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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-SWI/SNF interaction is responsive to DNA damage(a) BRIT1-SWI/SNF interaction is enhanced in the presence of DNA damage signaling. Lysates were prepared from cells transfected with empty vector (V) or Flag-BRIT (B) at the indicated time points after exposure to IR (10 Gy) and immunoprecipitated. (b) DNA-damage induced BRIT1-SWI/SNF interaction is dependent on ATM/ATR signaling. Cells were harvested 15 minutes after exposure to IR (10 Gy). (c) BAF170 is a substrate candidate for ATM/ATR (Top and Middle) and its phosphorylation is dependent on the presence of ATM/ATR (Bottom). (d) The mutation on BAF170 (S969) suppressed the recognition of BAF170 by the p-S/TQ antibody after IR. A series of mutations on BAF170 were generated to replace potential ATM/ATR target sites S/TQ to AQ. Chk2-pulldown by p-S/TQ was used as a positive control to show that the general binding activity of p-S/TQ to other ATM/ATR substrates was not affected in cells transfected with M7 mutant. (e) BAF170 (S969) is phosphorylated in vitro by ATM/ATR kinase assay. The sequence around BAF170 (S969) was cloned into GST vector and the phospho-mutant (S969A) was made in this vector.
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Figure 2: BRIT1-SWI/SNF interaction is responsive to DNA damage(a) BRIT1-SWI/SNF interaction is enhanced in the presence of DNA damage signaling. Lysates were prepared from cells transfected with empty vector (V) or Flag-BRIT (B) at the indicated time points after exposure to IR (10 Gy) and immunoprecipitated. (b) DNA-damage induced BRIT1-SWI/SNF interaction is dependent on ATM/ATR signaling. Cells were harvested 15 minutes after exposure to IR (10 Gy). (c) BAF170 is a substrate candidate for ATM/ATR (Top and Middle) and its phosphorylation is dependent on the presence of ATM/ATR (Bottom). (d) The mutation on BAF170 (S969) suppressed the recognition of BAF170 by the p-S/TQ antibody after IR. A series of mutations on BAF170 were generated to replace potential ATM/ATR target sites S/TQ to AQ. Chk2-pulldown by p-S/TQ was used as a positive control to show that the general binding activity of p-S/TQ to other ATM/ATR substrates was not affected in cells transfected with M7 mutant. (e) BAF170 (S969) is phosphorylated in vitro by ATM/ATR kinase assay. The sequence around BAF170 (S969) was cloned into GST vector and the phospho-mutant (S969A) was made in this vector.

Mentions: As BRIT1 is an early DNA damage response protein5,6, we next examined whether the BRIT1-SWI/SNF interaction is responsive to DNA damage. The interaction between BRIT1 and SWI/SNF was indeed enhanced 15 mins after DNA damage with ionizing radiation (IR) (Fig. 2a). To gain mechanistic insights into this DNA damage-enhanced BRIT1-SWI/SNF interaction, we first determined whether this interaction is dependent on ATM and/or ATR, two central kinases in the DNA-damage response network. No apparent change was observed when either ATM or ATR was depleted (Supplementary Fig. 2a, b). However, deficiency of both ATM and ATR abolished the damage-enhanced interaction without affecting the basal binding affinity (Fig. 2b). These results suggest that ATM/ATR kinases are required for the DNA-damage enhanced BRIT1-SWI/SNF interaction. ATM/ATR substrates share a common motif S/TQ. Interestingly, we identified BAF170 (not BAF155) as a potential ATM/ATR substrate, which could be pulled down by the phospho-S/TQ (p-S/TQ) antibody in an ATM/ATR-dependent manner (Fig. 2c). We then generated a series of mutations (serines/threonines to alanines) on BAF170 S/TQ sites and found that when S969 was mutated, there was a significantly decreased the p-S/TQ antibody-binding affinity (Fig. 2d). The sequences around this site are very similar to the sequences around BRCA1 (S1432), a known ATM/ATR target site12 (Supplementary Fig. 2c), which allowed us to detect p-BAF170 (S969) by the antibody against p-BRCA1 (S1432) but not the S969A mutant (Supplementary Fig. 2c). The phosphorylation of this site was further confirmed by the in vitro kinase assay (Fig. 2e).


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-SWI/SNF interaction is responsive to DNA damage(a) BRIT1-SWI/SNF interaction is enhanced in the presence of DNA damage signaling. Lysates were prepared from cells transfected with empty vector (V) or Flag-BRIT (B) at the indicated time points after exposure to IR (10 Gy) and immunoprecipitated. (b) DNA-damage induced BRIT1-SWI/SNF interaction is dependent on ATM/ATR signaling. Cells were harvested 15 minutes after exposure to IR (10 Gy). (c) BAF170 is a substrate candidate for ATM/ATR (Top and Middle) and its phosphorylation is dependent on the presence of ATM/ATR (Bottom). (d) The mutation on BAF170 (S969) suppressed the recognition of BAF170 by the p-S/TQ antibody after IR. A series of mutations on BAF170 were generated to replace potential ATM/ATR target sites S/TQ to AQ. Chk2-pulldown by p-S/TQ was used as a positive control to show that the general binding activity of p-S/TQ to other ATM/ATR substrates was not affected in cells transfected with M7 mutant. (e) BAF170 (S969) is phosphorylated in vitro by ATM/ATR kinase assay. The sequence around BAF170 (S969) was cloned into GST vector and the phospho-mutant (S969A) was made in this vector.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2714531&req=5

Figure 2: BRIT1-SWI/SNF interaction is responsive to DNA damage(a) BRIT1-SWI/SNF interaction is enhanced in the presence of DNA damage signaling. Lysates were prepared from cells transfected with empty vector (V) or Flag-BRIT (B) at the indicated time points after exposure to IR (10 Gy) and immunoprecipitated. (b) DNA-damage induced BRIT1-SWI/SNF interaction is dependent on ATM/ATR signaling. Cells were harvested 15 minutes after exposure to IR (10 Gy). (c) BAF170 is a substrate candidate for ATM/ATR (Top and Middle) and its phosphorylation is dependent on the presence of ATM/ATR (Bottom). (d) The mutation on BAF170 (S969) suppressed the recognition of BAF170 by the p-S/TQ antibody after IR. A series of mutations on BAF170 were generated to replace potential ATM/ATR target sites S/TQ to AQ. Chk2-pulldown by p-S/TQ was used as a positive control to show that the general binding activity of p-S/TQ to other ATM/ATR substrates was not affected in cells transfected with M7 mutant. (e) BAF170 (S969) is phosphorylated in vitro by ATM/ATR kinase assay. The sequence around BAF170 (S969) was cloned into GST vector and the phospho-mutant (S969A) was made in this vector.
Mentions: As BRIT1 is an early DNA damage response protein5,6, we next examined whether the BRIT1-SWI/SNF interaction is responsive to DNA damage. The interaction between BRIT1 and SWI/SNF was indeed enhanced 15 mins after DNA damage with ionizing radiation (IR) (Fig. 2a). To gain mechanistic insights into this DNA damage-enhanced BRIT1-SWI/SNF interaction, we first determined whether this interaction is dependent on ATM and/or ATR, two central kinases in the DNA-damage response network. No apparent change was observed when either ATM or ATR was depleted (Supplementary Fig. 2a, b). However, deficiency of both ATM and ATR abolished the damage-enhanced interaction without affecting the basal binding affinity (Fig. 2b). These results suggest that ATM/ATR kinases are required for the DNA-damage enhanced BRIT1-SWI/SNF interaction. ATM/ATR substrates share a common motif S/TQ. Interestingly, we identified BAF170 (not BAF155) as a potential ATM/ATR substrate, which could be pulled down by the phospho-S/TQ (p-S/TQ) antibody in an ATM/ATR-dependent manner (Fig. 2c). We then generated a series of mutations (serines/threonines to alanines) on BAF170 S/TQ sites and found that when S969 was mutated, there was a significantly decreased the p-S/TQ antibody-binding affinity (Fig. 2d). The sequences around this site are very similar to the sequences around BRCA1 (S1432), a known ATM/ATR target site12 (Supplementary Fig. 2c), which allowed us to detect p-BAF170 (S969) by the antibody against p-BRCA1 (S1432) but not the S969A mutant (Supplementary Fig. 2c). The phosphorylation of this site was further confirmed by the in vitro kinase assay (Fig. 2e).

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