PMC

H2AX is required for ZMYM3 recruitment to DSBs. (A,B) Endogenous H2AX and ZMYM3 interact independently from DNA damage. Interactions of endogenous proteins were performed by immunoprecipitation and Western blotting, as indicated. IR treatment (10 Gy for 3 h). (C) Schematic diagram of ZMYM3 domain organization and deletion constructs. (D,E) The N terminus of ZMYM3 is necessary and sufficient for chromatin binding. Interactions between H2AX and ZMYM3 were analyzed by streptavidin pull-down assays in HEK293T cells. Detection of endogenous H2AX was performed by Western blotting. (F) The N terminus of ZMYM3 is required for damage recruitment. Quantification of ZMYM3 and N-terminal deletion GFP-tagged protein recruitment to laser-induced DSBs were performed. Data represent mean ± SEM. n ≥ 10. (G) H2AX promotes ZMYM3 accumulation at damage sites. Representative images of GFP-ZMYM3 laser-induced DSB recruitment in U2OS and H2AX knockout were analyzed. Note that expression of H2AX wild type, but not H2AX-S139A, rescues ZMYM3 recruitment to DNA damage in H2AX knockout cells. (H) Quantification of G. Mean ± SEM. n ≥ 6. (***) P < 0.001 versus same treatment with control cells, Student's t-test.

ZMYM3 binds histones and DNA to facilitate damage recruitment. (A) The ZMYM3 DNA-binding domain and its conserved alignment across various species. Schematic diagram of the ZMYM3 domain structure; the deletion mutants used are indicated. (B) ZMYM3 binds dsDNA independently from DNA ends. MBP-ZMYM3 fragments were expressed, purified, and analyzed by EMSA on dsDNA probes with (left panel) and without (right panel) DNA ends (see the Materials and Methods). (C) ZMYM3 interactions with chromatin and DNA occur through separate, independent domains. Streptavidin pull-down assay of SFB-ZMYM3 N-terminal fragments stably expressed in HEK293T were performed followed by Western blot analysis of endogenous H2AX. (D) ZMYM3 interacts with H2A- or H2AX-containing nucleosomes. In vitro reconstituted nucleosomes were incubated with MBP-ZMYM3 fragments followed by Western blot analysis. (E,F) ZMYM3 chromatin and DNA-binding domains contribute to DNA damage interactions. Representative images of damage recruitment analysis of GFP-tagged ZMYM3 and derivative mutants in U2OS cells. Accumulation of ZMYM3 at damage sites from E are quantified in F. (***) P < 0.001 versus same treatment with control cells, Student's t-test.

ZMYM3 interacts with components of the BRCA1-A complex. (A) TAP and MS analysis of ZMYM3. ZMYM3 was purified from HEK293T cells, and purified complexes were analyzed by MS. ZMYM3-associated proteins are listed. (B) ZMYM3 interacts with BRCA1-A subcomplex members RAP80, ABRA1, and BRE. SFB-ZMYM3- and MYC-tagged protein was transiently expressed, purified with streptavidin, and analyzed by Western blotting. (C) Endogenous ZMYM3 interacts with RAP80 and ABRA1. RAP80 and ABRA1 were immunoprecipitated from HEK293T cells using specific antibodies followed by Western blotting with anti-ZMYM3 to detect protein associations. (D) ZMYM3 interaction domain mapping with the BRCA1-A subcomplex. SFB-ZMYM3 and mutants were cotransfected with myc-tagged RAP80, ABRA1, or BRE. Complexes were purified with streptavidin pull-down, and interactions were analyzed by Western blotting. (E) Detailed domain mapping of ZMYM3–RAP80 interactions. Streptavidin pull-down of SFB-ZMYM3 and deletion mutants was performed in cells expressing Myc-RAP80. (F) ZMYM3 specifically interacts with RAP80. Individual ZMYM proteins and RAP80 were cotransfected, and interactions were analyzed by streptavidin pull-down and Western blotting. (G) RAP80 domain structure and mutants. (H) Identification of the RAP80 domain required for ZMYM3 binding. Streptavidin pull-down of SFB-ZMYM3 was performed in cells expressing Myc-RAP80 or deletion derivatives. Interactions were determined by Western blotting with Flag and Myc to identify ZMYM3 and RAP80, respectively. (I,J) Damage recruitment of ZMYM3 mutants. GFP-tagged ZMYM3 wild type and variants were analyzed by laser damage in I and quantified in J. Mean ± SEM. n ≥ 6. (***) P < 0.001 versus same treatment with control cells, Student's t-test.

Identification of chromatin proteins involved in DNA DSB repair via HR. (A) Table of the top 10 proteins by peptide counts identified from histone H2AX variant TAP-MS samples. Proteins in red indicate known interactors. (B) HR screen of putative H2A variant-interacting proteins from A and Supplemental Figure S1D. HR efficiencies were obtained in cells transfected with siRNAs targeting all individual genes from B and analyzed as in A. CtIP and BRCA1 acted as positive controls. Data represent mean ± SD. n = 3. (C) Recruitment of GFP-tagged proteins to laser damage. Cells expressing the indicated GFP-tagged proteins were damaged and analyzed 15 min after damage by confocal microscopy. Dotted red lines indicate the laser path. (D) ZMYM3 interactions with H2A and its variants. Streptavidin pull-down of SFB-ZMYM3 was performed in HEK293T cells followed by Western blotting analysis. Flag detected SFB-ZMYM3, and endogenous core histone H2A and its variants were detected with specific antibodies. (*) P < 0.05; (**) P < 0.01 versus the same treatment with control cells, Student's t-test.

ZMYM3 antagonizes the BRCA1-A complex to promote HR. (A) ZMYM3 promotes HR and opposes RAP80 and ABRA1 inhibition of HR. DR-GFP reporter assays were performed after depletion or codepletion of the indicated proteins by siRNAs. Data represent mean ± SD. n = 3. (B) Confirmation of knockdown efficiency by Western blotting from experiments performed in A. (C) 53BP1 depletion rescues HR defects in ZMYM3-depleted cells. Experiments were performed as in A. (D) ZMYM3 knockout cells are sensitive to IR and PARP inhibitors compared with parental U2OS cells. Cells were challenged with IR or PARP inhibitor as indicated and were analyzed by colony formation assays. Data represent mean ± SD. n = 3. (E) Western blotting analysis of knockdown efficiency in cells transfected with BRCA1 siRNA. (F) Epistasis analysis of ZMYM3 and BRCA1. Wild-type and ZMYM3 knockout cells either alone or in combination with siBRCA1 were challenged with IR and PARP inhibitor followed by colony formation assays. (G) Chromosome aberration analyses in ZMYM3 knockout and BRCA1 knockdown cells. Experiments were performed as in E. (H) Complementation assay of ZMYM3 knockout cells. ZMYM3 knockout cells with empty vector or wild-type ZMYM3 were analyzed as in D. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001 versus same treatment with control cells, Student's t-test.

ZMYM3 promotes the recruitment of BRCA1 and its associated factor, ABRA1, to DSBs. (A) Analysis of endogenous RAP80 and γH2AX focus formation after 10 Gy of IR treatment for 3 h by immunofluorescence was performed in U2OS cells. Focus formation of RAP80 and γH2AX was analyzed in wild-type U2OS and compared with cells deleted for ZMYM3, RAP80, or H2AX. (Top panel) RAP80 foci after IR at the indicated time points were quantified from experiments performed in A (bottom panel). (B) ABRA1 and γH2AX damage-induced foci were analyzed as in A. (Bottom panel) Quantification of the total number of ABRA1 foci per cell in the indicated cell background after IR treatment. (C, bottom panel) U2OS and RAP80 knockout cells stably expressing GFP-ZMYM3 were subjected to laser-induced irradiation and quantified. (D) BRCA1 focus formation was analyzed by immunofluorescence after IR treatment. Experiments were performed as in A. (Right panel) Quantification of BRCA1 IRIFs as the percentage of cells with more than five foci. All quantification data are presented as mean ± SD. n = 2. (E) Analysis of RAD51 foci following IR by immunofluorescence. Parental and ZMYM3 knockout U2OS cells were costained with cyclin A, and RAD51 foci were quantified only in cyclin A-positive cells. (*) P < 0.05; (***) P < 0.001 versus same treatment in control cells, Student's t-test.

ZMYM3 localizes to DNA damage and promotes genome stability. (A) ZMYM3 localizes to DNA damage. Live imaging by confocal microscopy of GFP-tagged ZMYM3 following laser-induced DNA damage. (B) Endogenous ZMYM3 accrual at DNA damage sites. Cells were damaged as in A and analyzed by immunofluorescence 1 h after damage with anti-ZMYM3 antibodies. γH2AX marks DNA damage sites. (C,D) ZMYM3 knockout cells exhibit defective checkpoint activation and signaling following DNA damage. Cells were damaged with IR followed by FACS analysis with anti-H3 pS10 and propidium iodide to identify mitotic cells. Data represent mean ± SD. n = 3. For D, cells were IR-treated with the indicated dose and analyzed by Western blotting with the indicated antibodies 3 h after IR. (E,F) ZMYM3 knockout cells exhibit chromosome defects following DNA damage. Cells were either untreated or treated with IR and analyzed by metaphase spreads. Examples of chromosome aberrations are shown in E. Quantification of data from E in F. Data were the average of three independent experiments. Mean ± SD. n = 3. (G) ZMYM3 knockout cells displayed DNA damage signaling defects following CPT treatment. Samples from CPT treatments were analyzed by Western blotting with the indicated antibodies 1 h after treatment. (**) P < 0.01; (***) P < 0.001 versus same treatment with control cells, Student's t-test.