BRPF 3‐ HBO 1 regulates replication origin activation and histone H3K14 acetylation
2015; Springer Nature; Volume: 35; Issue: 2 Linguagem: Inglês
10.15252/embj.201591293
ISSN1460-2075
AutoresYunpeng Feng, Arsenios Vlassis, Céline Lopez‐Roques, Marie‐Eve Lalonde, Cristina González‐Aguilera, Jean‐Philippe Lambert, Sung‐Bau Lee, Xiaobei Zhao, Constance Alabert, Jens Vilstrup Johansen, Éric R. Paquet, Xiang‐Jiao Yang, Anne‐Claude Gingras, Jacques Côté, Anja Groth,
Tópico(s)Epigenetics and DNA Methylation
ResumoArticle30 November 2015free access BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation Yunpeng Feng Yunpeng Feng Biotech Research and Innovation Centre (BRIC) and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Arsenios Vlassis Arsenios Vlassis Biotech Research and Innovation Centre (BRIC) and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Céline Roques Céline Roques St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Oncology Axis-CHU de Québec Research Center, Quebec City, QC, Canada Search for more papers by this author Marie-Eve Lalonde Marie-Eve Lalonde St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Oncology Axis-CHU de Québec Research Center, Quebec City, QC, Canada Search for more papers by this author Cristina González-Aguilera Cristina González-Aguilera Biotech Research and Innovation Centre (BRIC) and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Jean-Philippe Lambert Jean-Philippe Lambert Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Search for more papers by this author Sung-Bau Lee Sung-Bau Lee Biotech Research and Innovation Centre (BRIC) and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Master Program for Clinical Pharmacogenomics and Pharmacoproteomics, School of Pharmacy, Taipei Medical University, Taipei, Taiwan Search for more papers by this author Xiaobei Zhao Xiaobei Zhao Bioinformatics Centre Department of Biology, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Constance Alabert Constance Alabert Biotech Research and Innovation Centre (BRIC) and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Jens V Johansen Jens V Johansen Biotech Research and Innovation Centre (BRIC) and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Eric Paquet Eric Paquet St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Oncology Axis-CHU de Québec Research Center, Quebec City, QC, Canada Search for more papers by this author Xiang-Jiao Yang Xiang-Jiao Yang Department of Medicine, McGill University Health Center, Montréal, QC, Canada Search for more papers by this author Anne-Claude Gingras Anne-Claude Gingras Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Search for more papers by this author Jacques Côté Corresponding Author Jacques Côté St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Oncology Axis-CHU de Québec Research Center, Quebec City, QC, Canada Search for more papers by this author Anja Groth Corresponding Author Anja Groth Biotech Research and Innovation Centre (BRIC) and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Yunpeng Feng Yunpeng Feng Biotech Research and Innovation Centre (BRIC) and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Arsenios Vlassis Arsenios Vlassis Biotech Research and Innovation Centre (BRIC) and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Céline Roques Céline Roques St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Oncology Axis-CHU de Québec Research Center, Quebec City, QC, Canada Search for more papers by this author Marie-Eve Lalonde Marie-Eve Lalonde St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Oncology Axis-CHU de Québec Research Center, Quebec City, QC, Canada Search for more papers by this author Cristina González-Aguilera Cristina González-Aguilera Biotech Research and Innovation Centre (BRIC) and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Jean-Philippe Lambert Jean-Philippe Lambert Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Search for more papers by this author Sung-Bau Lee Sung-Bau Lee Biotech Research and Innovation Centre (BRIC) and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Master Program for Clinical Pharmacogenomics and Pharmacoproteomics, School of Pharmacy, Taipei Medical University, Taipei, Taiwan Search for more papers by this author Xiaobei Zhao Xiaobei Zhao Bioinformatics Centre Department of Biology, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Constance Alabert Constance Alabert Biotech Research and Innovation Centre (BRIC) and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Jens V Johansen Jens V Johansen Biotech Research and Innovation Centre (BRIC) and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Eric Paquet Eric Paquet St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Oncology Axis-CHU de Québec Research Center, Quebec City, QC, Canada Search for more papers by this author Xiang-Jiao Yang Xiang-Jiao Yang Department of Medicine, McGill University Health Center, Montréal, QC, Canada Search for more papers by this author Anne-Claude Gingras Anne-Claude Gingras Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Search for more papers by this author Jacques Côté Corresponding Author Jacques Côté St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Oncology Axis-CHU de Québec Research Center, Quebec City, QC, Canada Search for more papers by this author Anja Groth Corresponding Author Anja Groth Biotech Research and Innovation Centre (BRIC) and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Author Information Yunpeng Feng1,8,‡, Arsenios Vlassis1,‡, Céline Roques2,‡, Marie-Eve Lalonde2, Cristina González-Aguilera1, Jean-Philippe Lambert3, Sung-Bau Lee1,4, Xiaobei Zhao5,9, Constance Alabert1, Jens V Johansen1, Eric Paquet2, Xiang-Jiao Yang6, Anne-Claude Gingras3,7, Jacques Côté 2 and Anja Groth 1 1Biotech Research and Innovation Centre (BRIC) and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark 2St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Oncology Axis-CHU de Québec Research Center, Quebec City, QC, Canada 3Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada 4Master Program for Clinical Pharmacogenomics and Pharmacoproteomics, School of Pharmacy, Taipei Medical University, Taipei, Taiwan 5Bioinformatics Centre Department of Biology, University of Copenhagen, Copenhagen, Denmark 6Department of Medicine, McGill University Health Center, Montréal, QC, Canada 7Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada 8Present address: The Institute of Genetics and Cytology, Northeast Normal University, Changchun, China 9Present address: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 418 525 4444, poste 15545; E-mail: [email protected] *Corresponding author. Tel: +45 3532 5538; E-mail: [email protected] The EMBO Journal (2016)35:176-192https://doi.org/10.15252/embj.201591293 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract During DNA replication, thousands of replication origins are activated across the genome. Chromatin architecture contributes to origin specification and usage, yet it remains unclear which chromatin features impact on DNA replication. Here, we perform a RNAi screen for chromatin regulators implicated in replication control by measuring RPA accumulation upon replication stress. We identify six factors required for normal rates of DNA replication and characterize a function of the bromodomain and PHD finger-containing protein 3 (BRPF3) in replication initiation. BRPF3 forms a complex with HBO1 that specifically acetylates histone H3K14, and genomewide analysis shows high enrichment of BRPF3, HBO1 and H3K14ac at ORC1-binding sites and replication origins found in the vicinity of TSSs. Consistent with this, BRPF3 is necessary for H3K14ac at selected origins and efficient origin activation. CDC45 recruitment, but not MCM2-7 loading, is impaired in BRPF3-depleted cells, identifying a BRPF3-dependent function of HBO1 in origin activation that is complementary to its role in licencing. We thus propose that BRPF3-HBO1 acetylation of histone H3K14 around TSS facilitates efficient activation of nearby replication origins. Synopsis A distinct origin activation role of HBO1 acetyltransferase provides new insight into how demarcation of chromatin surrounding transcription start sites affects the regulation of nearby replication origins. siRNA screen identifies chromatin regulators important for DNA replication, including the scaffold protein BRPF3. BRPF3 regulates origin firing by directing the acetyltransferase HBO1 to target histone H3K14 in chromatin surrounding replication origins. HBO1-BRPF3 complex function in origin activation is separate from and complementary to HBO1-JADE1 function in origin licensing. Reduced origin activation upon BRPF3 depletion protects cells against replication stress-induced DNA damage. Introduction In S phase of the cell cycle, the genome must be faithfully duplicated in order to maintain genome integrity across cell generations. To ensure complete replication of the entire human genome, replication initiates from thousands of origins in a well-defined manner. Tight regulation of replication initiation is central to maintain genome integrity and prevent tumorigenesis (Alver et al, 2014). Origin hyper-activation in response to oncogenic signalling can drive genome instability (Halazonetis et al, 2008; Alver et al, 2014), and cell type-specific deficits in origins at so-called fragile sites can contribute to DNA breakage and chromosomal rearrangements (Letessier et al, 2011; Debatisse et al, 2012). Initiation of DNA replication can be divided into three tightly regulated steps. First, ORC (origin recognition complex) binding specifies replication origins as cells exit mitosis (Bell & Dutta, 2002; MacAlpine & Bell, 2005; Mechali, 2010; Alabert & Groth, 2012). Second, in a process termed licencing, pre-RCs (pre-replication complexes) are assembled via CDT1- and CDC6-dependent loading of the hexameric MCM2-7 complex (Bell & Dutta, 2002; Remus & Diffley, 2009; Mechali, 2010). Third, in S phase, origins are activated by the dual action of CDK and DDK kinases that by phosphorylation of the pre-RC and accessory fork components recruit CDC45 and the GINS complex to activate the replicative helicase (Labib & Gambus, 2007; Remus & Diffley, 2009). To ensure that initiation happens only once per cell cycle, these events are restricted to specific phases of the cell cycle. However, to provide backup sites for replication initiation in case nearby replication forks are compromised, a large number of excess origins are licensed during the G1 phase (Woodward et al, 2006; Ge et al, 2007). Only a fraction of these origins are activated during S phase, and the rest remain dormant and are passively replicated by forks arriving from nearby active origins (reviewed in Alver et al, 2014). If fork progression is impaired due to DNA damage or reduced dNTP supply, nearby dormant origins can be activated. It is not clear what determines whether an origin is dormant or not. In part, which origins are activated or remain dormant appear to be stochastic, reflecting the intrinsic inefficiency of origin activation. Single-molecule analysis has established that checkpoint kinases control origin firing and dormant origin activation (Ge et al, 2007; Maya-Mendoza et al, 2007; Petermann et al, 2010b). Whether chromatin context directly influences origin efficiency remains unclear (Mechali, 2010), but chromosomal architecture is important for confining origin firing to a distinct spatiotemporal pattern during S phase (Gilbert et al, 2010; Pope et al, 2010). This replication-timing programme is cell type specific, and chromatin context provides a potential key to understand cell type-specific origin usage and thus replication timing (Gilbert et al, 2010; Pope et al, 2010). In general, active regions of the genome replicate early and silenced domains replicate late (Goren & Cedar, 2003). Recently, it was shown that replication timing correlates directly with the three-dimensional organization of the genome in topological domains (Pope et al, 2014), which translates into open and closed chromatin compartments. Mounting evidence indicates that chromatin environment contributes to origin specification and licensing. Genomewide analysis has revealed that regions of low nucleosome occupancy are preferred binding sites for ORC (Lubelsky et al, 2011). Features typical of active chromatin such as histone acetylation, the histone variant H3.3 and recruitment of chromatin remodellers generally demarcate ORC-binding sites (MacAlpine et al, 2010; Mechali, 2010). Methylation of histone H4 at lysine 20 can also facilitate ORC1 recruitment by serving as a recognition site for ORC1 and co-factors (Beck et al, 2012; Kuo et al, 2012). Subsequently, acetylation of histone H4 at lysine 5, 8 and 12 by the lysine acetyltransferase (KAT) HBO1 contributes to MCM2-7 loading (Miotto & Struhl, 2010). HBO1 is required for DNA replication and interacts with ORC1, Cdt1 and MCM2-7 (Iizuka & Stillman, 1999; Burke et al, 2001; Doyon et al, 2006; Miotto & Struhl, 2008, 2010). Notably, artificial tethering of the H4K20me1 enzyme SET8 and HBO1 can promote recruitment of ORC and MCM2-7 (Aggarwal & Calvi, 2004; Tardat et al, 2010; Chen et al, 2013). The HBO1 protein was initially characterized as the catalytic subunit of the main histone H4 acetyltransferase complex in human 293T cells, and it is enriched near the transcription start site of active genes (Doyon et al, 2006; Avvakumov et al, 2012). In this complex, PHD finger-containing subunits JADE1/2/3(PHF15/16/17) and ING4/5 enable HBO1 activity towards chromatin substrates and H3K4me3-bearing nucleosomes, respectively (Saksouk et al, 2009). It was recently discovered that HBO1 also exists in native complexes containing BRPF1/2 scaffold subunits instead of JADEs, shifting the specificity of chromatin acetylation to histone H3 instead of H4 (Mishima et al, 2011; Lalonde et al, 2013, 2014). HBO1 knockout in mice led to a dramatic reduction of histone H3 acetylation at K14 and severe defects in embryonic development (Kueh et al, 2011). BRPF1 has been shown to be important for brain development (You et al, 2015a,c), and deletion of the mouse BRPF1 gene causes embryonic lethality (You et al, 2015b). Similar to HBO1-knockdown erythroblasts, BRPF2-deficient mice exhibited impaired global H3K14 acetylation and decreased H3K14 acetylation at the promoters of dysregulated erythroid developmental regulator genes (Mishima et al, 2011). However, the function of BRPF3 remains uncharacterized. Given that H4 acetylation by HBO1-JADE is important for DNA replication (Doyon et al, 2006; Miotto & Struhl, 2008, 2010; Swarnalatha et al, 2012), whether BRPFs function with HBO1 in DNA replication is unclear. To uncover novel chromatin-based mechanisms controlling DNA replication, we established a siRNA screen based on the phenotype of cells lacking the ASF1 histone H3-H4 chaperone. ASF1-depleted cells fail to expose single-stranded DNA (ssDNA) at replication forks upon treatment with hydroxyurea (HU) (Groth et al, 2007). ssDNA accumulates as a result of uncoupling between the replicative helicase and DNA polymerases (Walter & Newport, 2000; Pacek & Walter, 2004; Cimprich & Cortez, 2008; Toledo et al, 2013), representing a hallmark of stalled replication forks as well as a very robust read-out for screening. We reasoned that lack of ssDNA accumulation in S-phase cells treated with HU could result from either a DNA unwinding defect or a reduced number of active forks (Groth et al, 2007; Mejlvang et al, 2014). Using a customized library targeting chromatin factors, we identified 20 factors required for HU-induced ssDNA accumulation. A subset of these was also required for efficient DNA replication, including the multi-domain scaffold protein BRPF3. We show that BRPF3 forms a complex with HBO1, which specifically acetylates H3K14 and is required for efficient activation of licenced origins in S phase. Results siRNA screen identifies candidate genes involved in DNA replication To identify chromatin factors involved in regulation of DNA replication, we performed a siRNA screen for proteins that are required for HU-induced ssDNA exposure similar to the ASF1 histone chaperone (Groth et al, 2007). We used a customized library containing three independent siRNAs targeting 219 putative chromatin factors (Table EV1). To monitor ssDNA exposure in S-phase cells, we used a reporter cell line stably expressing GFP-RPA1 together with RFP-PCNA (Mejlvang et al, 2014). To focus on DNA unwinding at stalled replication forks, rather than collapsed forks, we treated cells short term with HU (2 h) (Petermann et al, 2010a). Furthermore, we removed soluble proteins prior to fixation to measure chromatin-bound GFP-RPA1 and RFP-PCNA. siRNAs targeting ASF1 (a and b) were used as a positive control, efficiently inhibiting GFP-RPA1 accumulation as shown previously (Fig 1A, Groth et al, 2007; Mejlvang et al, 2014). We performed the screen in two biological replicas and ranked all genes in our library by the collective activities of the multiple siRNAs using redundant siRNA activity (RSA) analysis (Konig et al, 2007; Beck et al, 2010). The top hits were derived by the Fisher method, ranking genes with combined P-values (Kost & McDermott, 2002; Menzel et al, 2011) (Fig 1B and Table EV2). On this basis along with available literature, we selected 20 genes for further validation and assembled a sub-library containing the two highest scoring siRNAs targeting each candidate. We then carried out systematic functional analyses by high-content imaging. We focused on S-phase cells, and therefore, only PCNA-positive cells were included in the following assays. Firstly, we verified that siRNA depletion of our candidate genes attenuated GFP-RPA1 accumulation in response to HU (Fig 1C). siRNAs against ASF1 (a and b) and CDC45 were included as positive controls (Walter & Newport, 2000; Pacek & Walter, 2004; Groth et al, 2005, 2007; Toledo et al, 2013). Given that lack of HU-induced RPA1 accumulation may result from a cell cycle defect such as G1 arrest, we also quantified PCNA-positive cells. However, the proportion of PCNA-positive cells was largely unchanged upon siRNA depletion of the candidate genes (Appendix Fig S1A), arguing that they are either not required for S-phase entry or that the siRNA knockdown is partial. Importantly, this ruled out that impaired ssDNA accumulation was secondary to a cell cycle arrest, supporting that the 20 identified genes could play a role in DNA replication. Figure 1. siRNA screen identifies BRPF3 as a novel regulator of DNA replication Representative images of controls from the siRNA screen for HU-induced RPA accumulation. GFP-RPA1 and RFP-PCNA reporter cells were transfected with siRNAs and 48 h later treated with HU (3 mM) for 2 h prior to pre-extraction, fixation and imaging. Gene ranking based on siRNA scores from two independent screens. A total of 219 genes were targeted by three individual siRNAs and ranked based on P-values derived from RSA and Fisher's analysis. Selected hits are highlighted. Validation of selected genes by single-cell analysis of GFP-RPA1 intensity in pre-extracted cells. Only S-phase cells positive for RFP-PCNA were analysed. Median with interquartile range of relative intensity per cell is shown, n > 4,000. Mann–Whitney: ****P < 10−4, n.s. non-significant. One representative experiment out of two biological replicas is shown. Analysis of DNA synthesis rate. Cells were pulsed 15 min with EdU, and EdU intensity was analysed in S-phase cells positive for RFP-PCNA. EdU incorporation is shown relative to siCtrl. The average and standard deviation from three biological replicas is shown. n > 2,000. ANOVA t-tests: ****P < 10−4, ***P < 10−3, **P < 10−2, *P ≤ 0.05; n.s., non-significant. Data information: In (B, D), a, b, c denote independent siRNAs; Ctrl, control. Download figure Download PowerPoint Next, we measured the rate of DNA synthesis by quantifying ethynyl deoxyuridine (EdU) incorporation in siRNA-depleted cells. Here, siRNA against ASF1 (a and b) served as a positive control, repressing DNA replication as previously reported (Groth et al, 2007). Although the degree of inhibition varied, we found that several of the siRNAs significantly impaired EdU incorporation (Fig 1D). By correlating this phenotype with knockdown efficiency measured by RT-qPCR (Appendix Fig S2A–O), we identified six genes acting as positive regulators of DNA replication (AGFG2, BRPF3, HEMK2, KAT5, KAT8 and PRDM12). However, reduced DNA replication could be a secondary effect of high loads of DNA damage. To address this point, we screened for γH2AX, a hallmark of DNA damage signalling (Harrison & Haber, 2006; Harper & Elledge, 2007). However, none of the siRNAs significantly increased the γH2AX level, arguing that the identified factors are required for normal rates of DNA replication independent of DNA damage signalling (Appendix Fig S1B). BRPF3 is required for DNA replication and H3K14 acetylation We decided to focus on the high-ranking candidate, BRPF3 (bromodomain- and PHD finger-containing protein 3), whose function is largely unaddressed. The BRPF family of proteins contains typical domains found in chromatin proteins, including PHD fingers, a bromodomain and a chromo/Tudor-related PWWP domain (Doyon et al, 2006). BRPF3, along with its paralogs BRPF1 and BRPF2, was initially identified as potential scaffold proteins in large MOZ/MORF KAT complexes containing ING5 (Doyon et al, 2006). From our siRNA screen data, we noted that BRPF1 and BRPF2 were not required for ssDNA formation in response to HU (Appendix Fig S3A), suggesting that we could separate the function of these paralogs. Consistent with this, depletion of BRPF1 and BRPF2 with verified siRNAs did not affect DNA replication, whereas depletion of BRPF3 with multiple independent siRNAs reduced DNA synthesis by 30% without activating a DNA damage response (γH2AX, P-RPA and P-Chk1) (Fig 2A, Appendix Figs S3B and S4A). This reduction in DNA replication was highly consistent between different BRPF3 siRNAs and significant regardless of whether EdU intensities were compared across the total cell population or in S-phase cells (Appendix Fig S3C and D). Furthermore, the replication defect could be partially rescued by exogenous expression of siRNA-resistant BRPF3 (Fig 2B and Appendix Fig S3E). Thus, BRPF3, but not BRPF1 or BRPF2, regulates DNA replication. Figure 2. BRPF3-HBO1 regulates DNA replication and acetylates H3K14ac DNA replication measured by EdU incorporation. RPF-PCNA reporter cells were transfected with the indicated siRNAs and pulsed with EdU for 20 min. S-phase cells positive for RFP-PCNA were analysed. n > 150. Error bars, SD; n = 3 biological replicas. One-sample t-test, ***P < 0.001, **P < 0.01, n.s., non-significant. Complementation analysis of DNA synthesis. Stable cell lines expressing BRPF3 resistant to the BRPF3/a siRNA and control (lacZ-V5) were siRNA transfected and analysed for EdU incorporation as in (A). Error bars, SD; n = 6 biological replicas. Two-tailed t-test, **P < 10−2. Comparison of native BRPF1 and BRPF3 complexes purified from K562 cells. (left) Mass spectrometric analysis (spectral counts/total peptides identified) and (right) verification of associated protein by Western blot. Immunoprecipitation analysis of BRPF3 deletion mutants. One representative experiment out of two is shown. Histone acetyltransferase assay (HAT) with purified BRPF1/3 complexes on free histones and mononucleosomes. HAT assay with purified BRPF1/3 complexes on H3 peptides unmodified or acetylated (ac)/methylated (me) at K14 and K27, respectively. Mean ± SD is shown, n = 4. Analysis of histone acetylation in BRPF3-depleted cells. (left) Western blot of siRNA-treated U-2-OS cells. TSA treatment (1 h) was included as a positive control. 2x, double amount of extract loaded as in 1x. (right) H3K14 acetylation levels quantified relative to total H3. Error bars, SD; n = 3 biological replicates. One-sample t-test. Complementation analysis of H3K14ac. Total cell extracts of siRNA-treated lacZ-V5 and BRPF3-V5 (siBRPF3/a resistant) expressing cells were analysed by Western blotting. One representative experiment out of four biological replicas is shown. Download figure Download PowerPoint To gain molecular insight into the different function of the BRPF paralogs, we isolated BRPF1 and BRPF3 complexes from cells carrying a single-copy ZFN integrated transgene and identified interaction partners by mass spectrometry. This revealed that the two paralogs were part of distinct KAT complexes; BRPF3 associated exclusively with HBO1, while BRPF1 formed complex with MOZ/MORF as described previously (Doyon et al, 2006; Lalonde et al, 2013) (Fig 2C). These results were confirmed by co-immunoprecipitation analysis in U-2-OS cells (Fig 2D, Appendix Fig S3F and G). Analysis of BRPF3 deletion mutants showed that a small N-terminal region was required for HBO1 binding, while deletion of an internal region specific to BRPF3 (BRPF3Δinter) was dispensable (Fig 2D and Appendix Fig S3H). Of note, both BRPF3 mutants interact with ING5 (Fig 2C and D), consistent with previous data showing that the BRPF scaffold binds the KAT and ING proteins independently (Doyon et al, 2006; Ullah et al, 2008). HBO1 is part of the MYST family of KATs and has previously been linked to acetylation of histone H4 K5/8/K12, and histone H3 K14 and K23 (Doyon et al, 2006; Kueh et al, 2011; Mishima et al, 2011; Lalonde et al, 2013). In particular, it was recently proposed that the scaffold partner protein could determine the target specificity of HBO1 (Lalonde et al, 2013). We thus addressed the specificity of the BRPF3-HBO1 complex. The purified BRPF3 complex could acetylate both histones H3 and H4 when presented as core histones in solution, but on a mononucleosome substrate, the BRPF3 complex preferentially acetylated histone H3 (Fig 2E) as reported previously for the BRPF1 complex (Lalonde et al, 2013). BRPF3 complexes specifically acetylated H3 peptides spanning K4, K9 and K14 (1–21), but had no activity on an identical peptide already acetylated at K14 (Fig 2F). BRPF1 complexes showed activity against K14 in the same assay, but, in contrast to BRPF3, also acetylated peptides spanning K23 and K27 (21–44) regardless of whether K27 was blocked by methylation (Fig 2F). This shows that BRPF3 complexes preferentially target H3K14ac in vitro, and consistent with this, H3K14ac (but not H3K23ac or H4K5ac) was significantly reduced upon BRPF3 depletion in vivo (Fig 2G). Furthermore, the loss of H3K14ac could be rescued by expression of siRNA-resistant BRPF3 (Fig 2H). BRPF1 complexes could target both H3 K14 and K23 in vitro, but depletion of BRPF1 mainly affected H3K23ac levels in vivo (Appendix Fig S4A). Collectively these data suggest that acetylation of H3K14 by BRPF3-HBO1 could be important for DNA replication. In complex with another scaffold, JADE1, HBO1 can acetylate histone H4 (Doyon et al, 2006; Foy et al, 2008) and this has been shown to facilitate origin licensing (Miotto & Struhl, 2010). However, given that H4K5ac is not altered upon BRPF3 knockdown, we anticipated that the function of the BRPF3-HBO1 complex would be distinct from the role of HBO1-JADE1 in licensing. BRPF3 is regulating replication origin activation To understand whether BRPF3 regulates DNA replication at the level of elongation or new origin firing, we investigated replication at the single-molecule level by molecular DNA combing (Mejlvang et al, 2014). Newly synthesized DNA was labelled by consecutive pulses of IdU and CldU to allow identification of independent forks by selection of CldU tracks flanked by IdU. First, we measured inter-track distances to evaluate the number of active origins and used the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01) as a control for increased origin firing. Chk1 inhibition activates dormant origins and consistently inter-track distances were reduced (Ge et al, 2007; Maya-Mendoza et al, 2007; Petermann et al, 2010b) (Fig 3A). In contrast, BRPF3-depleted cells showed significantly longer inter-track distances as compared to control cells (Fig 3A). Concomitantly, the length of CIdU-labelled tracks was increased in cells lacking BRPF3 (Fig 3B and Appendix Fig S4B) and similar results were observed by DNA fibre assay (Appendix Fig S4C). Collectively, these data sugge
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