Methylated DNA-binding domain 1 and methylpurine–DNA glycosylase link transcriptional repression and DNA repair in chromatin

Sugiko Watanabe†‡, Takaya Ichimura†, Naoyuki Fujita†, Shu Tsuruzoe†, Izuru Ohki§, Masahiro Shirakawa§, Michio Kawasuji‡, and Mitsuyoshi Nakao†¶

†Department of Regeneration Medicine, Institute of Molecular Embryology and Genetics, and ‡First Department of Surgery, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan; and §Graduate School of Integrated Science, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama, Kanagawa 230-0045, Japan

Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved August 25, 2003 (received for review March 30, 2003) The methyl–CpG dinucleotide containing a symmetrical 5-methylcy- created by alkylating agents such as methylmethanesulfonate tosine (mC) is involved in regulation and genome stability. We (MMS) are 3-methyladenine (3-mA) (10%), 7-methylguanine (7- report here that -mediated transcriptional repressor mG) (65Ϸ80%), and O6-mG (0.3Ϸ7%). Among them, MPG methylated DNA-binding domain 1 (MBD1) interacts with methylpu- effectively removes 7-mG and 3-mA (21). The presence of these rine–DNA glycosylase (MPG), which excises damaged bases from modified bases in the genome leads to cell damage and death, due substrate DNA. MPG itself actively represses and has a to inappropriate DNA replication and genome instability with synergistic effect on gene silencing together with MBD1. Chromatin increased (22). In fact, Mpg knockout mice showed high immunoprecipitation analysis reveals the molecular movement of sensitivity to alkylation damage of their DNA (23). MBD1 and MPG in vivo:(i) The MBD1–MPG complex normally exists Evidence of multiple connections between transcription and on the methylated gene ; (ii) treatment of cells with alky- DNA repair has emerged from many studies. In particular, tran- lating agent methylmethanesulfonate (MMS) induces the dissocia- scription-coupled repair has been documented in exci- tion of MBD1 from the methylated promoter, and MPG is located on sion repair that is a main pathway involved in correcting bulky DNA both methylated and unmethylated promoters; and (iii) after com- lesions induced by UV light. The rate of repair in actively tran- pletion of the repair, the MBD1–MPG complex is restored on the scribed is significantly faster than in nontranscribed regions methylated promoter. Mobility-shift and structural analyses show of the genome (24). It has also been found that several lesions ؋ that the MBD of MBD1 binds a methyl–CpG pair (mCpG mCpG) but removed by BER are related to transcriptional status (25, 26), but not the methyl–CpG pair containing a single 7-methylguanine (N) ؋ the relationship between BER and transcription remains to be (mCpG mCpN) that is known as one of the major lesions caused by elucidated. Recently, TDG has been shown to function in tran- MMS. We further demonstrate that knockdown of MBD1 by specific scriptional control, through an interaction with transcription factors GENETICS small interfering RNAs significantly increases cell sensitivity to MMS. and coactivators (27–31). Human endonuclease III, one of the These data suggest that MBD1 cooperates with MPG for transcrip- DNA glycosylases, also interacts with nucleotide excision repair– tional repression and DNA repair. We hypothesize that MBD1 func- endonuclease XPG and the damage-inducible tions as a reservoir for MPG and senses the base damage in chromatin. Y box-binding 1 (32, 33). During the investigation of the MBD1-containing complex, we NA methylation at position 5 of within CpG dinucle- found that MBD1 specifically interacts with MPG. From the Dotides is the major epigenetic modification of mammalian observation of molecular movement of MBD1 and MPG in vivo,we genomes and is required for gene regulation, chromatin formation, found that MBD1 was dissociated from the methylated gene and genome stability (1). The methylation status of DNA is promoter under the conditions of MMS treatment, which may correlated with a wide range of phenomena during mammalian support that repair of specific DNA lesions within the genome relies development (2–4). On the other hand, aberrant methylation of on local perturbation of chromatin structure (34, 35). We discuss a tumor-associated gene promoters contributes directly to the pro- mechanistic link between DNA repair, gene repression, and chro- gression of some cells (5–7). In the nucleus, cytosine matin dynamics in response to base damage. methylation is specifically recognized by transacting factors such as the methyl–CpG-binding domain [methylated DNA- Materials and Methods binding domain (MBD) proteins] (1, 8). MBD1, MBD2, MBD3, Cell Culture, Transfection, and Immunoprecipitation. HeLa, NCL- and MeCP2 act as transcriptional repressors depending on the H1299, and SBC5 cells were cultured and introduced with plasmids presence of methyl–CpG pairs in chromatin (9–12). In addition, by using FuGENE6 (Boehringer Mannheim) or Lipofectamine 5-mC and cytosine have high rates due to spontaneous (Invitrogen) (36, 37). Immunoprecipitation of MBD1 and MPG hydrolytic , giving rise to and , respec- was done as described in Supporting Methods, which is published as tively (13). Two DNA glycosylases, MBD4 and thymine DNA supporting information on the PNAS web site, www.pnas.org. glycosylase (TDG), correct the resultant mismatches in the CpG context by excising thymine and uracil (14–16). Plasmids and Antibodies. For information on plasmids and anti- DNA glycosylases initiate (BER) by severing the glycosylic bonds of numerous damaged bases such as alkylated, bodies, see Supporting Methods. deaminated, and oxidized bases due to cellular metabolism and

exogenous agents (17). Thus, BER protects the genome against the This paper was submitted directly (Track II) to the PNAS office. mutagenic effects of a variety of DNA damage in the cell (18). Abbreviations: siRNA, small interfering RNA; MBD, methylated DNA-binding domain; TDG, Among the glycosylase family of proteins, methylpurine–DNA thymine DNA glycosylase; MPG, methylpurine–DNA glycosylase; MMS, methylmethanesul- glycosylase (MPG) catalyzes the excision of many kinds of base fonate; TRD, transcriptional repression domain; BER, base excision repair; mG, methylgua- substrates, including methylpurines, deaminated [hypoxan- nine; mC, methylcytosine. thine], oxidized [8-oxoguanine], and cyclic etheno adducts ¶To whom correspondence should be addressed. E-mail: [email protected]. on both adenine and guanine (19, 20). The majority of lesions © 2003 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.2131819100 PNAS ͉ October 28, 2003 ͉ vol. 100 ͉ no. 22 ͉ 12859–12864 Downloaded by guest on October 1, 2021 Fig. 1. Interaction of MBD1 with MPG. (A) Structure of MBD1 and MPG. MBD1 (isoform v3) contains the MBD, nuclear localization signal (NLS), cysteine-rich CXXC domains, and TRD (9, 37). TRD (amino acids 473–536) was used in a yeast two-hybrid screen as a bait. MPG has an enzymatically active site at glutamic acid (E) 125. In full-length and deletion of MPG, the presence and absence of MBD1 interaction in Fig. 2A are indicated by plus and minus, respectively. (B) Association between endogenous MBD1 and MPG in HeLa cells. (C) Direct binding of the TRD of MBD1 to MPG in vitro. Bacterially expressed (His)6-TRD of MBD1 and GST-MPG were used for nickel-NTA resin affinity chromatography (38). The arrowhead indicates the full-length MPG fused to GST.

In Vitro Binding and GST Pull-Down Assay. The in vitro binding assay Results was performed as described (ref. 38 and Supporting Methods). For MBD1 Interacts with MPG. To identify factors that interact with pull-down assay, GST and GST-fusion proteins (200 nM each) were MBD1, we performed a yeast two-hybrid screen by using the immobilized on glutathione-agarose beads and incubated with 250 C-terminal TRD as a bait (Fig. 1A). From a screening of Ϸ7 ϫ 106 ␮l of the lysates from HeLa cells. The lysates were prepared by using independent transformants of a human HeLa cDNA library, we a buffer [0.05% Nonidet P-40͞50 mM Tris⅐HCl (pH 8.0)͞100 mM isolated two independent cDNA clones encoding the MPG. NaCl͞protease inhibitors͞1 mM sodium orthovanadate]. To demonstrate an interaction between MBD1 and MPG in vivo, we investigated an association of these endogenous proteins in Luciferase Assay. For information on the luciferase assay, see HeLa cells. The cells were lysed and then subjected to immuno- Supporting Methods. precipitation with anti-MPG or anti-MBD1 antibodies. Western blot analysis showed that MBD1 was present in the immunopre- Growth Inhibition Assay. Cells were seeded at a density of 1 ϫ 105 cipitates with MPG but not in the control lane (Fig. 1B Upper). cells per well on six-well dishes and treated for1hwithMMS. After Endogenous MPG was coprecipitated with MBD1, but not with the incubation for 3 days, the cells were quantitatively counted, and the control antibodies (Fig. 1B Lower). results were shown as a ratio of the number of MMS-treated cells To demonstrate whether the TRD of MBD1 directly binds MPG, relative to that of untreated cells (percent of control growth) (39). (His)6-TRD of MBD1 and GST-fused MPG were bacterially prepared for affinity chromatography as described (38). The puri- Chemical Crosslinking and Chromatin Immunoprecipitation. At 48 h fied (His)6-TRD of MBD1 was incubated with GST or GST-MPG after transfection, cells (1 ϫ 106) were treated with 1 mM MMS for in the presence of the nickel-NTA resin (Fig. 1C). The GST-MPG 1 h. The cells were treated with dimethyl 3,3Ј-dithiobispropionimi- was released specifically from the resin bound by TRD of MBD1 date-2HCl (5 mM), rinsed, and then crosslinked by addition of (lane 5), indicating the association of MPG with the TRD of MBD1. 1% formaldehyde for 10 min. The cell lysates were prepared As a control, GST was not released from the same resin (lane 3), for chromatin immunoprecipitation and PCR amplification of and GST-MPG did not associate with the resin bound by unrelated the human p16 gene promoter (refs. 36 and 37 and Supporting (His)6-Ubc4, one of the ubiquitin-conjugating (lane 4). In addition, there were several degradation products of GST-MPG in Methods). the input (lane 1), because this protein was reported to have many protease hypersensitive sites (43). These degradation products did Electrophoretic Mobility-Shift Assay (EMSA). Oligodeoxynucleotides not bind the TRD of MBD1, suggesting that almost full-length of containing 7-mG were prepared by a primer extension method MPG is required for this interaction (as shown in Fig. 2). (refs. 40 and 41 and Supporting Methods). For the EMSA, binding reactions were carried out in 10 ␮l of a buffer [10 mM Tris⅐HCl (pH ͞ ͞ ͞ ␮ Regions of MPG for Interacting with MBD1. To determine the regions 8.0) 5 mM MgCl2 5mMDTT5% glycerol] by using 0.8 gof of MPG that are crucial to the interaction with TRD of MBD1, six GST-fused MBD of MBD1 and 200 nM of the 30-mer oligonucle- deletion mutants of FLAG-MPG (shown in Fig. 1A) were ex- otide duplex containing cytosine or mC in combination with G or pressed in HeLa cells. GST-fused TRD of MBD1 or GST was mG, for 30 min at room temperature. The reaction mixtures were immobilized on glutathione-agarose beads and incubated with the loaded on a 6% polyacrylamide gel, and the gel was stained with lysates from HeLa cells expressing the full-length or deletion SYBR green I (Molecular Probes). mutants of MPG. After repeated washings, the proteins bound on the beads were detected by anti-FLAG antibodies (Fig. 2A). The Small Interfering RNA (siRNA) Knockdown of MBD1. siRNA duplexes full-length MPG bound the TRD of MBD1 but not GST alone. The were designed for targeting mRNA encoding human MBD1 (Japan deletions of C-terminal regions of MPG did not associate with Bioservice, Saitama, Japan): 5Ј-GGCAUCUUGUGCUAUCCA- the TRD of MBD1 [⌬C1, -2, and -3 (data not shown)]. Further, GTT-3Ј and 5Ј-CUGGAUAGCACAAGAUGCCTT-3Ј. The the N-terminal deletions ⌬N2 and -3 bound the TRD of MBD1, siRNAs for lamin A͞C and GL3 were previously reported (42). but ⌬N1 lost the ability to bind it. Collectively, these re- The siRNAs were transfected into the cells by using Oligofectamine sults suggested that MPG directly interacts with TRD of MBD1 (Invitrogen). through both terminal regions of MPG.

12860 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.2131819100 Watanabe et al. Downloaded by guest on October 1, 2021 Fig. 2. Regions of MPG for interacting with MBD1. (A) Association of MPG with the TRD of MBD1. GST- fused TRD of MBD1 or GST was immobilized on gluta- thione agarose beads and incubated with the lysates from HeLa cells expressing full-length or deletion mu- tants of MPG. (B) Localization of MPG and MBD1 in the nucleus. The full-length or deletion mutants of FLAG- MPG were coexpressed with DsRed-fused MBD1.

To investigate the localization of MPG and MBD1 in the nucleus, these effects was very similar to that in the case of TDG and thyroid full-length or deletion mutants of MPG fused to FLAG and transcription factor-1 (30). However, FLAG-MPG mutants (⌬C1 fluorescent protein-tagged MBD1 were coexpressed and visualized and ⌬N1) did not affect the repression by TRD of MBD1 (Fig. 3C), by using a confocal laser-scanning microscope (Fig. 2B). Previous emphasizing the importance of the interaction between MBD1 and reports clarified that the punctate localization of MBD1 depends on MPG. These results suggested that MPG itself can actively repress the distribution of methyl–CpG sequences on the genome (9, 10, 36, transcription, and that MBD1 and MPG cooperate to enhance their 37). Full-length ⌬N2 and ⌬N3 of MPG colocalized with MBD1 at repressive activities. multiple foci in the nuclei. In contrast, both ⌬C1 and ⌬N1 mutants diffusely distributed throughout the nuclei and significantly weak- Molecular Dynamics of MBD1 and MPG Under MMS-Induced DNA ened the specific colocalization with MBD1. These results repre- Damage. The alkylating agent MMS is known to induce genome- sented Ͼ90% of the nuclei in the cells studied, suggesting that both wide base damage, including 7-mG, which is the major substrate for terminal regions of MPG are important for the association with MPG in vivo. To investigate the association of MBD1 and MPG MBD1. with chromosomal gene promoters under conditions of MMS treatment, we chose the p16 tumor suppressor gene in which MBD1 and MPG Cooperate for Transcriptional Repression. Molecules hypermethylation of the promoter-associated CpG island causes in the DNA repair system, like TDG, are actively involved in transcriptional repression in many (5, 7). NCI-H1299 cells transcriptional regulation (27, 28, 30, 31). To check whether MPG possess methylated p16 promoter, whereas the same DNA region modulates gene promoter activities, we expressed GAL4-MPG and is unmethylated in SBC-5 cells (36, 44, 45). We first tested a FLAG-MBD1 (Fig. 3A), GAL4-TRD of MBD1 and FLAG-MPG dose-dependent effect of MMS on the treated cells by using a cell (Fig. 3B), and GAL4-TRD of MBD1 and FLAG-MPG mutants growth inhibition assay (Fig. 4A). The growth inhibition usually (⌬C1 and ⌬N1) (Fig. 3C) in HeLa cells. The effects of these corresponds to the level of genome-wide base damage (39). Both GENETICS combinations were examined by using a Photinus pyralis luciferase cell lines were treated with MMS (0Ϸ3 mM) for 1 h and grown for reporter that contains five GAL4-binding elements just upstream 3 days, resulting in a similar growth inhibition in proportion to the of human SNRPN and p16 gene promoters. Both gene activities are MMS concentrations. Repeated experiments showed that MMS (1 known to be affected by the methylation status in cells (44). mM)-treated cells resumed their proliferation after temporary GAL4-MPG alone repressed transcription from both promoters in growth arrest (data not shown), indicating that this treatment is not a dose-dependent manner (Fig. 3A; FLAG mock). To assess the excessively toxic for the cells studied. functional implication of the MBD1-MPG association, we exam- We next investigated the dynamics of MBD1 and MPG on ined the effect of FLAG-MBD1 on the repression by GAL4-MPG. chromosomal genes in vivo (Fig. 4 B and C). For chromatin Expression of MBD1 enhanced the MPG-mediated repression of immunoprecipitation, HA-MBD1 and FLAG-MPG were equally both promoter activities (Fig. 3A; FLAG-MBD1). Next, we eluci- expressed in NCI-H1299 and SBC-5 cells, and these cells were then dated the effect of MPG on repression by GAL4-fused TRD of treated with 1 mM MMS for 1 h. Western blot analysis revealed that MBD1 (Fig. 3B). TRD efficiently repressed the luciferase activities exogenously expressed MBD1 and MPG as well as endogenous of both promoters in agreement with previous reports (9, 10). The ␤-tubulin were present at the indicated time points after MMS coexistence of GAL4-TRD of MBD1 and FLAG-MPG produced treatment (Fig. 4B). These cells were crosslinked with dimethyl a synergistically repressive effect on these promoters. The level of 3,3Ј-dithiobispropionimidate-2HCl and then with formaldehyde.

Fig. 3. Cooperation of MBD1 and MPG for transcrip- tional repression. GAL4-MPG and FLAG-MBD1 (A), GAL4-TRD of MBD1 and FLAG-MPG (B), and GAL4-TRD of MBD1 and FLAG-MPG mutants (C) were expressed in HeLa cells to examine their effects on a luciferase reporter that contains five GAL4-binding elements just upstream of human SNRPN or the p16 gene pro- moter. The cells were transfected with plasmids (1 ␮g) to express FLAG-tagged proteins, together with indi- cated amounts of plasmids for GAL4-fused proteins. FLAG-mock was used as a control. The luciferase ac- tivities from insertless GAL4-mock were normalized to 100. Values are given as means and standard devia- tions of results from more than three independent experiments.

Watanabe et al. PNAS ͉ October 28, 2003 ͉ vol. 100 ͉ no. 22 ͉ 12861 Downloaded by guest on October 1, 2021 Fig. 4. Molecular dynamics of MBD1 and MPG under MMS-induced DNA damage. (A) MMS-dependent growth inhibition of NCI-H1299 and SBC-5 cells. Cell numbers were examined after MMS treatment for 1 h and after incubation for 3 days. (B) Expression of MBD1, MPG, and ␤-tubulin in untreated and MMS-treated cells. At 48 h after transfection, both cell lines expressing HA-MBD1 and FLAG-MPG were treated with 1 mM MMS for 1 h. Western blot anal- ysis was done with anti-MBD1, anti-FLAG, and anti-␤-tubulin antibodies. (C) Associa- tion of MBD1 and MPG with chromosomal p16 gene promoters under MMS treat- ment. The p16 gene promoter is highly methylated in NCI-H1299 cells and unmeth- ylated in SBC-5 cells (36, 37). For chromatin immunoprecipitation, the cells expressing HA-MBD1 and FLAG-MPG were treated with 1 mM MMS for 1 h and incubated for the indicated times. The coprecipitated with the indicated antibodies were PCR-amplified by using a set of primers for p16 promoter sequences. Genomic DNAs in the input cell lysates before the immuno- precipitation were used as a control. All data are from more than three indepen- dent experiments.

Coprecipitated DNAs with the indicated antibodies were subjected CpG site (51). Therefore, it is of great interest to investigate to PCR amplification by using a set of primers for p16 promoter whether the ability of MBD to bind a methyl–CpG pair is affected sequences. The amplified sequence is 217 bp long, containing 23 by base damage. The major lesion created by methylating agents CpG dinucleotides (CϩG content 75.1%; CpG͞GpC ϭ 0.68). such as MMS is a 7-mG (21). Further, recent studies have indicated These CpG sites are highly methylated in NCI-H1299 cells (36, 37, that cytosine methylation at CpG* sequences enhances mitomycin- 44, 45). The MBD1–MPG complex was normally present on the induced alkylation of the guanine (*), compared with unmethylated methylated, but not unmethylated, p16 gene promoter (Fig. 4C; CpG sites (52). Because there is the possibility that chemical untreated). At3haftertreatment (referred to as the active stage guanine methylation by MMS occurs at a methyl–CpG site, we of BER (refs. 46 and 47, and Fig. 7A, which is published as analyzed whether MBD of MBD1 is able to bind the CpG pair supporting information on the PNAS web site), MBD1 was unex- containing a mG (Fig. 5). We first prepared four 30-base paired pectedly dissociated from the methylated promoter (Fig. 4C; 3 h). oligodeoxynucleotides containing CG ϫ CG, mCG ϫ mCG, This dissociation of MBD1 from the methylated promoter was also mCG ϫ mCmG, or CG ϫ CmG in a unique position by using a found at6hafterMMStreatment. In contrast, MPG existed on the primer extension reaction as described (Fig. 5A) (40, 41). An both methylated and unmethylated promoters, probably for exci- electrophoretic mobility-shift assay was performed by using these sion of damaged bases. At 24 h after treatment, the presumed double-stranded DNAs and bacterially expressed MBD of MBD1 terminal stage of the repair process (refs. 46 and 47, and Fig. 7A), (Fig. 5B) (10, 51). MBD specifically bound the oligodeoxynucle- MBD1 was restored on the methylated promoter (Fig. 4C;24h). otide-containing mCG ϫ mCG, whereas the DNA containing MPG was associated with the unmethylated promoter until 24 h mCG ϫ mCmG did not associate with MBD. The oligodeoxynucle- after treatment and was little found on the promoter regardless of otide containing CG ϫ CG or CG ϫ CmG also did not bind MBD. the methylation status at 80 h after treatment (Fig. 4C;80h). MPG This result suggested that the occurrence of a single mG in the may leave this promoter and target other damaged sites on the methyl–CpG pair abolished the ability of MBD1 to bind the genome at this stage. At 100 h after MMS treatment, the MBD1– methylated DNA. MPG complex was restored on the methylated, but not unmethyl- Structure analysis by NMR further supported the important ated, p16 gene promoter (Fig. 4C; 100 h). Additional analyses at the participation of the modified guanine bases in the association other chromosomal region, such as the imprinted SNRPN gene between MBD of MBD1 and the methyl–CpG dinucleotide (Fig. promoter, gave consistent results (data not shown). These obser- 5C). Two methyl groups of in the methyl–CpG site are vations suggested that MBD1 and MPG change their localization distinguished by major groove contacts made by five residues of the on the genome in response to MMS-induced base damage (see MBD: Val-20, Arg-22, Trp-34, Arg-44, and Ser-45. Among them, Discussion). Arg-22 and -44 also recognize the two guanine bases within the dinucleotide (a). The guanidiums of these residues are poised to Presence of 7-mG in a Methyl–CpG Pair Abolishes Methylated DNA- donate a hydrogen bond to the guanine bases in the methyl–CpG Binding Ability of MBD1. We focused on understanding our obser- site (b and d). Conversion of either of these guanine bases to 7-mG vation that MBD1 was dissociated from the methylated region by alkylation could significantly interfere with guanidium–G inter- under the damage caused by MMS. MBD, which is highly con- actions. The methyl group introduced to one of the guanine bases served among the MBD family of proteins, is reported to be would cause a steric clash with the guanidium group of Arg-22 (c); essential for their localization on methylated regions (10, 37, the methyl group of the guanine base also can interfere with 48–50). Our previous structural analyses by using NMR revealed ␦-methylene of Arg-44 (e). These arginine residues play integral that MBD residues not only distinguish the two methyl groups of roles in DNA recognition, because substitution of either these cytosines but also recognize the two guanine bases at the methyl– residues totally abolished DNA binding (10, 51). So far, these

12862 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.2131819100 Watanabe et al. Downloaded by guest on October 1, 2021 Fig. 6. MBD1 knockdown increases cell sensitivity to MMS. (A) Effectiveness of siRNA knockdown of MBD1. The cells were transfected with siRNAs target- ing mRNAs encoding MBD1 and lamin A͞C. MPG and ␤-tubulin are also detected. (B) Effect of MBD1 knockdown on cell growth inhibition by MMS. Growth inhibition assay was performed as in Fig. 4A. GL3 siRNA was used as a control (41). Values are given as means and standard deviations from five independent experiments. GENETICS used specific siRNAs capable of degrading target transcripts in a highly selective manner in vivo. Western blot analysis demonstrated the effectiveness of siRNA knockdown of MBD1 and lamin A͞C (Fig. 6A). Both siRNAs did not change the levels of MPG and ␤-tubulin. Under such conditions, we examined whether MBD1 can affect cell growth inhibition with MMS treatment. At 24 h after the transfection of siRNAs, these two cell lines were treated with indicated concentrations of MMS for 1 h and then incubated for 3 days. The growth inhibition by MMS moderately but significantly increased in the MBD1 knockdown cells, in contrast to the control cells (Fig. 6B). This result was comparable to the higher sensitivity to alkylation damage in MPG knockout cells (23). Finally, we quantified abasic sites in MMS treated control and MBD1- Fig. 5. Abrogation of methylated DNA binding of MBD by the presence of 7-mG in a methyl–CpG pair. (A) Construction of 30 base-paired oligode- knockdown cells by using aldehyde reaction probes (40). This result oxynucleotides containing 5-mC and͞or 7-mG. The unique dinucleotides, suggested that MBD1 knockdown disturbed the efficient repair of CG ϫ CG, mCG ϫ mCG, mCG ϫ mCmG, and CG ϫ CmG, were introduced into base lesions induced by MMS (Fig. 7B). Taken together, these data position (XG ϫ XY). mG or G was incorporated into position Y of the synthe- suggest that MBD1 in cooperation with MPG contributes to DNA sized strand. (B) Electrophoretic mobility-shift assay by using the oligode- repair response. oxynucleotides and MBD from MBD1. (C) Structural model of the interaction between MBD and methylated DNA. A proposed interaction between the Discussion methyl–CpG site and two arginine side chains at the interface, on the basis of In the present study, we reported that MBD1 specifically interacts NMR structure determination, is shown in A. The interactions between the with MPG. Both terminal regions of MPG were potentially critical guanine bases and the arginine side chains (b and d) can be perturbed by alkylations at N7 positions of the guanine bases (c and e). Arginine 22 and 44, for the association with the TRD of MBD1. The 3D structure of blue; DNA, red; hydrogen bond between arginine and guanine, white; methyl MPG complexed with the substrate DNA showed that MPG binds groups in methyl–CpG sequence, yellow. a DNA adduct without any requirements of additional proteins in vitro (53). It was, however, pointed out that MPG protein used for the structural analysis is an enzymatically active fragment that findings suggest that the incidence of damaged guanine in a lacked the N terminus (residues 1–79) of the protein. In living cells, methyl–CpG pair leads to dissociation of MBD from methylated interaction with MBD1 and other molecules is likely to contribute DNAs. to the functional regulation of MPG. In addition, in vitro gel-shift assays demonstrated that MPG can bind DNAs without sequence Increased Sensitivity to MMS in MBD1 Knockdown Cells. To demon- specificity and regardless of the presence of modified bases (Fig. 8A, strate the role of MBD1 in MPG-mediated DNA repair, we finally which is published as supporting information on the PNAS web

Watanabe et al. PNAS ͉ October 28, 2003 ͉ vol. 100 ͉ no. 22 ͉ 12863 Downloaded by guest on October 1, 2021 site). MPG seemed to interact with fully methylated DNAs and with are involved in DNA damage response. Similarly, MBD1-based MBD1 on the methylated DNAs (Fig. 8B). heterochromatin may serve as a reservoir for MPG that responds As in the cases of TDG and endonuclease III, MPG functions as to numerous base damage. a transcriptional repressor. By using a luciferase reporter analysis, Compared with 3-methyladenine, it has been thought that 7-mG we found that MPG-dependent repression is not alleviated by is relatively innocuous to cells because it appears not to directly deacetylase (HDAC) inhibitors (data not shown). TDG acts interfere with DNA replication (55). Our study, however, suggested as a repressor of the transcriptional factors, and TDG-mediated that 7-mG in a methyl–CpG pair can alter the chromatin structure repression was also resistant to the HDAC inhibitors (30). Our due to the inability of MBD1 to bind the damaged methyl–CpG recent investigations revealed that MPG physically binds transcrip- dinucleotide. Alternatively, the dissociation of MBD1 may result tional factor Sp1 and some components of TFIIH (data not shown). from unidentified chromatin change after DNA damage. In tran- These associations may play a role in MPG-mediated gene repres- scription-coupled repair, actively transcribed genes are repaired sion. The TRD of MBD1 produces strong transcriptional repres- significantly earlier than nontranscribed regions of genome (34). sion activities via interaction with MPG as well as recently identified However, it is also important that base damage in nontranscribed MBD1-containing chromatin associating factor (MCAF) (36). In- heterochromatin regions is properly repaired, because genome- terestingly, MPG stably interacts with TRD of MBD1 in the wide DNA damage directly induces abnormalities and presence of MCAF (data not shown). genomic instability (56). Thymine glycosylase MBD4 binds prefer- On the basis of our data in Fig. 4, we propose a model of dynamics entially to methyl–CpG ϫ TpG for the mismatch repair that of both proteins for the repair process. MBD1 and MPG normally originates from a methyl–CpG pair (16). On the other hand, MPG exist on the methylated promoter, probably for repression and in coexists with MBD1 in the methylated DNA regions and repairs readiness for DNA damage. On DNA damage by MMS that mostly base damage in both the methylated and unmethylated regions. As creates 7-mG, MBD1 is dissociated from the damaged methyl–CpG was the case of MBD4, the interaction of MBD1 with MPG may sites just like a sensor for the damaged bases. Because MBD1 lies function as one of the DNA repair system associated with methyl– in the bottom of the chromatin on methylated DNA regions, the CpG dinucleotides. Our study sheds light on the close link between dissociation of MBD1 would stimulate chromatin remodeling and BER, transcription, and chromatin dynamics. further release the molecules packed in the chromatin. At this stage, MPG distributes widely on the genome to remove the damaged We thank K. Kubo (Osaka Prefecture University, Osaka) for MPG- bases and inhibit transcription from these sites. After completion of expressing Escherichia coli, H. Ide and N. Saitoh for helpful suggestions, the repair, MBD1 returned on the repaired methyl–CpG sites and H. Saya for much appreciated support. This work was supported by together with MPG to reconstruct the repressive chromatin. Re- a Grant-in-Aid for Scientific Research on Priority Areas; by a Grant- cently, Martin et al. (54) proposed a model in which the telomeric in-Aid for 21st Century Center of Excellence Research from the Ministry heterochromatin serves as a reservoir for many chromatin factors of Education, Culture, Sports, Science and Technology; and by a grant such as Ku and the nucleosome-binding SIR proteins in yeast that from the Uehara Memorial Foundation (to M.N.).

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