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TRIM29 Regulates the Assembly of DNA Repair Proteins Into Damaged Chromatin

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TRIM29 Regulates the Assembly of DNA Repair Proteins Into Damaged Chromatin ARTICLE Received 4 Feb 2015 | Accepted 27 Apr 2015 | Published 22 Jun 2015 DOI: 10.1038/ncomms8299 TRIM29 regulates the assembly of DNA repair proteins into damaged chromatin Yasushi Masuda1, Hidehisa Takahashi1, Shigeo Sato2, Chieri Tomomori-Sato2, Anita Saraf2, Michael P. Washburn2,3, Laurence Florens2, Ronald C. Conaway2,4, Joan W. Conaway2,4 & Shigetsugu Hatakeyama1 Although DNA double-strand break (DSB) repair is mediated by numerous proteins accu- mulated at DSB sites, how DNA repair proteins are assembled into damaged chromatin has not been fully elucidated. Here we show that a member of the tripartite motif protein family, TRIM29, is a histone-binding protein responsible for DNA damage response (DDR). We found that TRIM29 interacts with BRCA1-associated surveillance complex, cohesion, DNA- PKcs and components of TIP60 complex. The dynamics of the TRIM29-containing complex on H2AX nucleosomes is coordinated by a cross-talk between histone modifications. TRIM29 binds to modified histone H3 and H4 tails in the context of nucleosomes. Furthermore, chromatin binding of TRIM29 is required for the phosphorylation of H2AX and cell viability in response to ionizing radiation. Our results suggest that TRIM29 functions as a scaffold protein to assemble DNA repair proteins into chromatin followed by efficient activation of DDR. 1 Department of Biochemistry, Hokkaido University Graduate School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo, Hokkaido 060-8638, Japan. 2 Stowers Institute for Medical Research, 1000 East, 50th Street, Kansas City, Missouri 64110, USA. 3 Department of Pathology and Laboratory Medicine, Kansas University Medical Center, Kansas City, Kansas 6616, USA. 4 Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, Kansas 66160, USA. Correspondence and requests for materials should be addressed to S. H. (email: [email protected]). NATURE COMMUNICATIONS | 6:7299 | DOI: 10.1038/ncomms8299 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8299 NA double-strand break (DSB) is a serious DNA lesion Results that is caused by various genotoxic stresses including TRIM29 forms a protein complex with DNA repair proteins. Dionizing radiation (IR), DNA replication errors and To clarify which types of cells express TRIM29, we performed alkylating reagents. Deficiency or inaccuracy of DSB repair leads western blot analysis with the lysates of several cell lines. to various diseases including cancer, neurodegeneration and Consistent with the prediction using RefExA and previous immunodeficiency1. To overcome the intense toxicity of DSBs reports26, TRIM29 was highly expressed in epithelial cancer cell and maintain genome stability, cells have developed elaborate lines including HeLa, HeLa S3 and SiHa cells but not in surveillance mechanisms to detect DNA lesions and recruit DNA HEK293T and U2OS cells (Supplementary Fig. 1a). Although repair proteins to the DSB sites2. In the first step of DDR, the exogenous TRIM29 has been shown to be localized in the MRE11-RAD50-NBS1 (MRN) complex and Ku70-Ku80 cytoplasm27, the subcellular localization of endogenous TRIM29 heterodimer bind to the DSB ends and activate ATM and remains to be elucidated. We fractionated the lysates of HeLa S3, DNA-PKcs, respectively3,4. Then histone H2AX at Ser 139 is HeLa and SiHa cells into cytoplasmic, nucleoplasmic and rapidly phosphorylated by ATM and DNA-PKcs in response to chromatin fractions as shown in Fig. 1a28 and determined the DSBs5. Following the induction of phosphorylated H2AX at Ser subcellular localization of TRIM29. Both full-length TRIM29 and 139 (gH2AX), many regulators of the DNA repair pathway a truncated variant of TRIM29 with a molecular weight of about accumulate at DSB sites and activate the DSB repair pathway6,7. 50 kDa were expressed in the chromatin fraction (Fig. 1b and In addition to these sensor factors of DSBs, various proteins Supplementary Fig. 1b). The truncated variant is likely to be an contribute to the DSB repair pathway. MSH2 and MSH6 have a amino-terminal truncated variant and to be translated from the fundamental role in promoting DNA mismatch repair (MMR) internal start codon methionine 143. Indeed, an N-terminal and are also involved in the recruitment of DSB repair factors to truncated variant that shares 99% homology with full-length the DSB sites8. Several DNA repair factors form a multiprotein TRIM29 residues Met 143 to Pro 588 has already been complex. For example, ATM, MRN complex and MSH2-MSH6 isolated29. Immunofluorescence staining of endogenous dimer are components of the BRCA1-associated surveillance TRIM29 revealed that the distribution of TRIM29 overlapped complex (BASC)9. Cooperative assembly of DNA repair proteins with the 4,6-diamidino-2-phenylindole (DAPI)-stained region of into chromatin plays a critical role in the maintenance of chromatin in HeLa S3 cells (Fig. 1c). TRIM29 knockdown in chromatin integrity. HeLa S3, HeLa and SiHa cells decreased immunofluorescence Accumulating evidence indicates that a variety of histone signals of TRIM29 in the DAPI-stained region (Supplementary modifications function as a molecular switch in the recruitment Fig. 1c,d). Next, we generated HeLa S3 and U2OS cells stably of DSB repair proteins to the DSB sites10,11. Dimethylated H4 at expressing carboxy-terminal enhanced green fluorescent protein Lys 20 (H4K20me2) facilitates the accumulation of 53BP1 at (EGFP)-tagged TRIM29 and investigated whether it is located on the DSB sites12,13. Conversely, acetylation of H4 at Lys 16 chromatin. The distribution of TRIM29-EGFP was overlapped (H4K16Ac) by TIP60 promotes the accumulation of BRCA1 by with the DAPI-stained region in HeLa S3 cells but not in U2OS inhibiting the recruitment of 53BP1 to the DSB sites13. cells (Supplementary Fig. 1e). These findings indicate that Dimethylated H3 at Lys 36 (H3K36me2) recruits NBS1 and TRIM29 is located on chromatin in epithelial cancer cells. Ku70 at the DSB sites14. SETD2-dependent trimethylated H3K36 To understand the function of TRIM29 in chromatin, we (H3K36me3) plays an important role in both DSB repair and generated HeLa S3 cells stably expressing FLAG-TRIM29, the MMR pathway15–17. The assembly of DNA repair proteins prepared nuclear extracts from the cells by Dignam’s method into chromatin is coordinated by a cross-talk between and then immunopurified FLAG-TRIM29 and TRIM29-asso- posttranslational modifications (PTMs) of histones. ciated proteins from the nuclear extracts (Fig. 1d). We identified The gene encoding ATM is responsible for ataxia telangiectasia many of TRIM29-binding proteins by multidimensional protein (AT), which is an autosomal recessive disorder with pleiotropic identification technology (MudPIT) mass spectrometry. The phenotypes, including neuronal degeneration, immune dysfunc- relative abundance of a particular protein was assessed by means tion, premature ageing and increased cancer risk18. Tripartite of a normalized spectral abundance factor, or NSAF30. MudPIT motif protein 29 (TRIM29) was identified as an AT group analysis revealed that TRIM29 formed a multiprotein complex D-complementing gene in a functional complementation study with DSB repair proteins (Fig. 1e and Supplementary Table 1). with an AT cell line by transfection with a human cosmid Many of these proteins have DNA-binding activity, raising the library19. The TRIM protein family is characterized by the possibility that formation of the TRIM29 complex is dependent presence of a tripartite motif composed of a RING finger domain, on DNA derived from nuclear extracts. To exclude this one or two B-box domains and a coiled-coil domain20. possibility, we treated nuclear extracts with benzonase, Knockdown of TRIM29 in SiHa, Panc1 and BxPC3 cell lines immunopurified TRIM29-binding proteins and then analysed increased radiosensitivity21,22. Moreover, phosphorylation of TRIM29-associated proteins. Agarose gel electrophoresis of the TRIM29 by ATM and MAPKAP kinase 2 (MK2) contributes to nuclear extracts revealed that nuclear extracts did not contain the radioresistance of cells22. Although TRIM29 has been shown DNA (Fig. 1f). Western blotting and silver staining showed that to interact with several proteins including protein kinase C the TRIM29 complex can be formed independently of DNA substrate, disheveled-2, p53 and TIP60 (refs 21,23–25), little is (Fig. 1g and Supplementary Fig. 2a). We also found that TRIM29 known about how TRIM29 enhances the radioresistance of cells. was copurified with ATM (Fig. 1g). BASC, DNA-PKcs, cohesin Here we show that TRIM29 is a histone-binding protein and TIP60 complex play an important role in DDR5,10,31,32, involved in the regulation of DDR. Our biochemical studies suggesting that formation of the TRIM29 complex is dependent reveal that TRIM29 forms a multiprotein complex with BASC on genotoxic stress. To address this possibility, we purified the components, cohesion, DNA-PKcs and TIP60 complex. The TRIM29 complex from nuclear extracts of cells treated with or dynamics of the TRIM29 complex on nucleosomes containing without IR and analysed TRIM29-associated proteins. There was H2AX are coordinated by H3K36me2/3, H4K16Ac and no significant difference in composition of the TRIM29 complex H4K20me2. Chromatin binding of TRIM29 is also required to after IR treatment (Supplementary Fig. 2b,c). promote DDR. We propose that TRIM29 functions as a scaffold protein incorporating DNA repair proteins into
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