Nucleosome positioning
A simple hypothesis to explain the observed specificity in ORC localization is that not all sequences are equally accessible and that chromatin organization (nucleosome positioning) is a key determinant of ORC localization. This hypothesis has been vigorously pursued in
S. cerevisiae. Early nucleosome mapping experiments found that the ACS of the ARS1 origin is flanked by well positioned nucleosomes (
Thoma et al., 1984). The positioning of these flanking nucleosomes is critical for origin function. If the nucleosomes are experimentally forced to encroach into the ACS of ARS1, origin function on an episome is compromised (
Simpson, 1990), presumably due to the loss of ORC binding. The nucleosome positioning observed on an ARS1 containing plasmid has also been confirmed in the chromosomal context at the endogenous ARS1 locus (
Lipford and Bell, 2001). Thus, the location and position of nucleosomes can influence ORC binding and origin function.
Recent advances in genomic technologies have made it possible to determine the location of nucleosomes throughout the genome in a variety of organisms (
Albert et al., 2007;
Lee et al., 2007;
Mavrich et al., 2008;
Yuan et al., 2005). In
S.cerevisiae (as well as metazoans), promoters have a characteristic pattern of nucleosome occupancy. This nucleosome occupancy pattern consists of an array of well positioned nucleosomes at the transcription start site (TSS) progressing into the gene body and a region of low nucleosome occupancy immediately upstream of the transcription start site (
Jiang and Pugh, 2009;
Segal et al., 2006;
Yuan et al., 2005). This nucleosome free region (NFR) is associated with actively transcribed genes and likely contains the cis-acting binding sites for regulatory factors. Examination of nucleosome positioning at broadly mapped ARS elements in the yeast genome revealed that origins of replication were typically devoid of nucleosomes with weakly positioned nucleosomes on either side of the ARS element (
Albert et al., 2007;
Field et al., 2008). Close examination of the positioning of nucleosomes around a more precisely identified ACS sequence within the ARS revealed a distinct and conserved pattern of nucleosome organization at almost all origins of replication (
Berbenetz et al., 2010;
Eaton et al., 2010). Therefore, the well defined nucleosome organization first described at a single locus (ARS1) is a feature common to the large majority of yeast replication origins.
The inevitable question – “which came first, the chicken or the egg”, immediately arises -- do origins of replication have a nucleosome free region because ORC is bound or does ORC bind because the region is nucleosome free? Although trans-acting factors clearly contribute to the distribution of nucleosomes throughout the genome (
Liu et al., 2006), there are also sequence determinants which influence where a nucleosome will reside (
Segal et al., 2006;
Kaplan et al., 2009). The Segal group used high-throughput sequencing to map the preferred nucleosomal locations in the yeast genome in the absence of any trans-acting factors (
Kaplan et al., 2009). Specifically, they reconstituted nucleosomes
in vitro by assembling histone octamers onto purified yeast genomic DNA to identify the preferred nucleosomal positions encoded by primary sequence. In this
in vitro derived catalog of nucleosome locations, the ACS matches, which were
bona fide ORC binding sites, were indeed nucleosome free and those ACS matches which did not appear to be functional had an increased level of nucleosomal occupancy (
Eaton et al., 2010). These data suggest that primary sequence contributes to the nucleosome occupancy and organization at ORC binding sites and is likely a determinant of ORC association. Clearly, the ACS sequence itself is not sufficient to position nucleosomes or recruit ORC
in vivo; instead, structural cues in the primary sequence surrounding the ACS (e.g., helical stability, nucleosome positioning signals) are likely important for nucleosome positioning and ORC binding. These structural properties of the DNA are also conserved between related fungi (
Nieduszynski et al., 2006).
It is not clear how ORC facilitates the precise nucleosome positioning observed at
S. cerervisiae origins of replication. The precise positioning can be reconstituted
in vitro with recombinant histones, ORC and an ATP dependent chromatin remodeling activity (
Eaton et al., 2010). Not only are precisely positioned nucleosomes a signature of yeast replication origins but they are also required for origin activity. Displacement of the ACS proximal nucleosome further upstream results in loss of pre-RC assembly and origin activity (
Lipford and Bell, 2001). Orc1 contains a conserved bromo-adjacent homology (BAH) domain (
Callebaut et al., 1999) which, in the case of the Sir3 BAH domain (
Onishi et al., 2007), is known to interact with histones and facilitate chromatin silencing (
Zhang et al., 2002b). Direct interactions between ORC and flanking nucleosomes mediated by the BAH domain of ORC may be involved in establishing the local chromatin organization at a subset of replication origins. Consistent with this hypothesis, the deletion of the Orc1 BAH domain decreases the affinity of ORC for a subset of DNA replication origins resulting in a disorganization of the surrounding nucleosomes (
Muller et al., 2010).
The chromatin flanking ORC binding sites is dynamic and undergoes active nucleosome turnover and replacement outside of S-phase (
Kaplan et al., 2008;
Rufiange et al., 2007). Newly synthesized histone H3 is acetylated on an internal lysine (K56) by the histone transferase Rtt109 prior to assembly into a nucleosome (
Driscoll et al., 2007). H3K56Ac is incorporated into chromatin by both replication dependent and independent mechanisms and serves as a marker for newly assembled nucleosomes. In the absence of the histone chaperone Asf1, which is required for assembling histone H3K56Ac into nucleosomes, the incorporation of histone H3 is decreased at origins of replication (
Rufiange et al., 2007). Similarly, the analysis of H3K56Ac through the cell cycle revealed that nucleosomes flanking early origins of replication were being dynamically replaced even during G1 (
Kaplan et al., 2008). It is unclear if ORC acts directly on the surrounding nucleosomes or indirectly by recruiting a chromatin remodeler or nucleosome positioning factor. In this regard it is noteworthy that genome-wide analysis revealed an enrichment of the chromatin remodeler, Ino80, at origins and stalled replication forks (
Shimada et al., 2008), suggesting that perhaps ORC and Ino80 together facilitate the precise nucleosome positioning and rapid histone exchange observed at origins of replication.
Mechanistically, origins of replication in
S. cerevisiae appear to be established in the context of chromatin in two distinct steps (). First, ORC binds accessible ACS motifs that are not occupied by a nucleosome. Downstream A-rich sequences from the ACS may also facilitate the targeting of ORC to specific sequences or may function to keep the downstream region nucleosome free (
Eaton et al., 2010). Following ORC binding there is a subsequent ORC-dependent remodeling of the nucleosome organization that results in precisely positioned nucleosomes flanking the ACS (
Berbenetz et al., 2010;
Eaton et al., 2010). The nucleosome remodeling is presumably mediated by an ATP-dependent chromatin remodeling activity although the specific chromatin remodeling activity utilized i
n vivo is currently unknown. The resulting precise nucleosome organization at yeast origins of replication may be critical for the subsequent loading of multiple MCM2-7 complexes or for downstream initiation events.
Nucleosome occupancy also appears to be a determinant for ORC binding in higher eukaryotes. Studies in
Drosophila reveal that almost two thirds of ORC binding sites occur near the transcription start sites of actively expressed genes (
MacAlpine et al., 2010). As in yeast, the region immediately upstream of promoters in
Drosophila is typically free of nucleosomes (
Mavrich et al., 2008;
Henikoff et al., 2009). Importantly, those ORC binding sites not at promoters are also nucleosome free (
MacAlpine et al., 2010), suggesting that in the absence of sequence specificity, ORC may be localizing to highly accessible regions of the genome. Recent studies in Chinese hamster cells also indicate that ORC localizes to nucleosome free regions within the
DHFR initiation zone (
Lubelsky et al., 2010).
In
Drosophila, ORC associated sequences reside in dynamic chromatin. ORC binding sites are enriched for the replication independent histone variant H3.3 (
MacAlpine et al., 2010). Histone H3.3 is assembled into nucleosomes outside of S-phase and marks regions of the genome undergoing active chromatin remodelling (actively expressed gene bodies, promoter regions, and enhancers) (
Ahmad and Henikoff, 2002;
Schwartz and Ahmad, 2005). Regions enriched for the histone variant H3.3 are also typically depleted for bulk nucleosomes, consistent with the dynamic nature of their occupancy (
Wirbelauer et al., 2005). The Henikoff group has directly examined the genome-wide kinetics of nucleosome turn-over in the fruit fly using an approach, termed CATCH-IT, to biotin label nascent histones (
Deal et al., 2010). Strikingly, they observed that nucleosomes within gene bodies, surrounding regulatory elements, and ORC binding sites are undergoing active nucleosome turn-over and exchange. Further investigation will be required to determine if regions of dynamic chromatin facilitate ORC binding or, alternatively, if ORC actively participates in the local remodeling of chromatin and nucleosome exchange.
Chromatin modifications and other factors
In both yeast and higher eukaryotes, DNA accessibility in the context of chromatin is important for the recruitment of ORC to specific locations in the genome. Less clear is the role of chromatin modifications and additional specificity factors (e.g. transcription factors) in recruiting ORC to the DNA or in facilitating pre-RC assembly. In metazoans ORC frequently co-localizes near the transcription start sites of actively transcribed genes (
Ladenburger et al., 2002;
MacAlpine et al., 2004) and multiple transcription factors have been implicated in interacting with ORC and components of the pre-RC including Myc (
Dominguez-Sola et al., 2007), E2F1, Rb (
Bosco et al., 2001), and Myb (
Beall et al., 2002). Together, these results suggest that transcription factors may act as specificity factors in recruiting ORC to the DNA.
During
Drosophila oogenesis ORC localizes to specific clusters of chorion genes in the polytene follicle cells surrounding the oocyte. Following the endocycles, which are regulated transitions between S and G1 without an intervening mitosis, the chorion loci undergo a developmentally programmed amplification event that is dependent on ORC and the cis-acting DNA elements
ACE3 and
Oriβ (
Austin et al., 1999). Thus, outside of the confines of the cell cycle, the chorion loci are specifically amplified up to 60-fold over the surrounding sequence (
Claycomb and Orr-Weaver, 2005). This amplification serves to increase the number of chorion gene templates and augments transcription of these loci. The transcription factors E2F1, Rb (
Bosco et al., 2001), and Myb (
Beall et al., 2002), all of which co-localize with ORC at the chorion locus on chromosome 3L, are required for the proper developmentally regulated amplification. However, the role of transcription factors as specificity factors for ORC localization is still unclear as mutants in Myb still exhibit proper ORC localization at the chorion locus (
Beall et al., 2002), suggesting that transcription factors may act redundantly to recruit ORC to the DNA. Alternatively, transcription factors, as part of larger complexes with transcriptional activating and repressive properties (
Georlette et al., 2007) may alter the local chromatin environment and regulate downstream replication initiation events.
Genome-wide analysis of the distribution of multiple site-specific transcription factors have revealed surprising overlap in the locations occupied by these transcription factors (
MacArthur et al., 2009;
modENCODE Consortium, 2010). These high occupancy (HOT) regions of the genome may either reside in a distinct chromatin environment that is promiscuous for DNA binding or there might be unexpected cooperativity in the recruitment of transcription factors to HOT regions. HOT regions share several hallmarks which may facilitate ORC binding. For example, they are frequently found near promoters of active genes, depleted for bulk nucleosomes, exhibit high nucleosome turnover and are enriched for specific activating marks. Not surprisingly, the likelihood of finding an ORC binding site increases with the number of transcription factors (or complexity) of the HOT regions (
modENCODE Consortium, 2010). HOT regions may simply represent extremely accessible DNA or, alternatively, the different transcription factors may act redundantly to recruit ORC to the DNA.
Recent genome-wide origin mapping experiments in mouse and human cells have shown that origins are enriched near active promoter elements most commonly at CpG islands (
Cadoret et al., 2008;
Sequeira-Mendes et al., 2009). Transcription may affect not only the local chromatin environment but also the local topology of the DNA.
In vitro, ORC binds preferentially to negatively super-coiled DNA (
Remus et al., 2004) which would also be found immediately upstream of actively transcribed genes. Finally, coupling ORC localization to promoter elements may facilitate coordinating the replication program and transcription programs. For example, an origin at the TSS ensures that replication and transcription forks proceed in same direction and will minimize head-on collisions between DNA polymerase and RNA polymerase.
Actively transcribed genes often have distinct local chromatin environments. For example, promoter regions are often marked by H3K4me2 and H3K9Ac (
Kouzarides, 2007). Activating chromatin marks such as histone acetylation have been clearly linked with the regulation of replication origins (see below); however, their direct role in recruiting ORC to the DNA is less clear. Perhaps the best evidence for the role of histone acetylation in recruiting ORC to the DNA comes from experiments at the
Drosophila chorion locus (
Aggarwal and Calvi, 2004). Not surprisingly, the increased transcription of the amplified chorion locus coincides with increased histone acetylation (
Aggarwal and Calvi, 2004;
Hartl et al., 2007). Conversely, depletion of Rpd3, a histone deacetylase, results in a global increase in histone acetylation levels, the initiation of non-specific replication throughout the genome, and a loss of ORC localization at the chorion locus (
Aggarwal and Calvi, 2004). Presumably, in the absence of Rpd3 many more sites become acetylated and can compete for ORC localization.
Segmentation of the
Drosophila genome into distinct chromatin states based on either chromatin binding proteins (
Filion et al., 2010) or histone modifications (
Kharchenko et al., 2010;
modENCODE Consortium, 2010) revealed that ORC is enriched in distinct chromatin environments. Filion and colleagues identified 5 chromatin states which they named after five colors. ORC was found to be enriched in `RED' chromatin, one of the two active euchromatin states. The enrichment of ORC in active `RED'chromatin is perhaps not surprising given that ORC is enriched in dynamic and accessible chromatin (
Deal et al., 2010) and that the hallmarks of `RED' chromatin include the chomatin remodelling factor Caf-1 and GAGA factor both of which facilitate nucleosome dynamics and turn-over (
Bulger et al., 1995;
Petesch and Lis, 2008). Kharchenko and colleagues (
Kharchenko et al., 2010;
modENCODE Consortium, 2010) segmented the genome into 9 and 30 state chromatin models using histone modification data and found ORC enriched in a subset of states associated with transcription start sites and highly enriched for nucleosome turn-over. Future experiments will be required to demonstrate a causal role for these chromatin environments in the selection and regulation of replication origins.
Surprisingly, there is little direct data demonstrating a role for specific chromatin modifications in recruiting ORC to potential origins of replication. However, recent experiments in both yeast (
Pappas et al., 2004) and mammalian (
Miotto and Struhl, 2010;
Tardat et al., 2010) systems have shown that the local chromatin environment at origins of replication can impact the assembly of the pre-RC complex. These experiments are exciting because they indicate an additional control mechanism for establishing replication origins downstream of ORC binding. Specifically, these experiments suggest that the recruitment of ORC may not be sufficient for pre-RC assembly and instead the local chromatin environment may be critical for helicase loading.
The NAD+ dependent histone deacetylase, Sir2, is required for the establishment and maintenance of silenced heterochromatin in the yeast genome (
Rusche and Lynch, 2009). Unexpectedly, Sir2 also has a negative role in regulating pre-RC formation at select origins of replication (
Pappas et al., 2004). Mutations that inactivate the deacetylase activity of Sir2 rescue a temperature sensitive mutant allele of CDC6, cdc6-4. Crampton and colleagues individually screened origins from two yeast chromosomes for sensitivity to a Sir2 mutant (
Crampton et al., 2008). They found that approximately 20% of the origins examined were sensitive to Sir2. This sensitivity to Sir2 was not dependent on chromatin silencing as Sir3 and Sir4 mutants did not affect the origins of replication. Sir2 activity appears to directly inhibit pre-RC formation at sensitive origins. For example, Mcm2-7 loading is blocked in cdc6-4 mutants at the non-permissive temperature, but in combination with a Sir2 mutant, Mcm2-7 loading is restored only at those sensitive origins. This effect is mediated by histone acetlyation as a histone H4 tail mutant that mimics acetylation on K16 via a glutamine residue (H4K16Q) is also able to rescue the cdc6-4 mutant. Thus, pre-RC assembly at a subset of yeast origins depends on the local acetylation of H4K16 which is negatively regulated by Sir2 activity. Only a subset of origins are sensitive to Sir2, suggesting that the local chromatin acetylation state is critical for pre-RC formation. Perhaps, the hypoacetylation of local nucleosomes impairs the precise nucleosome positioning required for helicase loading (
Lipford and Bell, 2001) and that due to the complexity of nucleosomal positioning cues (sequences, trans-acting factors, topology, etc.) different origins are differentially affected.
The histone acetyltransferase Hbo1 (histone acetylase binding to Orc1) was initially identified as a histone acetyltransferase (HAT) activity that interacts with multiple components of the pre-RC including Orc1, Mcm2 and Cdt1 (
Burke et al., 2001;
Iizuka et al., 2006). Hbo1 is the predominant HAT responsible for the bulk of histone H4 acetylation in the genome (
Doyon et al., 2006). Although Hbo1 interacts with Orc1 and Mcm2, it is recruited to origins of replication in a cell cycle dependent manner via its interaction with Cdt1 (
Miotto and Struhl, 2010).
Recent studies suggest the involvement of Hbo1 in pre-RC formation at origins of replication. Depletion of Hbo1 results in an impaired S-phase (
Doyon et al., 2006) (presumably due to decreased origin function), and in
Xenopus extracts Hbo1 is required for pre-RC formation (
Iizuka et al., 2006). Although in a more reconstituted
Xenopus system, Hbo1 was not necessary for pre-RC formation (
Gillespie et al., 2001). Recent experiments by Miotto and colleagues demonstrate that recruitment of a catalytically inactive HBO1 mutant to an origin of replication impairs Mcm2-7 loading (
Miotto and Struhl, 2010).
In vitro, Hbo1 can acetylate a variety of substrates including histone H4 as well as multiple members of the pre-RC including Orc2, Mcm2 and Cdc6 (
Iizuka et al., 2006); thus, it is unclear whether histone H4 acetylation or perhaps protein acetylation promotes pre-RC formation. In support of Hbo1's acetyltransferase activity being directed at histones, the relative abundance of H4 acetylation at origins of replication is specifically decreased at a few select origins. Also, the tethering of Hbo1 (Chameaue) to
Drosophila origins stimulates origin activity at the chorion locus (
Aggarwal and Calvi, 2004); however, it is unclear if this stimulation of replication activity is mediated by increased pre-RC assembly or increased activation of the origin.
It will be interesting to see if Hbo1 facilitates origin selection throughout the genome or if its effects are limited to specific origins of replication as is the case with Sir2 which impacts less than 20% of the origins (
Crampton et al., 2008). The ability to modulate histone acetylation levels at specific origins may represent a rheostat that is used to fine tune origin function in cooperation with the transcription program. The acetylation of neighboring histones likely facilitates histone exchange (a common property at origins) and perhaps the unwinding of the DNA in preparation for initiation.
Set8 (PR-Set7) is a cell cycle regulated histone methyltransferase that catalyzes the monomethylation of histone H4 on lysine 20 (H4K20me1) (
Fang et al., 2002;
Nishioka et al., 2002). The degradation of Set8 during S-phase is dependent on PCNA and mediated by the E3 ubiqutitin ligase CRL4
cdt2 (
Abbas et al., 2010;
Centore et al., 2010;
Oda et al., 2010). Loss of Set8 has myriad of phenotypes including: chromosome decondensation, delayed S-phase progression, G2 arrest, centrosome amplification and DNA damage (
Houston et al., 2008;
Jorgensen et al., 2007;
Karachentsev et al., 2005;
Oda et al., 2009;
Tardat et al., 2010). Interestingly, stabilization of Set8 results in premature chromatin compaction and a checkpoint-mediated G2 arrest (
Abbas et al., 2010;
Centore et al., 2010). Tardat and colleagues further demonstrated that stabilization of Set8 results in re-replication which is presumably mediated by the re-assembly of the pre-RC at H4K20me1 marked origins of replication (
Tardat et al., 2010). Tethering of Set8 to a specific genomic locus resulted in an increase in H4K20me1 and recruitment of pre-RC components. These results suggest that the monomethylation of H4K20me1 is conducive to pre-RC loading and origin function, and imply that the tight cell cycle regulation of Set8 may represent an additional control mechanism to ensure genomic stability.
Despite the differences in ORC specificity for conserved sequence elements between yeast and higher eukaryotes, the chromatin organization is remarkably similar (). ORC frequently co-localizes in the open and accessible chromatin upstream of transcription start sites. Transcription factors, chromatin remodeling activities and histone acetylation likely contribute to maintaining open chromatin. A signature of ORC associated sequences is the dynamic turn-over of nucleosomes at the ORC binding sites (
Deal et al., 2010;
Henikoff et al., 2009), although it is unclear whether this turn-over is dependent on or facilitates ORC binding. Finally, the assembly of the pre-RC at specific origins may be mediated by the HAT, Hbo1, which interacts with multiple pre-RC components (
Burke et al., 2001;
Iizuka et al., 2006).
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