ING Tumor Suppressor Proteins Are Critical Regulators of Chromatin Acetylation Required for Genome Expression and Perpetuation

Molecular Cell 21, 51–64, January 6, 2006 ª2006 Elsevier Inc.

DOI 10.1016/j.molcel.2005.12.007

ING Tumor Suppressor Proteins Are Critical Regulators of Chromatin Acetylation Required for Genome Expression and Perpetuation Yannick Doyon,1,5 Christelle Cayrou,1,5 Mukta Ullah,2 Anne-Julie Landry,1 Vale´rie Coˆte´,1 William Selleck,3 William S. Lane,4 Song Tan,3 Xiang-Jiao Yang,2 and Jacques Coˆte´1,* 1 Laval University Cancer Research Center Hoˆtel-Dieu de Que´bec (CHUQ) Que´bec City, Que´bec G1R 2J6 Canada 2 Molecular Oncology Group Department of Medicine McGill University Montreal, Que´bec H3A 1A1 Canada 3 Department of Biochemistry and Molecular Biology Pennsylvania State University University Park, Pennsylvania 16802 4 Harvard Microchemistry Facility Harvard University Cambridge, Massachusetts 02138

Summary Members of the ING family of tumor suppressors regulate cell cycle progression, apoptosis, and DNA repair as important cofactors of p53. ING1 and ING3 are stable components of the mSin3A HDAC and Tip60/NuA4 HAT complexes, respectively. We now report the purification of the three remaining human ING proteins. While ING2 is in an HDAC complex similar to ING1, ING4 associates with the HBO1 HAT required for normal progression through S phase and the majority of histone H4 acetylation in vivo. ING5 fractionates with two distinct complexes containing HBO1 or nucleosomal H3-specific MOZ/MORF HATs. These ING5 HAT complexes interact with the MCM helicase and are essential for DNA replication to occur during S phase. Our data also indicate that ING subunits are crucial for acetylation of chromatin substrates. Since INGs, HBO1, and MOZ/ MORF contribute to oncogenic transformation, the multisubunit assemblies characterized here underscore the critical role of epigenetic regulation in cancer development. Introduction Regulation of chromosome dynamics dictates the outcome of numerous nuclear processes such as transcription, DNA repair, recombination, and replication. It is central to cell homeostasis, as alterations in chromatin structure contribute to the development of cancer and other human diseases (Lund and van Lohuizen, 2004). Of particular significance, aberrant silencing of tumor suppressor genes in cancer cells is in part achieved by methylation and deacetylation of nucleosomal histones within their promoters. Furthermore, extensive loss of histone H4 Lys16 acetylation and Lys20 trimethylation *Correspondence: [email protected] 5 These authors contributed equally to this work.

is a feature of human cancer, and the analysis of global changes in histone modification patterns is envisaged as a prognostic marker in prostate cancer treatment (Fraga and Esteller, 2005). In support of this view, chromatin regulators like Brg1, Ini1, and p300/CBP are now considered as tumor suppressors, whereas Bmi1 and MLL display oncogenic features (Lund and van Lohuizen, 2004). These proteins are part of multisubunit assemblies that either covalently modify or remodel nucleosomes, basic units of chromatin fibers. The identification, isolation, and biochemical characterization of these protein machines is thus at the heart of our molecular understanding of gene expression as well as the maintenance and transfer of genetic information in eukaryotes. Among the histone modification repertoire, dynamic variations in histone acetylation governed by histone acetyltransferases (HATs) and deacetylases (HDACs) are detected during transcription regulation, DNA repair, recombination, and replication, suggesting that the activities responsible for the establishment of these marks are crucial for the regulation of these processes. The inhibitor of growth 1 (ING1) gene, the founding member of the ING tumor suppressor family, was initially identified as a critical cofactor for p53 in cell growth control (Campos et al., 2004). As for the other ING proteins, it contains a plant homeodomain (PHD) finger, a motif common to many chromatin-regulatory proteins. In Saccharomyces cerevisiae, the three ING proteins, namely Pho23, Yng1, and Yng2, are purified as stable components of the Sin3/Rpd3 HDAC, NuA3, and NuA4 HAT complexes, respectively (Howe et al., 2002; Nourani et al., 2001; Nourani et al., 2003). In human cells, the five ING proteins have been implicated in p53 function, control of cell growth/proliferation, and cancer (Campos et al., 2004). However, only two of them have been characterized biochemically. While ING1 is a component of two related Sin3/HDAC1/2 complexes, ING3 is found in the hNuA4/Tip60 HAT complex (Doyon et al., 2004; Kuzmichev et al., 2002; Skowyra et al., 2001). Thus, an interesting possibility is that ING2, ING4, and ING5 proteins could also be associated with specific histone modifying complexes, linking chromatin regulation with p53 function and tumor suppression. To address this, we carried out thorough biochemical characterization of the human ING-associated protein complexes and established their functional link to the control of chromatin acetylation through their exclusive association with specific HAT and HDAC complexes. These studies revealed new native HAT complexes harboring different specificities through their catalytic subunits HBO1, MOZ, and MORF. We also demonstrate the importance of these activities in cell cycle progression and identified HBO1 as the main source of histone H4 acetylation in vivo. Our data support the critical role of ING proteins for acetylation of nucleosomal histones. Importantly, we demonstrate that ING5 complexes associate with the MCM helicase and are essential for DNA replication in human cells, indicating a crucial role of chromatin acetylation during initiation and elongation

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Figure 1. Characteristic Features of the Human ING Family of Tumor Suppressor Proteins (A) Sequence alignment of the five human ING proteins. Three domains, represented by black boxes, are conserved. Based on sequence comparison, the INGs were subdivided in three different groups (ING1/2 in red, ING3 in blue, and ING4/5 in green). (B) TAP/FLAG-purified ING2 complex from HeLa S3 cells expressing FLAG-ING2-TAP was subjected to SDS-PAGE followed by silver staining (left panel). Interacting proteins identified by mass spectrometric analysis are indicated on the right (numbers of independent peptides obtained vary between three and 50 per indicated protein). Western blot analysis of the preparation shows the association of ING2 in a Brg1-HDAC1/2 complex. (C) Silver staining of TAP-purified ING3 complex (left panel) and Western blot analysis using antibodies specific to Tip60 complex subunits. Asterisks indicate nonspecific protein bands.

of DNA synthesis. Taken together, these results emphasize the significance of MYST family HATs and ING tumor suppressor proteins in genome expression, maintenance, and duplication and hence cancer biology. Results The Human ING Tumor Suppressor Proteins Can Be Separated into Three Groups Upon analysis of human ING protein amino acid sequences, three distinct conserved regions can be identified

(Figure 1A). The PHD finger region is highly conserved but is dispensable for both complex assembly and the catalytic role of ING proteins in nucleosome acetylation (Boudreault et al., 2003; Doyon et al., 2004; Nourani et al., 2001; Selleck et al., 2005). Studies on yeast Yng2 suggest that conserved domain I at the N terminus of ING proteins is also not required for complex assembly but is key to potentiate chromatin modification by the associated enzyme (Selleck et al., 2005). The role of conserved domain II is less clear, but work in yeast suggests that it is required for complex assembly (Selleck et al.,

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2005). Upon closer examination of these sequence homologies within domain II, it became apparent that human INGs could be divided in three groups, ING1/2, ING3, and ING4/5, suggesting three distinct types of association to protein complexes. With the goal of dissecting roles of the five human ING tumor suppressor proteins in the control of cell growth and proliferation, affinity purification was used to identify their associated proteins. Because of the inherent sequence similarity between ING proteins, epitope-tagged versions expressed by strong promoters in transient or even stable transfections led to nonspecific crossinteractions between ING partners (data not shown). As a result, we used a retroviral expression vector with a tetracyclin-regulated promoter to isolate clones with low expression levels, key to obtaining native protein complexes. Double- or triple-affinity purifications using TAP, FLAG, or HA tags were performed from HeLa cell nuclear extracts (see Figure S1A in the Supplemental Data available with this article online) (Doyon et al., 2004). ING2 Is a Stable Component of a mSin3A HDAC Complex Containing the Metastasis Suppressor Protein BRMS1 The major form of human ING1 has already been purified and shown to be part of the mSin3 HDAC complexes (Kuzmichev et al., 2002; Skowyra et al., 2001). We purified the human ING2 protein and found it associated with a very similar set of polypeptides classically found with mammalian Sin3 HDAC complexes, including the chromodomain-containing RBP1 and its paralog (Figure 1B). RBP1 allows recruitment of mSin3 HDAC complex by retinoblastoma tumor suppressor family pocket proteins to induce cell cycle arrest by repressing E2Fdependent transcription and DNA replication origins (Lai et al., 2001). Several peptides identified by mass spectrometry also corresponded to breast cancer metastasis suppressor-1 (BRMS1) and its paralog. BRMS1 suppresses metastasis of multiple human and murine cancer cells and has been found with RBP1 in mSin3 HDAC complexes (Meehan et al., 2004). A subset of ING1-mSin3 HDAC complex was also shown to be associated with Brg1-based SWI/SNF chromatin remodeling complexes (Kuzmichev et al., 2002), and we found similar results for ING2 (Figure 1B). ING2 negatively regulates cell proliferation through modulation of p53 acetylation in response to DNA damage, and the PHD finger region was implicated in this process as a nuclear receptor for phospholipid signaling (Campos et al., 2004; Gozani et al., 2003). Interestingly, BRMS1 function in metastasis suppression was also linked to phosphoinositide signaling (DeWald et al., 2005). The Majority of ING3 Is Associated with the hNuA4/Tip60 HAT Complex ING3 is also implicated in p53 function in transcription, cell cycle control, and apoptosis and is found mutated in cancer cells (Campos et al., 2004; Nagashima et al., 2003). We previously identified ING3 as a stable subunit of the human NuA4/Tip60 HAT complex and showed that it is required for acetylation of chromatin substrates (Doyon et al., 2004). Tip60 and its associated proteins are important cofactors for p53-, NF-kappaB-, Myc-,

E2F1-, and nuclear-receptor-dependent transcription activation; cell response to DNA damage, apoptosis; and metastasis suppression (Berns et al., 2004; Doyon and Coˆte´, 2004; Kim et al., 2005). An interesting issue is whether ING3 is part of other complexes. To address this, we affinity purified ING3 itself. As shown in Figure 1C, the associated proteins are known hNuA4/ Tip60 complex subunits, indicating that cellular ING3 is mainly, if not exclusively, associated with hNuA4/Tip60 HAT in vivo. ING4 Identifies a New Four-Subunit HAT Complex Containing HBO1 Candidate tumor suppressor ING4 was recently shown to restrain brain tumor growth and angiogenesis to suppress the loss of contact inhibition elicited by MYC or MYCN and to be frequently mutated in human cancer (Garkavtsev et al., 2004; Kim et al., 2004). Tandem affinity purification (TAP) of ING4 yielded only four major bands on gel (Figure 2A and Figure S1B). Tandem mass spectrometry analysis identified these bands as ING4, hEaf6, HBO1, and JADE1/2/3 paralogs (Figure 2B). hEaf6 is an uncharacterized protein homologous to a subunit of yeast NuA4 (nucleosome acetyltransferase of H4) HAT complex and also found in the human NuA4/Tip60 complex (Doyon et al., 2004). JADE1 (gene for apoptosis and differentiation in epithelia) is stabilized by the von Hippel-Lindau tumor suppressor and has itself tumor suppressor activity, and its expression is regulated during anteroposterior axis development (Tzouanacou et al., 2003; Zhou et al., 2004, 2005). It was recently shown that overexpression of JADE1 increases histone H4 acetylation in vivo (Panchenko et al., 2004). The MYST family HBO1 HAT protein (histone acetyltransferase binding to ORC1) has been implicated in transcription regulation by androgen receptor, replication origin function, and lymphomagenesis (Stedman et al., 2004; Utley and Coˆte´, 2003). Western analysis confirmed that ING3-Tip60 and ING4-HBO1 are distinct HAT complexes (Figure 2C). HAT assays on free histones did not show difference in specificity between Tip60-ING3 and HBO1ING4, but the use of native chromatin or recombinant nucleosomes clearly indicates that the two complexes have different specificities (Figure 2D). While Tip60ING3 targets nucleosomal histones H4 and H2A, HBO1ING4 prefers H4 but also H3 to a lesser extent. To confirm the specific association of these proteins, we performed a reciprocal purification using FLAGHBO1-TAP-expressing HeLa cells. Triple-affinity purification yielded the same HAT activity and four-band pattern, indicating that the majority of HBO1 HAT is associated with a similar complex in vivo. Mass spectrometry analysis identified the same proteins as in the ING4 purification, with the exception that ING5 was also detected and confirmed by Western blot (Figures 2E–2G) (see below). ING4 has been reported to activate the p21/WAF1 promoter and induce apoptosis in a p53-dependent manner and to interact with p53 in vivo (Shiseki et al., 2003). To confirm the physical and functional link between ING4 and HBO1, we tested the two proteins in the same p21-luciferase reporter assay in RKO and RKO-E6 cells (Figure 2H). As previously shown (Nagashima et al., 2003; Shiseki et al., 2003), expression of ING3 has a potent stimulatory effect in this assay, while

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Figure 2. The Purified ING4 Complex Contains the HBO1 Histone Acetyltransferase (A) Silver staining of the ING4-TAP complex. Interacting proteins identified by mass spectrometric analysis are indicated on the right (between eight and 66 peptides per indicated protein). (B) Schematic representation of the protein domains present in the identified subunits. JADE proteins are also known as PHF15/16/17. (C) Western blot analysis of TAP purifications of ING4 and Tip60 that shows two distinct complexes. (D) Fractions from Tip60- and ING4-TAP were tested for HAT activity specificity on human core histones, oligonucleosomes, or recombinant nucleosomes substrates. (E) Reciprocal purification of HBO1 complex (by TAP/FLAG triple affinity) was subjected to SDS-PAGE followed by silver staining. Interacting proteins identified by mass spectrometric analysis are indicated on the right (between two and 12 peptides per indicated protein). (F) Western blot analysis of purified HBO1-TAP. (G) Purified HBO1-TAP was assayed for HAT activity on core histones and oligonucleosomes. (H) Luciferase assays of p53-dependent transcription. Reporter construct containing the p21 promoter was cotransfected with the indicated expression vectors in RKO versus RKO-E6 cells and assayed for luciferase activity (standard deviations are based on three different experiments).

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Figure 3. HBO1 Is the Major Histone H4 Histone Acetyltransferase In Vivo and Is Required for Cell Cycle Progression (A) HAT specificity of purified ING4-TAP was tested on recombinant nucleosomes and analyzed by Western blot with the indicated antibodies. (B) 293T cells, cotransfected with the indicated pSUPER plasmids and caax-GFP, were sorted 48 hr posttransfection and subjected to acidic extraction. Histones were analyzed by Western blot with the indicated antibodies. (C) Cells prepared as in (B) but sorted 24 hr after transfection were subjected to MTT assay during 3 days after seeding to monitor growth. (D) Flow cytometry indicated the DNA content at 72 hr posttransfection of the GFP-positive cells described in (B). (E and F) MCF7 cells treated for 48 hr with the indicated pSUPER plasmids were marked with BrdU for 30 min and analyzed by flow cytometry for BrdU incorporation and (F) for analysis of DNA content to determined percentage of cells in S phase that incorporated BrdU (mean of three experiments). (G) Subcellular localization of HBO1 and Tip60 was determined by indirect immunofluoresence and confocal microscopy on MCF7 cells. Nuclei were visualized with DRAQ5. (H) Double staining for HBO1 (green) and Tip60 (red).

ING4 stimulates to a lesser extent, both in a strict p53dependent manner. Expression of HBO1 had an effect analogous to ING4, supporting their functional interaction. HBO1 Is Responsible for the Bulk of Histone H4 Acetylation In Vivo, Is Required for Cell Cycle Progression, and Localizes Differently from Tip60 Since both HBO1 and Tip60 complexes acetylate histone H4, we investigated functional differences between the two activities. Using recombinant histone H4, we found that HBO1- or ING4-purified complexes can acetylate all four lysine residues on H4 N-terminal domain, as does NuA4 (Figure S1C). We then used recombinant nucleosomes in a similar experiment and found that, in the context of chromatin, HBO1-ING4 acetylates lysines 5, 8, and 12 but not 16 (Figure 3A). In contrast, ING3-

Tip60 still acetylates all four lysine residues on the same substrate (see Figure 4F). To determine if these in vitro specificities correspond to the native targets in the cell, we used RNA interference to knock down HBO1 and Tip60 proteins in vivo. Figures S2A and S2B demonstrate the high efficiency of the shRNA-expressing vectors used in these experiments. Total histones were purified 48 hr posttransfection and analyzed by Western blot for acetylation of each H4 lysine residue (Figure 3B). When we compared the empty vector-transfected cell, it became evident that downregulation of HBO1 had a major impact on histone H4 acetylation in vivo (compare lanes 1 and 3). Acetylation of lysines 5, 8, and 12 was strongly reduced, while Ac-lysine 16 was not affected, corresponding exactly to the specificity detected in vitro (Figure 3A). Similar results were obtained with other RNAi reagents against HBO1 and in

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Figure 4. ING5 Associates with Three Different MYST Histone Acetytransferases: HBO1, MOZ, and MORF (A) TAP-purified ING5 protein was subjected to SDS-PAGE and visualized by SYPRO ruby staining. Proteins identified by tandem mass spectrometry are indicated (between two and 50 peptides per protein). (B) Schematic representation of protein domains present in the identified subunits. (C) Western blot analysis of purified ING4- and ING5-TAP complexes with indicated antibodies. (D) Purified ING5-TAP and ING4-TAP were tested for HAT activity on core histones or oligonucleosomes. (E) Purified ING4- and ING5-TAP fractions were immunoprecipitated with the indicated antibodies, and bound material was analyzed by HAT assay on chromatin substrate. (F) HAT specificity of ING3 and ING5 complexes was tested on recombinant nucleosomes with purified fractions and analyzed by Western blot with the indicated antibodies. (G) A luciferase reporter construct containing six Runx2 binding sites was cotransfected with the indicated expression vectors, and cell extracts were analyzed for luciferase activity (mean of three different experiments).

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MCF7 cells (data not shown). In comparison, Tip60 knockdown had a significantly smaller effect on the same residues. These surprising results indicate that the HBO1-JADE-ING-hEAF6 tetramer complexes are likely responsible for the majority of histone H4 acetylation higher eukaryotes. It also supports the recent finding that JADE1 overexpression increases H4 acetylation in vivo (Panchenko et al., 2004). To determine the impact of these activities on cell growth/proliferation, we also analyzed the same transfected cells for growth and cell cycle distribution. Figure 3C clearly demonstrates that cells stopped growing when Tip60 and HBO1 were depleted. FACS analysis indicates that both Tip60 and HBO1 are required for normal cell cycle progression (Figure 3D). Both Tip60 and HBO1 knockdown cells tend to accumulate in G2/M, a phenotype similar to several NuA4 mutants in yeast (Doyon and Coˆte´, 2004). In addition, loss of HBO1 seems to affect progression through the S phase, which would support its proposed role in replication. To confirm a link to DNA replication, we analyzed BrdU incorporation in HBO1 and Tip60 knockdown cells. As shown in Figure 3E, DNA synthesis is specifically affected in HBO1depleted cells (see also cell images in Figure S1D). Furthermore, FACS analysis showed that cells already in S phase also tend to incorporate less BrdU (Figure 3F). These data suggest that HBO1 is important for DNA synthesis throughout S phase, not only for initiation/firing of replication origins. To further analyze the functional difference between HBO1 and Tip60 complexes, we performed immunofluorescence analysis of endogenous proteins in cultured cells. We found that, while HBO1 is evenly distributed throughout the nucleoplasm, it is not present in the nucleoli (Figure 3G). In striking contrast, the majority of Tip60 proteins are detected in the nucleoli. The double-labeling experiment clearly shows that the two histone H4 HATs are in distinct nuclear compartments (Figure 3H). These data support our finding that HBO1 is the major source of nuclear H4 acetylation in vivo. They are also in agreement with a report implicating Tip60 in ribosomal gene transcription and the impact of homologous esa1 mutation on nucleolus structure in yeast (Halkidou et al., 2004; Utley and Coˆte´, 2003). ING5 Is Present in a Similar HBO1 HAT Complex but Also in H3-Specific HAT Complexes Containing MOZ/MORF Leukemogenic Proteins Finally, we purified the ING5 protein, the last human paralog, expecting to obtain a complex similar to ING4HBO1, as suggested by the HBO1 purification (Figures 2E and 2F). However, in addition to the four expected bands, higher molecular weight bands were also observed (Figure 4A). Tandem mass spectrometry analysis identified the 150 kDa band as a mixture of BRPF1/2/3 paralogs (bromodomain- and PHD finger-containing; BRPF1 and BRPF2 are also known as BR140 and BRD1, respectively). This family of proteins is rich in domains typically found in chromatin-associated factors, including PHD fingers, bromodomain, and chromo/Tudor-related PWWP domain (Figure 4B). The nematode BRPF homolog has been shown to affect development and regulate transcription (Chamberlin and Thomas, 2000; Chang et al., 2003). Two weaker bands above

200 kDa were identified as the MYST family closely related HAT proteins MOZ (monocytic leukemia zinc finger protein) and MORF (MOZ-related factor) (Utley and Coˆte´, 2003). The specific association of MOZ/MORF with ING5 but not ING4 was confirmed by Western analysis (Figure 4C; the MORF antibody crossreacts with MOZ). MOZ and MORF genes are often translocated in acute myeloid leukemia cells, producing fusion proteins with CBP/p300 HATs, or TIF2, a member of the p160 family of coactivators (Yang, 2004). It is thought that aberrant targeting/acetylation by the fusion proteins is responsible for oncogenic transformation. Indeed, MOZ and MORF are coactivators of the Runx1/2 (AML1/3) transcription factors, and the MOZ-CBP fusion protein inhibits Runx1-dependent transcription (Kitabayashi et al., 2001; Pelletier et al., 2002). MOZ and MORF have also been shown to regulate HOX expression and development in Zebrafish and mouse (Miller et al., 2004; Thomas et al., 2000). To determine if the presence of MOZ/MORF affects the HAT specificity of the ING5 complexes, we compared it with ING4 (Figure 4D). While the activities of ING4 and ING5 complexes were similar on free histones, ING5 did not show a similar preference for H4 on chromatin, instead equally targeting nucleosomal H3 and H4. We hypothesized that purified ING5 was in fact a mixture of two distinct HAT complexes, one with HBO1-JADE and the other one with MOZ/MORFBRPF. To test this, we did immunoprecipitations with HBO1 and MORF antibodies on our purified fractions and assayed the bound material for nucleosomal HAT activity (Figure 4E). The results clearly indicate that HBO1-ING5 association is independent of MOZ/MORF and is responsible for the H4-specific HAT activity in purified ING5. In contrast, MOZ/MORF-ING5 association targets only histone H3 in the assay (compare lanes 5 and 6 with 9 and 10). Altogether, these data demonstrate that ING5 is present in two independent native HAT complexes with different histone specificity, with HBO1-JADE for H4 and with MOZ/MORF-BRPF for H3 (BRPF proteins are considered specific to MOZ/ MORF, since they were not obtained in the HBO1 purification; see Figure 2E). We also mapped the specific lysines acetylated by ING5 complexes on chromatin (Figure 4F). As for the ING4 complex, HBO1-dependent H4 acetylation occurs at lysines 5, 8, and 12 but not 16 (ING3-Tip60 is shown for comparison). MOZ/MORFdependent acetylation of H3 occurs at lysine 14 but is undetected at lysines 9 or 18. Such H3 specificity is different than classical H3 HATs like Gcn5/SAGA/PCAF but is similar to the yeast NuA3 (nucleosome acetyltransferase of H3) complex (Howe et al., 2001) (see Discussion). Finally, since MOZ and MORF are known coactivators of Runx1/2-dependent transcription, we tested if this could be functionally linked to their association with ING5-BRPF. Luciferase reporter assays demonstrate that expression of ING5 stimulates Runx2-dependent transcription to levels similar to what is obtained with MOZ (Figure 4G). In contrast, ING4 does not function as a coactivator of Runx2 while ING3 is intermediate. These data support the idea that MOZ/MORF function in the cell through their presence in a complex with ING5 and BRPF proteins. In agreement with this idea, coexpression of MOZ, BRPF1, and ING5 in the Runx2

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reporter assay leads to higher gene expression (Figure S3A). Transient overexpression of these three proteins does lead to formation of a trimeric HAT complex in vivo, BRPF1 bridging ING5 and MOZ together (Figure S3B). ING5 but Not ING4 Complexes Play Essential Roles in DNA Replication Studies on the HBO1 HAT protein and its Drosophila homolog suggest that this activity is implicated at the origins of DNA replication. HBO1 was cloned in two-hybrid screens with origin recognition complex (ORC) and minichromosome maintenance (MCM) proteins, which play an essential role in eukaryotic DNA replication through formation of a prereplicative complex at origins of replication (Burke et al., 2001; Iizuka and Stillman, 1999). ORC1, MCM2, and HBO1 were also shown to interact in vitro and in extracts. Furthermore, HBO1 binds along ORC and MCM proteins to the Kaposi’s sarcoma-associated herpesvirus latent replication origin in vivo, an association that correlates with local H4 hyperacetylation and is required for viral DNA replication (Stedman et al., 2004). Finally, in Drosophila, chromatin hyperacetylation occurs at specific origins coincidently with ORC binding, and the HBO1 homolog, Chameau, increases origin activity (Aggarwal and Calvi, 2004). Mass spectrometry analysis of our purified fractions provided support for such a functional link between HBO1 and protein binding replication origins. In the ING5-purified fraction, we obtained significant numbers of peptide hits for the helicase forming MCM4, MCM6, and MCM7 proteins (four to five hits each). Western analysis confirmed this substoichiometric association and also identified MCM2 as another interacting protein (Figure 5A). Surprisingly, no MCM proteins were detected in the ING4HBO1 fraction (or the mock purification), indicating that this association is specific to the ING5 complexes. To determine if the MCM helicase was indeed interacting with an HBO1-ING5 complex, we analyzed our purified HBO1 fraction and indeed found substoichiometric amount of MCM proteins (Figure 5B) as well as peptide hits for replication protein A (data not shown). On the other hand, no ORC proteins were detected in any of our fractions. Our previous HBO1 knockdown experiment with shRNA-expressing vectors suggested that passage through S-phase and DNA synthesis were affected, supporting a role in DNA replication (Figures 3D–3F). We repeated the experiment using more efficiently transfected synthetic siRNA. FACS analysis 48 hr posttransfection indicates a clear accumulation of cells in S phase (Figure 5C). Analysis of histone acetylation levels in these cells again demonstrates the critical role of HBO1 for H4 acetylation at lysines 5, 8, and 12 in vivo (Figure 5D; >75% drop of Western signals). Altogether, these results support an important role for the HBO1-JADE-ING5 HAT complex during DNA replication in cooperation with the MCM complex. To assert the specific roles of ING4 and ING5 proteins in their respective HAT complexes, we efficiently and specifically knocked down each protein in vivo using vector-based or synthetic RNAi reagents (Figures S2C and S2D). We then evaluated effect on DNA synthesis by measuring BrdU incorporation. As seen in Figure 5E, shRNA-mediated knockdown of ING4 has a relatively

small effect on the percentage of BrdU-positive cells. In contrast, ING5 knocked down cells almost completely lack DNA synthesis. FACS analysis of these cells provided striking results, since ING5 knocked down cells were clearly unable to complete S phase, showing almost no cells left in G2/M (Figure 5F). In contrast, knockdown of ING4 led the cells to specifically accumulate in G2/M. Similar results were obtained with siRNA-mediated knockdowns and with other cell lines (data not shown). The different roles of ING4 and ING5 are further depicted by analysis of cells in S phase (Figure 5G). In control and ING4 knockdown cells, most cells in S phase have active DNA synthesis. In contrast, the high proportion of cells in S phase in the ING5 knockdown show no BrdU incorporation, hence a strong defect in DNA synthesis (Figures 5F and 5G). These results clearly indicate that ING5 HAT complexes are essential for DNA replication, not only for initiation but also for replication fork movement. Since the cell cycle and BrdU phenotype is stronger than what was seen with HBO1 knockdown, these data suggest that both MOZ/MORF-ING5 and HBO1-ING5 HAT complexes are important for DNA replication. In fact, the cell cycle phenotype of HBO1 knockdown cells seems to reflect a mix of both ING4 and ING5 phenotypes, in agreement with their physical association (compare Figures 5C and 5F). ING Proteins Play a Conserved Catalytic Role in Acetylation of Chromatin Substrates Studies on yeast INGs and human ING3 led us to propose that part of ING protein function is to enable their associated HAT/HDAC enzyme function on chromatin substrates (Boudreault et al., 2003; Doyon et al., 2004; Howe et al., 2002; Nourani et al., 2003; Selleck et al., 2005). Most HAT/HDAC enzymes are able on their own to modify free histones in solution but fail to function on chromatin substrates. To test this hypothesis, we immunoprecipitated HBO1 complexes from cells treated with siRNAs against ING4, ING5, and HBO1 itself. As shown in Figure 6A, recovery of HBO1 protein was similar between cell extracts, except for the HBO1 knockdown cells, as expected. These results indicate that depletion of ING4 and ING5 proteins does not significantly affect HBO1 protein stability in vivo. When these fractions were tested in HAT assays on free histones, INGdepleted HBO1 complexes showed a 25% decrease in activity, compared to 75% for the HBO1 knockdown control (Figure 6B). We then performed HAT assays on chromatin substrates and calculated the ratio of activity in comparison to free histones. As shown in Figure 6C, HBO1 depletion, even though significantly decreasing yield of HAT activity, does not change its ratio on chromatin versus free histones. In contrast, ING4- and ING5-depleted complexes are specifically affected in their ability to acetylate nucleosomal histones. HBO1 complexes depleted of both ING4 and ING5 subunits show an even greater inability to modify chromatin (>5-fold). These results clearly indicate that ING4 and ING5 proteins are required in HBO1 complexes in order to efficiently modify chromatin. These results were further confirmed by purifying recombinant HBO1 HAT complexes from bacteria, similarly to what was done before for Tip60 and yEsa1 (Boudreault et al., 2003; Doyon et al., 2004; Selleck et al., 2005). Again, the presence of

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Figure 5. The ING5 HAT Complexes Interact with the MCM Helicase and Are Essential for DNA Replication (A) Purified ING4-TAP and ING5-TAP fractions were compared for presence of MCM proteins by Western blot with indicated antibodies. (B) Purified HBO1-TAP was analyzed by Western blot with anti-MCM2. (C) Flow cytometry analysis indicates the DNA content of 293T cells at 48 hr posttransfection with the indicated siRNA. (D) Acid extracted histones from cells as in (C) were analyzed by Western blot. (E–G) MCF7 cells treated for 48 hr with the indicated pSUPER plasmids were marked with BrdU for 30 min and analyzed by flow cytometry for BrdU incorporation and (F) analyzed for DNA content to determine the percentage of cells in S phase that incorporated BrdU (G) (mean of two experiments).

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Figure 6. ING4 and ING5 Subunits Are Required for HBO1 to Acetylate Chromatin Substrates HBO1 complexes from transduced HeLa S3 cells expressing FLAG-HBO1-TAP (or nontagged) and transfected with the indicated siRNAs were immunoprecipitated with antiFLAG M2 resin, and bound material was analyzed for HBO1 recovery (anti-FLAG Western) (A), HAT activity on free histones (direct counts) (B), and ratio of HAT activity on oligonucleosomes versus free histones (results are presented relative to the siRNA control sample set to 1) (C).

ING4 was found to have a role in stimulating HBO1 HAT activity toward chromatin, although to a smaller extent (Figures S3C and S3D). While ING proteins allow HAT enzymes to function on chromatin, they do not dictate histone tail specificity. This was tested by purifying recombinant Tip60-EPC1-ING complexes from bacteria (Doyon et al., 2004) in which ING3 was changed for ING4. Comparison of HAT specificity on nucleosomes showed similar histone H4 and H2A acetylation (data not shown). Altogether, these results strongly support the model in which part of ING protein function is to assist HAT/HDAC enzymes in the modification of chromatin fibers. It establishes the ING family of tumor suppressors as critical regulators of chromatin acetylation in vivo. Discussion In the present study, we characterized the stable molecular environment of each human member of the ING family of tumor suppressor proteins. We found that, like the three yeast INGs, each human ING protein is associated with a specific HDAC or HAT complex. Previous work on yeast Pho23, Yng1, and Yng2 and human ING3 showed that a critical role of ING proteins is to enable its associated HAT or HDAC enzyme to modify chromatin substrates (Boudreault et al., 2003; Doyon et al., 2004; Howe et al., 2002; Nourani et al., 2003; Selleck et al., 2005). Human ING1 and ING2 are purified as stable components of mSin3 HDAC complexes, like yeast Pho23. Purified ING3 is a subunit of the histone H4/H2A-specific Tip60 HAT complex, analogous to Yng2 in yeast NuA4 (Figure 7). ING4 was purified with a relatively simple four-subunit H4-specific HAT complex containing HBO1, whereas ING5 was found in two distinct complexes, one targeting histone H4 through HBO1 and the other targeting H3 through MOZ/MORF. The simple subunit structure of ING4 and ING5 com-

plexes is reminiscent of the yeast NuA3 complex (Figure 7). This yeast H3-specific HAT complex contains the Eaf6 protein, like NuA4, but also Nto1, a double PHD finger protein related to human JADE and BRPF proteins (L. Howe, personal communication; N. Lacoste and J.C., unpublished data) (see Figure S4C). Based on the similar H3 lysine specificity of the human MOZ/ MORF-ING5 and yeast NuA3 HAT complexes, we suggest that these are functional homologs (Figure 4F) (Howe et al., 2001). A similar hypothesis could be made based on the structure and HAT specificity of the HBO1-ING4/5 complexes. HBO1 seems responsible for global acetylation of histone H4 in vivo (Figures 3B and 5D). This is also the case for the piccolo NuA4 complex in yeast (Boudreault et al., 2003). This activity is a separate subcomplex of NuA4 containing Esa1, Epl1, Yng2, and Eaf6 (N. Lacoste and J.C., unpublished data). This tetrameric composition and HAT specificity suggest that HBO1-ING4/5 complexes may be functionally related to piccolo NuA4 in global nucleosomal H4 acetylation. We have previously shown that yeast Epl1 bridges Yng2 and Esa1 together, enabling acetylation of chromatin substrates (Boudreault et al., 2003), and that EPC1 does the same with Tip60 and ING3 in human cells (Doyon et al., 2004). Based on the simplicity of the ING4 and ING5 complexes, we speculate that a similar subunit exists in these complexes to bridge MYST HAT and ING proteins together. Accordingly, JADE1 and BRPF1 are required for ING4 and ING5 to associate with HBO1 and MOZ, respectively (Figure S3B; data not shown). In support of such a model Epl1, Nto1, EPC, JADE, and BRPF proteins contain two domains of related amino acid composition that could be responsible for these interactions (Figure S4). Yeast Dep1 and human BRMS1 proteins even contain a region of some sequence similarity with one of the domains, suggesting that this one is an interface for ING protein association. It

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Figure 7. Schematic Representation of the Different Histone Acetyltransferases and Deacetylases Complexes that Contain a Member of the Yeast or Human ING Family of Proteins

will be interesting to test this model of similar structure/ subunit interfaces. The work presented here also demonstrated that a critical function of ING proteins is to allow its associated HAT enzyme to modify nucleosomal histones (Figure 6 and Figures S3C and S3D). Based on our previous work on yeast INGs and human ING3, we propose that all ING proteins play similar catalytic roles in eukaryotes, in association with a specific HAT or HDAC complex. We found that a subset of ING5-HBO1 complex associates with the MCM proteins. This clearly supports the functional link previously suggested for HBO1 in DNA replication. Histone H4 and H3 acetylation has been shown to regulate the time of replication origin firing in yeast and Drosophila (Aggarwal and Calvi, 2004; Vogelauer et al., 2002). An independent study also found that HBO1 is a positive regulator of prereplicative complex assembly and is required for MCM proteins to associate with chromatin (Iizuka et al., 2005). Recruitment of ING5 HAT complexes associated with MCM helicase is an attractive model that could explain local hyperacetylation at origins of replication. The question remains whether the MOZ/MORF-ING5 complex also binds MCMs and regulates replication. This is very likely, since DNA replication defects due to ING5 depletion are much stronger than what is seen with HBO1 alone, arguing that both HBO1-ING5 and MOZ/MORF-ING5 HAT complexes are

important regulators of DNA synthesis. The structural and functional homology with the yeast NuA3 complex supports this idea. NuA3 has been physically and functionally linked to yeast FACT complex (Spt16-Pob3), which is involved in transcription elongation and DNA replication (Utley and Coˆte´, 2003). A recent report has also found the NuA3 catalytic subunit, Sas3, in a distinct protein complex linked to DNA polymerase e and containing two proteins with a bromodomain and a PWWP domain (Tackett et al., 2005). Interestingly, these two domains are present in MOZ/MORF-associated BRPF proteins but are missing in NuA3’s Nto1 (Figure S4C). Directly testing the role of MOZ or MORF in replication might be difficult because of the putative redundancy between the two proteins. Nevertheless, it will be important to investigate this link and to dissect the precise role of HBO1-ING complex during DNA replication. Another important piece of information provided by our data is that ING5 HAT complexes are required for DNA synthesis in cells that have already initiated replication (Figures 5F and 5G). These results suggest that histone H3 and H4 HAT complexes also function at advancing replication forks, allowing DNA synthesis/elongation in the chromatin environment. Through our biochemical analysis of human ING tumor suppressor proteins, we identified new native multisubunit HAT complexes. The HBO1, MOZ, and MORF

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proteins were discovered several years ago but were never characterized as part of a stable multisubunit HAT complex like hGCN5 (STAGA, TFTC), PCAF, Tip60, or Elongator complexes. We showed here that the HBO1 HAT protein is always part of a multisubunit assembly in vivo and responsible for the bulk of H4 acetylation in the two cell lines we analyzed. The stable physical link between HBO1 and ING proteins implicates a functional link with the p53 tumor suppressor protein and control of cell proliferation. On a related note, a MOZ leukemogenic fusion protein was shown to inhibit p53-dependent transcription, in agreement with our data linking this HAT to an ING tumor suppressor protein (Kindle et al., 2005). Our data suggest two distinct roles for ING4- and ING5-HBO1 HAT complexes. While ING5-HBO1 is important for DNA replication, ING4-HBO1 plays different roles, as depicted by a defect in G2/M passage. In conclusion, we have characterized the molecular interactions of human ING tumor suppressor proteins, which provided mechanistic insights on their function in cell proliferation control. These five proteins are each key components of specific HAT or HDAC multisubunit complexes. Their catalytic function in chromatin modification establishes them as critical regulators of chromatin acetylation in vivo. With such an important function, it is not surprising that each identified ING complex and their components are intimately linked to different forms of cancer, at the onset as well as for tumor growth (angiogenesis) and metastasis (e.g., Garkavtsev et al. [2004] and Zhou et al. [2005]). These protein complexes are added to the growing number of chromatin-related activities that link cancer to epigenetic regulation. Experimental Procedures Plasmid and Reagents To generate mammalian TAP-tag C-terminal fusion retroviral expression vectors, the HBO1 and ING2/3/4/5 cDNAs were amplified along with N-terminal FLAG or HA epitope by PCR from pcDNA constructs and subcloned into pRevTre-TAP as previously described (Doyon et al., 2004). Details on the cloning procedure are available in Supplemental Data. The list of antibodies used in this study is provided in the Supplemental Data. Retroviral Infection of Cell Lines and Purification of TAP-Tagged Complexes The production of retrovirally transduced HeLa S3 tet-off cell lines, purification of TAP-tagged native complexes (from w4.5 3 109 cells), and identification of specific subunits by tandem mass spectrometry were performed as previously described (Doyon et al., 2004). When indicated, a third affinity purification step was performed with anti-HA or FLAG resin (see Figures S1A and S1B) and mock purifications were performed with nontransduced HeLa S3 cells. RNA Interference The mammalian expression vector pSUPER (OligoEngine) was used for expression of shRNAs in 293T and MCF7 cells. Details about sequences, cloning, and efficiency are provided in Supplemental Experimental Procedures. The same sequences were also used to synthesize siRNAs (Dharmacon). For in vivo mapping of HAT specificity, 293T cells were cotransfected with 12 mg pSUPER, pSUPER-Tip60, or pSUPER-HBO1a and 0.8 mg of caax-GFP (gift of J. Lavoie). Posttransfection (48 hr), 150,000 GFP-positive cells were sorted and subjected to acidic extraction. Partially purified histones (1 mg) were loaded on 18% SDS-PAGE and analyzed by Western blotting on independent membranes.

Cell Cycle, BrdU Incorporation, and Growth Analysis Cell-cycle analysis was performed in 293T cells after cotransfection with 12 mg of the pSUPER-Tip60 or pSUPER-HBO1a and 1.2 mg of caax-GFP. Posttransfection (24 hr or 48 hr), cells were fixed 30 min in ice-cold 70% ethanol. The fixed cells were washed once with PBS, treated with RNase A (0.2 mg/ml) for 30 min at 37ºC, and stained with PI (30 mg/ml). GFP-positive cells were analyzed by using Coulter EPICS XL-MCL flow cytometer and MultiCycle software (Phoenix). siRNAs (8 mg) were transfected with oligofectamine (Qiagen) according to the manufacturer’s instructions. Posttransfection (48 hr) cells were subjected to cell-cycle analysis as above on whole cellular populations. MCF7 cells were treated with BrdU (10 mM) during 30 min, 48 hr after transfection with 12 mg of the pSUPER-ING4 or pSUPER-ING5. The fixed cells were washed once with PBS, treated with HCl 2N 30 min at 37ºC, incubated with anti-BrdU-FITC (e-bioscience) during 30 min, and stained with PI (30 mg/ml). Whole population, BrdU-positive and -negative MCF7 cells were analyzed by using Coulter EPICS XL-MCL flow cytometer and MultiCycle software (Phoenix). Cell growth was determined by MTT assay using standard procedure (Sigma). GFP-positive cells were sorted, seeded 24 hr posttransfection with different pSUPER, and tested by MTT assay 24, 48, or 72 hr after seeding. Other Techniques Detailed procedures for HAT assays, luciferase reporter assays, and immunoprecipitations have been described (Doyon et al., 2004; Nagashima et al., 2003; Nourani et al., 2001; Pelletier et al., 2002). Assays to determine the site specificity of ING complexes were performed on recombinant Xenopus nucleosome core particles (xNCP) (Selleck et al., 2005). Briefly, active fractions were incubated with 1 mg of xNCP and 50 mM acetyl-CoA in a typical HAT assay for 1 hr at 30ºC. Histone acetylation was analyzed by Western blot using antibodies specific for acetylated Lys 5/8/12/16 on histone H4 and Lys14/9–18 on histone H3. For indirect immunofluorescence analysis, MCF7 cells were fixed with 2% paraformaldehyde and permeabilized with 0.1% Triton X-100. Primary antibodies (goat polyclonal anti-HBO1 at 1:40 or rabbit polyclonal anti-Tip60 at 1:200) were applied to the cells for 2 hr. After washing with PBS, Alexa-conjugated (488 nm or 568 nm) secondary antibodies (Molecular Probes) were applied for 1 hr. Nuclei were stained with DRAQ5 (Alexis), and coverslips were mounted and analyzed by confocal microscopy. Supplemental Data Supplemental Data include four figures, Supplemental Experimental Procedures, and Supplemental References and can be found with this article online at http://www.molecule.org/cgi/content/full/21/1/ 51/DC1/. Acknowledgments We thank W. Wang, J. Lucchesi/E. Smith, and Y. Makino for antibodies; B. Hnatkovich and L. Gaudreau for recombinant histones/ nucleosomes; B. Clarke and M. Zhou for help creating bacterial expression vectors; and M.M. Smith/M. Iizuka for recombinant HBO1 vector. We are grateful to M.M. Smith, L. Howe, T. Kusch, and R. Shiekhattar for sharing unpublished results and stimulating discussions. This work was supported by grants from the Canadian Institutes of Health Research (CIHR) and GenomeCanada/GenomeQuebec to J.C., the National Cancer Institute of Canada (NCIC) to X.-J.Y., and the National Institutes of Health (NIH) (to S.T.). Y.D. holds a CIHR/Canada Graduate Scholarship. S.T. is a Pew Scholar in the Biomedical Sciences, and J.C. is a CIHR Investigator. Received: July 19, 2005 Revised: October 31, 2005 Accepted: December 7, 2005 Published: January 5, 2006 References Aggarwal, B.D., and Calvi, B.R. (2004). Chromatin regulates origin activity in Drosophila follicle cells. Nature 430, 372–376. Berns, K., Hijmans, E.M., Mullenders, J., Brummelkamp, T.R., Velds, A., Heimerikx, M., Kerkhoven, R.M., Madiredjo, M., Nijkamp, W.,

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