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• W2745177530 abstract "Article4 August 2017free access Transparent process Histone H4K20 tri-methylation at late-firing origins ensures timely heterochromatin replication Julien Brustel Institut de Recherche en Cancérologie de Montpellier (IRCM), INSERM U1194, Institut Régional du Cancer (ICM), Montpellier, France University of Montpellier, Montpellier, France Search for more papers by this author Nina Kirstein Research Unit Gene Vectors, Helmholtz Zentrum München, Munich, Germany Search for more papers by this author Fanny Izard Institut de Recherche en Cancérologie de Montpellier (IRCM), INSERM U1194, Institut Régional du Cancer (ICM), Montpellier, France University of Montpellier, Montpellier, France Search for more papers by this author Charlotte Grimaud Institut de Recherche en Cancérologie de Montpellier (IRCM), INSERM U1194, Institut Régional du Cancer (ICM), Montpellier, France University of Montpellier, Montpellier, France Search for more papers by this author Paulina Prorok Institute of Human Genetics (IGH), CNRS, Montpellier, France Search for more papers by this author Christelle Cayrou Institute of Human Genetics (IGH), CNRS, Montpellier, France Search for more papers by this author Gunnar Schotta orcid.org/0000-0003-4940-6135 Biomedical Center Munich, Planegg-Martinsried, Germany Search for more papers by this author Alhassan F Abdelsamie Research Unit Gene Vectors, Helmholtz Zentrum München, Munich, Germany Search for more papers by this author Jérôme Déjardin Institute of Human Genetics (IGH), CNRS, Montpellier, France Search for more papers by this author Marcel Méchali Institute of Human Genetics (IGH), CNRS, Montpellier, France Search for more papers by this author Giuseppe Baldacci Institut Jacques Monod, UMR7592, CNRS and University Paris-Diderot, Paris, France Search for more papers by this author Claude Sardet Institut de Recherche en Cancérologie de Montpellier (IRCM), INSERM U1194, Institut Régional du Cancer (ICM), Montpellier, France University of Montpellier, Montpellier, France Search for more papers by this author Jean-Charles Cadoret Corresponding Author [email protected] orcid.org/0000-0002-3827-5208 Institut Jacques Monod, UMR7592, CNRS and University Paris-Diderot, Paris, France Search for more papers by this author Aloys Schepers Corresponding Author [email protected] orcid.org/0000-0002-5442-5608 Research Unit Gene Vectors, Helmholtz Zentrum München, Munich, Germany Search for more papers by this author Eric Julien Corresponding Author [email protected] orcid.org/0000-0003-2976-666X Institut de Recherche en Cancérologie de Montpellier (IRCM), INSERM U1194, Institut Régional du Cancer (ICM), Montpellier, France University of Montpellier, Montpellier, France Search for more papers by this author Julien Brustel Institut de Recherche en Cancérologie de Montpellier (IRCM), INSERM U1194, Institut Régional du Cancer (ICM), Montpellier, France University of Montpellier, Montpellier, France Search for more papers by this author Nina Kirstein Research Unit Gene Vectors, Helmholtz Zentrum München, Munich, Germany Search for more papers by this author Fanny Izard Institut de Recherche en Cancérologie de Montpellier (IRCM), INSERM U1194, Institut Régional du Cancer (ICM), Montpellier, France University of Montpellier, Montpellier, France Search for more papers by this author Charlotte Grimaud Institut de Recherche en Cancérologie de Montpellier (IRCM), INSERM U1194, Institut Régional du Cancer (ICM), Montpellier, France University of Montpellier, Montpellier, France Search for more papers by this author Paulina Prorok Institute of Human Genetics (IGH), CNRS, Montpellier, France Search for more papers by this author Christelle Cayrou Institute of Human Genetics (IGH), CNRS, Montpellier, France Search for more papers by this author Gunnar Schotta orcid.org/0000-0003-4940-6135 Biomedical Center Munich, Planegg-Martinsried, Germany Search for more papers by this author Alhassan F Abdelsamie Research Unit Gene Vectors, Helmholtz Zentrum München, Munich, Germany Search for more papers by this author Jérôme Déjardin Institute of Human Genetics (IGH), CNRS, Montpellier, France Search for more papers by this author Marcel Méchali Institute of Human Genetics (IGH), CNRS, Montpellier, France Search for more papers by this author Giuseppe Baldacci Institut Jacques Monod, UMR7592, CNRS and University Paris-Diderot, Paris, France Search for more papers by this author Claude Sardet Institut de Recherche en Cancérologie de Montpellier (IRCM), INSERM U1194, Institut Régional du Cancer (ICM), Montpellier, France University of Montpellier, Montpellier, France Search for more papers by this author Jean-Charles Cadoret Corresponding Author [email protected] orcid.org/0000-0002-3827-5208 Institut Jacques Monod, UMR7592, CNRS and University Paris-Diderot, Paris, France Search for more papers by this author Aloys Schepers Corresponding Author [email protected] orcid.org/0000-0002-5442-5608 Research Unit Gene Vectors, Helmholtz Zentrum München, Munich, Germany Search for more papers by this author Eric Julien Corresponding Author [email protected] orcid.org/0000-0003-2976-666X Institut de Recherche en Cancérologie de Montpellier (IRCM), INSERM U1194, Institut Régional du Cancer (ICM), Montpellier, France University of Montpellier, Montpellier, France Search for more papers by this author Author Information Julien Brustel1,2,‡, Nina Kirstein3,‡, Fanny Izard1,2,‡, Charlotte Grimaud1,2, Paulina Prorok4, Christelle Cayrou4, Gunnar Schotta5, Alhassan F Abdelsamie3, Jérôme Déjardin4, Marcel Méchali4, Giuseppe Baldacci6, Claude Sardet1,2, Jean-Charles Cadoret *,6,‡, Aloys Schepers *,3,†,‡ and Eric Julien *,1,2,‡ 1Institut de Recherche en Cancérologie de Montpellier (IRCM), INSERM U1194, Institut Régional du Cancer (ICM), Montpellier, France 2University of Montpellier, Montpellier, France 3Research Unit Gene Vectors, Helmholtz Zentrum München, Munich, Germany 4Institute of Human Genetics (IGH), CNRS, Montpellier, France 5Biomedical Center Munich, Planegg-Martinsried, Germany 6Institut Jacques Monod, UMR7592, CNRS and University Paris-Diderot, Paris, France †Present address: Helmholtz Zentrum München, Institute for Diabetes and Obesity, Monoclonal Antibody Facility, Neuherberg, Germany ‡These authors contributed equally to this work as first authors ‡These authors contributed equally to this work as senior authors *Corresponding author. Tel: +33 1 57 27 80 74; E-mail: [email protected] *Corresponding author. Tel: +49 89 31871509; E-mail: [email protected] *Corresponding author. Tel: +33 4 67 61 45 14; E-mail: [email protected] EMBO J (2017)36:2726-2741https://doi.org/10.15252/embj.201796541 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 Among other targets, the protein lysine methyltransferase PR-Set7 induces histone H4 lysine 20 monomethylation (H4K20me1), which is the substrate for further methylation by the Suv4-20h methyltransferase. Although these enzymes have been implicated in control of replication origins, the specific contribution of H4K20 methylation to DNA replication remains unclear. Here, we show that H4K20 mutation in mammalian cells, unlike in Drosophila, partially impairs S-phase progression and protects from DNA re-replication induced by stabilization of PR-Set7. Using Epstein–Barr virus-derived episomes, we further demonstrate that conversion of H4K20me1 to higher H4K20me2/3 states by Suv4-20h is not sufficient to define an efficient origin per se, but rather serves as an enhancer for MCM2-7 helicase loading and replication activation at defined origins. Consistent with this, we find that Suv4-20h-mediated H4K20 tri-methylation (H4K20me3) is required to sustain the licensing and activity of a subset of ORCA/LRWD1-associated origins, which ensure proper replication timing of late-replicating heterochromatin domains. Altogether, these results reveal Suv4-20h-mediated H4K20 tri-methylation as a critical determinant in the selection of active replication initiation sites in heterochromatin regions of mammalian genomes. Synopsis H4K20 methylation by subsequent action of PR-Set7 and Suv4-20h methyltransferases does not by itself specify replication origins in mammalian cells, but enhances licensing and timely activation at defined heterochromatin origins. Replacing histone H4 lysine 20 with alanine partially impairs S-phase progression in mammalian cells. PR-Set7-induced DNA re-replication requires appropriate levels of histone H4K20 methylation. H4K20me1-to-H4K20me3 switch enhances pre-replication complex formation and activation only at defined origin sequences. Suv4-20h-mediated H4K20me3 is enriched at a subset of late-firing origins. Timely heterochromatin replication requires H4K20me3-mediated LRWD1/ORCA recruitment at a subset of late-firing origins. Introduction Eukaryotic DNA replication is initiated at thousands of chromosomal sites, known as replication origins, which are scattered along the genome and activated in a well-defined order during S-phase (Rhind & Gilbert, 2013; Renard-Guillet et al, 2014). To ensure that replication origins are activated only once per cell cycle, the initiation of replication is separated into three tightly regulated steps (Remus & Diffley, 2009; Ding & MacAlpine, 2011; Leonard & Méchali, 2013). First, origins are selected by the binding of the hexameric origin recognition complex (ORC), as cells exit mitosis. In a second step termed “replication licensing”, ORC serves as a scaffold for the subsequent association of CDC6 and CDT1, which together coordinate the loading of the MCM2-7 complex on chromatin to form the pre-replication complex (pre-RC) (Siddiqui et al, 2013). Finally, in S-phase, CDK and DDK kinases trigger pre-RC activation, resulting in the association of additional components and establishment of active replication forks (Bell & Botchan, 2013). Once origins have fired, the recruitment of pre-RC components on chromatin is inhibited until the onset of the following mitosis (Arias & Walter, 2007). Importantly, to provide a backup system when cells undergo replication stress, a large excess of origins is licensed during G1-phase and only a fraction of them are transformed in active replication initiation sites during S-phase (Blow et al, 2011). To date, the mechanisms that dictate whether an origin will be active or not in unperturbed S-phase are unclear. Although metazoan replication origins tend to be enriched in GC-rich sequences and G4 quadruplex structures, they do not share a clear consensus sequence (Besnard et al, 2012; Fragkos et al, 2015; Prioleau & MacAlpine, 2016). Chromatin features, such as nucleosome positioning and histone modifications, are also involved in the selection of replication initiation sites (Smith and Aladjem, 2014). Hence, origin activation follows a spatiotemporal program that is highly connected to the regulation of chromatin states, with origins in transcriptionally active chromatin firing earlier than those in repressed chromatin (McGuffee et al, 2013; Pope & Gilbert, 2013; Pope et al, 2014; Fragkos et al, 2015). Typically, early-firing origins are characterized by their proximity to a combination of activating chromatin marks, including acetylation of histone H3 and H4 tails and methylation at lysines (K) 4 and 79 of histone H3 (Picard et al, 2014; Cayrou et al, 2015; Smith et al, 2016). In contrast, little is known about chromatin modifications that control the formation and activity of late origins in heterochromatin regions. Lysine 20 of histone H4 (H4K20) is the major methylated residue on the H4 tail that can exist as mono (H4K20me1)-, di (H4K20me2)-, or tri-methylation (H4K20me3) (Brustel et al, 2011; Jørgensen et al, 2013). While H4K20me2 is the predominant H4K20 methyl state found in 80% of total histone H4, H4K20me1 and H4K20me3 are less abundant and typically enriched in transcriptionally active and silent chromatin, respectively (Beck et al, 2012a). Recently, biochemical analysis performed with histone peptides revealed that H4K20me2 and H4K20me3 might serve as binding sites for mammalian ORC1 and its cofactors (Oda et al, 2010; Vermeulen et al, 2010; Beck et al, 2012b; Kuo et al, 2012), suggesting a potential role of these two histone marks in replication origin selection and activity. Consistent with this hypothesis, artificial recruitment of the H4K20me1 methyltransferase PR-Set7 (also known as SET8, SETD8, and KMT5A) at a specific locus can induce the recruitment of several ORC components in a manner depending on the presence of Suv4-20h1/h2 (also known as KMT5B and KMT5C), the enzymes that catalyze H4K20me2/3 from H4K20me1 (Schotta et al, 2008; Beck et al, 2012b). In addition, PR-Set7 is targeted for proteasome degradation during S-phase by the PCNA-CRL4cdt2 ubiquitin complex and failure to degrade PR-Set7 triggers DNA re-replication in Suv4-20h1-expressing cells (Tardat et al, 2010; Beck et al, 2012b). However, it remains unclear whether these enzymes have a broad role in the regulation of DNA replication origins or restrained to specific genomic regions. Furthermore, several puzzles remain including the fact that loss of Suv4-20h and H4K20me2/3 only leads to a partial reduction in cell growth during mouse development (Schotta et al, 2008). Likewise, H4K20 methylation is not essential for DNA replication initiation in Drosophila cells (McKay et al, 2015; Li et al, 2016) and H4K20 enzymes have also non-histone substrates (Takawa et al, 2012), which also question on the role, if any, of H4K20 methylation in the control of DNA replication origins. In order to clarify these issues, we have investigated further the functions of H4K20 methylation and the associated H4K20 enzymes in the regulation of DNA replication. By expressing a histone H4 mutant unable to undergo methylation on lysine 20, we first provide evidence that unlike Drosophila, H4K20 methylation states are mandatory for proper DNA replication and contribute to DNA re-replication induced by PR-Set7 stabilization in mammalian cells. Although facilitating ORC recruitment on chromatin, we show that the conversion of H4K20me1 to higher H4K20me states is not sufficient to define an efficient origin per se but rather serves as an enhancer for MCM2-7 loading and replication activation at defined origins. In line with this, we reveal that Suv4-20h-mediated H4K20me3 stimulates the binding of ORCA-associated protein LRWD1/ORCA and pre-RC complex at a subset of late-firing origins, which is essential for the timely replication of heterochromatin during late S-phase. Results H4K20 methylation is critical for S-phase progression and PR-Set7 replication licensing functions As shown in human U2OS cells (Appendix Fig S1), loss of mammalian PR-Set7 results in improper S-phase progression, which is in part attributed to defects in replication origin activity (Jørgensen et al, 2007; Tardat et al, 2007). To provide evidence that this S-phase phenotype is directly caused by a decrease in the levels of H4K20 methylation rather than defects in the methylation of other PR-Set7 targets, we examined the cell cycle of human U2OS cells transduced with a high titer of retroviral vectors encoding a FLAG-tagged histone H4K20A mutant carrying a lysine to alanine substitution at position 20 (K20A). Cells transduced with a virus encoding a FLAG-tagged wild-type histone H4WT were used as controls. As shown in Fig 1A and B, immunoblot analysis showed that both FLAG-tagged H4WT and H4K20A proteins were expressed at similar levels and were efficiently incorporated into chromatin 3 days after retroviral infection (Fig 1A, lanes 3 and 6), thereby leading to the replacement of half of the pool of the endogenous histone H4 (Fig 1B, top panel). Incorporation of FLAG-H4K20A mutant into chromatin slightly increased PR-Set7 levels but led to a specific decrease in the global levels of the three methylated H4K20 states, with a stronger reduction for H4K20me1 and H4K20me3 (Fig 1B). Cell-cycle profiles of FLAG-H4WT and FLAG-H4K20A cells were then analyzed the same day by measuring the DNA content according to low (gates 1) and high (gates 2) levels of FLAG-H4 expression (Fig 1C; gates 1 and 2, respectively). We noticed that the amount of cells with the highest levels of H4K20A (i.e., with the strongest decrease in H4K20me) was low compared to FLAG-H4WT cells (Fig 1C, upper panels, compare gates 1 and 2), suggesting that the H4K20A mutation is relatively toxic. Conspicuously, whereas cells with low levels of H4K20A displayed a cell-cycle profile similar to FLAG-H4WT cells (Fig 1C, gates 1), cells expressing high levels of FLAG-H4K20A accumulate in S-phase, suggesting that they fail to complete DNA replication as previously described in PR-Set7-depleted cells (Tardat et al, 2007; Appendix Fig S1). Figure 1. H4K20 mutation affects S-phase progression and prevents DNA re-replication induced by PR-Set7 stabilization Immunoblot analysis of histone H4 and FLAG-tagged histone H4 protein levels in FLAG-H4WT and FLAG-H4K20A U2OS cells and subjected to biochemical fractionation: Cytosolic (S1) and nuclear (S2) are soluble supernatants and P3 is the chromatin-enriched fraction. MEK1 was used as a control of soluble components and HCF-1 protein was used for control of chromatin fraction. Immunoblot analysis of PR-Set7 and the levels of acetylation and methylation of endogenous H4 and FLAG-tagged H4 in FLAG-H4WT and FLAG-H4K20A U2OS cells. FACS analysis of DNA content and FLAG signal in FLAG-H4WT or FLAG-H4K20A cells. DNA content was analyzed according to the low (gate 1) and high (gate 2) levels of FLAG-tagged histone H4 proteins. FACS analysis of DNA content in cells expressing similar levels (gate 1) of FLAG-tagged histone H4WT and H4K20A upon expression of the PR-Set7PIPmut and PR-Set7PIPmut+SETmut mutants. Quantitation of re-replicating parental (No FLAG), FLAG-H4WT, and H4K20A cells upon PR-Set7PIPmut expression. Data are means ± SD, n = 3. (*) Statistical significance with P < 0.05 (t-test). Download figure Download PowerPoint Our results suggest that appropriate H4K20 methylation levels may be required for PR-Set7 functions in the licensing of replication origins. To verify this, we measured by FACS the levels of re-replication induced by the proteolytic-resistant PR-Set7PIPmut mutant in cell populations expressing similar and non-deleterious levels of FLAG-tagged H4WT or FLAG-tagged H4K20A, 3 days before infection with retrovirus encoding PR-Set7PIPmut mutant (Fig 1C, gate 1). PR-Set7PIPmut contains mutations in the PCNA binding domain that inhibit CRL4cdt2-mediated degradation and induce PR-Set7 stabilization (Tardat et al, 2010; Beck et al, 2012b). The resulting re-replication phenotype is caused by repeated PR-Set7-induced replication origin licensing (Tardat et al, 2010), which can be observed by the appearance of cells with DNA content greater than 4N, 2 days after PR-Set7PIPmut expression (Fig 1D, panel a). As shown in Fig 1D, expression of the PR-Set7PIPmut, but not of the catalytic inactive PR-Set7PIPmut+SETmut mutant, caused the appearance of ~30% of re-replicated cells in both parental and FLAG-tagged H4WT cells (Fig 1D, compare panels a–d). In contrast, the level of re-replication upon PR-Set7PIPmut expression was greatly reduced in FLAG-tagged H4K20A cells (Fig 1D, panels e–f) correlating with the reduced amount of H4K20 residues in these cells. These results provide the evidence that H4K20 methylation is indeed a critical downstream effector of PR-Set7 replication licensing functions. H4K20 methylation is an enhancer of origin efficiency K20 methylation enhances histone H4 interaction with ORC in vitro (Beck et al, 2012b), suggesting that this histone mark might be sufficient to induce DNA replication at specific loci in vivo. To test this possibility, we examined the impact of H4K20 methylation on the replication of Epstein–Barr virus-derived (EBV) episomes from origin DNA sequences that involve cellular pre-RC complex for licensing. As illustrated in Fig 2A, the replication and stability of EBV episomes require the binding of the EBV-encoded protein EBNA1 to two DNA elements: (i) the family of repeats (FR) element that ensures the segregation and stability of episomes during cell division and (ii) the dyad symmetry (DS) element that serves as replication origin in human cells by co-recruiting ORC to this DNA sequence (Hammerschmidt & Sugden, 2013). To assess whether PR-Set7-induced H4K20 methylation on chromatin induces origin activity by its own, a GAL4-binding UAS sequence was introduced into a DS-deleted EBV plasmid named FR-UAS, which is incompetent for replication but not for EBN1A-mediated segregation. We then examined the rescue of the replication competence of this plasmid in HEK293 cells stably expressing EBNA1 and either GAL4 DNA-binding domain (GAL4), the wild-type GAL4-PR-Set7WT, or the methylase-deficient GAL4-PR-Set7SETmut fusion proteins (Fig EV1A). HEK293 cell line was chosen because it is suitable for transfection and EBV episome replication (Gerhardt et al, 2006). As observed previously (Beck et al, 2012b), quantitative chromatin immunoprecipitation (ChIP-qPCR) showed that GAL4-PR-Set7 binding at the UAS site, but not of the GAL4 or GAL4-PR-Set7SETmut proteins, led to a local increase in H4K20me1 followed by its conversion to higher H4K20me states, as indicated by the appearance of high H4K20me3 enrichment at this locus (Fig EV1B). To measure episome replication efficiency, low molecular weight DNA was harvested 6 days after plasmid transfection and the number of replicated episomes was quantified by bacterial transformation after DpnI digestion to eliminate the bacterial-derived plasmids initially transfected into GAL4 cell lines. The results are shown in Fig 2B. As controls, similar experiments were performed with the wild-type FR-DS plasmid and the FR-ORIRDH plasmid (Fig 2A), which contains the 300-bp ORC-binding DNA fragment of the human replication origin (ORIRDH) of the RDH gene instead of the DS element (Gerhardt et al, 2006). The results are shown in Fig 2B. Compared to the FR-DS plasmid, the FR-ORIRDH plasmid displayed a low replication competence in all cell lines (Fig 2B), which is representative of the low efficiency of short mammalian origin DNA sequences to define an origin by their own (Gerhardt et al, 2006). We noticed that the replication of the FR-UAS plasmid was almost undetectable in GAL4 and GAL4-PR-Set7SETmut cells but not in GAL4-PR-Set7-expressing cells, where this plasmid exhibited a low replication similar to that of the FR-ORIRDH plasmid (Fig 2B). These results indicate that a local increase in H4K20 methylation, as observed with mammalian DNA sequences, is not sufficient to define a fully competent origin of replication. Figure 2. H4K20 methylation enhances origin formation and activity Schematic representation of EBN1A protein and studied EBV-derived plasmids with the relative position of DNA fragments amplified during ChIP-qPCR experiments. Quantitation of replicating FR-DS, FR-UAS, and FR-ORIRDH plasmids in cells expressing EBNA1 and either GAL4, GAL4-PR-Set7, or GAL4-PR-Set7SETmut. Data are means ± SEM (n = 4) with control FR-DS plasmid arbitrarily set as one in every cell line (gray bars). Quantitation of replicating FR-UAS-ORIRDH and FR-ORIRDH in the same cell lines as above. Data are means ± SEM (n = 4) relative to FR-ORIRDH (black bars). (*) Statistical significance (paired two-tailed t-test) with P < 0.05. ChIP-qPCR analysis at ORIRDH, UAS, and FR sequences in different GAL4 cell lines transfected with FR-ORIRDH or FR-UAS-ORIRDH plasmids using antibodies as indicated. Data are means ± SEM (n = 4) as fold enrichment of the each antibody relative to isotype IgG control. (*) Statistical significance (paired two-tailed t-test) with P < 0.05. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. H4K20 methylation promotes the replication of EBV-derived episomes Expression levels of GAL4, GAL4-PR-Set7, and GAL4-PR-Set7SETmut in EBNA1-expressing HEK293 cell lines. Immunoblot analysis with GAL4 antibody. ChIP-qPCR analysis of the FR-UAS plasmid with anti-GAL4, anti-H4K20me1, and anti-H4K20me3 antibodies. Values are depicted relative to isotype control. Data are means ± SEM (n = 3). Replication efficiency of FR-DS and FR-UAS-DS plasmids in GAL4, GAL4-PR-Set7, and Gal4-PRset7SETmut cells. Replication efficiencies are depicted relative to the FR-DS plasmid arbitrarily set as 100% in every cell line. Data are means ± SEM (n = 4). ChIP-qPCR analysis at the FR, UAS, and DS sites with anti-GAL4, anti-MCM3, anti-H4K20me1, and anti-H4K20me3 using chromatin from GAL4, GAL4-PR-Set7, and GAL4-PR-Set7SETmut cells expressing EBNA1 and transfected with similar amounts of FR-DS or FR-UAS-DS plasmids. The y-axis represents the relative fold enrichment of the specific antibody versus isotype control. Data are means ± SEM (n = 5). Download figure Download PowerPoint Since origin efficiency might depend on the presence of multiple origin determinants (Méchali et al, 2013; Valton et al, 2014), we next examined the effect of inducing H4K20 methylation in the vicinity of the ORIRDH origin. For this, we created the FR-UAS-ORIRDH plasmid (Fig 2A) and tested the replication competence of this plasmid as described above. As shown in Fig 2D, the ORIRDH efficiency in FR-UAS-ORIRDH plasmid was fourfold increased in GAL4-PR-Set7 cells, whereas it remained unchanged in GAL4 and GAL4-PRSet7SETmut cells (Fig 2C). ChIP-qPCR analysis showed that this stimulation of ORIRDH activity GAL4-PR-Set7 cells was associated with an accumulation of H4K20me1/me3 and a higher efficiency of MCM2-7 loading, as measured by the significant increase in the levels of MCM3 at the UAS and ORIRDH sequences in FR-UAS-ORIRDH plasmid (Fig 2D). Importantly, the same enhancer effect on MCM2-7 loading and origin activity was also observed for the viral origin sequence oriP in a FR-UAS-DS plasmid transfected in GAL4-PR-Set7-expressing cells (Fig EV1B and C), confirming the ability of PR-Set7-induced H4K20 methylation to stimulate replication initiation at defined origins. Taken together, these results show that PR-Set7-induced H4K20 methylation is likely not sufficient to promote efficient origin function but rather serves to strengthen the activity of defined origins by enhancing the recruitment and/or stability of pre-RC complexes at these origins. PR-Set7-mediated stimulation of origin activity depends on the further methylation of H4K20me1 by Suv4-20h1/h2 Since the ORC shows a higher affinity for H4K20me2/3 than for H4K20me1 in vitro (Oda et al, 2010; Vermeulen et al, 2010; Kuo et al, 2012), we asked whether the stimulatory effect of H4K20 methylation on origin activity depends on the switch from H4K20me1 to higher H4K20me states induced by Suv4-20h1/h2 methyltransferases. To address this question in vivo, we repeated the replication assay with FR-ORIRDH and FR-UAS-ORIRDH plasmids in GAL4-PR-Set7 cells in the presence of A-196, a chemical inhibitor of Suv4-20h1/h2. Immunoblot analysis of whole-cell extracts confirmed the specificity of the A-196 treatment, which led to a strong reduction in the levels of H4K20me2/3 and the subsequent increase in H4K20me1 without an impact on the levels of histone H3 tri-methylation at lysines 4, 9, and 27 (Appendix Fig S2). Consistent with these results, ChIP-qPCR analysis showed that H4K20me1, but not H4K20me3, was highly enriched at the UAS and ORIRDH sequences in GAL4-PR-Set7-expressing cells treated with A-196 (Fig 3A). In this experimental context, the FR-UAS-ORIRDH and FR-ORIRDH plasmids displayed a similar replication activity without any significant difference in the levels of MCM3 loading at the ORIRDH origin (Fig 3B and C). These results confirm that PR-Set7-mediated H4K20me1 is only a prerequisite for replication origin stimulation (Beck et al, 2012b), which depends on additional methylation of H4K20me1 by Suv4-20h enzymes. Figure 3. PR-Set7 improves origin activity through Suv4-20h activity on H4K20me1 ChIP-qPCR analysis of H4K20me1 and H4K20me3 levels at ORIRDH, UAS, and FR in the HEK293 EBNA1+ Gal4-PR-Set7-expressing cell line in the absence (left panel) and presence of the Suv4-20h inhibitor A196 (right panels). Data are means ± SEM (n = 4). ChIP-qPCR analysis of MCM3 levels at ORIRDH, UAS, and FR in EBNA1/Gal4-PR-Set7-expressing cell lines in the absence (left panel) and presence of A196 (right panel). Data are means ± SEM (n = 4). Quantitation of replicating FR-ORIRDH (black) and FR-UAS-ORIRDH (striped) plasmids in EBNA1/GAL4-PR-Set7-expressing cells in the presence and absence of A196. The replication efficiency is relative to ORIRDH in untreated cells. Data are means ± SEM (n = 4). Data information: Significance was determined employing an unpaired two-tailed t-test. *P < 0.05. Download figure Download PowerPoint Loss of Suv4-20h activity on H4K20me1 specifically impairs heterochromatin replication timing We next sought to determine whether the ability of Suv4-20h and higher H4K20 methylation states to enhance replication origin activity plays an essential role in the replication of all the genome or only at specific chromatin regions. To address this question, we examined at genome-wide levels the spatiotemporal replication program of mouse embryonic fibroblast (MEF364.2) line derived from Suv4-20h2−/−, Suv4-20h1−/flox, Cre-ER embryos and in which the remaining knoc" @default.
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• W2745177530 title "Histone H4K20 tri‐methylation at late‐firing origins ensures timely heterochromatin replication" @default.
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• W2745177530 doi "https://doi.org/10.15252/embj.201796541" @default.
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