© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 417-429 doi:10.1242/jcs.178889

RESEARCH ARTICLE Orc5 induces large-scale chromatin decondensation in a GCN5-dependent manner Sumanprava Giri, Arindam Chakraborty, Kizhakke M. Sathyan, Kannanganattu V. Prasanth and Supriya G. Prasanth*

ABSTRACT the efficiency of origin usage and firing (Brown et al., 1991; In eukaryotes, origin recognition complex (ORC) establish Ferguson and Fangman, 1992; Simpson, 1990; Stevenson and the pre-replicative complex (preRC) at the origins, and this is Gottschling, 1999). Histone acetylation is known to play a key role in Drosophila essential for the initiation of DNA replication. Open chromatin the regulation of origins of DNA replication in yeast and , structures regulate the efficiency of preRC formation and replication and there is accumulating evidence that the deacetylation of histones initiation. However, the molecular mechanisms that control chromatin negatively affects origin activity (Aggarwal and Calvi, 2004; Groth structure, and how the preRC components establish themselves on et al., 2007a; Knott et al., 2009b; Unnikrishnan et al., 2010; the chromatin remain to be understood. In human cells, the ORC is a Vogelauer et al., 2002). Furthermore, the replication timing of β highly dynamic complex with many separate functions attributed to the -globin domain in human cells is also modulated by sub-complexes or individual subunits of the ORC, including histone modifications at the origin (Goren et al., 2008). There is heterochromatin organization, telomere and centromere function, accumulating evidence that histone acetyl transferases act as positive Drosophila centrosome duplication and cytokinesis. We demonstrate that human regulators of replication origins in yeast and as well as Orc5, unlike other ORC subunits, when ectopically tethered to a human cells (Groth et al., 2007b; Knott et al., 2009a). In yeast, chromatin locus, induces large-scale chromatin decondensation, GCN5p (also known as KAT2A in humans), a histone acetyl predominantly during G1 phase of the cell cycle. Orc5 associates transferase (HAT), has been found to positively stimulate DNA with the H3 histone acetyl transferase GCN5 (also known as KAT2A), replication by negating the inhibitory effect of the histone and this association enhances the chromatin-opening function of deacetylases (Espinosa et al., 2010; Vogelauer et al., 2002). Orc5. In the absence of Orc5, histone H3 acetylation is decreased at Further, Hat1p and its partner Hat2p interact with the ORC (Suter Drosophila the origins. We propose that the ability of Orc5 to induce chromatin et al., 2007). In , the HATs Chameau (Chm) and CBP unfolding during G1 allows the establishment of the preRC at the (Nejire) stimulate origin activity (Aggarwal and Calvi, 2004; origins. McConnell et al., 2012). In human cells, HBO1 (also known as KAT7), another HAT, associates with ORC and is required for the KEY WORDS: Orc5, GCN5, Chromatin decondensation, Acetylation, loading of the minichromosome maintenance complex (MCM) onto Origins chromatin and for replication fork progression (Iizuka et al., 2006; Iizuka and Stillman, 1999; Miotto and Struhl, 2008, 2010). A recent INTRODUCTION study has pointed out that the acetylation of some histone lysine In eukaryotes, the initiation of DNA replication requires the residues depends on the binding of ORC to the origin and that the coordinated action of a multiprotein pre-replication complex acetylation is at its maximum on the nucleosomes adjacent to one (preRC) at the origins (Bell and Dutta, 2002). The origin side of the major initiation site (Liu et al., 2012). How the ORC recognition complex (ORC), a six-subunit complex, binds to regulates such chromatin modifications and how the chromatin replication origins during the G1 phase of the cell cycle, and this is structure at origins is organized remain to be defined. followed by a sequential assembly of other preRC components The ORC comprises six subunits, and in human cells they are (Bell and Stillman, 1992). In addition to their role in replication highly dynamic. The largest subunit, Orc1 is degraded at the end of initiation, ORC subunits contribute to other cellular processes G1, and rebinding of the to chromatin is an obligatory step including transcriptional silencing, heterochromatin organization, for the establishment of the preRC in G1 (Méndez et al., 2002). The sister chromatid cohesion, centrosome duplication, telomere smallest subunit of ORC, Orc6, binds to the ORC in a transient maintenance and cytokinesis (Sasaki and Gilbert, 2007). manner and also has independent roles in cytokinesis (Bernal and Open chromatin structures are known to regulate the efficiency of Venkitaraman, 2011; Prasanth et al., 2002). Orc2, Orc3, Orc4 and preRC formation, thereby facilitating replication initiation (Papior Orc5 in human cells constitute the core ORC and are associated with et al., 2012). However, the molecular mechanisms that affect each other throughout the cell cycle (Dhar et al., 2001; Vashee et al., chromatin structure and how the preRC components establish 2001). Orc1, Orc4 and Orc5 are members of the AAA+ family of themselves on the chromatin remain to be understood. The ATPases and contain consensus motifs. Mutations in the ATP- accessibility of the replication factors is influenced by the binding sites on Orc4 and Orc5 impair complex assembly, whereas chromatin structure, and the chromatin architecture dictates the ATP binding of Orc1 is dispensable (Ranjan and Gossen, 2006; Siddiqui and Stillman, 2007). Orc2 and Orc3 also have a structure Department of Cell and Developmental Biology, University of Illinois at Urbana- that is common to AAA+ proteins, but they do not possess a Champaign, 601S Goodwin Avenue, Urbana, IL 61801, USA. consensus ATP-binding motif. *Author for correspondence ([email protected]) Multiple subunits of human ORC – including Orc1, Orc2, Orc3 and Orc5 – and the protein ORC-associated (ORCA) have roles in

Received 10 August 2015; Accepted 27 November 2015 heterochromatin organization (Giri et al., 2015; Prasanth et al., Journal of Cell Science

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2010; Shen et al., 2010). In yeast, Orc5 has distinguishable were generated (Fig. 2A), and their ability to cause chromatin functions in replication initiation and silencing (Dillin and Rine, unfolding was examined (Fig. 2B). As described earlier, full-length 1997). Further, in Drosophila and humans, the loss of multiple ORC YFP–LacI–Orc5 caused decondensation of 81% of the CLTon locus subunits leads to segregation defects (Pflumm and (Fig. 2C). The Orc5 truncation mutants 1–100, 101–200, 201–300 Botchan, 2001; Prasanth et al., 2004b). In this manuscript, we report and 301–400 failed to mediate chromatin unfolding, but the that Orc5 has a distinct function in chromatin unfolding. Ectopic N-terminal truncation 301–435 mutant resulted in chromatin tethering of Orc5 to a chromatin locus leads to dramatic chromatin unfolding in ∼40% of cells (Fig. 2B,C). The decondensation decondensation, predominantly during G1 phase of the cell cycle. upon tethering YFP–LacI–Orc5 (301–400) was in the range 0.5– This chromatin-opening role of Orc5 requires the activity of the 1.0 µm2, whereas tethering of YFP–LacI–Orc5 (301–435aa) HAT GCN5. We propose that the Orc5 subunit of the ORC plays a resulted in decondensation in the range 2.5–19.0 µm2 (Fig. 2D). key role in mediating large-scale chromatin-opening during G1 that, Our results indicate that the last 35 amino acid residues at the in turn, facilitates the loading of other preRC components onto the C-terminus of Orc5 are crucial for its chromatin decondensation origins. function. Upon examination of the C-terminal 400–435 residues, we found RESULTS that it was enriched with acidic residues (Fig. S1A). It has Ectopic tethering of Orc5 induces large-scale chromatin previously been reported that targeting of acidic activators to decondensation heterochromatic chromatin domains can cause large-scale To investigate the chromatin changes that occur when preRC chromatin decondensation (Carpenter et al., 2005). We generated proteins, including ORC proteins, bind to origins, we tethered a mutant of Orc5 where multiple aspartic acid residues were individual subunits of the ORC to a heterochromatic locus using an replaced by alanine residues (Fig. S1A). However, this mutant, in vivo human U2OS osteosarcoma cell system (CLTon) (Fig. 1A). when tethered to the locus, showed similar levels of chromatin This reporter carries a stably integrated 200-copy transgene array decondensation, suggesting that the ‘acidic domain’ within Orc5 is with lac operator repeats, and this heterochromatic locus is not required for decondensation (Fig. S1B). We next determined visualized through the stable expression of Cherry–lac-repressor whether the ATP-binding ability of Orc5 is required for its (Cherry–LacI). Upon transcriptional activation of this reporter chromatin-unfolding function. We generated Walker A mutant locus, through the addition of doxycycline, this locus shows (K43A) and an arginine finger mutant (R166A) (Fig. S1A), and chromatin decondensation (Janicki et al., 2004; Shen et al., 2010). tethered these to the CLTon locus (Fig. S1C). The extent of We generated triple-fusion proteins of YFP–LacI–ORCs, and these chromatin decondensation upon tethering these mutants was were tethered to the CLTon locus. Targeting YFP–LacI to this locus comparable to that of the wild-type Orc5, suggesting that the showed association of LacI with the heterochromatic CLTon locus ATP-binding ability of Orc5 is also dispensable for its chromatin (Fig. 1Ba). Surprisingly, tethering of YFP–LacI–Orc5 caused decondensation function. dramatic decondensation at the CLTon locus, whereas none of the other ORC subunits, including Orc1, Orc2, Orc3, Orc4 and Orc6, Orc5 associates with the histone acetyl transferase GCN5 caused any changes to the chromatin architecture at the locus Histone acetylation, catalyzed by various HATs, is linked with the (Fig. 1Ba). 81% of YFP–LacI–Orc5-tethered cells showed open chromatin state and is known to facilitate transcription decondensation of the heterochromatic locus (Fig. 1Bb). (Narlikar et al., 2002). Because Orc5 was found to induce Furthermore, the extent of decondensation upon tethering Orc5 to chromatin decondensation, we asked whether it achieves this the locus was determined by calculating the area of the decondensed through its association with known HATs. We transfected T7–Orc5 chromatin. Measurement of the area of decondensation upon into U2OS cells and performed immunoprecipitation with an tethering Orc5 revealed a range of chromatin decondensation, antibody against T7 (Fig. 3A). We found that T7–Orc5 interacted ranging 2–35 µm2 (Fig. 1Bc), whereas the control YFP–LacI cells strongly with endogenous GCN5 (Fig. 3A). Next, we co-transfected showed condensed loci with sizes in the range 0.2–1.3 µm2 T7–Orc5 and Flag–GCN5, and performed immunoprecipitation (Fig. 1Bc). The average area of the U2OS nuclei was found to be with an antibody against Flag. Orc5 and GCN5 were found to 360±101 µm2 (n=52 cells). Based on the area of decondensation, we interact with one another (Fig. 3B). A reverse immunoprecipitation categorized the Orc5-mediated decondensation phenotype into experiment recapitulated the interaction (Fig. 3E, full-length T7– three categories: medium (2–6µm2), large (6–10 µm2) and very Orc5 lane). Further, tethering of GCN5 to the CLTon locus also large (10–35 µm2) (Fig. 1C). The tethering of Orc5 to the locus showed strong recruitment of Orc5 to the site, corroborating the resulted in 37%, 34% and 29% of cells showing medium, large and immunoblot results (Fig. 3C). very large ranges of decondensation, respectively. We next mapped the region within Orc5 that associates with We investigated the role of Orc5 in chromatin decondensation by GCN5. We generated several truncation mutants of Orc5 (Fig. 3D) utilizing another system, in this case a CHO-derived A03 cell line and co-transfected each of these with Flag–GCN5. that contains 90 Mb of a homogenously staining region generated Immunoprecipitation with the antibody against T7 revealed that through stable integration and amplification of the LacO-DHFR full-length Orc5 and the 100–435 fragment efficiently associated vector (Li et al., 1998). Tethering Orc5 to the A03 locus also with GCN5 (Fig. 3E), suggesting that the first 100 amino acids resulted in dramatic decondensation of this locus (Fig. 1D). The within the N-terminus of Orc5 are dispensable for GCN5 binding. decondensation upon tethering of Orc5 was in the range 4.5– Orc2 was found to bind to full-length Orc5 and to fragments 100– 27 µm2, whereas tethering of YFP–LacI resulted in decondensation 435 and 300–435 (Fig. 3D,E). These results suggest that Orc2 and in the range 0.6–1.2 µm2. GCN5 could associate with Orc5 simultaneously and that the We next determined the minimum domain of Orc5 that is required binding might not be mutually exclusive. Because the T7–Orc5 for its ability to mediate chromatin decondensation. Triple-fusion 100–435 mutant could bind to GCN5, we tested the ability of this Orc5 truncation mutants (including fusions comprising amino acid mutant to cause chromatin decondensation. We tethered it to the residues 1–100, 101–200, 201–300, 301–400 and 301–435 of Orc5) CLTon locus (Fig. 3Fa) and found that it could cause chromatin Journal of Cell Science

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Fig. 1. Orc5 causes chromatin decondensation. (A) Schematic of the heterochromatic locus in U2OS 2-6-3 CLTon cells. The copy numbers for the indicated regions are shown. (Ba) Chromatin decondensation upon tethering YFP–LacI and the indicated YFP–LacI-tagged ORC proteins to the heterochromatic locus of CLTon cells. Insets represent 200% magnifications of the boxed regions. Scale bar: 10 µm. (Bb) The percentage of cells with open loci upon tethering YFP–LacI or the indicated YFP–LacI-tagged ORC proteins to the heterochromatic locus of CLTon cells. Error bars represent s.d., n=3 independent experiments (100 loci examined upon tethering each construct). ***P<0.001 (Student’s t-test). (Bc) Area of heterochromatic loci upon tethering YFP–LacI and YFP–LacI–Orc5. Error bars represent s.d., n=3 independent experiments (area of 25–50 loci measured upon tethering each construct). ****P<0.0001 (Student’s t-test). Lines represent the median, and the boxes represent the 25–75th percentiles. (C) Chromatin decondensation upon tethering YFP–LacI and YFP–LacI–Orc5 to the heterochromatic locus of CLTon cells. Insets represent 200% magnifications of the boxed regions. Scale bar: 10 µm. (D) Chromatin decondensation upon tethering YFP–LacI and the – indicated YFP LacI-tagged Orc proteins to the heterochromatic locus of AO3 cells. Insets represent 200% magnifications of the boxed regions. Scale bar: 10 µm. Journal of Cell Science

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Fig. 2. The last 35 amino acids of Orc5 are necessary for its function in chromatin decondensation. (A) Schematic representation of various truncation mutants of Orc5 containing a T7-epitope on the N-terminus. The specific domains that can cause decondensation are indicated with ‘+’. AAs, amino acid residues. (B) Chromatin decondensation upon tethering YFP–LacI and various truncation mutants of YFP–LacI–Orc5 to the heterochromatic locus of CLTon cells. Insets represent 200% magnifications of the boxed regions. Scale bar: 10 µm. (C) The percentage of cells with open loci upon tethering either YFP–LacI or various truncation mutants of YFP–LacI–Orc5 to the heterochromatic locus of CLTon cells. Error bars represent s.d., n=3 independent experiments (100 loci examined upon tethering each construct). **P<0.01, ***P<0.001 (Student’s t-test). The YFP–LacI and YFP–LacI–Orc5 data from Fig. 1Bb has been replotted here for ease of comparison with the YFP–LacI-tagged Orc5 mutants. (D) Area of heterochromatic loci upon tethering YFP–LacI, YFP–LacI–Orc5 and YFP–LacI–GCN5. Error bars represent s.d., n=3 independent experiments (areas of 25–50 loci measured upon tethering each construct). ***P<0.001, ****P<0.0001 (Student’s t-test). Lines represent the median, and the boxes represent the 25–75th percentiles. The YFP–LacI and YFP–LacI–Orc5 data from – Fig. 1Bc has been replotted here for ease of comparison with the YFP LacI-tagged Orc5 mutants. Journal of Cell Science

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Fig. 3. Orc5 interacts with GCN5. (A) Semi-endogenous immunoprecipitation (IP) in U2OS cells expressing T7–Orc5 and analysis by immunoblotting for T7 and GCN5. (B) Immunoprecipitation in U2OS cells expressing T7–Orc5 and Flag–GCN5 by using an anti-Flag antibody and analysis by immunoblotting for T7 and Flag. (C) Tethering of YFP–LacI–GCN5 to the CLTon locus shows recruitment of Orc5 to the site. Insets represent 200% magnifications of the boxed regions. Scale bar: 10 μm. (D) Schematic representation of various truncation mutants of Orc5 containing a T7-epitope on the N-terminus. The specific domains that can associate with GCN5 and Orc2 based on immunoprecipitation and immunoblotting analysis (E) are depicted as ‘+’. (E) Immunoprecipitation from U2OS cells expressing various T7-tagged Orc5 mutants and Flag–GCN5 using an antibody against T7 and immunoblotting for T7, Flag and Orc2. (Fa) Chromatin decondensation upon tethering YFP–LacI–Orc5 (100–435aa) to the heterochromatic locus of CLTon cells. Insets represent 200% magnifications of the boxed regions. Scale bar: 10 µm. (Fb) Area of heterochromatic loci upon tethering YFP–LacI, YFP–LacI–Orc5(100-435aa) and full-length YFP–LacI–Orc5. Error bars represent s.d., n=3 independent experiments (areas of 25–50 loci measured upon tethering each construct). ****P<0.0001 (Student’s t-test). Lines represent the median, and the boxes represent the 25–75th – – – – – –

percentiles. The YFP LacI and YFP LacI Orc5 data from Fig. 1Bc has been replotted here for ease of comparison with the YFP LacI Orc5(100 435aa) mutant. Journal of Cell Science

421 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 417-429 doi:10.1242/jcs.178889 decondensation comparable to that of the full-length protein and PCNA. MCM and PCNA show distinct temporal patterns of (Fig. 3Fb). It is interesting to note that the fragment of chromatin loading (Prasanth et al., 2004a). MCM, the replication Orc5 comprising amino acids 300–435 did not interact with Flag– , loads onto chromatin in G1, shows spatio-temporal GCN5 (Fig. 3E), but did cause partial chromatin decondensation patterns during S phase and is gradually lost by the end of S (Fig. 2B–D). phase, whereas PCNA, the clamp, is associated with chromatin only We next examined the status of various chromatin marks at the during S phase. Based on the patterns of MCM and PCNA loading, Orc5-tethered locus. Immunofluorescence analysis using an it is possible to discern whether the cell is in G1 or S phase of the antibody against trimethylated histone 3 (H3) at residue K9 cell cycle (G1 cells are MCM+ and PCNA−, whereas S-phase cells (H3K9me3) showed robust accumulation of this mark at the LacI- are PCNA+). We observed that the YFP–LacI–Orc5-expressing containing heterochromatic locus (Fig. S3A). However, upon cells with the most dramatic chromatin decondensation at the tethering Orc5 to the locus, H3K9me3 was distinctly devoid at CLTon locus were positive for MCM staining (Fig. 5Aa). At the these sites (Fig. S3A). Because Orc5 associates with GCN5, we same time, YFP–LacI–Orc5-tethered cells in S phase (PCNA+ examined whether H3 acetylation accumulated in Orc5-tethered cells) showed a lower degree of chromatin decondensation cells. We did not observe robust accumulation of acetylated H3 (Fig. 5Ab, inset 3). The most dramatic decondensation of the marks in the highly decondensed Orc5-tethered cells; however, the CLTon loci happened in MCM+ PCNA− cells (Fig. 5Ab, inset1 and control cells were clearly devoid of this mark at the condensed 2, and Fig. 5Ac). These results suggest that the Orc5-mediated CLTon locus (Fig. S3Ba–Bc). decondensation occurs predominantly during G1 phase of the cell We then evaluated whether GCN5 could itself cause chromatin cycle. decondensation in our CLTon assay. YFP–LacI–GCN5 was Because we observed a decrease in the extent of Orc5-mediated tethered to the locus, and the status of chromatin architecture at chromatin decondensation in cells that lacked GCN5, we examined the CLTon locus was evaluated (Fig. 4Aa). GCN5 could also if this was due to an effect of cell cycle perturbation. We evaluated mediate chromatin decondensation; however, this decondensation the cell cycle profile of control and GCN5-depleted cells with flow looked visually different from that observed for Orc5. Tethering cytometry (Fig. S2Aa,Ab) and observed a marginal increase in YFP–LacI–Orc5 caused considerable large-scale decondensation, the G1 population of cells upon loss of GCN5. Because the whereas tethering YFP–LacI–GCN5 caused a smaller scale ‘puffy’ Orc5-mediated chromatin decondensation is most dramatic in G1, chromatin appearance, but still significant decondensation when the loss of the decondensation in the absence of GCN5 is not due to compared to that of YFP–LacI control (Fig. 4Aa). Although the perturbation of cell cycle progression. We propose that GCN5 and range of chromatin decondensation upon tethering YFP–LacI–Orc5 Orc5 cooperate in mediating this chromatin decondensation. varied from 2 to 35 µm2, the extent was much smaller for YFP– Chromatin changes are related to the efficiency of DNA LacI–GCN5, which showed decondensation in the range 3–9.5 µm2 replication. We therefore profiled the replication timing of the (Fig. 4Ab). locus upon tethering YFP–LacI–Orc5. To determine the stage of S phase during which the locus replicates, we performed Orc5-mediated chromatin decondensation is GCN5- immunofluorescence analysis of PCNA. PCNA shows distinct dependent localization patterns during S phase, and based on the PCNA To gain an insight into the functional relevance of the Orc5 and patterns, S phase can be categorized into early, mid or late stages GCN5 interaction, we evaluated whether GCN5 is required for the (O’Keefe et al., 1992). We tethered YFP–LacI or YFP–LacI–Orc5 Orc5-mediated chromatin decondensation. We depleted GCN5 to the locus and examined the accumulation of PCNA at the locus using small interfering (si)RNA in CLTon cells (Fig. 4B) and (Fig. 5Ba,Bb, Fig. S4). The replication timing of the CLTon locus examined whether tethering of Orc5 could still induce chromatin was discerned by examining the PCNA pattern of the cell nucleus. decondensation. Remarkably, in GCN5-depleted cells, we observed Upon tethering YFP–LacI, the locus did not show any accumulation a significant decrease (32% decrease, **P<0.01, Student’s t-test) in of PCNA in early S-phase (Fig. 5Ba). In late S-phase, we could the extent of Orc5-mediated chromatin decondensation (Fig. 4C–E). observe robust accumulation of PCNA at the locus, indicating In control cells, 84.3% of the cells showed chromatin that the YFP–LacI-labeled locus replicated in late S-phase decondensation upon tethering Orc5. By contrast, this number (Fig. 5Ba,Bb). This wasn’t surprising owing to the inherent reduced to 52% upon loss of GCN5 (Fig. 4C). To better understand heterochromatic nature of the locus. Next, we tethered YFP–LacI– this reduction in Orc5-mediated chromatin decondensation, we Orc5 to the locus and examined the replication timing of the locus scored CLTon cells based on medium or large decondensation of the (Fig. 5Bb; Fig. S4). We could observe robust accumulation of heterochromatic locus. Upon loss of GCN5, there was a striking PCNA at the locus preferentially in early S-phase and not in late S- reduction in the percentage of cells showing large decondensation phase, indicating that the replication timing of the locus shifted to (from 44% in control cells to 16% in GCN5-knockdown cells) early S-phase upon tethering of Orc5. Note that even the few (Fig. 4D). This result was corroborated by examining the area of the dramatically decondensed Orc5-tethered loci that we observed loci in control and GCN5-depleted cells. The area of the CLTon during S phase replicated early (Fig. S4). locus varied from 2 to 35 µm2 in control cells and 1 to 7 µm2 in GCN5-depleted cells (Fig. 4E). Loss of Orc5 causes a reduction in histone acetylation at origins Orc5-mediated chromatin decondensation occurs Histone acetylation is required for origin activation during S phase predominantly during G1 phase (Unnikrishnan et al., 2010), and it has also been shown to regulate We next determined whether the Orc5-mediated chromatin the timing of replication origin firing (Vogelauer et al., 2002). We decondensation occurred throughout the cell cycle or was asked whether Orc5-mediated chromatin-opening facilitates histone restricted to a specific stage of the cell cycle. To investigate this, acetylation in cooperation with GCN5 at the origins of replication. we tethered YFP–LacI–Orc5 to the CLTon locus and performed We conducted acetylated H3 chromatin immunoprecipitation (ChIP) double fluorescence labeling of MCM (through labeling of MCM3) in control and Orc5-depleted cells (Fig. 6A), and found a significant Journal of Cell Science

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Fig. 4. Orc5 causes decondensation in a GCN5-dependent manner. (Aa) Chromatin decondensation upon tethering YFP–LacI, YFP–LacI–Orc5 and YFP–LacI–GCN5 to the heterochromatic locus of CLTon cells. Insets represent 200% magnifications of the boxed regions. Scale bar: 10 µm. (Ab) Area of heterochromatic loci upon tethering YFP–LacI, YFP–LacI–Orc5 and YFP–LacI–GCN5. Error bars represent s.d., n=3 independent experiments (areas of 25–50 loci measured upon tethering each construct). ****P<0.0001 (Student’s t-test). The YFP–LacI and YFP–LacI–Orc5 data in Fig. 1Bc has been replotted here for ease of comparison with that for YFP–LacI–GCN5. (B) Immunoblot showing efficient siRNA-mediated knockdown of GCN5. (C) The percentage of YFP–LacI–Orc5-tethered cells with open loci in control and GCN5-knockdown (GCN5 si) cells. Error bars represent s.d., n=3 independent experiments (100 loci examined upon tethering each construct). **P<0.01 (Student’s t-test). (D) Extent of decondensation upon tethering YFP–LacI–Orc5 in control and GCN5-knockdown cells. Error bars represent s.d., n=3 independent experiments (100 loci examined upon tethering each construct). (E) Area of heterochromatic loci upon tethering YFP–LacI and YFP–LacI–Orc5 (in control and GCN5-knockdown cells). Error bars represent s.d., n=3 independent experiments (area of 25–50 loci measured upon tethering each construct). ****P<0.0001 (Student’s t-test). Lines represent the median, and the boxes represent the 25–75th percentiles. The YFP–LacI and YFP–LacI–Orc5 data in Fig. 1Bc has been replotted here for ease of comparison with the decondensation caused by YFP–LacI–Orc5 upon GCN5 knockdown. Journal of Cell Science

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Fig. 5. Orc5 causes maximal chromatin decondensation in G1 and early replication upon tethering to heterochromatic locus. (Aa) Immunofluorescent labeling of MCM3 upon tethering CFP–LacI–Orc5 to heterochromatic loci in CLTon cells. Scale bar: 10 µm. (Ab) Immunofluorescent labeling of PCNA and MCM3 upon tethering CFP–LacI–Orc5 to heterochromatic loci in CLTon cells. Scale bar: 10 µm. Insets represent 200% magnifications of the boxed regions. (Ac) Percentage of CLTon loci showing dramatic decondensation upon tethering YFP–LacI–Orc5 in cells that are PCNA+ or MCM3+. Error bars represent s.d., n=3 independent experiments (50 PCNA+ and 50–100 MCM3+ cells used for analysis). (Ba) PCNA immunofluorescence upon tethering YFP–LacI to CLTon loci. Scale bar: 10 µm. Note the accumulation of PCNA at the locus in late S-phase. (Bb) Percentage of YFP–LacI or YFP–LacI–Orc5-tethered CLTon loci replicating in

early and late S-phase. Error bars represent s.d., n=3 independent experiments (100 cells used for analysis). Journal of Cell Science

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Fig. 6. Orc5 regulates histone acetylation in a subset of origins. (A) Immunoblot showing efficient siRNA-mediated knockdown of Orc5 (Orc5 si). (B) Acetylated H3 ChIP analysis at lamin B1, MCM5 and lamin B2 gene origins and non-origins in U2OS cells after transfection with control siRNA or Orc5- knockdown. Error bars represent s.d., n=3. *P<0.1, **P<0.01 (Student’s t-test). (C) Percentage of cells that were PCNA+, or MCM3+ and PCNA− in U2OS cells after transfection with control siRNA or Orc5-knockdown. Error bars represent s.d., n=3 independent experiments (1000 cells used for analysis). (D) Cartoon depicting Orc5-mediated chromatin opening. H3Ac, acetylated H3; S, S phase. Journal of Cell Science

425 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 417-429 doi:10.1242/jcs.178889 reduction in the acetylation of H3 at select origins – including those Hbo1 is an H4-specific HAT that interacts with the human ORC of lamin B1, MCM5 and lamin B2 – but not at distal sites (either and MCM proteins (Iizuka and Stillman, 1999). It is required for upstream or downstream) of the specific origins (Fig. 6Ba–Bd) or replication licensing (Iizuka et al., 2006) and is known to associate other origins – including those of AKNA, TRPM1, MCM6 and with replication origins (Miotto and Struhl, 2008). It has recently MCM3 (data not shown). We next wanted to determine whether the been demonstrated that Hbo1-mediated hyperacetylation of H4 reduced acetylation of origins causes inefficient origin firing and increases Mcm2-7 loading onto chromatin (Miotto and Struhl, replication defects in S phase. Therefore, we depleted Orc5 and 2010). It is generally believed that histone acetylation loosens up the performed double immunofluorescence staining of MCM and compacted chromatin, thereby increasing the accessibility for PCNA to examine the cell cycle profile upon loss of Orc5 (Fig. 6C). loading of the MCM helicase complex, or acts as a molecular tag We observed a 21% reduction in S-phase cells (PCNA+ cells) from to which the helicase is tethered (Chadha and Blow, 2010). Hbo1 58 to 37%, and a concomitant increase in G1 (MCM+ PCNA− association to origins is dependent on Cdt1, and it has been shown cells) from 28% to 46% upon loss of Orc5, indicating that cells that Cdt1 can modulate chromatin accessibility through temporal accumulated in G1 in the absence of Orc5. recruitment of Hbo1 to origins (Miotto and Struhl, 2008). Our results suggest that Orc5 might make the chromatin more Interestingly, ectopic tethering of Cdt1 is also known to induce accessible for the establishment of preRCs during G1 at specific large-scale chromatin unfolding at a transgene array (Wong et al., origins (Fig. 6D), and this in turn is important for efficient origin 2010). It is well established that the origins comprise open firing and DNA replication in S phase. Our results provide new chromatin during G1 and then become less accessible as cells exit insights into the specific role of one of the ORC subunits in out of G1 phase (Djeliova et al., 2002; Pemov et al., 1998). The facilitating chromatin opening for the establishment of preRCs. replication origins are known to exhibit temporal dynamics in chromatin structure, with a highly open structure during G1 and DISCUSSION more closed architecture during S phase. Elegant work, using a Replication of DNA occurs once and only once per cell division quantitative-PCR-based approach on DNase-1-treated chromatin cycle. The licensing of replication origins requires the sequential samples, has shown that endogenous replication origins, including binding of the ORC, and Cdt1 that is then needed for the those of MCM4 and lamin, display a more open chromatin structure loading of the Mcm2-7 complex onto the chromatin (Bell and Dutta, during G1 than in S phase (Wong et al., 2010). 2002). ORC, comprising six subunits, serves as the landing pad In addition, tethering of replication protein Cdc45 leads to for the assembly of this multiprotein complex at the origins of chromatin decondensation in a Cdk2-dependent manner replication. The contribution of individual subunits in this process (Alexandrow and Hamlin, 2005). Such decondensation is remains to be understood. We demonstrate that the Orc5 subunit is mediated by phosphorylation of H1. The phosphorylation of H1, unique, and when tethered ectopically to a transgene array, induces in turn, is Cdk2-dependent and leads to chromatin decondensation large-scale chromatin decondensation. It associates with the HAT in S phase. These observations point towards a role for Cdc45 in GCN5, which acetylates H3. The ability of Orc5 to cause chromatin replication fork progression by regulating large-scale chromatin decondensation requires GCN5 (Fig. 6D), and the loss of Orc5 changes (Alexandrow and Hamlin, 2005). Our observations of the causes decreased acetylation at specific origins. Our data suggest CLTon locus – which reveal that Orc5 can efficiently cause that there could be multiple mechanisms for the chromatin chromatin decondensation, predominantly during the G1 phase of decondensation that is mediated by Orc5 during G1, a GCN5- the cell cycle – provide a strong indication that similar events occur dependent mechanism involving residues 100–200 of Orc5 and a at endogenous origins, whereby Orc5 might cause local chromatin GCN5-independent mechanism involving the last 35 amino acids of changes in collaboration with GCN5. Another piece of evidence to Orc5. Once the chromatin is decondensed during G1, it facilitates support this idea comes from a recent study that shows preferential origin licensing, and it is likely that Orc5 and GCN5 cooperate at association of Orc5 with acetylated H3 at K27 (Ji et al., 2015) as select origins and that Orc5 cooperates with other ‘unknown factors’ compared to several other markers of activation, such as at other origins. trimethylated H3 at K4, dimethylated H3 at K79 and GCN5 is a global regulator of gene expression (Baker and trimethylated H3 at K36. We propose that at an endogenous locus Grant, 2007; Robert et al., 2004). More recently, GCN5 has been at which ORC binds, the amount of Orc5 present would be found to associate with yeast origins, albeit weakly (Espinosa sufficient for a smaller scale of decondensation, which might not be et al., 2010), and positively regulates DNA replication by observable at the resolution of light microscopy. But that counteracting the inhibitory effects of HDACs. In addition, decondensation would be sufficient for the cognate cellular GCN5-mediated acetylation of H3 lysine residues has also been function, such as replication initiation. proposed to function in replication-coupled nucleosome assembly Orc5 is one of the ORC subunits, and ATP binding to Orc5 is (Burgess et al., 2010). We propose that Orc5 at origins helps the involved in efficient ORC formation (Siddiqui and Stillman, 2007; recruitment of GCN5 to these sites and that this in turn facilitates Takahashi et al., 2004). Orc5 has also been implicated in silencing at the opening of chromatin, thus enabling the loading of other the HML and MHR loci in yeast and in heterochromatin preRC components. organization in human cells (Dillin and Rine, 1997; Prasanth The acetylation of histones H3 and H4 is known to be et al., 2010). The gene encoding Orc5 maps to chromosome 7q22 dynamically regulated around the origins of replication that and is frequently deleted in adult acute myeloid leukemia, facilitate origin firing (Unnikrishnan et al., 2010). Furthermore, myelodysplastic syndrome, uterine leiomyomas and malignant acetylation of H4 at residue K79 is enriched at origins; acetylation of myeloid diseases (Fröhling et al., 2001; Quintana et al., 1998). We H4 at residue K16 is enriched at early-firing origins and also limits have observed dose-dependent chromatin decondensation at the the spread of heterochromatin at origins. Acetylation of H3 at CLTon locus, and this was directly correlated with the expression residues K9 or K14, and of H4 at residues K5, K8 or K12 is enriched level of Orc5. This finding is supported by the fact that tethering at active origins, and these modifications are believed to promote of Orc5 to the CLTon locus can cause dramatic chromatin firing (Dorn and Cook, 2011). decondensation. However, tethering of other ORC subunits does Journal of Cell Science

426 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 417-429 doi:10.1242/jcs.178889 not, despite the fact that Orc5 can be recruited to the locus by other 100 nM using Lipofectamine RNAiMax (Invitrogen). This was followed by ORC subunits. Our data imply that excessive levels of Orc5 in the collection of cells for ChIP analysis 24 h after the second knockdown. cell could result in aberrant chromatin decondensation and cause For GCN5 knockdown, CLTon cells were grown to 30% confluence on genomic instability. coverslips followed by two rounds of knockdown with a control siRNA against the luciferase gene (Shen et al., 2010) or with an siRNA against GCN5 (Palhan et al., 2005), 24 h apart at a final concentration of 40 nM. MATERIALS AND METHODS YFP–LacI–Orc5 was transfected into cells while carrying out the second pEGFP-LacI vector was a kind gift from Dr Miroslav Dundr (Rosalind round of knockdown, and the cells were fixed for microscopy analysis 24 h Franklin University of Medicine and Science, North Chicago, IL) (Kaiser later. The first round of knockdown was performed using Lipofectamine et al., 2008) and used to generate the pEYFP-LacI vector. YFP-LacI-Orc1, RNAimax (Invitrogen), and the second round of knockdown with -Orc2, -Orc3, -Orc4, -Orc5 and -Orc6 were cloned by amplifying and inserting transfection of the YFP-tagged constructs was performed using the ORC proteins into the pEYFP-LacI vector. YFP–LacI–Orc5 D414A, Lipofectamine 2000 (Invitrogen). D426A,D433A; YFP–LacI–Orc5 K43A; YFP–LacI–Orc5 R166A were generated using site-directed mutagenesis (Stratagene) with YFP–LacI–Orc5 wild type as template. T7–Orc5 full length and truncations were cloned by Immunoprecipitations and immunoblots – For co-immunoprecipitation experiments, co-transfections were performed amplifying and inserted into pCGT vector. Flag GCN5 was a kind gift from – – Brian Freeman (University of Illinois at Urbana–Champaign, Champaign, IL). in U2OS cells. Flag HATs and T7 Orc5 were transiently transfected, and cells were lysed, 24 h post-transfection, in immunoprecipitation buffer (50 mM HEPES pH 7.9, 10% Glycerol, 200 mM NaCl, 0.1% Triton X-100, Cell culture 1 mM CaCl ) supplemented with the protease and phosphatase inhibitors. U2OS osteosarcoma cells were grown in high glucose Dulbecco’s modified 2 4U of MNase (Sigma) was then added per 10-cm plate of sample, followed Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum by rocking at room temperature for 20 min. EDTA (final concentration (FBS; Hyclone). U2OS-2-6-3 CLTon cells were cultured in DMEM 5 mM) was added to stop the reaction, and samples were centrifuged at supplemented with 10% Tet-system-approved FBS (Clonetech). 12,500 rpm (Eppendorf centrifuge 5424), for 5 min at 4°C. The supernatant was pre-cleared. Pre-clearing was performed using Gammabind Sepharose Immunofluorescence and fluorescent protein visualization beads for 1 h, and the lysates were incubated with appropriate antibody – For visualizing YFP LacI-tagged proteins, cells were fixed with 2% overnight. For pulldown of the antibody-bound complexes, agarose beads formaldehyde in PBS (pH 7.4) for 15 min at room temperature, followed by were washed in the same immunoprecipitation buffer and were incubated permeabilization with 0.5% Triton X-100 in PBS for 7 min on ice and by with lysate containing antibodies for 2 h. This was followed by three washes blocking. For immunofluorescent labeling of H3K9me3, acetylated H3, of the pulled-down complexes, and finally denaturation of the pulled-down MCM3 and PCNA, cells were pre-extracted before fixing with 0.5% Triton proteins by the addition of Laemmli buffer and incubation in a heat block for X-100 in cytoskeletal buffer (CSK; 100 mM NaCl, 300 mM sucrose, 3 mM 10 min. The complexes were then analyzed by western blotting. MgCl2, 10 mM PIPES at pH 6.8) for 5 min on ice followed by fixing with For semi-endogenous immunoprecipitation, the cells were initially lysed 1% formaldehyde in PBS (pH 7.4) for 5 min at room temperature and then in IP-100 buffer (50 mM Tris pH 7.5, 100 mM NaCl, 1 mM MgCl2, 0.2% blocking. This was followed by blocking for 30 min with 1% normal goat NP-40, 1% glycerol supplemented with protease and phosphatase serum in PBS. After that, cells were incubated for 1 h with primary antibody inhibitors). The lysate was then sonicated for 2 min with Bioruptor in a humidified chamber, followed by incubation with secondary antibody Power-up (Diagenode) using 30-s-on and 30-s-off cycles. Benzonase for 25 min. Nuclei were then stained with DAPI and mounted using (250 U/107 cells) was then added to the lysate, and the sample was incubated Vectashield (Vector Laboratories). The following antibodies were used for at room temperature for 30 min with rotation. The reaction was stopped immunofluorescence: anti-H3K9me3 (1:200, Millipore 07-523), anti- by adding EDTA (final concentration 5 mM), and the salt concentration acetylated-H3 (1:500, Millipore 06-599), anti-MCM3 (1:300, 738 rabbit was raised to 200 mM with NaCl. The sample was then pre-cleared, and polyclonal antibody) and anti-PCNA mAbPC10 (1:150, PC10 monoclonal subsequent steps of immunoprecipitation were performed as explained in antibody). the previous paragraph. All subsequent washes were performed with IP-200 buffer (50 mM Tris pH 7.5, 200 mM NaCl, 1 mM MgCl2, 0.2% NP-40, 1% Microscopy glycerol supplemented with protease and phosphatase inhibitors). For observing cells and obtaining statistics (area of decondensation, and For immunoprecipitations and immunoblotting, the following antibodies PCNA and MCM3 staining), we used a Zeiss Axioimager z1 fluorescence were used: anti-Flag M2 (1:500, Sigma), anti-T7 (1:5000, Novagen), anti- microscope (Carl Zeiss Inc.) equipped with chroma filters (Chroma ORC2 polyclonal antibody 205-6 (1:1000) and anti-GCN5 (1:200, Cell Technology). The images were acquired using a 60×, 1.4NA oil Signaling). immersion objective. Digital imaging was performed using an Hamamatsu ORCA cooled CCD camera. Axiovision software (Zeiss) was Chromatin immunoprecipitation used for processing the images. Control and Orc5-knockdown cells were crosslinked for 10 min at room A delta vision optical sectioning deconvolution instrument (Applied temperature through addition of formaldehyde (Sigma) to culture medium to precision) on an Olympus microscope was also used for observing cells and a final concentration of 1%. The reaction was stopped by addition of glycine for obtaining the images used in the manuscript. The images were acquired at a final concentration of 0.125 M. Fixed cells were then washed twice using a 60×, 1.4NA oil immersion objective. softWoRx imaging workstation (quickly) in chilled PBS and harvested with PBS. Chromatin was prepared was used to calculate the area of the decondensed locus. The ‘Edit Polygon’ using two subsequent extraction steps (10 min at 4°C) with Buffer 1 (50 mM tool was used as it is suitable for calculating irregular-area statistics. The HEPES with KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, extent of overlap of H3K9me3 with YFP–LacI or YFP–LacI–Orc5 was 0.5% NP-40, 0.25% Triton X-100) and Buffer 2 (200 mM NaCl, 1 mM measured by using the ‘Colocalization’ tool and defining a region of interest EDTA, 0.5 mM EGTA, 10 mM Tris-HCl pH 8). Nuclei were then pelleted (ROI); the Pearson coefficient of colocalization was then determined. Line by centrifugation, resuspended in SDS lysis buffer (50 mM Tris-HCl pH 8, profiles for H3K9me3 and acetylated H3 immunofluorescence were 0.1% SDS, 1% NP-40, 0.1% sodium deoxycholate, 10 mM EDTA, 150 mM generated by using the ‘Line Profile’ tool with the band value set to 2. NaCl) and subjected to sonication with a Bioruptor Power-up instrument (Diagenode) for 45 min (three times of 15 min of sonication, each cycle siRNA-mediated depletion of Orc5 comprising 30-s on and 30-s off). This yielded genomic DNA fragments U2OS cells were grown to 30% confluence, followed by two rounds of that were 300 bp in length. The obtained chromatin was then precleared with knockdown with a control siRNA against the luciferase gene (Shen et al., Protein A/G ultralink beads (53133, Pierce) for 1 h at 4°C, and 2010) or with an siRNA against Orc5 (5′-UGACUUUGUUCGCUUGU- immunoprecipitation with the anti-acetylated-H3 antibody and rabbit IgG

UUUU-3′) (Prasanth et al., 2010), 24 h apart at a final concentration of was performed overnight at 4°C. Immune complexes were pulled down by Journal of Cell Science

427 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 417-429 doi:10.1242/jcs.178889 adding pre-blocked protein A/G ultralink beads, and the mixture was Bell, S. P. and Dutta, A. (2002). DNA replication in eukaryotic cells. Annu. Rev. incubated for 2 h at room temperature. Beads with immune complexes Biochem. 71, 333-374. bound to them were washed once with low-salt buffer (0.1% SDS, 1% Triton Bell, S. P. and Stillman, B. (1992). ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature 357, 128-134. X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8, 150 mM NaCl), once with Bernal, J. A. and Venkitaraman, A. R. (2011). A vertebrate N-end rule degron high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris- reveals that Orc6 is required in mitosis for daughter cell abscission. J. Cell Biol. HCl pH 8, 500 mM NaCl), once with LiCl wash buffer (10 mM Tris-HCl 192, 969-978. pH 8.0, 1% sodium deoxycholate, 1% NP-40, 250 mM LiCl, 1 mM EDTA) Brown, J. A., Holmes, S. G. and Smith, M. M. (1991). The chromatin structure of and twice with Tris-EDTA. Beads were then eluted twice (10 min each) in Saccharomyces cerevisiae autonomously replicating sequences changes during Tris-EDTA+1% SDS+0.1% NaHCO at 65°C, and the cross-links were then the cell division cycle. Mol. Cell. Biol. 11, 5301-5311. 3 Burgess, R. J., Zhou, H., Han, J. and Zhang, Z. (2010). A role for Gcn5 in reversed overnight at 65°C. DNA from the eluted material was then treated replication-coupled nucleosome assembly. Mol. Cell 37, 469-480. with RNase (10 µg/ml) for 1 h at 37°C followed by 2 h of treatment with Carpenter, A. E., Memedula, S., Plutz, M. J. and Belmont, A. S. (2005). Common proteinase K (4 μl 0.5 M EDTA, 8 μl 1 M Tris-HCl pH 6.9, 1 μl proteinase effects of acidic activators on large-scale chromatin structure and transcription. K 20 mg/ml) at 42°C. DNA was isolated by using the QIAquick PCR Mol. Cell. Biol. 25, 958-968. purification kit (Qiagen) resuspended in elution buffer, and quantitative Chadha, G. S. and Blow, J. J. (2010). Histone acetylation by HBO1 tightens replication licensing. Mol. Cell 37, 5-6. PCR was performed using PowerSYBR Green PCR Master mix (Applied Dhar, S. K., Delmolino, L. and Dutta, A. (2001). Architecture of the human origin Biosystems) and analyzed on a 7300 PCR System (Applied Biosystems). recognition complex. J. Biol. Chem. 276, 29067-29071. ChIP–quantitative-PCR results were analyzed and plotted as percentages Dillin, A. and Rine, J. (1997). Separable functions of ORC5 in replication initiation (%) of immunoprecipitation/input signal (% input). and silencing in Saccharomyces cerevisiae. Genetics 147, 1053-1062. 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CAACCTCATGTTCTCT-3′; lamin B2 origin −2.2 Kb forward 5′-GAGA- Ferguson, B. M. and Fangman, W. L. (1992). A position effect on the time of ′ ′ replication origin activation in yeast. Cell 68, 333-339. ATCACTTGAACCCAGGAG-3 , reverse 5 -CCACTAGCTGCCAAGCT- Fröhling, S., Nakabayashi, K., Scherer, S. W., Dohner, H. and Dohner, K. (2001). TAT-3′; lamin B2 origin forward 5′-TGAGGAGTCCCTCAGATCTTT- Mutation analysis of the origin recognition complex subunit 5 (ORC5L) gene in A-3′, reverse 5′-CAGAATCCGATCATGCACCT-3′; MCM4−5 Kb for- adult patients with myeloid leukemias exhibiting deletions of chromosome band ward 5′-TTCACATCCACCCAGCTTATC-3′, reverse 5′-AGAGCATTC- 7q22. Hum. Genet. 108, 304-309. TTCCCCTGATG-3′; MCM4 origin forward 5′-TTGGGTGGCTACTTG- Giri, S., Aggarwal, V., Pontis, J., Shen, Z., Chakraborty, A., Khan, A., Mizzen, C., GTGTT-3′, reverse 5′-TAGGCCCCTCGCTTGTTT-3′. Prasanth, K. V., Ait-Si-Ali, S., Ha, T. et al. (2015). The preRC protein ORCA organizes heterochromatin by assembling histone H3 lysine 9 methyltransferases on chromatin. eLife 4, 332. Acknowledgements Goren, A., Tabib, A., Hecht, M. and Cedar, H. (2008). DNA replication timing of the We thank members of the Prasanth laboratory for discussions and suggestions. We human beta-globin domain is controlled by histone modification at the origin. thank Drs B. Freeman, C. Mizzen (phosphorylated histone H1 antibodies, University Dev. 22, 1319-1324. of Illinois at Urbana–Champaign, Champaign, IL), M. Dundr, D. Spector (Cold Spring Groth, A., Corpet, A., Cook, A. J. L., Roche, D., Bartek, J., Lukas, J. and Harbor Laboratory, Cold Spring Harbor, NY) and B. Stillman (Cold Spring Harbor Almouzni, G. (2007a). Regulation of replication fork progression through histone Laboratory, Cold Spring Harbor, NY) for providing reagents and suggestions. We supply and demand. Science 318, 1928-1931. Groth, A., Rocha, W., Verreault, A. and Almouzni, G. (2007b). 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S.G., K.V.P. and S.G.P. designed the experiments and wrote the Ji, X., Dadon, D. B., Abraham, B. J., Lee, T. I., Jaenisch, R., Bradner, J. E. and manuscript Young, R. A. (2015). Chromatin proteomic profiling reveals novel proteins associated with histone-marked genomic regions. Proc. Natl. Acad. Sci. USA 112, Funding 3841-3846. Kaiser, T. E., Intine, R. V. and Dundr, M. (2008). De novo formation of a subnuclear This work was supported by NSF-CMMB-IGERT and F31 [grant number body. Science 322, 1713-1717. CA180616]; a National Institutes of Health (NIH) fellowship to S.G.; American Knott, S. R. V., Viggiani, C. J. and Aparicio, O. M. (2009a). To promote and Cancer Society [grant number RSG 11-174-01RMC]; NIH [grant number protect: coordinating DNA replication and transcription for genome stability. 1RO1GM088252] award to K.V.P.; and National Science Foundation career [grant Epigenetics 4, 362-365. number 1243372] and NIH [grant number 1RO1GM099669] awards to S.G.P. Knott, S. R. 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