p300 Acetylase and Thymine DNA Glycosylase Links DNA Repair and Transcription

Molecular Cell, Vol. 9, 265–277, February, 2002, Copyright 2002 by Cell Press

Association of CBP/p300 Acetylase and Thymine DNA Glycosylase Links DNA Repair and Transcription Marc Tini,1,2,4,5 Arndt Benecke,2 Soo-Joong Um,2,6 Joseph Torchia,3 Ronald M. Evans,1 and Pierre Chambon2 1 Gene Expression Laboratory Howard Hughes Medical Institute The Salk Institute for Biological Studies 10010 North Torrey Pines Road La Jolla, California 92037 2 Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire CNRS/INSERM/ULP Colle`ge de France, BP163 67404 Illkirch Cedex France 3 Department of Pharmacology and Toxicology Cancer Research Laboratories London Regional Cancer Center The University of Western Ontario London, Ontario N6A 4L6 Canada

Summary DNA repair in chromatin is subject to topological constraints, suggesting a requirement for chromatin modification and remodeling activities. Thymine DNA glycosylase (TDG) initiates repair of G/T and G/U mismatches, commonly associated with CpG islands, by removing thymine and uracil moieties. We report that TDG associates with transcriptional coactivators CBP and p300 and that the resulting complexes are competent for both the excision step of repair and histone acetylation. Furthermore, TDG stimulates CBP transcriptional activity in transfected cells and reciprocally serves as a substrate for CBP/p300 acetylation. Remarkably, this acetylation triggers release of CBP from DNA ternary complexes and also regulates recruitment of repair endonuclease APE. These observations reveal a potential regulatory role for protein acetylation in base mismatch repair and a role for CBP/ p300 in maintaining genomic stability. Introduction In vertebrate genomes, CpG islands are associated with regulatory regions of genes, and methylation of the C5 position of cytosines (meC) mediates gene silencing via specification of repressive chromatin structure (reviewed in Ng and Bird, 1999). The CpG dinucleotide is highly susceptible to mutation, contributing to approximately 30% of all germline 4

Correspondence: [email protected] Present address: Department of Pharmacology and Toxicology, Cancer Research Laboratories, The University of Western Ontario, London, Ontario N6A 4L6, Canada. 6 Present address: Department of Bioscience and Biotechnology, Sejong University, 98, Kunja-Dong, Kwangjin-Ku, Seoul 143-747, Republic of Korea. 5

mutations (Cooper and Youssoufian, 1988). Cytosines and, in particular, methylated cytosines (meC) are susceptible to high mutation rates due to spontaneous hydrolytic deamination that generates uracil and thymine, respectively (Lindahl, 1993). Uncorrected G/T and G/U mispairs may alter both coding and regulatory sequences. Hence, repair of these mispairs is necessary to maintain stable patterns of gene expression. The key enzymes in these repair processes are DNA glycosylases that recognize and excise the mispaired thymine and uracil moieties from the ribose ring, generating an abasic residue (reviewed in Scha¨rer and Jiricny, 2001). The predominant pathway for correction of an abasic site involves the creation of a single nucleotide gap by apurinic/apyrimidinic endonuclease I (APE also known as HAP1, Ref-1, and APEX) that cleaves the phosphodiester bond 5⬘ of the missing base and DNA polymerase ␤ (Pol␤), which removes the deoxyribose 5⬘ phosphate (Scha¨rer and Jiricny, 2001). Repair is completed by Pol␤ and ligase, which replace the missing nucleotide and reestablish the continuity of the DNA strand. Two mammalian thymine DNA glycosylases have been identified (TDG and MBD4) (Hendrich et al., 1999; Neddermann et al., 1996) containing distinct glycosylase domains that preferentially excise G/T and G/U mismatches in the CpG context. TDG has also been demonstrated to mediate excision of 3,N4-ethenocytosine, a promutagenic and genotoxic adduct produced by metabolites of carcinogens ethyl carbamate and vinyl chloride (Medina et al., 1998; Saparbaev and Laval, 1998). The transcriptional machinery relies on chromatinmodifying enzymes and remodeling complexes to alter chromatin structure and gain access to DNA (reviewed in Urnov and Wolffe, 2001); similarly, an increasing body of evidence suggests that the DNA repair machinery requires chromatin remodeling activities for repair of DNA lesions (Meijer and Smerdon, 1999). Moreover, since G/T and G/U mispairs occur frequently within CpG islands, repair of these lesions may be coupled to transcription, as in nucleotide excision repair mediated by general transcription factor TFIIH (de Laat et al., 1999). The observation that TDG associates with transcription factors (Chevray and Nathans, 1992; Um et al., 1998) is consistent with this possibility. CREB binding protein (CBP) (Chrivia et al., 1993) and p300 (Eckner et al., 1994) integrate diverse signaling pathways by acting as coactivators for a number of sequence-specific transcription factors, including CREB, AP-1 (Jun and Fos), nuclear receptors, and the tumor suppressor p53 (reviewed in Goodman and Smolik, 2000). When brought to DNA, CBP/p300 are believed to activate transcription through chromatin remodeling via intrinsic histone acetyltransferase (HAT) activity (Bannister and Kouzarides, 1996; Ogryzko et al., 1996) and interactions with components of the basal transcription machinery (e.g., TBP, TFIIB, RNA pol II) (see Goodman and Smolik, 2000). We demonstrate that CBP/p300 form a physical and functional complex with TDG, directly linking chromatin modifying activity and DNA repair. In addition, we estab-

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Figure 1. CBP-TDG Complexes Form In Vivo and Cleave G/T Mispairs (A) Whole-cell extracts prepared from COS-1 cells transfected with TDG and/or HA-tagged CBP expression vectors were subjected to immunoaffinity selection using a monoclonal HA antibody. The immunoprecipitates were analyzed by immunoblotting with either a CBPspecific antibody (top panel) or a TDG-specific antibody (bottom panel). The relative mass of size markers is indicated in kilodaltons (kDa). (B) G/T oligonucleotide cleavage analysis of CBP-TDG complexes. The above immunoprecipitates were incubated with radioactively labeled duplex oligonucleotide (sequence is indicated at bottom of panel) containing a G/T mismatch. Cleavage of the mismatched thymine releases a 12 nucleotide fragment following alkali treatment and PAGE analysis. A 12 nucleotide size marker (12-mer) was included in lane 1.

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lish that TDG is an effective transcriptional coactivator, as well as a CBP/p300 substrate. Accordingly, we provide evidence for the reciprocal modulation of transcription and DNA repair and suggest TDG acetylation as a plausible molecular switch coordinating these functions. Results CBP Forms Complexes with TDG In Vivo that Can Recognize G/T Mispairs As modification of CpG islands is linked to both gene regulation and chromatin modulation, we explored the potential links between TDG, the enzyme specialized in maintenance of CpG islands, and chromatin remodeling factors. CBP was identified as a potential partner of TDG by coimmunoprecipitation and GST (glutathione S-transferase)-fusion protein interaction assays. Expression vectors for TDG and hemagglutinin (HA) epitope-tagged CBP were transfected into COS-1 cells, and whole-cell extracts were subjected to immunoaffinity selection using anti-HA antibody coupled to sepharose. Immunoprecipitates were analyzed by immunoblotting with either a CBP- or TDG-specific antibody. TDG was coprecipitated from extracts prepared from cells transfected with both expression vectors but not from extracts of cells expressing either CBP or TDG vectors alone (Figure 1A, compare lane 6 with lanes 4 and 5), indicating that CBP-TDG complexes can be formed in intact cells. We tested these immunoprecipitates for the ability to cleave a radioactively end-labeled G/T mispaired duplex oligonucleotide. Thymine-cleaving activity could be readily detected only in immunoprecipitates derived from cells coexpressing HA-tagged CBP and TDG (Figure 1B, lane 4, compare with lanes 2 and 3). Furthermore, we examined the nuclear distribution of cotransfected fluorescent protein-tagged CBP (YFP-CBP) and TDG (CFP-TDG) in NIH3T3 cells. Generally, expression of YFP-CBP generated a granular nuclear pattern (Figure 1C, I), whereas diffuse nuclear staining was observed with CFP-TDG (Figure 1C, III). Coexpression resulted in a distinctive macro-granular pattern for both YFP-CBP and CFP-TDG in which the proteins colocalized (Figure 1C, V–VII). To exclude the possibility that the CBP-TDG interaction might result from protein overexpression, HeLa cell nuclear extracts were initially fractionated on a P11 phosphocellulose column using a step gradient with increasing salt concentrations. Immunoblot analysis of the P11 fractions indicated that the vast majority of TDG was eluted in 0.5 KCl buffer (data not shown), which also contained part of the CBP cellular pool. This TDGcontaining fraction was subjected to further purification by DEAE-Sepharose followed by gel filtration chromatography using a Sephacryl S-300 column. TDG consis-

tently resolved into two major components on the sizeexclusion column, a peak eluting at a mass greater than 1.5 MDa and a second peak eluting at approximately 400 kDa. In contrast, CBP was found only in the fractions corresponding to the 1.5 MDa TDG complex. Importantly, immunoprecipitation of the pooled CBP-TDG containing S300 fractions using a TDG-specific antibody indicates that CBP is present in TDG-containing complexes (Figure 1E), in agreement with immunoprecipitation experiments using crude nuclear extracts (data not shown). HAT and CH3 Domains of CBP/p300 Interact with Distinct Regions of TDG Potential interaction domains were localized using subcloned segments of CBP bacterially expressed as fusion proteins with GST (Figure 2A) and radioactively labeled TDG produced in vitro by transcription/translation. Initially, TDG was found to interact with two overlapping segments of CBP corresponding roughly to the CH3 (amino acids [aa] 1621–1877) and HAT domains (aa 1098–1758) (see Figure 2B). To further delineate the interaction domain(s), overlapping GST fusion proteins were constructed spanning the entire CH3 region. TDG interacted strongly with two carboxy-terminal fragments of CH3 (GST-CH3.C and GST-CH3.D) (Figure 2C, lanes 5 and 6) and only weakly with GST-CH3.B (lane 4), indicating the existence of at least one strong binding site within the 50 residues shared by CH3.C and CH3.D (aa 1777–1827). As this region is distinct from the HAT domain, CBP appears to contain at least two separate TDG interaction sites. The CH3 region interacts with RNA helicase A (RHA) (aa 1805–1891) (Nakajima et al., 1997), TFIIB (aa 1680–1812) (Kwok et al., 1994), and PCAF (aa 1801–1880) (Yang et al., 1996). All three factors bound strongly to GST-CH3.C and GST-CH3.D (Figure 2C, lanes 5 and 6) as previously described; however, PCAF also significantly bound to GST-CH3.B (lane 4). Hence, TDG, RHA, TFIIB, and PCAF interact with a region of CBP that has been shown to be important for the formation of transcriptionally competent complexes (Nakajima et al., 1997). Deletion mutants of mouse TDG were produced in vitro and used for interaction studies with bacterially expressed GST-HAT and GST-CH3 fusion proteins (Figure 2E). TDG residues 32–91 were necessary for binding to the HAT domain but dispensable for interaction with CH3, while interaction of the latter with TDG required both residues 92–121 and 273–421. The requirement for both amino- and carboxy-terminal residues for CH3 binding was confirmed by yeast two-hybrid analysis (Figure 3D), and their importance was evaluated in the context of full-length CBP using recombinant baculovirus-expressed protein. Deletion of residues 1–121 led to a reduction in binding, whereas lack of residues 273–421

(C) Nuclear colocalization of transfected TDG and CBP. NIH3T3 cells were transfected with either YFP-CBP (panels I and II) or CFP-TDG (panels III and IV) and in combination (panels V–VII). Nuclear fluorescence was visualized on a deconvolution microscope. (D) Partial purification of CBP-TDG complexes. HeLa cell nuclear extracts were biochemically fractionated using the indicated chromatographic steps. Fractions from size exclusion column (S300) were immunoblotted for CBP and TDG. Molecular mass standards are indicated. (E) Coimmunoprecipitation of CBP and TDG from partially purified fractions. Pooled aliquots from S300 column fractions 19–21 were immunoprecipitated with anti-TDG IgG or normal IgG as control.

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Figure 2. Mapping of CBP-TDG Interaction Domains (A) Schematic representation of CBP showing protein interacting domains. Three cysteine-histidine (CH)-rich regions are indicated along with transcriptional activation domains (AD) located within the amino-terminal region (N-AD, aa 228–461) and the carboxy-terminal region (C-AD, aa 1960–2158) (Swope et al., 1996). The bromo (B) domain (aa 1108–1170) is also shown. (B) Binding of in vitro-translated TDG to discrete domains of CBP. GST interaction assays were carried out with the indicated CBP fusion proteins. (C) In vitro binding of TDG, TFIIB, RHA, and PCAF to overlapping fragments of CH3. [35S]-labeled TDG and RHA produced in vitro, along with recombinant TFIIB and PCAF, were used. (D) Schematic representation of mouse TDG showing enzymatic and interaction domains. Residues 123–371 of mouse TDG are sufficient for G/U cleavage activity, whereas G/T cleavage requires an additional 57 aa extension (aa 67–371) rich in hydrophilic residues (Gallinari and Jiricny, 1996; Neddermann et al., 1996). Yeast two-hybrid analysis was performed on the CBP CH3 domain fused to the lexA DNA binding domain and TDG deletions fused to the VP16 activation domain. Deletions not analyzed are designated ND (not determined). [35S]-labeled full-length TDG and deletion mutants were analyzed for binding to full-length recombinant CBP (D), GST-HAT, and GST-CH3 (E). “I” represents 10% of input TDG, while “C” indicates binding to the GST control and “B” binding to GST-fusion.

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had little effect (see Figure 2D). These observations indicate that the amino- and carboxy-terminal interfaces of TDG are each sufficient for association with full-length CBP.

(Sheng et al., 1991) containing a serine to alanine mutation at amino acid 133 did not respond to cotransfected CBP and TDG, consistent with the failure of this mutant to bind CBP (Parker et al., 1996).

TDG Stimulates CBP-Dependent Transcription and Is Associated with Euchromatin We compared the nuclear distribution of fluorescent protein-tagged MBD4 (EGFP-MBD4) (Hendrich and Bird, 1998) and TDG (YFP-TDG) in transfected NIH3T3 cells. As previously reported, EGFP-MBD4 is localized in distinct nuclear foci that stain intensely with DAPI and correspond to heterochromatin (Figure 3A, IV–VI). Interestingly, YFP-TDG appears to be absent from heterochromatin as demonstrated by the prominent blue staining within a green background in the merged YFP/DAPI image (Figure 3A, III). The striking difference in nuclear distribution of MBD4 and TDG is consistent with the presence of a methyl CpG binding domain in MBD4 and indicates that these enzymes, although possessing similar DNA repair specificities, function in distinct nuclear compartments. Hence, TDG may function within transcriptionally active regions associated with euchromatin. The effect of TDG on CBP transcriptional activity was investigated using a GAL4 DNA binding domain (DBD) fusion of CBP and a GAL reporter gene. Cotransfection of TDG with GAL4DBD-CBP in a number of cell lines (COS-1, MCF-7, and JEG-3) stimulated in a dose-dependent manner the activity of the reporter gene (Figure 3B and data not shown) but had negligible effects on the GAL4DBD-SP1Q (Seipel et al., 1992) chimeric transactivator. TDG does not appear to possess intrinsic activation function as GAL4DBD-TDG is transcriptionally inactive even upon overexpression of CBP (Figure 3B). Deletion of carboxy-terminal residues (aa 273–421) completely abrogated stimulation of CBP-dependent transcription, while deletion of the amino-terminal hydrophilic region (aa 1–121) had negligible effects (Figure 3C). Therefore, the carboxy-terminal interface of TDG appears to play a more dominant role in interactions with CBP in vivo compared to the amino-terminal interface. Interestingly, an enzymatically deficient mutant of TDG (N151A) (Hardeland et al., 2000) retained the ability to stimulate transcription, thereby indicating that the transcription and DNA repair functions of TDG are separable. We next examined whether TDG could stimulate CREB-dependent transcription in a CBP-dependent manner, as CREB is activated by the cyclic AMP pathway through phosphorylation of serine 133 and recruitment of CBP (Parker et al., 1996). In these assays, we used a deletion mutant of CREB lacking the leucine zipper (GAL-CREB⌬LZ) (Sheng et al., 1991), since in c-Jun this structure has been shown to interact with TDG in yeast two-hybrid assays (Chevray and Nathans, 1992). Consistent with previous reports (Kwok et al., 1994), the transcriptional activity of GAL-CREB⌬LZ was stimulated by expression of CBP and protein kinase ␣ catalytic subunit (PKA). Cotransfection of TDG expression vector resulted in a dose-dependent enhancement of the reporter gene activity, thus confirming that TDG acts as a positive regulator of CBP-dependent transcription (Figure 3D). A mutant of CREB (GAL-CREB⌬LZM1)

CBP-TDG Complexes Bind DNA and Are Competent for G/T-U Repair and Histone Acetylation CBP-TDG complexes appear to bind DNA as they retain the ability to cleave mispaired thymines (Figure 1B). The association of CBP and TDG may provide chromatin modifying activity at sites of G/T-U repair to facilitate access of other components of this repair pathway. To explore this possibility, we assessed both the ability of CBP-TDG complexes to mediate histone acetylation and cleavage of mispairs. We used the avidin/biotin coupled DNA (ABCD) precipitation assay (Glass et al., 1990) to investigate ternary complex formation between TDG and either full-length CBP or the HAT and CH3 fragments. The DNA binding properties of bacterially expressed TDG were tested using either normally paired (G/C) or mispaired (G/T, G/U) duplex oligonucleotides. Binding assays were carried out in the presence of trace amounts of the respective [32P]-labeled oligonucleotide to allow monitoring of cleavage activity. TDG bound to all three oligonucleotides but exhibited higher affinity for mispaired DNA (Figure 4A, lanes 3–5). The expected 12 nucleotide cleavage product was detected only with oligonucleotides bearing G/T or G/U mispairs, thus confirming that recombinant TDG was biologically active in this assay. Full-length CBP did not bind directly to either G/T or G/U mispaired DNA (Figure 4B, lanes 6 and 8) but could be recruited to DNA by TDG (Figure 4B, lanes 9 and 10). Similarly, the separate TDG-interacting domains of CBP (HAT and CH3) bound to either G/T or G/U mispaired DNA only in the presence of TDG (data not shown). As highly purified proteins were used in these assays, these data confirm that TDG directly associates with each CBP/p300 interaction domain. The effect of CBP on TDG glycosylase activity was addressed by performing G/T and G/U cleavage assays in vitro with recombinant TDG in the presence of highly purified baculovirus-expressed CBP. The ability of TDG to cleave G/T and G/U mispairs was not affected even when 4-fold molar excess of CBP (Figure 4C, lanes 5 and 10) was added in the repair assay. Similarly, analysis of the CBP HAT activity using a biotinylated histone H4 peptide (Ait-Si-Ali et al., 1998) revealed that a large molar excess of TDG (30-fold) caused only a small (25%) decrease in HAT activity (Figure 4D). These observations indicate that TDG-CBP complexes can recognize G/T-U mispairs and retain the ability to acetylate histones. TDG Is Acetylated In Vivo and In Vitro by CBP/p300 The HAT domain of CBP/p300 has been shown to acetylate both histones and nonhistone proteins (for review see Chen et al., 2001). TDG contains lysine residues within the hydrophilic amino-terminal domain that constitute potential acetylation sites; therefore, the acetylation state of FLAG epitope-tagged TDG was examined in vivo by labeling transfected MCF-7 cells with [3H] sodium acetate and immunoprecipitation with antiFLAG antibody (Figure 5A). A band corresponding to acetylated TDG could be detected in transfected cells

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Figure 3. TDG Associates with Euchromatin and Stimulates CBP-Dependent Transcription (A) Distinct nuclear distribution of glycosylases MBD4 and TDG. NIH3T3 cells were transfected with EGFP-MBD4 and YFP-TDG expression vectors. Nuclear fluorescence (EGFP, YFP, and DAPI) was visualized (magnification 150⫻) using appropriate filters. (B) MCF-7 cells were transfected with GAL4-DBD reporter plasmid (1 ug) (see below) and expression vectors (250 ng) for Gal4DBD, Gal4DBDCBP, Gal4DBD-SP1Q, and Gal4DBD-TDG in combination with TDG (50, 250, 1000 ng) and CBP (500 ng) expression vectors. (C) Differential requirement for amino- and carboxy-terminal residues of TDG for transcriptional stimulation. Wild-type TDG (1000 ng) and

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Figure 4. CBP-TDG Complexes Promote G/T and G/U Cleavage and Histone Acetylation (A) Recombinant TDG binds to paired (G/C) and mispaired (G/T, G/U) oligonucleotides and specifically cleaves mispaired thymine and uracil. Avidin-biotin coupled DNA binding (ABCD) assay was performed with each biotinylated double-stranded oligonucleotide (refer to Figure 1B for sequence) and recombinant TDG (top panel). The respective radiolabeled oligonucleotides were also included in the binding reactions to monitor cleavage activity (bottom panel). (B) ABCD assay showing TDG-dependent recruitment of baculovirus-expressed full-length CBP to oligonucleotides containing either G/T or G/U mispairs. (C) CBP does not alter cleavage activity of TDG. G/T and G/U cleavage assays were performed in the presence of TDG (12 ng) and increasing amounts (75, 150, 300 ng) of purified baculovirus-expressed CBP. (D) TDG binding preserves the HAT activity of CBP. A biotinylated H4 peptide was incubated in the presence of CBP, increasing amounts of TDG, and [14C] acetyl CoA. Acetylated peptide was recovered using streptavidin beads and quantified by scintillation counting.

but not in mock-transfected cells, indicating that TDG is acetylated in vivo. Furthermore, TDG could be directly acetylated in vitro by CBP and p300 in the presence of [14C] AcCoA (Figure 5C, lanes 3 and 4). Similar results were obtained with bacterially expressed GST-HAT (data not shown). Deletion of the first 91 residues of TDG did not significantly alter the level of acetylation, whereas deletion of 121 residues reduced acetylation

to background levels (Figure 5D). These data confirm that the hydrophilic amino-terminal region is a target for acetylation. Identification of acetylated lysine residues was carried out by in vitro acetylation of synthetic peptides corresponding to residues 68–91 (P1) and 91–121 (P2) in the presence of [14C] acetyl CoA, separation of acetylated species by HPLC, and quantification of radioactivity as-

truncation mutants TDG122-421 and TDG32-272 were transfected with Gal4DBD-CBP (250 ng). Glycosylase-deficient mutant N151A was also tested. (D) TDG activates the GAL-CREB⌬LZ chimeric activator but not GAL-CREBM1⌬LZ bearing a mutation in the CBP interaction domain. Four hundred nanograms of GAL-CREB⌬LZ, 1 ␮g of CBP, and 100 ng of PKA␣ expression vectors were transfected where indicated along with TDG expression vector. The luciferase reporter plasmid contains five copies of the GAL4 DNA binding site fused to the core of the ␤ globin promoter.

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Figure 5. Acetylation of TDG by CBP/p300 (A) In vivo acetylation of TDG. MCF-7 cells transfected with FLAG epitope-tagged TDG as well as mock-transfected cells were metabolically labeled with [3H] sodium acetate. Total cell extracts were immunoprecipitated with anti-FLAG immuno-affinity beads, followed by Western blot analysis (left panel) or autoradiography (right panel). (B) Coomassie staining of proteins used for in vitro acetylation studies. (C) TDG is acetylated by both CBP and p300 in vitro. Recombinant CBP and p300 (150 ng) were incubated with TDG (500 ng) in the presence of [14C] AcCoA. Proteins were then analyzed by SDS-PAGE and visualized by autoradiography. (D) CBP/p300 acetylate a basic amino-terminal domain of TDG. Acetylation reactions were carried out with intact TDG and amino-terminal deletions (TDG92-421 and TDG122-421). Coomassie staining of recombinant TDG amino-terminal deletions is shown on the left panel. (E) Identification of acetylated lysine residues. Peptides corresponding to residues 68–91 (P1) and residues 91–121 (P2) were acetylated in vitro with CBP and [14C] acetyl CoA. Acetylated species were purified by HPLC and subjected to Edman degradation. Graphs show the amount of radioactivity associated with each round of sequencing.

sociated with each residue following Edman degradation. Strong acetylation sites were identified at lysine residues 94, 95, and 98 with P2 and unexpectedly also at residue 70 with P1 (Figure 5E). TDG Acetylation Leads to Release of CBP and Prevents Recruitment of APE We next explored the potential influence of TDG acetylation on DNA binding and repair of G/T and G/U mispairs. The effect of acetylation on the formation of CBP-TDG mispaired DNA ternary complexes was examined using the ABCD assay. Addition of AcCoA sharply decreased the amount of CBP recovered in the complex, whereas the level of TDG remained unchanged (Figure 6A, lanes 7–10). To determine whether TDG acetylation affected DNA binding and the excision step of repair, G/T and G/U cleavage and DNA binding assays were performed following acetylation of TDG in vitro. These studies did not detect significant differences in DNA

binding or cleavage activity of acetylated TDG (data not shown). Complete repair of G/T-U mismatches requires TDG, APE, DNA polymerase ␤ (pol␤), and ligase (Waters et al., 1999). The different repair steps are believed to be coordinated by sequential recruitment of each component to the repair site. Thus, interactions between pol␤ and APE (Bennett et al., 1997) and pol␤ and ligase (Prasad et al., 1996) have been demonstrated, while interaction between TDG and APE has been inferred on the basis of reaction kinetics (Waters et al., 1999). Direct association between recombinant TDG and APE was demonstrated using the GST interaction assay and mapped to residues 92–121 (Figure 6B), which are also required for interaction with CH3 (Figure 2). We shall refer to this region as AID (Acetyltransferase/APE Interaction Domain). The effect of TDG acetylation on recruitment of APE was examined using the GST-based interaction assay.

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Figure 6. Acetylation of TDG Blocks both CBP and APE Interactions (A) Acetylation of TDG releases CBP from DNA ternary complexes. ABCD assays were carried out with TDG and CBP on G/T and G/U oligonucleotides in the presence or absence of AcCoA. (B) Interaction of TDG with APE requires residues 92–121. Intact TDG or amino-terminal truncations 92–421 and 122–421 were tested for binding to GST-APE affinity beads. Degradation products of TDG are evident when recombinant proteins are prepared by nondenaturing conditions as opposed to denaturing conditions (as in [C]). (C) Acetylated TDG does not recruit APE. Interaction assays were carried out with GSTAPE coupled to glutathione beads and either unmodified TDG (lane 4) or acetylated TDG (lane 6). TDG was acetylated in vitro with CBP and depleted of acetylase by immunoprecipitation. Approximately 150 ng of TDG was used in the binding reactions.

TDG was acetylated in vitro and CBP removed by immunodepletion using an anti-FLAG antibody affinity matrix. Surprisingly, while unmodified TDG bound readily to GST-APE beads, no binding could be detected with acetylated TDG (Figure 6C, lane 6). These results were also confirmed by performing interaction studies with APE on acetylated TDG immobilized on nickel sepharose beads (data not shown). Hence, CBP through acetylation of AID can modulate interactions of TDG with APE and therefore regulate the second step in this DNA repair pathway. Discussion The mechanisms underlying DNA damage recognition and repair in chromatin are poorly understood; however, chromatin remodeling is a common hallmark of DNA repair, transcription, and DNA replication. To the best of our knowledge, TDG is the first example of a repair enzyme involved in detection of primary DNA lesions that interacts directly with histone acetyltransferase CBP/p300 and represents a new class of HAT substrate. The striking feature of the CBP/p300-TDG interaction is that the complex retains enzymatic activities associated with each component and therefore has the potential to function in both DNA repair and transcription. CBP/p300 as Chromatin Remodeling Cofactors in TDG-Mediated DNA Repair The mechanisms by which DNA glycosylases locate damaged bases within the genome are not known. However, it has been suggested that they scan DNA for

modified bases that fit into the active site (Verdine and Bruner, 1997). Hence, TDG as a DNA binding factor may function similarly to sequence-specific transcription factors that recruit chromatin remodeling factors to promoter regions. In eukaryotes, repair of specific DNA lesions within the genome is associated with local perturbation of chromatin structure (Meijer and Smerdon, 1999). Presumably, local chromatin remodeling facilitates the repair process, consistent with studies indicating that access to DNA lesions is impeded by chromatin structure (Wellinger and Thoma, 1997) and that histone hyperacetylation is associated with more efficient DNA repair (Ramanathan and Smerdon, 1989). Similarly, a TIP60 acetylase cellular complex has been recently identified and implicated in double-strand break DNA repair (Ikura et al., 2000). The demonstration that CBP-TDG complexes can recognize mispairs, mediate excision, and acetylate histone tails supports a model whereby CBP/p300 coupled to TDG mediate local chromatin remodeling to facilitate further processing of the abasic site (see Figure 7, I). The recent finding that p300 associates with proliferating cell nuclear antigen (PCNA) and flap endonuclease I (FEN1), factors required for both DNA replication and repair, suggests a central role of CBP/p300 in these processes (Hasan et al., 2001a, 2001b). PCNA and FEN1 are essential for an alternative pathway to short-patch repair that corrects abasic sites and other DNA lesions generating a 2–6 nucleotide repair patch (long-patch repair) (Scha¨rer and Jiricny, 2001). Our findings, together with these observations, are consistent with multiple functions of CBP/p300 in base excision repair. The transcriptional stimulatory properties of TDG and

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Figure 7. Putative Roles of CBP/p300-TDG Complexes in Repair and Transcription CBP/p300 recruitment to mispairs by TDG may facilitate repair by promoting local chromatin remodeling (left panel). Displacement of CBP/ p300 by APE would then be required for complete repair. Since TDG acetylation appears to exert an inhibitory effect on recruitment of accessory factor APE, further repair may be contingent on inhibition of this acetylation or require the action of deacetylases. Alternatively, TDG may be recruited to promoter regions as part of CBP/p300 complexes or directly by sequence-specific transcription factors (TF) (right panel). The association of TDG with transcription factors would allow TDG to transiently uncouple from CBP/p300 and allow the recruitment of APE. Following repair, acetylation of TDG would prevent further interactions with APE but allow transcriptional activation. TDG acetylation may therefore act as a molecular switch to coordinate DNA repair and transcriptional functions.

its reported association with transcription factors (Chevray and Nathans, 1992; Um et al., 1998) suggest that CBP/p300-TDG complexes may link transcription and DNA repair. In this scenario, CBP/p300-TDG or TDG may be recruited to promoter regions by transcription factors to first mediate the repair of regulatory regions and subsequently to promote transcription (Figure 7, II). This would ensure that transcriptionally active genes are repaired prior to transcription. Hence, the transcriptional role of TDG may be linked to repair, which would be consistent with the association of numerous other factors with the CH3 domain (Goodman and Smolik, 2000). Interestingly, p53 has been shown to promote repair of abasic sites by APE and pol␤ in vitro, thus providing further evidence for the involvement of transcription factors in this repair pathway (Zhou et al., 2001). TDG Acetylation Modulates Molecular Interactions Acetylation-dependent alterations in DNA binding have been reported for a number of transcription factors

(Chen et al., 2001). In contrast, TDG acetylation appears not to affect binding and cleavage of G/T-U mispaired DNA but leads to the release of CBP/p300 from the DNAbound complex and abrogates interaction with APE. This induced release is consistent with the identification of acetylated lysine within the 92–121 region that serves as an interaction interface. These observations suggest a direct regulatory role for acetylation in TDG-dependent DNA repair and imply that formation of stable CBP/p300TDG complexes in vivo may require additional factors to initially inhibit acetyltransferase activity. Accordingly, a number of studies have indicated that the HAT activity of CBP/p300 and other acetyltransferases can be regulated by phosphorylation as well as through binding of viral and cellular proteins including transcription factors (Ait-Si-Ali et al., 1998; Barlev et al., 1998; Chakravarti et al., 1999; Hamamori et al., 1999; Soutoglou et al., 2001). The abrogation of the APE interaction following TDG acetylation suggests a plausible role in suppression of APE-dependent repair by blocking the second cleavage

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step that follows removal of the mispaired base. Although histone hyperacetylation has been linked to enhanced DNA repair (Meijer and Smerdon, 1999), there is also some evidence that acetylation may negatively regulate DNA repair (Stoilov et al., 2000). Since CBP/ p300 HAT activity appears to be regulated in a cell cycledependent manner (Ait-Si-Ali et al., 1998), acetylationdependent regulation of TDG-mediated repair may also display cell cycle dependence. In addition, TDG acetylation may promote the recruitment of other factors such as SWI-SNF remodeling complexes, which are known to preferentially recognize acetylated lysine residues (Zhang et al., 2001). Transfection studies have demonstrated that TDG activates GAL-CBP and GAL-CREB in a CBP-dependent manner. Interestingly, the amino-terminal interface (aa 92–121) for CBP/p300 and APE recruitment is not required for transcriptional stimulation. In contrast, either deletion or acetylation of this region prevents recruitment of APE. These findings suggest that acetylation has a selective inhibitory effect on DNA repair and therefore may serve as a molecular switch between the repair and transcriptional functions of TDG (Figure 7). Acetylation of TDG following initial repair would prevent further recruitment of the DNA repair machinery while maintaining the transcriptional activation function of TDG. However, we cannot exclude a more general role of TDG in transcriptional regulation. Along these lines, both chicken and human TDG have been reported to possess weak demethylase activity for meCpG in vitro (Zhu et al., 2001, 2000). Further studies are required to determine whether this activity plays a role in transcriptional activation. Genetic Instability and Cancer: Links to CBP/p300 and TDG CpG sites located within tumor suppressor genes are hotspots for somatic mutations believed to have a causative role in the etiology of cancer (Greenblatt et al., 1994). It has been suggested that the prevalence of C to T mutations found in key regulatory genes in cancer cells may reflect dysfunction in TDG-mediated repair or increased susceptibility to deamination damage (Robertson and Jones, 1997). However, no evidence for G/T repair defects have been found in cells derived from tumors, and no mutations in the tdg gene have been found to date. In contrast, mutations in the cbp and p300 genes have been found in a variety of tumors including myeloid leukemia, colorectal, and gastric carcinoma (Giles et al., 1998). Interestingly, mutations of cbp are associated with the Rubinstein-Taybi syndrome, a disorder characterized by developmental abnormalities and susceptibility to malignancy (Petrij et al., 1995). Recent studies have extended these observations by demonstrating that mice heterozygous for cbp are susceptible to hematopoietic tumors with late onset and characteristic loss of heterozygosity associated with tumor suppressor genes (Kung et al., 2000). Malignant transformations associated with mutations in the cbp and p300 genes are believed to be due to defects in the critical signaling pathways that require these coactivators (Petrij et al., 1995; Tanaka et al., 1997). Thus, our findings suggest that alteration of putative tumor suppressor

CBP and p300 may deregulate TDG-coupled repair and contribute to genomic instability commonly associated with cancer. Experimental Procedures Plasmids Plasmid constructs were verified by sequencing and details are available on request. Hemagglutinin A (HA)-tagged CBP, GAL-CBP, and TDG were expressed in either pSG5 (Green et al., 1988) or pCMX mammalian expression vectors. Constructs lacking the amino-terminal region of TDG were fused to the SV40 nuclear localization signal (NLS) to replace the natural NLS contained within this region. TDG bacterial expression vector was constructed using the pET15 vector (Novagen). Baculovirus expression vectors for CBP and PCAF are based on the pAcSGHis vector (PharMingen) and a derivative bearing the FLAG epitope. Bacterial expression vectors encoding GST fusions of CBP domains were made using the pGEX2TK vector (Pharmacia). A GST fusion bearing residues 1098–1758 of CBP has been previously described (Bannister and Kouzarides, 1996). Mouse TDG mutant N151A was designed on the basis of previous analysis of human TDG (Hardeland et al., 2000). Transfections COS-1 and MCF-7 were maintained in Dulbecco’s minimal essential medium supplemented with 5% and 10% fetal bovine serum, respectively. Cells were seeded onto six-well dishes and were generally transfected using the calcium phosphate procedure or Targefect transfection reagent (Targeting Systems, San Diego). Approximately 1 ␮g of luciferase-based reporter plasmid and 100–1000 ng of pSG5based or pCMX-based expression vectors were used. Cells were washed and supplemented with fresh medium 12–16 hr later and assayed for luciferase and ␤-galactosidase activity the next day. Transfection experiments were performed at least three times in duplicate, and representative experiments are shown. Immunoprecipitations and Biochemical Fractionation Whole-cell extracts prepared from transfected Cos-1 were precleared by incubating 1 ml (typically about 2.5 mg protein) with 100 ␮l (packed bead volume) of protein G-Sepharose (Pharmacia) for 1 hr at 4⬚C. After centrifugation, supernatants were transferred to a fresh tube containing 7.5 ␮l (packed bead volume) of protein G Sepharose loaded with anti-HA monoclonal antibody and incubated at 4⬚C for 3 hr. Immunoprecipitates were washed five times with 200 ␮l of IP buffer (50 mM Tris-HCl [pH 7.9], 150 mM NaCl, 10% glycerol, 0.1% NP40, 1 mM DTT, 0.5 mM PMSF, proteinase inhibitors cocktail) and resuspended in 2⫻ Laemmli buffer and subjected to SDS-PAGE. Proteins were electroblotted onto nitrocellulose membranes and identified using a rabbit polyclonal antibody against CBP (Santa Cruz) and a mouse monoclonal TDG antibody (Um et al., 1998). Biochemical fractionation of HeLa cell nuclear extracts were carried out as previously described (Underhill et al., 2000). Fractions from the size-exclusion column (S300) were enriched approximately 150- to 200-fold in TDG relative to unfractionated nuclear extracts. Immunoprecipitation of the S300 TDG-containing fractions was carried out with pooled 200 ␮l aliquots using TDGspecific rabbit IgG and normal IgG as control. Protein Expression, Purification and GST-Based Interaction Assay Protein purifications from pGEX-based (Pharmacia) and pET-based (Novagen) bacterial expression vectors were performed according to manufacturer’s recommendations using gluthatione and nickel affinity chromatography, respectively. Baculovirus-expressed CBP was partially purified using nickel affinity chromatography (Novagen) or purified to single band homogeneity using FLAG antibody affinity matrix according to manufacturer indications (Sigma). GST-based interaction assays were performed by incubating in NETN buffer (50 mM Tris HCl [pH 7.9], 100 mM NaCl, 1 mM EDTA, and 1 mM DTT) (1 hr at 4⬚C) glutathione sepharose beads containing recombinant GST-fusions with either recombinant target protein (100–1000 ng) or 2 ␮l of rabbit reticulocyte lysate (Promega) containing [35S] radiolabeled protein. The beads were subsequently washed four times with

Molecular Cell 276

NETN buffer, and bound proteins were fractionated by SDS-PAGE and visualized by autoradiography or immunoblotting. TFIIB was revealed by immunoblotting with a specific rabbit polyclonal antibody, while PCAF was detected with a polyhistidine monoclonal antibody (Sigma). ABCD Assays Approximately 1 ␮g of G/T and G/U mismatch containing doublestranded oligonucleotides (see Figure 1) was incubated for 30 min at room temperature with 10 ␮l of a slurry of streptavidin-coated paramagnetic beads (MagneSphere, Promega) in ABCD buffer (50 mM Tris-Cl [pH 7.9], 150 mM NaCl, 10% glycerol, 5 mM MgCl2, 0.1% NP40, and 0.5 mM DTT) and 600 ng of purified bacterially expressed TDG, as well as approximately half-molar amounts of purified baculovirus-expressed full-length CBP or purified bacterially expressed CBP fragments. Total reaction volume was 50 ␮l. Beads were washed five times with 200 ␮l of ABCD buffer, and bound proteins were analyzed by immunoblotting. Protein Acetylation Assays TDG (500 ng) was incubated with approximately 200 ng of purified full-length CBP/p300 in a total volume of 25 ␮l of buffer (20 mM HEPES [pH 7.8], 1 mM EDTA, 1 mM DTT, 10 mM sodium butyrate, and 10% glycerol) in the presence of 1.5 ␮M of 14C acetyl CoA and incubated for 30 min at 30⬚C, before electrophoresis on an 8% SDSpolyacrylamide gel. The gel was subsequently fixed and treated with amplifying solution (Amersham) and exposed to film. Coomassie staining was performed to confirm equal loading of protein. In vitro acetylation assays using biotinylated H4 peptide and in vivo acetylation (3 hr labeling) were performed as previously described (Ait-SiAli et al., 1998; Gu and Roeder, 1997). Approximately 5 ␮g of P1 and P2 peptides were acetylated with 1 ␮g of CBP and 4 ul of [14C]AcCoA (50 mCi/mmol). TDG Cleavage Assay Cleavage assays were performed essentially as previously described (Neddermann et al., 1996). In brief, 25–50 ng of recombinant TDG and approximately 5 ng of radiolabeled duplex oligonucleotide containing either a G/T or a G/U mismatch (see Figure 1) in cleavage buffer (25 mM HEPES-KOH [pH 7.8], 1 mM EDTA, and 1 mM DTT) were incubated in a final volume of 20 ␮l for 30 min at 30⬚C. Following precipitation, the DNA was resuspended in 0.1 M NaOH and incubated at 90⬚C for 30 min. The cleavage products were visualized by autoradiography and quantified by phosphoimaging following denaturing polyacrylamide gel electrophoresis. When addressing the role of protein acetylation, the reactions without the DNA were incubated at 30⬚C for 90 min in the presence of cold AcCoA. G/T and G/U oligonucleotides were then added and processed as described above. Microscopy Cells grown on chamber slides were fixed with 4% formaldehyde for 20 min at room temperature, rinsed in PBS, and mounted in vectashield medium containing DAPI (Vector Labs). Z-series images (100–150⫻ magnification) of 0.5 ␮m thickness were obtained using an Olympus IX-70 deconvolution microscope and Deltavision software (Applied Precision). Acknowledgments We thank Richard Goodman, Tony Kouzarides, Mark Kelley, Brian Hendrich, Adrian Bird, Michael Greenberg, and Yoshihiro Nakatani for providing plasmids; Jean-Marie Garnier, Damian Wilpitz, and Michelle Hon for technical help; and Chris Park and Wolfgang Fischer for HPLC and peptide sequencing. We are grateful to Richard Lin, Chi-hao Lee, and Ruth Yu for critical reading of the manuscript. M.T. was a recipient of Human Frontier Science Program, Association pour la Recherche sur le Cancer (ARC) fellowships and a research associate of the Howard Hughes Medical Institute. A.B. was a recipient of a Marie-Curie long-term fellowship from the European Commission. S.U. was the recipient of fellowships from the ARC, INSERM, and Universite´ Louis Pasteur. J.T. is funded by the NCI of Canada. R.M.E. is an Investigator of the Howard Hughes Medical

Institute at the Salk Institute for Biological Studies and March of Dimes Chair in Molecular and Developmental Biology. This work was supported by funds from the Centre Nationale de la Recherche Scientifique (CNRS), the Institut National de la Sante´ et de la Recherche Me´dicale (INSERM), the Hoˆpital Universitaire de Strasbourg, the College de France, and ARC. Received August 20, 2001; revised January 11, 2002. References Ait-Si-Ali, S., Ramirez, S., Barre, F.-X., Dkhissi, F., Magnaghi-Jaulin, L., Girault, J.A., Robin, P., Knibiehler, M., Pritchard, L.L., Ducommun, B., et al. (1998). Histone acetyltransferase activity of CBP is controlled by cycle-dependent kinases and oncoprotein E1A. Nature 396, 184–186. Bannister, A.J., and Kouzarides, T. (1996). The CBP co-activator is a histone acetyltransferase. Nature 384, 641–643. Barlev, N.A., Poltoratsky, V., Owen-Hughes, T., Ying, C., Liu, L., Workman, J.L., and Berger, S.L. (1998). Repression of GCN5 histone acetyltransferase activity via bromodomain-mediated binding and phosphorylation by the ku-DNA-dependent protein kinase complex. Mol. Cell. Biol. 18, 1349–1358. Bennett, R.A.O., Wilson, D.M., III, Wong, D., and Demple, B. (1997). Interaction of human apurinic endonuclease and DNA polymerase ␤ in the base excision repair pathway. Proc. Natl. Acad. Sci. USA 94, 7166–7169. Chakravarti, D., Ogryzko, V., Kao, H.-Y., Nash, A., Chen, H., Nakatani, Y., and Evans, R.M. (1999). A viral mechanism for inhibition of p300 and PCAF acetyltransferase activity. Cell 96, 393–403. Chen, H., Tini, M., and Evans, R.M. (2001). HATs on and beyond chromatin. Curr. Opin. Cell Biol. 13, 218–224. Chevray, P.M., and Nathans, D. (1992). Protein interaction cloning in yeast: identification of mammalian proteins that react with the leucine zipper of Jun. Proc. Natl. Acad. Sci. USA 89, 5789–5793. Chrivia, J.C., Kwok, R.P., Lamb, N., Hagiwara, M., Montminy, M.R., and Goodman, R.H. (1993). Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365, 855–859. Cooper, D.N., and Youssoufian, H. (1988). The CpG dinucleotide and human genetic disease. Hum. Genet. 78, 151–155. de Laat, W.L., Jaspers, N.G.J., and Hoeijmakers, J.H.J. (1999). Molecular mechanisms of nucleotide excision repair. Genes Dev. 13, 768–785. Eckner, R., Ewen, M.E., Newsome, D., Gerdes, M., DeCaprio, J.A., Lawrence, J.B., and Livingston, D.M. (1994). Molecular cloning and functional analysis of the adenovirus E1A-associated 300-kD protein (p300) reveals a protein with properties of a transcriptional adaptor. Genes Dev. 8, 869–884. Gallinari, P., and Jiricny, J. (1996). A new class of uracil-DNA glycosylases related to human thymine-DNA glycosylase. Nature 383, 735–738. Giles, R.H., Peters, D.J.M., and Breuning, M.H. (1998). Conjunction dysfunction: CBP/p300 in human disease. Trends Genet. 14, 178–183. Glass, C.K., Devary, O.V., and Rosenfeld, M.G. (1990). Multiple cell type-specific proteins differentially regulate target sequence recognition by the ␣ retinoic acid receptor. Cell 63, 729–738. Goodman, R.H., and Smolik, S. (2000). CBP/p300 in cell growth, transformation, and development. Genes Dev. 14, 1553–1577. Green, S., Issemann, I., and Sheer, E. (1988). A versatile eukaryotic expression vector for protein engineering. Nucleic Acids Res. 16, 369. Greenblatt, M.S., Bennett, W.P., Hollstein, M., and Harris, C.C. (1994). Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res. 54, 4855–4878. Gu, W., and Roeder, R.G. (1997). Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 90, 595–606. Hamamori, Y., Sartorelli, V., Ogryzko, V., Puri, P.L., Wu, H.-Y., Wang,

Association of TDG with CBP/p300 277

J.Y.J., Nakatani, Y., and Kedes, L. (1999). Regulation of histone acetyltransferases p300 and PCAF by the bHLH protein twist and adenoviral oncoprotein E1A. Cell 96, 405–413. Hardeland, U., Bentele, M., Jiricny, J., and Schar, P. (2000). Separating substrate recognition from base hydrolysis in human thymine DNA glycosylase by mutational analysis. J. Biol. Chem. 275, 33449– 33456. Hasan, S., Hassa, P.O., Imhof, R., and Hottiger, M.O. (2001a). Transcription coactivator p300 binds PCNA and may have a role in DNA repair synthesis. Nature 410, 387–391. Hasan, S., Stucki, M., Hassa, P.O., Imhof, R., Gehrig, P., Hunziker, P., Hubscher, U., and Hottiger, M.O. (2001b). Regulation of human flap endonuclease-1 activity by acetylation through the transcriptional coactivator p300. Mol. Cell 7, 1221–1231. Hendrich, B., and Bird, A. (1998). Identification and characterization of a family of mammalian methyl-CpG binding proteins. Mol. Cell. Biol. 18, 6538–6547. Hendrich, B., Hardeland, U., Ng, H.-H., Jiricny, J., and Bird, A. (1999). The thymine glycosylase MBD4 can bind to the product of deamination at methylated CpG sites. Nature 401, 301–304. Ikura, T., Ogryzko, V.V., Grigoriev, M., Groisman, R., Wang, J., Horikoshi, M., Scully, R., Qin, J., and Nakatani, Y. (2000). Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis. Cell 102, 463–473. Kung, A.L., Rebel, V.I., Bronson, R.T., Ch’ng, L.E., Sieff, C.A., Livingston, D.M., and Yao, T.P. (2000). Gene dose-dependent control of hematopoiesis and hematologic tumor suppression by CBP. Genes Dev. 14, 272–277. Kwok, R.P.S., Lundblad, J.R., Chrivia, J.C., Richards, J.P., Ba¨chinger, H.P., Brennan, R.G., Roberts, S.G.E., Green, M.R., and Goodman, R.H. (1994). Nuclear protein CBP is a coactivator for the transcription factor CREB. Nature 370, 223–226.

between DNA replication methylation and repair. Am. J. Hum. Genet. 61, 1220–1224. Saparbaev, M., and Laval, J. (1998). 3,N4-ethenocytosine, a highly mutagenic adduct, is a primary substrate for Escherichia coli double-stranded uracil-DNA glycosylase and human mismatch-specific thymine-DNA glycosylase. Proc. Natl. Acad. Sci. USA 95, 8508–8513. Scha¨rer, O.D., and Jiricny, J. (2001). Recent progress in the biology, chemistry and structural biology of glycosylases. Bioessays 23, 270–281. Seipel, K., Georgiev, O., and Schaffner, W. (1992). Different activation domains stimulate transcription from remote (‘enhancer’) and proximal (‘promoter’) positions. EMBO J. 11, 4961–4968. Sheng, M., Thompson, M.A., and Greenberg, M.E. (1991). CREB: a Ca2⫹-regulated transcription factor phosphorylated by calmodulindependent kinases. Science 252, 1427–1430. Soutoglou, E., Viollet, B., Vaxillaire, M., Yaniv, M., Pontoglio, M., and Talianidis, I. (2001). Transcription factor-dependent regulation of CBP and P/CAF histone acetyltransferase activity. EMBO J. 20, 1984–1992. Stoilov, L., Darroudi, R., Meschini, R., van der Schans, G., Mullenders, L.H.F., and Natarajan, A.T. (2000). Inhibition of repair of X-rayinduced DNA double-strand breaks in human lymphocytes exposed to sodium butyrate. Int. J. Radiat. Biol. 76, 1485–1491. Swope, D.L., Mueller, C.L., and Chrivia, J.C. (1996). CREB-binding protein activates transcription through multiple domains. J. Biol. Chem. 271, 28138–28145. Tanaka, Y., Naruse, I., Maekawa, T., Masuya, H., Shiroishi, T., and Ishii, S. (1997). Abnormal skeletal patterning in embryos lacking a single CBP allele: a partial similarity with Rubinstein-Taybi syndrome. Proc. Natl. Acad. Sci. USA 94, 10215–10220.

Lindahl, T. (1993). Instability and decay of the primary structure of DNA. Nature 362, 709–715.

Um, S., Harbers, M., Benecke, A., Pierrat, B., Losson, R., and Chambon, P. (1998). Retinoic Acid receptors interact physically and functionally with the T:G mismatch-specific thymine-DNA glycosylase. J. Biol. Chem. 273, 20728–20736.

Medina, B.H., Fraenkel-Conrat, H., and Singer, B. (1998). A 55-KDa protein isolated from human cells shows DNA glycosylase activity toward 3,N4-ethenocytosine and the G/T mismatch. Proc. Natl. Acad. Sci. USA 95, 13561–13566.

Underhill, C., Qutob, M.S., Yee, S.P., and Torchia, J. (2000). A novel nuclear receptor corepressor complex, N-CoR, contains components of the mammalian SWI/SNF complex and the corepressor KAP-1. J. Biol. Chem. 275, 40463–40470.

Meijer, M., and Smerdon, M.J. (1999). Accessing DNA damage in chromatin: insights from transcription. Bioessays 21, 596–603.

Urnov, F.D., and Wolffe, A.P. (2001). Chromatin remodeling and transcriptional activation: the cast (in order of appearance). Oncogene 20, 2991–3006.

Nakajima, T., Uchida, C., Anderson, S.F., Lee, C.-G., Hurwitz, J., Parvin, J.D., and Montminy, M. (1997). RNA helicase A mediates association of CBP with RNA polymerase II. Cell 90, 1107–1112.

Verdine, G.L., and Bruner, S.D. (1997). How do DNA repair proteins locate damaged bases in the genome? Chem. Biol. 4, 329–334.

Neddermann, P., Gallinari, P., Lettieri, T., Schmid, D., Truong, O., Hsuan, J.J., Wiebauer, K., and Jiricny, J. (1996). Cloning and expression of human G/T mismatch-specific thymine-DNA glycosylase. J. Biol. Chem. 271, 12767–12774.

Waters, T.R., Gallinari, P., Jiricny, J., and Swann, P.F. (1999). Human thymine DNA glycosylase binds to apurinic sites in DNA but is displaced by human apurinic endonuclease 1. J. Biol. Chem. 274, 67–74.

Ng, H.-H., and Bird, A. (1999). DNA methylation and chromatin modification. Curr. Op. Genet. Dev. 9, 158–163.

Wellinger, R.E., and Thoma, F. (1997). Nucleosome structure and positioning modulate nucleotide excision repair in the non-transcribed strand of an active gene. EMBO J. 16, 5046–5056.

Ogryzko, V.V., Schiltz, R.L., Russanova, V., Howard, B.H., and Nakatani, Y. (1996). The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87, 953–959. Parker, D., Ferreri, K., Nakajima, T., LaMorte, V.J., Evans, R., Koerber, S.C., Hoeger, C., and Montminy, M.R. (1996). Phosphorylation of CREB at Ser-133 includes complex formation with CREBbinding protein via a direct mechanism. Mol. Cell. Biol. 16, 694–703. Petrij, F., Giles, R.H., Dauwerse, H.G., Saris, J.J., Hennekam, R.C.M., Masuno, M., Tommerup, N., van Ommen, G.-J.B., Goodman, R.H., Peters, D.J.M., and Breuning, M.H. (1995). Rubinstein-Taybi syndrome caused by mutations in the transcriptional co-activator CBP. Nature 376, 348–351. Prasad, R., Singhal, R.K., Srivastava, D.K., Molina, J.T., Tomkinson, A.E., and Wilson, S.H. (1996). Specific interaction of DNA polymerase ␤ and DNA ligase I in a multiprotein base excision repair complex from bovine testis. J. Biol. Chem. 271, 16000–16007. Ramanathan, B., and Smerdon, M.J. (1989). Enhanced DNA repair synthesis in hyperacetylated nucleosomes. J. Biol. Chem. 264, 11026–11034. Robertson, K.D., and Jones, P.A. (1997). Dynamic interrelationships

Yang, X.-J., Ogryzko, V.V., Nishikawa, J.-I., Howard, B.H., and Nakatani, Y. (1996). A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A. Nature 382, 319–324. Zhang, W., Kadam, S., Emerson, B.M., and Bieker, J.J. (2001). Sitespecific acetylation by p300 or CREB binding protein regulates erythroid Kruppel-like factor transcriptional activity via its interaction with the SWI-SNF complex. Mol. Cell. Biol. 21, 2413–2422. Zhou, J., Ahn, J., Wilson, S.H., and Prives, C. (2001). A role for p53 in base excision repair. EMBO J. 20, 914–923. Zhu, B., Zheng, Y., Hess, D., Angliker, H., Schwarz, S., Siegmann, M., Thiry, S., and Jost, J.P. (2000). 5-methylcytosine-DNA glycosylase activity is present in a cloned G/T mismatch DNA glycosylase associated with the chicken embryo DNA demethylation complex. Proc. Natl. Acad. Sci. USA 97, 5135–5139. Zhu, B., Benjamin, D., Zheng, Y., Angliker, H., Thiry, S., Siegmann, M., and Jost, J.P. (2001). Overexpression of 5-methylcytosine DNA glycosylase in human embryonic kidney cells EcR293 demethylates the promoter of a hormone-regulated reporter gene. Proc. Natl. Acad. Sci. USA 98, 5031–5036.