with Sidak’s multiple comparison test was performed on data from three independent replicates with n (left to right) = 184, 166, 247, 223, 256, 247, 387, 348 cells; mean ± s.d. All scale bars are 10 µm.

Source data

Extended Data Fig. 3 Nuclear lamina defects caused by SETD2 loss arise from events dysregulated during G2/M.

a, Western blot analysis of indicated proteins from control and SETD2-KD RPE cells that are either arrested in G0/G1 or growing asynchronously (Async). b, Representative images of control and SETD2-KD RPE cells that are either proliferating (Async) or arrested in G0/G1, immunostained with anti-lamin A/C antibody. DNA counterstained with DAPI. Scale bars, 10 µm. c, Quantification of nuclear defects observed in b. Two-tailed unpaired Student’s t-test was performed from two independent biological replicates with n (left to right) = 357, 318, 395, 424 cells; mean ± s.d. d, Representative flow cytometry analysis of control and SETD2-KD RPE cells growing asynchronously. e, Representative flow cytometry analysis of control and SETD2-KD RPE cells arrested in G0/G1. Y-axis is EdU intensity and x-axis DNA content. f, Immunoblot analysis of indicated proteins in two independent replicates of control and SETD2-KD HKC cells. g, Quantitative analysis of γH2AX intensity in untreated (control) and dTAG-47 treated SETD2–FKBP HKC cells that were synchronized by the double thymidine block method and released for indicated timepoints. Integrated intensity per nucleus was measured from n (left to right) = 2,349, 2,180, 2,044, 2,061, 3,188, 3,191 cells. Kruskal–Wallis with Dunn’s multiple comparisons test was performed on data from two independent biological replicates; medians in red.

Source data

Extended Data Fig. 4 SETD2 loss impairs lamin phosphorylation during G2/M.

a, Representative time-lapse images of control and SETD2-depleted HKC expressing Emerald-lamin A (green). DNA was labelled with SiR-Hoechst (red). Arrowheads point to partially depolymerized lamin filaments observed throughout mitosis. b, Representative flow cytometry analysis of control and SETD2-KD cells to determine cell-cycle distribution. Y-axis is EdU intensity and x-axis DNA content. c, Quantitation of flow cytometry analysis data showing cell-cycle distribution of control and SETD2-KD cells. Two-tailed unpaired Student’s t-test was performed from three independent biological replicates; mean ± s.d. d, Quantitative analysis of immunofluorescence data from control and SETD2-KD RPE cells. Integrated fluorescence intensity of either S22ph- or S392ph-lamin A signal from mitotic cells normalized to integrated intensity of pan lamin A/C signal in control (n = 505, S22ph; n = 390, S392ph) and shSETD2 (n = 490, S22ph; n = 382, S392ph) cells from two independent biological replicates. Two-tailed non-parametric Mann–Whitney U-test was performed; medians in red. e, Quantitative analysis of immunofluorescence data from control and Setd2-KO MEF cells. Integrated fluorescence intensity of either S22ph- or S392ph-lamin A signal from mitotic cells normalized to integrated intensity of pan lamin A/C signal in control (n = 515, S22ph; n = 407, S392ph) and Setd2-KO (n = 457, S22ph; n = 327, S392ph) cells from two independent biological replicates. Two-tailed non-parametric Mann–Whitney U-test was performed; medians in red. f, Representative images of LMNA KO human fibroblasts cells expressing either WT, S- > A (S22A; S392A) or S- > D (S22D; S392D) mutant lamin A. Cells were immunostained with lamin A/C antibody (red) and DNA counterstained with DAPI. All scale bars are 10 µm. g, qRT-PCR analysis of SETD2 and Cyclin B1 mRNA levels during G2/M, normalized to Actin mRNA. X-axis is time (h) post release from a double thymidine block, similar to Fig. 3e. Y-axis is fold change relative to 0 hr timepoint. Paired Student’s t-test comparing different timepoints for each gene was performed from two independent biological replicates.

Extended Data Fig. 5 Non-enzymatic function of SETD2 in maintenance of nuclear morphology.

a, Immunoblot analyses of indicated proteins from control, Tamoxifen (Setd2-KO) and SETD2i (EPZ-719)-treated MEF cells for 48 h. b, Representative images of control, Setd2-KO and SETD2i-treated MEF cells immunostained with lamin B1 antibody. c, Quantitative analysis of nuclear defects from b. Two-tailed unpaired Student’s t-test was performed from two independent biological replicates; mean ± s.d.; DNA counterstained with DAPI. d, Representative images of control, SETD2-KD or SETD2i (EPZ-719) treated HKC cells for 2 d or 6 d, immunostained with lamin B1 antibody. DNA counterstained with DAPI. Scale bars, 10 µm.

Source data

Extended Data Fig. 6 The N terminus of SETD2 regulates nuclear lamina stability.

a, Immunoblot analysis for the indicated proteins following immunoprecipitation of endogenous SETD2 from asynchronous or nocodazole-arrested mitotic cells, showing SETD2 associates with CDK1 more robustly during mitosis. b, Representative images of Proximity Ligation Assay (PLA) analysis using anti-lamin B1 and anti-CDK1 antibodies in SETD2-KD cells expressing either WT-SETD2 or tSETD2. PLA pairs are green and DNA was counterstained with DAPI. c, Representative images of SETD2-depleted cells expressing either WT SETD2 or the indicated N-terminal mutants, immunostained with lamin B1 (green). SETD2 (red) visualized using Halo ligand JFX-549. DNA was counterstained with DAPI. Scale bars, 10 µm.

Source data

Extended Data Fig. 7 SETD2 N terminus enhances CDK1-mediated lamin phosphorylation and is required to suppress ccRCC tumour growth.

a, Long exposure of western blot analyses (from Fig. 6a) with anti-SUMO antibody following in vitro GST pulldown assays using either GST-tagged lamin A N-terminus (NT) or C-terminus (CT) or free GST (control). b, Immunoblot analyses using indicated antibodies following GST pulldown assays using either GST-tagged CDK1/Cyclin A2 complex or free GST (control). Anti-SUMO antibody was used to detect SUMO-tagged SETD2 (B/C) proteins. Asterisk indicates cross-reacting band of His–SUMO-B–GB1 in the anti-GST immunoblot. This is because the GB1 (B1 domain of Protein G) exhibits significant affinity towards most immunoglobulin IgG. c, Immunoblot analysis of indicated proteins following an in vitro kinase assay using lamin A-NT as substrate and CDK1/Cyclin B1 as kinase in the presence of either SETD2-B or SETD2-C or a control His–SUMO-GB1 protein. Red asterisk highlights cross-reacting band of His–SUMO-B–GB1 due to aforementioned reasons. d, Western blot with indicated antibodies from UMRC2 cells expressing either Halo (control) or Halo-3xFlag tagged WT-SETD2 or tSTED2 or BC-tSETD2. e, Representative images of UMRC2 cells expressing either Halo (control) or Halo-tagged WT SETD2 or tSETD2 or BC-tSETD2, immunostained with pan-lamin A/C antibody. DNA counterstained with DAPI; scale bar, 10 µm. f, Representative images of the full 35 mm cell culture well with 3D colony growth assay of UMRC2 cells expressing the indicated transgenes; scale bar is 10 mm. g, Xenograft tumour growth measurements (volume) of UMRC2 cells expressing the indicated transgene (n = 9). Related to Fig. 6j; mean ± s.e.m.

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Extended Data Fig. 8

Flow analysis gating strategy.

Supplementary information

Reporting Summary

Peer Review File

Supplementary Table 1

SETD2–APEX2 mass-spectometry data.

Source data

Source Data

Unprocessed western blots for all figures.

Source Data

Numerical source data for all figures.

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Khan, A., Zhang, C., Nguyen, P.G. et al. A SETD2–CDK1–lamin axis maintains nuclear morphology and genome stability. Nat Cell Biol 27, 1327–1341 (2025). https://doi.org/10.1038/s41556-025-01723-9

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