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BRIT1/MCPH1 links chromatin remodelling to DNA damage response.

Peng G, Yim EK, Dai H, Jackson AP, Burgt Iv, Pan MR, Hu R, Li K, Lin SY - Nat. Cell Biol. (2009)

Bottom Line: However, the mechanism mediating their recruitment to DNA lesions remains largely unknown.This increase in binding affinity provides a means by which SWI-SNF can be specifically recruited to and maintained at DNA lesions.Loss of BRIT1 causes impaired chromatin relaxation as a result of decreased association of SWI-SNF with chromatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, The University of Texas M. D. Anderson Cancer Center, Houston, 77054, USA.

ABSTRACT
To detect and repair damaged DNA, DNA-damage-response proteins need to overcome the barrier of condensed chromatin to gain access to DNA lesions. ATP-dependent chromatin remodelling is one of the fundamental mechanisms used by cells to relax chromatin in DNA repair. However, the mechanism mediating their recruitment to DNA lesions remains largely unknown. BRIT1 (also known as MCPH1) is an early DNA-damage-response protein that is mutated in human primary microcephaly. Here we report a previously unknown function of BRIT1 as a regulator of the ATP-dependent chromatin remodelling complex SWI-SNF in DNA repair. After damage to DNA, BRIT1 increases its interaction with SWI-SNF through ATM/ATR-dependent phosphorylation on the BAF170 subunit. This increase in binding affinity provides a means by which SWI-SNF can be specifically recruited to and maintained at DNA lesions. Loss of BRIT1 causes impaired chromatin relaxation as a result of decreased association of SWI-SNF with chromatin. This explains the decreased recruitment of repair proteins to DNA lesions and the reduced efficiency of repair in BRIT1-deficient cells, resulting in impaired cell survival after DNA damage. Our findings therefore identify BRIT1 as a key molecule that links chromatin remodelling with response to DNA damage in the control of DNA repair, and its dysfunction contributes to human disease.

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BRIT1 promotes DNA repair and chromatin relaxation in two human MCPH lymphoblastoid cell lines (MCPH #1 and MCPH #2)(a) Impaired DNA repair analyzed by comet assay after exposure of cells to IR. Percentage of cells with intact DNA (tail moment less than 2) in cells without IR exposure was set as 1. At least 100 cells were scored in each sample and each value represents the mean ± SEM of three independent experiments; Student’s t-test. Representative images are shown in Supplementary Fig. 7b. (b) Impaired cell survival in MCPH #1 cells exposed to DSB-inducing agents. Cells were pre-treated with the DNA replication inhibitor aphidicolin (Aphi) where indicated, and then treated with etoposide (Left) or camptothecin (Right). The graphs represent the mean ± SD of three independent experiments (c) Requirement of BRIT1 for foci formation of DNA repair proteins. (Top) Immunostaining of p-RPA foci. Scale bar is 10 µM. (Bottom) Quantification of p-RPA and Rad51 foci formation from three independent experiments (mean ± SD). At least 50 cells were scored in each sample for each experiment. (d) Reduced association of SWI/SNF to chromatin. (Top) Representative Western blots. (Bottom) Densitometry analyses of indicated protein values normalized against ORC2. Each value represents the mean ± SD of three independent experiments; Student’s t-test. (e) Impaired chromatin relaxation analyzed by MNase sensitivity assay. Cells were exposed to the DSB inducing agent neocarzinostatin (400 ng/mL). (Left) Representative image. (Right) Quantification of average nucleosome size. Each value represents the mean ± SEM of three independent experiments; Student’s t-test.
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Figure 5: BRIT1 promotes DNA repair and chromatin relaxation in two human MCPH lymphoblastoid cell lines (MCPH #1 and MCPH #2)(a) Impaired DNA repair analyzed by comet assay after exposure of cells to IR. Percentage of cells with intact DNA (tail moment less than 2) in cells without IR exposure was set as 1. At least 100 cells were scored in each sample and each value represents the mean ± SEM of three independent experiments; Student’s t-test. Representative images are shown in Supplementary Fig. 7b. (b) Impaired cell survival in MCPH #1 cells exposed to DSB-inducing agents. Cells were pre-treated with the DNA replication inhibitor aphidicolin (Aphi) where indicated, and then treated with etoposide (Left) or camptothecin (Right). The graphs represent the mean ± SD of three independent experiments (c) Requirement of BRIT1 for foci formation of DNA repair proteins. (Top) Immunostaining of p-RPA foci. Scale bar is 10 µM. (Bottom) Quantification of p-RPA and Rad51 foci formation from three independent experiments (mean ± SD). At least 50 cells were scored in each sample for each experiment. (d) Reduced association of SWI/SNF to chromatin. (Top) Representative Western blots. (Bottom) Densitometry analyses of indicated protein values normalized against ORC2. Each value represents the mean ± SD of three independent experiments; Student’s t-test. (e) Impaired chromatin relaxation analyzed by MNase sensitivity assay. Cells were exposed to the DSB inducing agent neocarzinostatin (400 ng/mL). (Left) Representative image. (Right) Quantification of average nucleosome size. Each value represents the mean ± SEM of three independent experiments; Student’s t-test.

Mentions: Comet assays demonstrated a significantly reduced DSB repair efficiency in BRIT1 LCLs (Fig. 5a, Supplementary Fig. 7b). Consistent with this, BRIT1 LCL also exhibited increased sensitivity to the topoisomerase inhibitors camptothecin and etoposide, which generate DSBs during S phase, a cell cycle phase in which lesions are predominantly repaired by HR26. This increased sensitivity was consistent with DSB generation during S-phase as the effects were abrogated when cells were treated with the DNA replication inhibitor aphidicolin (Fig. 5b). In addition, increased sensitivity to IR-induced DNA damage was observed in BRIT1 LCLs arrested in G1 phase, a cell cycle exclusively utilizing NHEJ to repair DSBs (Supplementary Fig. 7c). Together, our data suggested that BRIT1 LCL might have impaired cell survival as a result of generated DSBs being un-repaired because of both the defective HR and NHEJ repair. Furthermore, repair foci formation was also impaired in these cells with significantly reduced recruitment of RPA and Rad51 (Fig. 5c). These results were further confirmed by our detection of a decreased association of DNA repair proteins to chromatin in patients’ cells, while total protein levels were unaffected (Supplementary Fig. 7d–f).


BRIT1/MCPH1 links chromatin remodelling to DNA damage response.

Peng G, Yim EK, Dai H, Jackson AP, Burgt Iv, Pan MR, Hu R, Li K, Lin SY - Nat. Cell Biol. (2009)

BRIT1 promotes DNA repair and chromatin relaxation in two human MCPH lymphoblastoid cell lines (MCPH #1 and MCPH #2)(a) Impaired DNA repair analyzed by comet assay after exposure of cells to IR. Percentage of cells with intact DNA (tail moment less than 2) in cells without IR exposure was set as 1. At least 100 cells were scored in each sample and each value represents the mean ± SEM of three independent experiments; Student’s t-test. Representative images are shown in Supplementary Fig. 7b. (b) Impaired cell survival in MCPH #1 cells exposed to DSB-inducing agents. Cells were pre-treated with the DNA replication inhibitor aphidicolin (Aphi) where indicated, and then treated with etoposide (Left) or camptothecin (Right). The graphs represent the mean ± SD of three independent experiments (c) Requirement of BRIT1 for foci formation of DNA repair proteins. (Top) Immunostaining of p-RPA foci. Scale bar is 10 µM. (Bottom) Quantification of p-RPA and Rad51 foci formation from three independent experiments (mean ± SD). At least 50 cells were scored in each sample for each experiment. (d) Reduced association of SWI/SNF to chromatin. (Top) Representative Western blots. (Bottom) Densitometry analyses of indicated protein values normalized against ORC2. Each value represents the mean ± SD of three independent experiments; Student’s t-test. (e) Impaired chromatin relaxation analyzed by MNase sensitivity assay. Cells were exposed to the DSB inducing agent neocarzinostatin (400 ng/mL). (Left) Representative image. (Right) Quantification of average nucleosome size. Each value represents the mean ± SEM of three independent experiments; Student’s t-test.
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Figure 5: BRIT1 promotes DNA repair and chromatin relaxation in two human MCPH lymphoblastoid cell lines (MCPH #1 and MCPH #2)(a) Impaired DNA repair analyzed by comet assay after exposure of cells to IR. Percentage of cells with intact DNA (tail moment less than 2) in cells without IR exposure was set as 1. At least 100 cells were scored in each sample and each value represents the mean ± SEM of three independent experiments; Student’s t-test. Representative images are shown in Supplementary Fig. 7b. (b) Impaired cell survival in MCPH #1 cells exposed to DSB-inducing agents. Cells were pre-treated with the DNA replication inhibitor aphidicolin (Aphi) where indicated, and then treated with etoposide (Left) or camptothecin (Right). The graphs represent the mean ± SD of three independent experiments (c) Requirement of BRIT1 for foci formation of DNA repair proteins. (Top) Immunostaining of p-RPA foci. Scale bar is 10 µM. (Bottom) Quantification of p-RPA and Rad51 foci formation from three independent experiments (mean ± SD). At least 50 cells were scored in each sample for each experiment. (d) Reduced association of SWI/SNF to chromatin. (Top) Representative Western blots. (Bottom) Densitometry analyses of indicated protein values normalized against ORC2. Each value represents the mean ± SD of three independent experiments; Student’s t-test. (e) Impaired chromatin relaxation analyzed by MNase sensitivity assay. Cells were exposed to the DSB inducing agent neocarzinostatin (400 ng/mL). (Left) Representative image. (Right) Quantification of average nucleosome size. Each value represents the mean ± SEM of three independent experiments; Student’s t-test.
Mentions: Comet assays demonstrated a significantly reduced DSB repair efficiency in BRIT1 LCLs (Fig. 5a, Supplementary Fig. 7b). Consistent with this, BRIT1 LCL also exhibited increased sensitivity to the topoisomerase inhibitors camptothecin and etoposide, which generate DSBs during S phase, a cell cycle phase in which lesions are predominantly repaired by HR26. This increased sensitivity was consistent with DSB generation during S-phase as the effects were abrogated when cells were treated with the DNA replication inhibitor aphidicolin (Fig. 5b). In addition, increased sensitivity to IR-induced DNA damage was observed in BRIT1 LCLs arrested in G1 phase, a cell cycle exclusively utilizing NHEJ to repair DSBs (Supplementary Fig. 7c). Together, our data suggested that BRIT1 LCL might have impaired cell survival as a result of generated DSBs being un-repaired because of both the defective HR and NHEJ repair. Furthermore, repair foci formation was also impaired in these cells with significantly reduced recruitment of RPA and Rad51 (Fig. 5c). These results were further confirmed by our detection of a decreased association of DNA repair proteins to chromatin in patients’ cells, while total protein levels were unaffected (Supplementary Fig. 7d–f).

Bottom Line: However, the mechanism mediating their recruitment to DNA lesions remains largely unknown.This increase in binding affinity provides a means by which SWI-SNF can be specifically recruited to and maintained at DNA lesions.Loss of BRIT1 causes impaired chromatin relaxation as a result of decreased association of SWI-SNF with chromatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, The University of Texas M. D. Anderson Cancer Center, Houston, 77054, USA.

ABSTRACT
To detect and repair damaged DNA, DNA-damage-response proteins need to overcome the barrier of condensed chromatin to gain access to DNA lesions. ATP-dependent chromatin remodelling is one of the fundamental mechanisms used by cells to relax chromatin in DNA repair. However, the mechanism mediating their recruitment to DNA lesions remains largely unknown. BRIT1 (also known as MCPH1) is an early DNA-damage-response protein that is mutated in human primary microcephaly. Here we report a previously unknown function of BRIT1 as a regulator of the ATP-dependent chromatin remodelling complex SWI-SNF in DNA repair. After damage to DNA, BRIT1 increases its interaction with SWI-SNF through ATM/ATR-dependent phosphorylation on the BAF170 subunit. This increase in binding affinity provides a means by which SWI-SNF can be specifically recruited to and maintained at DNA lesions. Loss of BRIT1 causes impaired chromatin relaxation as a result of decreased association of SWI-SNF with chromatin. This explains the decreased recruitment of repair proteins to DNA lesions and the reduced efficiency of repair in BRIT1-deficient cells, resulting in impaired cell survival after DNA damage. Our findings therefore identify BRIT1 as a key molecule that links chromatin remodelling with response to DNA damage in the control of DNA repair, and its dysfunction contributes to human disease.

Show MeSH
Related in: MedlinePlus