Chromatin unfolding by Cdt1 regulates MCM loading via opposing functions of HBO1 and HDAC11-geminin

The efficiency of metazoan origins of DNA replication is known to be enhanced by histone acetylation near origins. Although this correlates with increased MCM recruitment, the mechanism by which such acetylation regulates MCM loading is unknown. We show here that Cdt1 induces large-scale chromatin decondensation that is required for MCM recruitment. This process occurs in G₁, is suppressed by Geminin and requires HBO1 HAT activity and histone H4 modifications. HDAC11, which binds Cdt1 and replication origins during S phase, potently inhibits Cdt1-induced chromatin unfolding and re-replication, suppresses MCM loading and binds Cdt1 more efficiently in the presence of Geminin. We also demonstrate that chromatin at endogenous origins is more accessible in G₁ relative to S phase. These results provide evidence that histone acetylation promotes MCM loading via enhanced chromatin accessibility. This process is regulated positively by Cdt1 and HBO1 in G₁ and repressed by Geminin-HDAC11 association with Cdt1 in S phase and represents a novel form of replication licensing control.Key words: Cdt1, HBO1, HDAC11, chromatin, DNA replication     

A Cdt1-geminin complex licenses chromatin for DNA replication and prevents rereplication during S phase in Xenopus

Initiation of DNA synthesis involves the loading of the MCM2-7 helicase onto chromatin by Cdt1 (origin licensing). Geminin is thought to prevent relicensing by binding and inhibiting Cdt1. Here we show, using Xenopus egg extracts, that geminin binding to Cdt1 is not sufficient to block its activity and that a Cdt1-geminin complex licenses chromatin, but prevents rereplication, working as a molecular switch at replication origins. We demonstrate that geminin is recruited to chromatin already during licensing, while bulk geminin is recruited at the onset of S phase. A recombinant Cdt1-geminin complex binds chromatin, interacts with the MCM2-7 complex and licenses chromatin once per cell cycle. Accordingly, while recombinant Cdt1 induces rereplication in G1 or G2 and activates an ATM/ATR-dependent checkpoint, the Cdt1-geminin complex does not. We further demonstrate that the stoichiometry of the Cdt1-geminin complex regulates its activity. Our results suggest a model in which the MCM2-7 helicase is loaded onto chromatin by a Cdt1-geminin complex, which is inactivated upon origin firing by binding additional geminin. This origin inactivation reaction does not occur if only free Cdt1 is present on chromatin.     

Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin

To maintain genome integrity, eukaryotic cells initiate DNA replication once per cell cycle after assembling prereplicative complexes (preRCs) on chromatin at the end of mitosis and during G1. In S phase, preRCs are disassembled, precluding initiation of another round of replication. Cdt1 is a key member of the preRC and its correct regulation via proteolysis and by its inhibitor Geminin is essential to prevent premature re-replication. Using quantitative fluorescence microscopy, we study the interactions of Cdt1 with chromatin and Geminin in living cells. We find that Cdt1 exhibits dynamic interactions with chromatin throughout G1 phase and that the protein domains responsible for chromatin and Geminin interactions are separable. Contrary to existing in vitro data, we show that Cdt1 simultaneously binds Geminin and chromatin in vivo, thereby recruiting Geminin onto chromatin. We propose that dynamic Cdt1-chromatin associations and the recruitment of Geminin to chromatin provide spatio-temporal control of the licensing process.     

Cdt1 Interactions in the Licensing Process: A Model for Dynamic Spatio-temporal Control of Licensing

Within each cell cycle, a cell must ensure that the processes of selection of replication origins (licensing) and initiation of DNA replication are well coordinated to prevent re-initiation of DNA replication from the same DNA segment during the same cell cycle. This is achieved by restricting the licensing process to G1 phase when the prereplicative complexes (preRCs) are assembled onto the origin DNA, while DNA replication is initiated only during S phase when de novo preRC assembly is blocked. Cdt1 is an important member of the preRC complex and its tight regulation through ubiquitin-dependent proteolysis and binding to its inhibitor Geminin ensure that Cdt1 will only be present in G1 phase, preventing relicensing of replication origins. We have recently reported that Cdt1 associates with chromatin in a dynamic way and recruits its inhibitor Geminin onto chromatin in vivo. Here we discuss how these dynamic Cdt1-chromatin interactions and the local recruitment of Geminin onto origins of replication by Cdt1 may provide a tight control of the licensing process in time and in space.     

MCM9 binds Cdt1 and is required for the assembly of prereplication complexes

Prereplication complexes (pre-RCs) define potential origins of DNA replication and allow the recruitment of the replicative DNA helicase MCM2-7. Here, we characterize MCM9, a member of the MCM2-8 family. We demonstrate that MCM9 binds to chromatin in an ORC-dependent manner and is required for the recruitment of the MCM2-7 helicase onto chromatin. Its depletion leads to a block in pre-RC assembly, as well as DNA replication inhibition. We show that MCM9 forms a stable complex with the licensing factor Cdt1, preventing an excess of geminin on chromatin during the licensing reaction. Our data suggest that MCM9 is an essential activating linker between Cdt1 and the MCM2-7 complex, required for loading the MCM2-7 helicase onto DNA replication origins. Thus, Cdt1, with its two opposing regulatory binding factors MCM9 and geminin, appears to be a major platform on the pre-RC to integrate cell-cycle signals.     

Proteolysis of DNA replication licensing factor Cdt1 in S-phase is performed independently of geminin through its N-terminal region

Licensing of replication origins is carefully regulated in a cell cycle to maintain genome integrity. Using an in vivo ubiquitination assay and temperature-sensitive cell lines we demonstrate that Cdt1 in mammalian cells is degraded through ubiquitin-dependent proteolysis in S-phase. siRNA experiments for Geminin indicate that Cdt1 is degraded in the absence of Geminin. The N terminus of Cdt1 is required for its nuclear localization, associates with cyclin A, but is dispensable for the association of Cdt1 with Geminin in cells. This region is responsible for proteolysis of Cdt1 in S-phase. On the other hand, the N terminus-truncated Cdt1 is stable in S-phase, and associates with the licensing inhibitor, Geminin. High level expression of this form of Cdt1 brings about cells having higher DNA content. Proteasome inhibitors stabilize Cdt1 in S-phase, and the protein is detected in the nucleus in a complex with Geminin. This form of Cdt1 associates with chromatin as tightly as that of G1-cells, when no Geminin is detected. Our data show that proteolysis and Geminin binding independently inactivate Cdt1 after the onset of S-phase to prevent re-replication.     

BRPF3‐HBO1 regulates replication origin activation and histone H3K14 acetylation

During DNA replication, thousands of replication origins are activated across the genome. Chromatin architecture contributes to origin specification and usage, yet it remains unclear which chromatin features impact on DNA replication. Here, we perform a RNAi screen for chromatin regulators implicated in replication control by measuring RPA accumulation upon replication stress. We identify six factors required for normal rates of DNA replication and characterize a function of the bromodomain and PHD finger‐containing protein 3 (BRPF3) in replication initiation. BRPF3 forms a complex with HBO1 that specifically acetylates histone H3K14, and genomewide analysis shows high enrichment of BRPF3, HBO1 and H3K14ac at ORC1‐binding sites and replication origins found in the vicinity of TSSs. Consistent with this, BRPF3 is necessary for H3K14ac at selected origins and efficient origin activation. CDC45 recruitment, but not MCM2‐7 loading, is impaired in BRPF3‐depleted cells, identifying a BRPF3‐dependent function of HBO1 in origin activation that is complementary to its role in licencing. We thus propose that BRPF3‐HBO1 acetylation of histone H3K14 around TSS facilitates efficient activation of nearby replication origins.     

Chromatin remodeler sucrose nonfermenting 2 homolog (SNF2H) is recruited onto DNA replication origins through interaction with Cdc10 protein-dependent transcript 1 (Cdt1) and promotes pre-replication complex formation

From late mitosis to the G(1) phase of the cell cycle, ORC, CDC6, and Cdt1 form the machinery necessary to load MCM2-7 complexes onto DNA. Here, we show that SNF2H, a member of the ATP-dependent chromatin-remodeling complex, is recruited onto DNA replication origins in human cells in a Cdt1-dependent manner and positively regulates MCM loading. SNF2H physically interacted with Cdt1. ChIP assays indicated that SNF2H associates with replication origins specifically during the G(1) phase. Binding of SNF2H at origins was decreased by Cdt1 silencing and, conversely, enhanced by Cdt1 overexpression. Furthermore, SNF2H silencing prevented MCM loading at origins and moderately inhibited S phase progression. Although neither SNF2H overexpression nor SNF2H silencing appeared to impact rereplication induced by Cdt1 overexpression, Cdt1-induced checkpoint activation was inhibited by SNF2H silencing. Collectively, these data suggest that SNF2H may promote MCM loading at DNA replication origins via interaction with Cdt1 in human cells. Because efficient loading of excess MCM complexes is thought to be required for cells to tolerate replication stress, Cdt1- and SNF2H-mediated promotion of MCM loading may be biologically relevant for the regulation of DNA replication.     

Cell cycle regulation of chromatin at an origin of DNA replication

Selection and licensing of mammalian DNA replication origins may be regulated by epigenetic changes in chromatin structure. The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) uses the cellular licensing machinery to regulate replication during latent infection of human cells. We found that the minimal replicator sequence of OriP, referred to as the dyad symmetry (DS), is flanked by nucleosomes. These nucleosomes were subject to cell cycle-dependent chromatin remodeling and histone modifications. Restriction enzyme accessibility assay indicated that the DS-bounded nucleosomes were remodeled in late G1. Remarkably, histone H3 acetylation of DS-bounded nucleosomes decreased during late G1, coinciding with nucleosome remodeling and MCM3 loading, and preceding the onset of DNA replication. The ATP-dependent chromatin-remodeling factor SNF2h was also recruited to DS in late G1, and formed a stable complex with HDAC2 at DS. siRNA depletion of SNF2h reduced G1-specific nucleosome remodeling, histone deacetylation, and MCM3 loading at DS. We conclude that an SNF2h-HDAC1/2 complex coordinates G1-specific chromatin remodeling and histone deacetylation with the DNA replication initiation process at OriP.     

Structural insights into the Cdt1-mediated MCM2-7 chromatin loading

Initiation of DNA replication in eukaryotes is exquisitely regulated to ensure that DNA replication occurs exactly once in each cell division. A conserved and essential step for the initiation of eukaryotic DNA replication is the loading of the mini-chromosome maintenance 2-7 (MCM2-7) helicase onto chromatin at replication origins by Cdt1. To elucidate the molecular mechanism of this event, we determined the structure of the human Cdt1-Mcm6 binding domains, the Cdt1(410-440)/MCM6(708-821) complex by NMR. Our structural and site-directed mutagenesis studies showed that charge complementarity is a key determinant for the specific interaction between Cdt1 and Mcm2-7. When this interaction was interrupted by alanine substitutions of the conserved interacting residues, the corresponding yeast Cdt1 and Mcm6 mutants were defective in DNA replication and the chromatin loading of Mcm2, resulting in cell death. Having shown that Cdt1 and Mcm6 interact through their C-termini, and knowing that Cdt1 is tethered to Orc6 during the loading of MCM2-7, our results suggest that the MCM2-7 hexamer is loaded with its C terminal end facing the ORC complex. These results provide a structural basis for the Cdt1-mediated MCM2-7 chromatin loading.     

Recombinant Cdt1 induces rereplication of G2 nuclei in Xenopus egg extracts

A crucial regulation for maintaining genome integrity in eukaryotes is to limit DNA replication in S phase to only one round. Several models have been proposed; one of which, the licensing model, predicted that formation of the nuclear membrane restricts access to chromatin to a positive replication factor. Cdt1, a factor binding to origins and recruiting the MCM2-7 helicase, has been identified as a component of the licensing system in Xenopus and other eukaryotes. Nevertheless, evidence is missing demonstrating a direct role for unscheduled Cdt1 expression in promoting illegitimate reinitiation of DNA synthesis. We show here that Xenopus Cdt1 is absent in G2 nuclei, suggesting that it might be either degraded or exported. Recombinant Cdt1, added to egg extracts in G2, crosses the nuclear membrane, binds to chromatin, and relicenses the chromosome for new rounds of DNA synthesis in combination with chromatin bound Cdc6. The mechanism involves rebinding of MCM3 to chromatin. Reinitiation is blocked by geminin only in G2 and is not stimulated by Cdc6, demonstrating that Cdt1, but not Cdc6, is limiting for reinitiation in egg extracts. These results suggest that removal of Cdt1 from chromatin and its nuclear exclusion in G2 is critical in regulating licensing and that override of this control is sufficient to promote illegitimate firing of origins.     

Human Cdt1 lacking the evolutionarily conserved region that interacts with MCM2-7 is capable of inducing re-replication

Replication initiation must be a carefully regulated process to avoid genomic instability caused by aberrant replication. In eukaryotic cells, distinct steps of protein loading (origin licensing) and replication activation are choreographed such that a cell can replicate only once per cell cycle. The first proteins recruited to the origins form the pre-replication complex. Of these proteins, Cdt1 is of interest, as it is the focus of several pathways to control replication initiation. It is degraded by two different pathways, mediated by the interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) or with cyclin-Cdk2 and inhibited by geminin once cells are in S-phase, presumably to prevent reloading of pre-replication complexes once S-phase has begun. Although the requirement of Cdt1 in loading MCM2-7 is known, the mechanism by which overexpressed Cdt1 stimulates re-replication is unclear. In this study we have designed various mutations in Cdt1 to determine which portion of Cdt1 is important for re-replication, providing insight into possible mechanisms. Surprisingly, we found that mutants of Cdt1 that do not interact with MCM2-7 are able to induce re-replication when overexpressed. The re-replication is not due to titration of geminin from endogenous Cdt1 and is not accompanied by stabilization of endogenous Cdt1. Additionally, the N-terminal one-third of Cdt1 is sufficient to induce re-replication. The N terminus contains the PCNA- and cyclin-interacting motifs, and deletion of both motifs simultaneously in the overexpressed Cdt1 prevents re-replication. These findings suggest that exogenous Cdt1 induces re-replication by de-repressing endogenous Cdt1 through the titration of PCNA and cyclin.     

The Involvement of Acidic Nucleoplasmic DNA-binding Protein (And-1) in the Regulation of Prereplicative Complex (pre-RC) Assembly in Human Cells

DNA replication in all eukaryotes starts with the process of loading the replicative helicase MCM2–7 onto chromatin during late mitosis of the cell cycle. MCM2–7 is a key component of the prereplicative complex (pre-RC), which is loaded onto chromatin by the concerted action of origin recognition complex, Cdc6, and Cdt1. Here, we demonstrate that And-1 is assembled onto chromatin in late mitosis and early G₁ phase before the assembly of pre-RC in human cells. And-1 forms complexes with MCM2–7 to facilitate the assembly of MCM2–7 onto chromatin at replication origins in late mitosis and G₁ phase. We also present data to show that depletion of And-1 significantly reduces the interaction between Cdt1 and MCM7 in G₁ phase cells. Thus, human And-1 facilitates loading of the MCM2–7 helicase onto chromatin during the assembly of pre-RC.     

Activation of a human chromosomal replication origin by protein tethering

The specification of mammalian chromosomal replication origins is incompletely understood. To analyze the assembly and activation of prereplicative complexes (pre-RCs), we tested the effects of tethered binding of chromatin acetyltransferases and replication proteins on chromosomal c-myc origin deletion mutants containing a GAL4-binding cassette. GAL4^DBD (DNA binding domain) fusions with Orc2, Cdt1, E2F1 or HBO1 coordinated the recruitment of the Mcm7 helicase subunit, the DNA unwinding element (DUE)-binding protein DUE-B and the minichromosome maintenance (MCM) helicase activator Cdc45 to the replicator, and restored origin activity. In contrast, replication protein binding and origin activity were not stimulated by fusion protein binding in the absence of flanking c-myc DNA. Substitution of the GAL4-binding site for the c-myc replicator DUE allowed Orc2 and Mcm7 binding, but eliminated origin activity, indicating that the DUE is essential for pre-RC activation. Additionally, tethering of DUE-B was not sufficient to recruit Cdc45 or activate pre-RCs formed in the absence of a DUE. These results show directly in a chromosomal background that chromatin acetylation, Orc2 or Cdt1 suffice to recruit all downstream replication initiation activities to a prospective origin, and that chromosomal origin activity requires singular DNA sequences.     

The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication

The initiation of eukaryotic DNA replication involves origin recruitment and activation of the MCM2-7 complex, the putative replicative helicase. Mini-chromosome maintenance (MCM)2-7 recruitment to origins in G1 requires origin recognition complex (ORC), Cdt1, and Cdc6, and activation at G1/S requires MCM10 and the protein kinases Cdc7 and S-Cdk, which together recruit Cdc45, a putative MCM2-7 cofactor required for origin unwinding. Here, we show that the Xenopus BRCA1 COOH terminus repeat-containing Xmus101 protein is required for loading of Cdc45 onto the origin. Xmus101 chromatin association is dependent on ORC, and independent of S-Cdk and MCM2-7. These results define a new factor that is required for Cdc45 loading. Additionally, these findings indicate that the initiation complex assembly pathway bifurcates early, after ORC association with the origin, and that two parallel pathways, one controlled by MCM2-7, and the other by Xmus101, cooperate to load Cdc45 onto the origin.     

Expression of Cdc18/Cdc6 and Cdt1 during G2 phase induces initiation of DNA replication

Cdc18/Cdc6 and Cdt1 are essential initiation factors for DNA replication. In this paper we show that expression of Cdc18 in fission yeast G2 cells is sufficient to override the controls that ensure one S phase per cell cycle. Cdc18 expression in G2 induces DNA synthesis by re-firing replication origins and recruiting the MCM Cdc21 to chromatin in the presence of low levels of Cdt1. However, when Cdt1 is expressed together with Cdc18 in G2, cells undergo very rapid, uncontrolled DNA synthesis, accumulating DNA contents of 64C or more. Our data suggest that Cdt1 may potentiate re-replication by inducing origins to fire more persistently, possibly by stabilizing Cdc18 on chromatin. In addition, low level expression of a mutant form of Cdc18 that cannot be phosphorylated by cyclin-dependent kinases is not sufficient to induce replication in G2, but does so only when co-expressed with Cdt1. Thus, regulation of both Cdc18 and Cdt1 in G2 plays a crucial role in preventing the re-initiation of DNA synthesis until the next cell cycle.     

Recruitment of Xenopus Scc2 and cohesin to chromatin requires the pre-replication complex

Cohesin is a multi-subunit, ring-shaped protein complex that holds sister chromatids together from the time of their synthesis in S phase until they are segregated in anaphase. In yeast, the loading of cohesin onto chromosomes requires the Scc2 protein. In vertebrates, cohesins first bind to chromosomes as cells exit mitosis, but the mechanism is unknown. Concurrent with cohesin binding, pre-replication complexes (pre-RCs) are assembled at origins of DNA replication through the sequential loading of the initiation factors ORC, Cdc6, Cdt1 and MCM2-7 (the 'licensing' reaction). In S phase, the protein kinase Cdk2 activates pre-RCs, causing origin unwinding and DNA replication. Here, we use Xenopus egg extracts to show that the recruitment of cohesins to chromosomes requires fully licensed chromatin and is dependent on ORC, Cdc6, Cdt1 and MCM2-7, but is independent of Cdk2. We further show that Xenopus Scc2 is required for cohesin loading and that binding of XScc2 to chromatin is MCM2-7 dependent. Our results define a novel pre-RC-dependent pathway for cohesin recruitment to chromosomes in a vertebrate model system.     

Licensing for DNA replication requires a strict sequential assembly of Cdc6 and Cdt1 onto chromatin in Xenopus egg extracts

Replication origins are licensed for a single initiation event by the loading of Mcm2-7 proteins during late mitosis and G1. Sequential associations of origin recognition complex, Cdc6 and Mcm2-7 are essential for completion of the licensing. Although Cdt1 also binds to the chromatin when the licensing reaction takes place, whether the binding is a requirement for Cdt1 to function is unclear. To analyze the relevance of the chromatin association of Cdt1, we carried out chromatin transfer experiments using either immunodepleted Xenopus egg extracts or purified proteins. Licensing assay and immunoblotting analyses indicated that Cdt1 could only license DNA replication and load Mcm2-7 onto DNA when it binds to chromatin that has already associated with Cdc6. These results provide evidence supporting that Cdc6 and Cdt1 must bind to chromatin in a strict order for DNA licensing to occur.     

Characterization of conserved arginine residues on Cdt1 that affect licensing activity and interaction with Geminin or Mcm complex

All organisms ensure once and only once replication during S phase through a process called replication licensing. Cdt1 is a key component and crucial loading factor of Mcm complex, which is a central component for the eukaryotic replicative helicase. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent rereplication. Here, we address the mechanism of DNA licensing using purified Cdt1, Mcm and Geminin proteins in combination with replication in Xenopus egg extracts. We mutagenized the 223th arginine of mouse Cdt1 (mCdt1) to cysteine or serine (R-S or R-C, respectively) and 342nd and 346th arginines constituting an arginine finger-like structure to alanine (RR-AA). The RR-AA mutant of Cdt1 could not only rescue the DNA replication activity in Cdt1-depleted extracts but also its specific activity for DNA replication and licensing was significantly increased compared to the wild-type protein. In contrast, the R223 mutants were partially defective in rescue of DNA replication and licensing. Biochemical analyses of these mutant Cdt1 proteins indicated that the RR-AA mutation disabled its functional interaction with Geminin, while R223 mutations resulted in ablation in interaction with the Mcm2～7 complex. Intriguingly, the R223 mutants are more susceptible to the phosphorylation-induced inactivation or chromatin dissociation. Our results show that conserved arginine residues play critical roles in interaction with Geminin and Mcm that are crucial for proper conformation of the complexes and its licensing activity.