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ZNF280A links DNA double-strand break repair to human 22q11.2 distal deletion syndrome

Nature Cell Biology volume 27, pages 1006–1020 (2025)Cite this article

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Abstract

DNA double-strand breaks (DSB) are among the most deleterious forms of DNA damage and, if unresolved, result in DNA mutations and chromosomal aberrations that can cause disease, including cancer. Repair of DSBs by homologous recombination requires extensive nucleolytic digestion of DNA ends in a process known as DNA-end resection. In recent years, progress has been made in understanding how this process is initiated, but the later stages of this process—long-range DNA-end resection—are not well understood. Many questions remain in terms of how the DNA helicases and endonucleases that catalyse this process are regulated, a key step to avoiding spurious activity in the absence of breaks. The importance of DNA-end resection in human disease is highlighted by several human genetic syndromes that are caused by mutations or deficiencies in key proteins involved in this process. Here, using high-throughput microscopy coupled with a cDNA ‘chromORFeome’ library, we identified ZNF280A as an uncharacterized chromatin factor that is recruited to breaks and essential for DNA DSB repair. Lack of ZNF280A drives genomic instability and substantial sensitivity to DNA-damaging agents. Mechanistically, we demonstrate that ZNF280A promotes long-range DNA-end resection by facilitating the recruitment of the BLM–DNA2 helicase–nuclease complex to DNA DSB sites, enhancing efficiency of the enzymatic activity of this complex at DNA damage sites. ZNF280A is therefore essential for DNA-end resection and DNA repair by homologous recombination. Importantly, ZNF280A is hemizygously deleted in a human genetic condition, 22q11.2 distal deletion syndrome. Features of this condition include congenital heart disease, microcephaly, immune deficiency, developmental delay and cognitive deficits—features that are associated with other human syndromes caused by defects in genes involved in DNA repair. Remarkably, cells from individuals with a 22q11.2 distal deletion have defects in DNA-end resection and homologous recombination, resulting in increased incidence of genomic instability. These phenotypes are rescued by reintroduction of ZNF280A, providing evidence of defective DNA repair as a potential mechanistic explanation for several clinical features associated with this human condition.

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Fig. 1: An uncharacterized chromatin factor ZNF280A is recruited to sites of DNA damage.
Fig. 2: ZNF280A is required for DNA DSB repair.
Fig. 3: ZNF280A is a critical mediator of HR.
Fig. 4: ZNF280A promotes DNA-end resection.
Fig. 5: ZNF280A interacts with and recruits BLM–DNA2 to DSB sites and enhances their enzymatic function to promote DNA-end resection.
Fig. 6: ZNF280A haploinsufficiency causes genomic instability and links defected DNA repair to clinical features in human 22q11.2 distal deletion syndrome.

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Data availability

All other data supporting the findings of this study are available from the corresponding author on reasonable request. DNA sequencing data are available via NCBI, accession code PRJNA1236402. Source data are provided with this paper.

References

  1. Ciccia, A. & Elledge, S. J. The DNA damage response: making it safe to play with knives. Mol. Cell 40, 179–204 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Clarke, T. L. & Mostoslavsky, R. DNA repair as a shared hallmark in cancer and ageing. Mol. Oncol. https://doi.org/10.1002/1878-0261.13285 (2022).

  3. Jackson, S. P. & Bartek, J. The DNA-damage response in human biology and disease. Nature 461, 1071–1078 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Hoeijmakers, J. H. J. DNA damage, aging, and cancer. N. Engl. J. Med. 361, 1475–1485 (2009).

    Article  CAS  PubMed  Google Scholar 

  5. Rich, T., Allen, R. L. & Wyllie, A. H. Defying death after DNA damage. Nature 407, 777–783 (2000).

    Article  CAS  PubMed  Google Scholar 

  6. Scully, R., Panday, A., Elango, R. & Willis, N. A. DNA double-strand break repair-pathway choice in somatic mammalian cells. Nat. Rev. Mol. Cell Biol. 20, 698–714 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Lamarche, B. J., Orazio, N. I. & Weitzman, M. D. The MRN complex in double-strand break repair and telomere maintenance. FEBS Lett. 584, 3682–3695 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Rogakou, E. P., Boon, C., Redon, C. & Bonner, W. M. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell Biol. 146, 905–916 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Jin, S. Double-strand break repair by Ku70 requires heterodimerization with Ku80 and DNA binding functions. EMBO J. 16, 6874–6885 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Noordermeer, S. M. et al. The shieldin complex mediates 53BP1-dependent DNA repair. Nature 560, 117–121 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ghezraoui, H. et al. 53BP1 cooperation with the REV7-shieldin complex underpins DNA structure-specific NHEJ. Nature 560, 122–127 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gupta, R. et al. DNA repair network analysis reveals shieldin as a key regulator of NHEJ and PARP inhibitor sensitivity. Cell 173, 972–988 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Botuyan, M. V. et al. Structural basis for the methylation state-specific recognition of histone H4-K20 by 53BP1 and Crb2 in DNA repair. Cell 127, 1361–1373 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Fradet-Turcotte, A. et al. 53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark. Nature 499, 50–54 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Mailand, N. et al. RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins. Cell 131, 887–900 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Stewart, G. S. et al. The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage. Cell 136, 420–434 (2009).

    Article  CAS  PubMed  Google Scholar 

  17. Mirman, Z. et al. 53BP1-RIF1-shieldin counteracts DSB resection through CST- and Polα-dependent fill-in. Nature 560, 112–116 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Tang, J. et al. Acetylation limits 53BP1 association with damaged chromatin to promote homologous recombination. Nat. Struct. Mol. Biol. 20, 317–325 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Clarke, T. L. et al. PRMT5-dependent methylation of the TIP60 coactivator RUVBL1 is a key regulator of homologous recombination. Mol. Cell 65, 900–916.e7 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Cejka, P. & Symington, L. S. DNA end resection: mechanism and control. Annu Rev. Genet 55, 285–307 (2021).

    Article  CAS  PubMed  Google Scholar 

  21. Sartori, A. A. et al. Human CtIP promotes DNA end resection. Nature 450, 509–514 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Huertas, P., Cortés-Ledesma, F., Sartori, A. A., Aguilera, A. & Jackson, S. P. CDK targets Sae2 to control DNA-end resection and homologous recombination. Nature 455, 689–692 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Huertas, P. & Jackson, S. P. Human CtIP mediates cell cycle control of DNA end resection and double strand break repair. J. Biol. Chem. 284, 9558–9565 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Levikova, M., Klaue, D., Seidel, R. & Cejka, P. Nuclease activity of Saccharomyces cerevisiae Dna2 inhibits its potent DNA helicase activity. Proc. Natl Acad. Sci. USA 110, E1992–E2001 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Pinto, C., Kasaciunaite, K., Seidel, R. & Cejka, P. Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases. eLife 5, e18574 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  26. He, Y. J. et al. DYNLL1 binds to MRE11 to limit DNA end resection in BRCA1-deficient cells. Nature 563, 522–526 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Taylor, A. M. R. et al. Chromosome instability syndromes. Nat. Rev. Dis. Prim. 5, 64 (2019).

    Article  PubMed  Google Scholar 

  28. Qvist, P. et al. CtIP mutations cause Seckel and Jawad syndromes. PLoS Genet. 7, e1002310 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Ceppi, I. et al. CtIP promotes the motor activity of DNA2 to accelerate long-range DNA end resection. Proc. Natl Acad. Sci. USA 117, 8859–8869 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. McDonald-McGinn, D. M. et al. 22q11.2 deletion syndrome. Nat. Rev. Dis. Prim. 1, 15071 (2015).

    Article  PubMed  Google Scholar 

  31. Martinez-Pastor, B. et al. Assessing kinetics and recruitment of DNA repair factors using high content screens. Cell Rep. 37, 110176 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Korwek, Z. et al. Inhibition of ATM blocks the etoposide-induced DNA damage response and apoptosis of resting human T cells. DNA Repair 11, 864–873 (2012).

    Article  CAS  PubMed  Google Scholar 

  33. Luzhna, L., Kathiria, P. & Kovalchuk, O. Micronuclei in genotoxicity assessment: from genetics to epigenetics and beyond. Front. Genet. 4, 131 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Farmer, H. et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434, 917–921 (2005).

    Article  CAS  PubMed  Google Scholar 

  35. Bryant, H. E. et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 434, 913–917 (2005).

    Article  CAS  PubMed  Google Scholar 

  36. Pilié, P. G., Tang, C., Mills, G. B. & Yap, T. A. State-of-the-art strategies for targeting the DNA damage response in cancer. Nat. Rev. Clin. Oncol. 16, 81–104 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Pilié, P. G., Gay, C. M., Byers, L. A., O’Connor, M. J. & Yap, T. A. PARP inhibitors: extending benefit beyond BRCA-mutant cancers. Clin. Cancer Res. 25, 3759–3771 (2019).

    Article  PubMed  Google Scholar 

  38. Bunting, S. F. et al. 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell 141, 243–254 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Smith, J., Tho, L. M., Xu, N. & Gillespie, D. A. The ATM–Chk2 and ATR–Chk1 pathways in DNA damage signaling and cancer. Adv. Cancer Res. 108, 73–112 (2010).

    Article  CAS  PubMed  Google Scholar 

  40. Zhou, Y., Caron, P., Legube, G. & Paull, T. T. Quantitation of DNA double-strand break resection intermediates in human cells. Nucleic Acids Res. 42, e19 (2014).

    Article  CAS  PubMed  Google Scholar 

  41. Wang, X. et al. ZNF280A promotes proliferation and tumorigenicity via inactivating the Hippo-signaling pathway in colorectal cancer. Mol. Ther. Oncolytics 12, 204–213 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Peng, G. et al. Human nuclease/helicase DNA2 alleviates replication stress by promoting DNA end resection. Cancer Res. 72, 2802–2813 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Yousefzadeh, M. et al. DNA damage—how and why we age? eLife 10, e62852 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Burnside, R. D. 22q11.21 deletion syndromes: a review of proximal, central, and distal deletions and their associated features. Cytogenet. Genome Res. 146, 89–99 (2015).

    Article  PubMed  Google Scholar 

  45. Cercek, A. et al. PD-1 blockade in mismatch repair-deficient, locally advanced rectal cancer. N. Engl. J. Med. 386, 2363–2376 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. D’Andrea, A. D. Mechanisms of PARP inhibitor sensitivity and resistance. DNA Repair 71, 172–176 (2018).

    Article  PubMed  Google Scholar 

  47. Dias, M. P., Moser, S. C., Ganesan, S. & Jonkers, J. Understanding and overcoming resistance to PARP inhibitors in cancer therapy. Nat. Rev. Clin. Oncol. 18, 773–791 (2021).

    Article  PubMed  Google Scholar 

  48. Kamaliyan, Z. & Clarke, T. L. Zinc finger proteins: guardians of genome stability. Front. Cell Dev. Biol. 12, 1448789 (2024).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Mraz, M. et al. The origin of deletion 22q11 in chronic lymphocytic leukemia is related to the rearrangement of immunoglobulin lambda light chain locus. Leuk. Res. 37, 802–808 (2013).

    Article  CAS  PubMed  Google Scholar 

  50. del Baldo, G. et al. Rhabdoid tumor predisposition syndrome: from clinical suspicion to general management. Front. Oncol. 11, 586288 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Stevens, T., van der Werff Ten Bosch, J., de Rademaeker, M., van den Bogaert, A. & van den Akker, M. Risk of malignancy in 22q11.2 deletion syndrome. Clin. Case Rep. 5, 486–490 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  52. O’Driscoll, M., Dobyns, W. B., van Hagen, J. M. & Jeggo, P. A. Cellular and clinical impact of haploinsufficiency for genes involved in ATR signaling. Am. J. Hum. Genet. 81, 77–86 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  53. de Ru, M. H. et al. Interstitial deletion in 3q in a patient with blepharophimosis–ptosis–epicanthus inversus syndrome (BPES) and microcephaly, mild mental retardation and growth delay: clinical report and review of the literature. Am. J. Med. Genet. A 137, 81–87 (2005).

    PubMed  Google Scholar 

  54. Cardoso, C. et al. Refinement of a 400-kb critical region allows genotypic differentiation between isolated lissencephaly, Miller–Dieker syndrome, and other phenotypes secondary to deletions of 17p13.3. Am. J. Hum. Genet. 72, 918–930 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Ewart, A. K. et al. Hemizygosity at the elastin locus in a developmental disorder, Williams syndrome. Nat. Genet. 5, 11–16 (1993).

    Article  CAS  PubMed  Google Scholar 

  56. Yang, X. et al. A public genome-scale lentiviral expression library of human ORFs. Nat. Methods 8, 659–661 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Anand, R., Pinto, C. & Cejka, P. Methods to study DNA end resection I: recombinant protein purification. Methods Enzymol. 600, 25–66 (2018).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank all the members of the Mostoslavsky lab and Clarke lab for helpful discussions and critical reading of the manuscript. MMI laser cut technology was provided by the Program in Membrane Biology Microscopy Core, which is partially supported by the Boston Area Diabetes Endocrinology Research Center (grant no. P30DK057521) and the Massachusetts General Hospital (MGH) Center for the Study of Inflammatory Bowel Disease (grant no. P30DK043351). T.L.C. was supported by a Charles King Trust Postdoctoral Fellowship and a Fund for Medical Discovery Postdoctoral Fellowship from Massachusetts General Hospital. T.L.C. is the Nancy L.R. Bucher Assistant Professor of Pathology and Laboratory Medicine at Boston University and is currently funded by an NIH R00 Pathway to Independence Award (award no. R00ES034443-04) and The Karin Grunebaum Cancer Research Foundation Faculty Fellowship. R.M. is the Laurel Schwartz Endowed Chair in Oncology. This work was supported by NIEHS grant no. R21ES027931 and a Krantz Breakthrough Award to R.M.

Author information

Authors and Affiliations

  1. The Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA

    Thomas L. Clarke, Hyo Min Cho, Tribhuwan Yadav, Giorgia G. Silveira, Ruben Boon, Nana Yaa A. Amoh, Belen Machin, Tiziano Bernasocchi, Dua Ashfaq, Josefina Mendez, José Del Río Pantoja, Giuliana Sardi Rogines, Lee Zou & Raul Mostoslavsky

  2. The Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Thomas L. Clarke, Hyo Min Cho, Giorgia G. Silveira, Ruben Boon, Nana Yaa A. Amoh, Belen Machin, Tiziano Bernasocchi, José Del Río Pantoja & Raul Mostoslavsky

  3. Department of Pathology and Laboratory Medicine, Boston University Chobanian and Avedisian School of Medicine, Boston, MA, USA

    Thomas L. Clarke & Zeeba Kamaliyan

  4. Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana, Bellinzona, Switzerland

    Ilaria Ceppi & Petr Cejka

  5. Department of Molecular Genetics and Microbiology, School of Medicine, Duke University, Durham, NC, USA

    Boya Gao & Li Lan

  6. Molecular Oncology Program, Spanish National Cancer Research Center (CNIO), Madrid, Spain

    Barbara Martinez-Pastor

  7. The 22q and You Center, Division of Human Genetics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Blaine T. Crowley, Daniel E. McGinn, Victoria Giunta, Oanh Tran, Elaine H. Zackai, Beverly S. Emanuel & Donna M. McDonald-McGinn

  8. Perelman School of Medicine of the University of Pennsylvania, Philadelphia, PA, USA

    Elaine H. Zackai, Beverly S. Emanuel & Donna M. McDonald-McGinn

  9. Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA

    Lee Zou

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  1. Thomas L. Clarke
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Contributions

R.M. and T.LC. designed the study and wrote the manuscript. T.L.C., H.M.C., I.C., T.Y., G.G.S., N.Y.A.A., B.M., B.G., R.B., D.A., J.M., Z.K., T.B., B.M.-P., J.D.R.P. and G.S.R. conducted experiments. R.M., L.L, L.Z., T.L.C. and P.C. supervised experiments. B.T.C., D.E.M., V.G., O.T., E.H.Z., B.S.E. and D.M.M.-M. collected and generated cell lines from individuals with 22q11.2 deletion syndrome and provided clinical data. All authors read and edited the manuscript.

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Correspondence to Thomas L. Clarke or Raul Mostoslavsky.

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Extended data

Extended Data Fig. 1 A newly identified chromatin factor ZNF280A is recruited to sites of DNA damage.

(a) Schematic linear architecture of ZNF280A protein. (b) Representative image of V5-tagged ZNF280A colocalization with γH2AX in U2OS cells, 40 min following UV-laser micro irradiation. Scale bar, 10um. (c) Chromatin fractionation experiment in U2OS cells following 10 Gy ionizing radiation. Unprocessed blots are available in source data.

Source data

Extended Data Fig. 2 ZNF280A is required for DNA double-strand break repair.

(a) RT-PCR analysis of znf280a (ZNF280A) mRNA transcripts in HeLa cells treated with non-targeting or ZNF280A-targeting siRNA sequences. N = 3, error represents S.E.M. (b) Representative images of clonogenic survival assay in HeLa cells, in response to increasing doses (Gy) of ionizing radiation. (c) RT-PCR analysis of znf280a (ZNF280A) mRNA transcripts in U2OS cells treated with non-targeting or two independent ZNF280A-targeting siRNA sequences. N = 3, error represents S.E.M. (d) Representative images of clonogenic survival assay in U2OS cells, in response to increasing doses (μM) of Etoposide. (e) Immunoblot demonstrating knockdown of endogenous ZNF280A protein and over-expression of V5-ZNF280A in U2OS cells. (f) Quantitation of the % of HeLa cells with >10 53BP1 foci, 48 h following 3 Gy ionizing radiation, as depicted in Fig. 2j. N = 3, error represents S.E.M. *p = <0.05, **p = <0.005. two-tailed t-test. (g) Quantitation of the number of chromosomal aberrations per metaphase in untreated cells, similar to that depicted in Fig. 2l, m. N = 3; error represents S.E.M. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 3 ZNF280A is a critical mediator of homologous recombination.

(a) Representative images of Rad51 foci in U2OS cells treated with siCTRL, or siZNF280A-targeting sequences, 6 h following exposure to 3 Gy ionizing radiation. Scale bar, 10um. (b) Quantitation of the number of Rad51 foci per nucleus in U2OS cells, 6 h following 3 Gy ionizing radiation. N = 3; (siCTRL n = 310; siZNF280A #1 n = 318; siZNF280A #2 n = 129). error represents S.E.M; ****p = <0.0001. two-tailed t-test. (c) Quantitation of the % of U2OS cells with >10 Rad51 foci per cells as depicted in Extended Data Fig. 3A. Each point represents the average of each biological repeat. N = 3; error represents S.E.M; **p = <0.005. two-tailed t-test. (d) RT-PCR analysis of znf280a (ZNF280A) mRNA transcripts in HeLa cells treated with non-targeting or ZNF280A-targeting siRNA sequences. N = 3, error represents S.E.M. (e) Representative image of Rad51 foci in siCTRL, siZNF280A or siZNF280A U2OS cells reconstituted with wild type ZNF280A cDNA, 6hrs following exposure to 3 Gy ionizing radiation. Scale bar, 10um. (f) Quantification of number of Rad51 foci per nucleus in siCTRL, siZNF280A or siZNF280A U2OS cells reconstituted with wild type ZNF280A cDNA, 6hrs following exposure to 3 Gy ionizing radiation. N = 3; (pLX304 + siCTRL n = 202; pLX304 + siZNF280A n = 352; V5-ZNF280A + siZNF280A n = 295). error represents S.E.M; ****p = <0.0001. two-tailed t-test. (g) Representative images of Rad51 foci in HeLa cells treated with siCTRL, or siZNF280A-targeting sequences, 6 h following exposure to 3 Gy ionizing radiation. Scale bar, 10um. (h) Quantitation of the number of Rad51 foci per nucleus in HeLa cells, 6 h following 3 Gy ionizing radiation. N = 3; (siCTRL n = 103; siZNF280A #1 n = 102; siZNF280A #2 n = 138). error represents S.E.M; ****p = <0.0001. two-tailed t-test. (i) RT-PCR analysis of znf280a (ZNF280A) mRNA transcripts in U2OS cells expressing pLX304 empty vector or pLX304 (v-5)-ZNF280A as depicted in Fig. 3g, h. N = 3; error represents S.E.M. (j) Representative images of clonogenic survival assay in HeLa cells in response to increasing doses (μM) of Olaparib, as quantified in Extended Data Fig. 3K. (k) Quantitation of clonogenic survival assay in HeLa cells in response to increasing doses (μM) of Olaparib. N = 3; error represents S.E.M; *p = <0.05. ***p = <0.0005. two-tailed t-test. (l) Representative images of clonogenic survival assay in HeLa cells in response to increasing doses (μM) of Olaparib, as quantified in Extended Data Fig. 3M. (m) Quantitation of clonogenic survival assay in HeLa cells in response to increasing doses (μM) of Olaparib. N = 3; error represents S.E.M; ***p = <0.0005. two-tailed t-test. (n) Representative image of radial chromosome in ZNF280A-depleted HeLa cells, 24 h post 2 Gy ionizing radiation. Scale bar, 10um. (o) Quantification of the number of radial chromosomes in ZNF280A-depleted HeLa cells, 24 h post 2 Gy ionizing radiation. N = 3; error represents S.E.M; *p = <0.05. **p = <0.005. two-tailed t-test. (p) Contour blots from FACSDiva software, demonstrating gating strategy for determining GFP positivity in siRNA-treated U2OS DR-GFP cells transfected with a DNA empty vector or Sce1 restriction enzyme. (q) Representative images of Rad51 foci in Mitosin positive (late S/G2 phase) U2OS cells, 6 h following 3 Gy ionizing radiation. Scale bar, 10um. Source numerical data and unprocessed blots are available in source data.

Extended Data Fig. 4 ZNF280A promotes DNA end-resection.

(a) RT-PCR analysis of znf280a (ZNF280A) mRNA transcripts in Panc-1 cells treated with non-targeting or ZNF280A-targeting siRNA sequences. N = 3, error represents S.E.M. (b) Immunoblot analysis of ZNF280A protein levels in shCTRL or shZNF280A HeLa cells. (c) Immunoblot analysis of RPA phosphorylation at serine 4 and serine 8 (s4/8) in shZNF280A HeLa cells treated with 1μM Camptothecin for the indicated timepoints. (d) Immunoblot analysis of ZNF280A protein levels in HeLa cells treated with siCTRL, or an siZNF280A-targeting sequence. (e) Immunoblot analysis of RPA phosphorylation at serine 4 and serine 8 (s4/8) in siCTRL or siZNF280A HeLa cells treated with 1μM Camptothecin for the indicated timepoints. (f) Schematic of native BrdU experiment, represented in Figs. 4f, g. (g) RT-PCR analysis of znf280a (ZNF280A) and brca1 mRNA transcripts in U2OS AsiSi cells treated with non-targeting, ZNF280A, or BRCA1-targeting siRNA sequences. N = 3; error represents S.E.M. (h) Representative images of 53BP1 foci in Mitosin positive (late S/G2 phase) U2OS cells, 24 h following 3 Gy ionizing radiation. Scale bar, 10um. (i) Quantification of number of 53BP1 foci per cell from Extended Data Fig. 4H. N = 3; (siCTRL untreated n = 129; siCTRL irradiated n = 119; siZNF280A untreated n = 35; siZNF280A irradiated n = 37). error represents S.E.M; ****p = <0.0001. two-tailed t-test. (j) Immunoblot analysis of ZNF280A and Rad51 protein levels in U2OS cells treated with siCTRL, or an siZNF280A-targeting sequence. GAPDH is used as a loading control. (k) Representative images of clonogenic survival assay in U2OS cells in response to increasing doses (μM) of Camptothecin, as quantified in Fig. 4k. (l) Representative images of clonogenic survival assay in HeLa cells in response to increasing doses (μM) of Camptothecin, as quantified in Fig. 4l. (m) Representative images of clonogenic survival assay in shCTRL or shZNF280A HeLa cells in response to increasing doses (μM) of Camptothecin. (n) Quantitation of clonogenic survival assay in shCTRL or shZNF280A HeLa cells in response to increasing doses (μM) of Camptothecin. N = 3; error represents S.E.M; *p = <0.05, **p = <0.005, ***p = <0.0005. two-tailed t-test (o) Immunoblot analysis of ZNF280A and V5 protein expression in pLX304-ZNF280A infected U2OS cells. GAPDH is used as a loading control. (p) Quantitation of the number of Rad51 foci per nucleus in U2OS cells expressing pLX304 empty vector or pLX304 (v-5)-ZNF280A, 6 h following 3 Gy ionizing radiation. N = 3; (pLX304 n = 113; V5-ZNF280A n = 106). error represents S.E.M. (q) Representative images of clonogenic survival assay in U2OS cells expressing pLX304 empty vector or pLX304 (v-5)-ZNF280A, in response to increasing doses of Camptothecin (μM). (r) Quantitation of clonogenic survival assay in U2OS cells expressing pLX304 empty vector or pLX304 (v-5)-ZNF280A, in response to increasing doses of Camptothecin. N = 6; error represents S.E.M; *p = <0.05. **p = <0.005. two-tailed t-test. (s) Schematic of Flag-tagged ZNF280A constructs. Source numerical data and unprocessed blots are available in source data. Schematic made using Biorender.

Source data

Extended Data Fig. 5 ZNF280A interacts with and recruits BLM-DNA2 to double-strand breaks sites and enhances their enzymatic function to promote DNA end-resection.

(a) Representative image of Mre-11 and 53BP1 foci colocalization at Fok-1-induced DNA double-strand breaks in Fok1-lac-1 U2OS cells. Scale bar, 10um. (b) Quantitation of Mre-11 and 53BP1 foci colocalization at Fok-1-induced DNA double-strand breaks in Fok1-lac-1 U2OS cells. N = 3. Error represents S.E.M. (c) RT-PCR analysis of znf280a (ZNF280A) mRNA transcripts in U2OS-Lac1-Fok1 cells treated with non-targeting or two independent ZNF280A-targeting siRNA sequences. N = 6; error represents S.E.M. (d) Quantification of Mre11 foci in siZNF280A-depleted U2OS cells, 15 min post 3 Gy IR. (siCTRL n = 232; siCTRL + IR n = 232; siZNF280A n = 232; siZNF280A + IR n = 232). Error represents S.E.M. NS p = >0.05. two-tailed t-test. (e) Chromatin fractionation experiment in U2OS cells treated with siCTRL or siCTIP following 10 Gy ionizing radiation. Whole cell extracts and chromatin fractions are shown. H3 and GAPDH are provided as loading controls for chromatin fractions and whole cell extracts, respectively. (f) Quantification of GFP-ZNF280A signal intensity at UV-laser stripe in siCTRL or siCTIP U2OS cells. (g) Co-immunoprecipitation of V5-tagged ZNF280A demonstrating interaction with endogenous BLM in HEK293T cells. (h) Co-immunoprecipitation of V5-tagged ZNF280A demonstrating interaction with endogenous DNA2 in HEK293T cells. (i) Co-immunoprecipitation of V5-tagged ZNF280A demonstrating interaction with endogenous BLM in HEK293T cells, that is increased 1 h following 10 Gy ionizing radiation. (j) Representative images of epistasis clonogenic survival assay in U2OS cells in response to increasing doses (μM) of Olaparib, as quantified in Fig. 5h. (k) Representative images of epistasis clonogenic survival assay in U2OS cells in response to increasing doses (μM) of Olaparib, as quantified in Fig. 5i. (l) RT-PCR analysis of znf280a (ZNF280A) or dna2 (DNA2) mRNA transcripts in U2OS cells treated with non-targeting or ZNF280A-targeting siRNA sequences. N = 2; error represents S.D. Representative knockdown analysis for clonogenic survival assay in Fig. 5i. (m) RT-PCR analysis of znf280a (ZNF280A) or blm (BLM) mRNA transcripts in U2OS cells treated with non-targeting or ZNF280A-targeting siRNA sequences. N = 4; error represents S.E.M. Representative knockdown analysis for clonogenic survival assay in Fig. 5h. (n) Representative images of Rad51 foci in U2OS treated with siRNA to ZNF280A or DNA2, 6hrs following exposure to 3 Gy ionizing radiation. Scale bar, 10um. (o) Quantitation of the number of Rad51 foci per nucleus in U2OS treated with siRNA to ZNF280A or DNA2, 6hrs following exposure to 3 Gy ionizing radiation, as depicted in Extended Data Fig. 5N. N = 3; error represents S.E.M; ****p = <0.0001. two-tailed t-test. (p) Quantitation of the % cells with >10 Rad51 foci per nucleus in U2OS treated with siRNA to ZNF280A or DNA2, 6hrs following exposure to 3 Gy ionizing radiation, as depicted in Extended Data Fig. 5N. Each data point represents the average from each independent biological replicate. N = 3; error represents S.E.M; **p = <0.005. two-tailed t-test. (q) Immunoblot analysis of Flag-tagged ZNF280A construct expression in U2OS cells. (r) Quantitation of the number of Rad51 foci per nucleus in U2OS cells expressing empty vector, Flag-ZNF280A Δ1-200, or Flag-ZNF280A Δ Zinc Finger, 6hrs following exposure to 3 Gy ionizing radiation. error represents S.E.M; ****p = <0.0001. two-tailed t-test. (s) A representative experiment showing DNA unwinding by ZNF280A in the presence of human RPA (15 nM) on oligonucleotide-based Y-structured (25 nt/25 bp) DNA. The reaction buffer contains 100 mM NaCl. (t) A representative experiment showing DNA unwinding by BLM (0.25 nM) in the absence or presence of ZNF280A and with human RPA (15 nM) on oligonucleotide-based Y-structured (25 nt/25 bp) DNA. The reaction buffer contains 150 mM NaCl. (u) Quantitation of overall DNA unwinding from experiments such as shown in Extended Data Fig. 5T. N = 3; error bars represent SEM. **P < 0.01, two-tailed t-test. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 6 ZNF280A haploinsufficiency causes genomic instability and links defected DNA repair to clinical features in human 22q11.2 distal deletion syndrome.

(a) Immunoblot analysis of ZNF280A protein levels in 22q11.2 deletion patient lymphocytes. (b) Schematic of chromosome 22q11.2 locus including cytogenetic bands and locus control regions (LCRs) and the approximate genomic loci recognized by MLPA probes. (c) Comparison of the % of patients who exhibit microcephaly with deletions in either 22q11.2 LCR22A-LCR22D or 22q11.2 LCR22D-LCR22E (N = 20) regions. (d) Comparison of the % of Female patients who exhibit microcephaly with deletions in either 22q11.2 LCR22A-LCR22D or 22q11.2 LCR22D-LCR22E regions (N = 13). (e) Comparison of the % of Male patients who exhibit microcephaly with deletions in either 22q11.2 LCR22A-LCR22D or 22q11.2 LCR22D-LCR22E regions (N = 7). (f) Representative image of gel from T7E1 endonuclease assay, confirming partial deletion of ZNF280A in U2OS cells. pKO = partial knockout. (g) Amino acid sequence of sgCTRL or sgZNF280APartial KO depicting the introduction of premature STOP codon via frame shift. (h) DNA sequence of sgZNF280APartial KO U2OS cells, depicting partial deletion of ZNF280A. (i) RT-PCR analysis of znf280a mRNA transcript in sgCTRL or sgZNF280APartial KO U2OS cells. (j) Immunoblot analysis of ZNF280A protein levels in sgCTRL or sgZNF280APartial KO U2OS cells. GAPDH is used as a loading control. (k) Quantitation of the fold change in γH2AX and 53BP1 foci in 22q11.2 distal deletion patient cell lines, as depicted in Fig. 6c, d. Each data point represents the average from each independent biological replicate. S.E.M; *p = <0.05. two-tailed t-test. (l) Quantitation of % of cells with >5 Rad51 foci per nucleus in 22q11.2 distal deletion patient cell lines, 6hrs following 3 Gy ionizing radiation, as depicted in Fig. 6g, h. Each data point represents the average from each independent biological replicate. N = 3; error represents S.E.M. Source numerical data and unprocessed blots are available in source data. Schematic made using Biorender.

Source data

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Clarke, T.L., Cho, H.M., Ceppi, I. et al. ZNF280A links DNA double-strand break repair to human 22q11.2 distal deletion syndrome. Nat Cell Biol 27, 1006–1020 (2025). https://doi.org/10.1038/s41556-025-01674-1

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