Origin recognition complex subunit 1


Transcriptional Regulation

Throughout the cell cycle of Saccharomyces cerevisiae, the level of origin recognition complex (ORC) is constant and ORCs are bound constitutively to replication origins (See Drosophila Orc2 for more information on the ORC). Replication is regulated by the recruitment of additional factors such as Cdc6. ORC components are widely conserved, and it generally has been assumed that they are also stable factors bound to origins throughout the cell cycle. In this report, it is shown that the level of the ORC1 subunit changes dramatically throughout Drosophila development. The accumulation of ORC1 is regulated by E2F-dependent transcription. In embryos, ORC1 accumulates preferentially in proliferating cells. In the eye imaginal disc, ORC1 accumulation is cell cycle regulated, with high levels in late G1 and S phase. In the ovary, the sub-nuclear distribution of ORC1 shifts during a developmentally regulated switch from endoreplication of the entire genome to amplification of the chorion gene clusters. Furthermore, it has been found that overexpression of ORC1 alters the pattern of DNA synthesis in the eye disc and the ovary. Thus, replication origin activity appears to be governed in part by the level of ORC1 in Drosophila (Asano, 1999).

Late in embryonic development, most cells enter an extended quiescent period, resuming DNA synthesis upon hatching. However, replication persists in three tissues (brain, ventral nerve cord, anterior- and posterior-midgut), and the mRNAs of E2F-regulated genes (such as Ribonucleotide reductase, RNR2) accumulate in these tissues. To determine whether transcription of ORC1 is regulated by E2F, the distribution of ORC1 mRNA was examined in wild-type and E2F- embryos by in situ hybridization. The distributions of ORC1 and RNR2 mRNAs are essentially the same in wild-type embryos at stage 13. Moreover, accumulation of either RNR2 or ORC1 mRNA is largely dependent on E2F function at this stage of development. Thus, transcription of ORC1 is E2F-dependent in the embryo. It was next determined whether E2F regulates ORC1 transcription in imaginal disc cells, which have canonical four-phase cell cycles. Accumulation of ORC1 mRNA is induced following overexpression of E2F (~4-fold). By comparison, accumulation of three other E2F-regulated mRNAs (PCNA, RNR1 and RNR2) is induced to a similar extent in these experiments (Asano, 1999).

To determine whether the regulation described above is mediated by the direct action of E2F, the ORC1 promoter was isolated. Within the 400 nt upstream of the major transcriptional start site, four candidate E2F binding sites with similarity to the canonical site in the adenovirus E2 promoter (TTTCGCGC) were identified by inspection, two at approximately -340 nt and two overlapping sites at -13 nt. Characterization of other E2F-responsive promoters has shown that binding sites close to the transcriptional start site frequently play a predominant role in regulation, and thus a focus was placed on the overlapping sites at -13. Drosophila E2F has been shown to bind to the ORC1 promoter just upstream of the start site of transcription. To test the role of these E2F sites in vivo, transcriptional reporter genes were prepared in which either the wild-type ORC1 promoter or a mutant derivative bearing substitutions within the proximal E2F binding sites drives the expression of a cDNA encoding an unstable Ftz-GFP-Myc tag fusion protein. Activity of the ORC1 promoter is dependent on the integrity of the E2F binding sites at -13 nt. In flies bearing the wild-type promoter construct, fusion protein is detectable in cells throughout most regions of the imaginal discs. However, in flies bearing a mutant promoter construct, essentially no fusion protein is detectable in any of the imaginal discs. These observations suggest that E2F acts directly by binding to the ORC1 promoter and stimulating transcription. Furthermore, the spatiotemporal pattern of ORC1 promoter activation within two specialized groups of cells in the eye and wing imaginal discs supports the idea that E2F couples transcription of ORC1 to cell cycle progression (Asano, 1999).

During the third larval instar, a developmentally regulated cell cycle transition takes place as a wave of differentiation sweeps across the eye imaginal disc. The wave front is marked by the morphogenetic furrow. During differentiation, four regions can be identified: (1) anterior to the morphogenetic furrow (including the antennal disc), undifferentiated cells cycle asynchronously; (2) as they enter the furrow, cells arrest in an extended G1 phase; (3) immediately posterior to the furrow, some cells are recruited into ommatidial pre-clusters and begin neural differentiation while others synchronously enter S phase, and (4) posterior to this synchronous wave of S phase, most cells cease cycling and terminally differentiate. The pattern of ORC1 promoter activity in the eye imaginal disc suggests that it is turned on late in G1, near the G1-S boundary. In particular, the ORC1 promoter is activated in a random pattern among the asynchronous cells in the anterior region of the disc: turned off as cells enter the morphogenetic furrow and G1, turned on in cells as they emerge from the furrow late in G1 phase, and then turned off in the quiescent cells in the posterior region of the eye. Another developmentally programmed cell cycle arrest has recently been described in the wing imaginal disc. At the dorsoventral boundary of the disc, Notch and wingless signaling establish a zone of non-proliferating cells (ZNC) in which no S phase is detectable. Cells throughout the posterior ZNC and in the center of the anterior ZNC arrest late in G1, at a point when expression of Cyclin E can drive them into S; flanking cells in the anterior ZNC arrest in G2. Among the cells of the ZNC, the ORC1 promoter is active only in G1-arrested cells and not in those arrested in G2. These observations support the idea that activation of E2F in G1 stimulates transcription of ORC1 in a variety of cell types (Asano, 1999).

In Drosophila, many genes have been shown to be transcriptionally regulated by E2F during the G1-S transition. These include Cyclin E, RNR, Polalpha, PCNA, MCM2 and MCM3. However, only in the case of Cyclin E has it been shown that protein levels are cell cycle regulated, presumably at least in part as a consequence of E2F action. In the other cases, either the protein distribution has not been reported or the protein level is constant throughout the cell cycle. Therefore, to determine whether the level of ORC1 is modulated as a result of E2F-dependent regulation, antibodies were prepared that specifically recognize the protein, and its distribution was examined in embryos and imaginal discs. Antisera were prepared by immunizing animals with glutathione S-transferase (GST) fusion proteins bearing three different portions of ORC1. The distribution of ORC1 was examined during embryonic development. The first 13 nuclear division cycles that occur in a syncitium are parasynchronous. However, upon formation of the cellular blastoderm and the onset of gastrulation, this synchrony breaks down. Subsequent cell divisions are synchronous within cohorts of adjacent cells, but cell cycles within adjacent 'mitotic domains' are out of register. During the first 13 synchronous cell cycles, maternally synthesized ORC1 is uniformly distributed among the embryonic nuclei. However, coincident with the formation of the cellular blastoderm and the onset of gastrulation, the ORC1 distribution changes dramatically, such that different nuclei contain very different levels of protein. For example, mesodermal precursors along the ventral midline, which comprise one of the mitotic domains, accumulate relatively high levels of protein at the onset of gastrulation. During germ band extension, ORC1 levels are highest among the mitotically active neuroblasts and in domains of epidermal precursor cells. Later, in stage 13 embryos, when most cells in the embryo are cell cycle arrested, ORC1 accumulates preferentially in cells of the nervous system and midgut that continue to cycle. In summary, the level of ORC1 in the Drosophila embryo is not constant as is the case in S. cerevisiae. Instead, the protein is developmentally regulated such that high levels of protein are found in proliferating cells (Asano, 1999).

Two lines of evidence suggest that E2F-dependent transcriptional regulation is responsible (at least in part) for this differential accumulation of ORC1. (1) In stage 13 E2F- embryos, essentially no ORC1 is detectable. (Analysis of E2F-dependence in earlier embryonic stages is confounded by the maternal supply of E2F.) (2) The pattern of ORC1 accumulation is mirrored by the patterns of ORC1 promoter activity and ORC1 mRNA accumulation during embryonic development. Therefore, it is concluded that E2F-dependent transcriptional regulation, at least in part, couples ORC1 accumulation to proliferation. The distribution of ORC1 was examined in eye-antennal imaginal discs, where a developmentally regulated cell cycle transition takes place as the morphogenetic furrow sweeps across the disc. The level of ORC1 changes dramatically during this G1-S transition. The level of protein initially is low among the G1-arrested cells in the furrow. As cells emerge from the furrow late in G1, the level of ORC1 rises. Following the completion of S phase, ORC1 levels fall, returning to the basal level seen in the furrow. Two additional observations suggest that these changes in ORC1 levels are not peculiar to cells in and around the morphogenetic furrow. (1) Cells with high and low levels of ORC1 are randomly interspersed in the anterior region of the eye disc and the antennal disc where cells cycle asynchronously. (2) Within the ZNC of the wing imaginal disc, cells arrested in G1 accumulate high levels of ORC1, whereas G2-arrested cells do not. High levels of ORC1 accumulate in cells 3-4 rows to the posterior of the furrow and S phase begins in cells 5-6 rows to the posterior. Since a new row of cells emerges from the furrow every 1.5 h, this suggests that ORC1 accumulates ~1.5-3 h before the onset of S phase. ORC1 levels fall only after the completion of S phase. In summary, the level of ORC1 is cell cycle regulated, with peak accumulation during late G1 and throughout S phase. Further overexpression studies show that the abundance of ORC1 regulates DNA synthesis. In wild-type discs, cells within the morphogenetic furrow never incorporate BrdU, and cells in the posterior region of the disc do so only rarely at this stage of development. However, in HS-ORC1 discs, some cells in both of these regions incorporate BrdU and thus appear to have entered S phase either prematurely or inappropriately. Ectopic ORC1 has no effect on either the onset or duration of the synchronous S phase among cells that emerge from the furrow. Nor does ectopic ORC1 have any noticeable effect on the proliferation of imaginal discs, the intensity of labeling at different BrdU concentrations or the growth rate of transgenic animals. Furthermore, the observation that endogenous ORC1 levels rise in anticipation of entry into S phase in the eye disc is consistent with the idea that high levels of ORC1 promote DNA synthesis rather than the opposite. As is the case in the imaginal discs, activity of the ORC1 promoter is E2F-dependent in the ovary (Asano, 1999).

Coordination of replication and transcription along a Drosophila chromosome: Origin recognition complex is localized to specific chromosomal sites, many of which coincide with early activating origins

The mechanisms by which metazoan origins of DNA replication are defined, regulated, and influenced by chromosomal events remain poorly understood. To gain insights into these mechanisms, a systematic approach was developed using a Drosophila high-resolution genomic microarray to determine replication timing, identify replication origins, and map protein-binding sites along a chromosome arm. A high-density genomic microarray was developed that covers the left arm of Drosophila chromosome 2 (representing 20% of Drosophila euchromatic sequence) with 11,243 nearly contiguous 1.5-kb PCR products. Because almost 90% of the nonrepetitive euchromatic sequence from chromosome 2L is represented on this array, it was possible to investigate replication timing at both inter- and intra-genic sequences. A defined temporal pattern of replication was identified that correlates with the density of active transcription. These data indicate that the influence of transcription status on replication timing is exerted over large domains (greater thatn 100 kb) rather than at the level of individual genes. This study identified 62 early activating replication origins across the chromosome by mapping sites of nucleotide incorporation during hydroxyurea arrest. Using genome-wide location analysis, it was demonstrated that the origin recognition complex (ORC) is localized to specific chromosomal sites, many of which coincide with early activating origins. The molecular attributes of ORC-binding sites include increased AT-content and association with a subset of RNA Pol II-binding sites. Based on these findings, it is suggested that the distribution of transcription along the chromosome acts locally to influence origin selection and globally to regulate origin activation (MacAlpine, 2004 ).

The replication timing data revealed clear early and late-replicating domains. These domains were often sharply defined by the density of transcription along the chromosome. The density of RNA Pol II along the chromosome was an order of magnitude greater at the earliest replicating sequences as compared with late-replicating regions. These differences suggest that the molecular architecture of the chromosome may define both the transcription and replication profiles of the chromosome. These transcriptionally active and early replicating domains may be physically marked by a change in chromatin structure that allows for increased access to both replication and transcription factors. It is possible that these domains are defined or restricted by elements of higher order chromosome structure, such as matrix attachment regions, transcriptional insulators, or chromatin loops. However, the state of the chromatin, whether euchromatic or heterochromatic, cannot be the sole determinant for origin activation, since there are examples of efficient heterochromatic origins in S. pombe. Interestingly, the gene-sparse, late-replicating regions identified in Kc cells overlap with late and under-replicated regions found in polytene salivary chromosomes. Taken together, these data suggest that the temporal program of replication is defined by chromatin structure and conserved in different Drosophila cell types (MacAlpine, 2004).

Hydroxylurea (HU) was used to arrest cells early in S phase and to restrict BrdU incorporation to sites overlapping and immediately adjacent to early origins of replication. Using this approach, this study identified 62 sites along the chromosome arm that are used as early replication origins. A recent study observed a change in the local pattern of origin usage at the adenylate deaminase2 locus in response to HU treatment. Although use of HU could have affected the set of origins identified, it is thought that this is unlikely: (1) the pattern of early and late-replicating regions observed using an HU-based protocol is similar to the pattern seen by others using approaches that did not involve replication inhibitors; (2) these studies used arresting concentrations of HU, unlike the hamster cell studies, in which lower concentrations of HU that slowed but did not completely arrest replication were used. Given that only a limited number of sites of BrdU incorporation are found under these arresting conditions, it is likely that only the earliest replicating origins are able to initiate before the intra-S-phase checkpoint prevents other origins from initiating. Finally, even if these origins represent only a subset of the origins along the chromosome, they are a valuable new resource, given the paucity of metazoan origins available prior to these studies (MacAlpine, 2004).

The findings provide clear evidence that ORC is localized to specific chromosomal regions. Consistent with the role of ORC as an essential initiator, this complex is found localized to the majority of early replicating origins, most often at or near the apex of BrdU incorporation. Although ORC was not detected at 27% of the early origins, it is not believed that these represent sites of ORC independent initiation, but rather a limitation of the ChIP technology. By no means have all of the ORC-binding sites along the chromosome been exhaustively identified, since many ORC sites are likely to be occluded from antibody access by additional chromatin-binding complexes. In addition, many ORC-binding sites are likely present in the repetitive and low-complexity sequences that are necessarily omitted from the array (MacAlpine, 2004).

In contrast to S. cerevisiae, where ORC binds discretely to single sites along the chromosome, significant clustering of ORC is seen along the chromosome. Almost 20% of the identified ORC-associated sequences were immediately adjacent to other ORC-associated sequences. This clustering of ORC along the chromosome was also observed at extra chromosomal copies of the ACE3 locus in amplifying follicle cells. Because the ORC-associated sequences often span greater than 3 kb, trivial factors cannot be ruled out such as shear size of the chromatin immunoprecipitated DNA. These clusters of ORC-associated sequence may represent unique chromatin environments favorable to ORC binding, or polymerization of ORC on the DNA following a nucleation event at a specific site (MacAlpine, 2004).

The type of ORC association may influence the nature of replication initiation at a particular locus. It was found that 36% of the early origins contain clusters of three or more ORC-binding sites. For example, at oriDalpha, ORC was continuously associated with a 10-kb region that overlapped a broad region of BrdU incorporation. Interestingly, the analysis of replication intermediates by two-dimensional gel electrophoresis in this region revealed multiple initiation sites over the entire region. In contrast, at the origin identified upstream of the chorion locus, three separate peaks of BrdU incorporation were each marked by distinct ORC-binding sites. This form of ORC association with origins may be analogous to the human lamin B2 locus, where replication initiates at a discrete site. Thus, the origin at oriD and the origin upstream of the chorion locus may represent two distinct types of origins, those defined by broad domains of ORC binding and those associated with more discrete ORC-binding sites (MacAlpine, 2004).

Despite finding ORC at specific regions along the chromosome, the exact mechanism that leads to ORC localization remains to be determined. There are multiple molecular characteristics of the sites of ORC localization, including increased AT-content, noncoding DNA, and RNA Pol II association. These molecular predictors of ORC association could be directly involved in ORC DNA binding, could bind to one or more factors that facilitate ORC localization, or could be required for another origin-related function (e.g., DNA unwinding). It is important to note, however, that none of these attributes are individually sufficient to identify ORC-binding sites. For example, high AT-content by itself is insufficient to define an ORC-binding site, since many sequences on the array have AT-content greater than 62%, but are not represented in the ORC-associated sequences. However, ORC was seemingly excluded from sequences with low AT-content, suggesting that increased AT-content is necessary, but not sufficient for ORC association. Indeed, it has not been possilbe to identify a consensus sequence within the 491 ORC-bound DNA sequences. The lack of a consensus sequence is consistent with the observation that metazoan ORC has only limited sequence specificity in vitro. It is proposed that the attributes that have been identified cooperate to define sites of ORC localization. It is certain that there are additional determinants that were not identified in these studies. For example, the topology of DNA can strongly influence Drosophila ORC binding. Nevertheless, the availability of numerous known Drosophila ORC-binding sites associated with origins of replication will greatly facilitate future studies of ORC localization and origin function (MacAlpine, 2004).

Because only a small subset of origins are likely to initiate in the presence of HU, it is not surprising that only a subset of ORC-binding sites are associated with the early replicating regions. It is anticipated that many of the remaining ORC-binding sites are associated with origins that fire later in S phase. The methods used in this study did not allow the confident identification of late or inefficient origins. However, studies in S. cerevisiae have shown that abrogation of the intra-S-phase checkpoint results in the activation of late-replication origins in the presence of HU, suggesting that a similar approach could be useful for identifying late-activating metazoan origins. In addition, it is possible that a subset of the ORC-binding sites that were identified are involved in other functions, such as gene regulation or the establishment of heterochromatin (MacAlpine, 2004).

It is proposed that the frequent colocalization of ORC and RNA Pol II reflects a connection between transcription and ORC localization. Although it is possible that there is a direct interaction between ORC and RNA Pol II, no such interaction was observed in coimmunoprecipitation assays. In addition, the majority of the sites of RNA Pol II association do not interact with ORC. An alternative hypothesis is that ORC localization is, at least in part, facilitated by a subset of the transcription factors that serve to localize RNA Pol II. Indeed, previous studies have shown that both Drosophila E2F1 and Myb interact with ORC; however, ORC is still localized to the chorion locus during amplification in Myb mutants and mutants of E2F1 that do not interact with ORC. One possible explanation for these findings is that Myb and E2F1 act redundantly to recruit ORC throughout the genome. It is proposed that ORC, like RNA Pol II, can be recruited by many different transcription factors, which would lead to the frequent colocalization with RNA Pol II, but not any particular transcription factor. These factors could recruit ORC by direct interaction or by establishing a chromatin domain that is conducive to ORC recruitment (MacAlpine, 2004).

These findings support a connection between the molecular architecture of the chromosome and the replication process at two levels: (1) the frequent colocalization of ORC and RNA Pol II leads to the hypothesis that nearby transcription factor-binding sites influence the earliest steps of origin selection by facilitating ORC localization and subsequently pre-RC formation; (2) the decision of when each origin initiates replication during S phase (which is mechanistically separate from ORC localization and the assembly of pre-RCs in G1 phase) is connected to transcriptional status in a more global manner. The more transcriptionally active the chromosomal region, the greater the likelihood that replication initiation will occur early in S phase within that domain. The findings indicate that transcriptional status is integrated over broad regions (greater than 100 kb) of the chromosome (rather than individual genes) to determine the time of replication of each chromosomal locus. Further exploration of the connection between higher order chromosome structure and DNA replication will provide insights into the coordination of the molecular events that must occur to propagate and maintain genomic information (MacAlpine, 2004).

Transcriptional regulation of the Drosophila orc2 gene by the DREF pathway

DNA replication-related element (DRE) and the DRE-binding factor (DREF) play an important role in regulating DNA replication-related genes such as PCNA and DNA polymerase alpha in Drosophila. Overexpression of DREF in developing eye imaginal discs induces ectopic DNA synthesis and apoptosis, which results in rough eyes. To identify genetic interactants with the DREF gene, a screen was carried out for modifiers of the rough eye phenotype. One of the suppressor genes identified was the Drosophila orc2 gene. A search for known transcription factor recognition sites revealed that the orc2 gene contains three DREs, named DRE1 (+14 to +21), DRE2 (-205 to -198), and DRE3 (-709 to -702). Band mobility shift analysis using Kc cell nuclear extracts detected the specific complex formed between DREF and the DRE1 or DRE2. Specific binding of DREF to genomic region containing the DRE1 or DRE2 was further demonstrated by chromatin immunoprecipitation assays, suggesting that these are the genuine complexes formed in vivo. The luciferase assay in Kc cells indicated that the DRE sites in the orc2 promoter are involved in a transcriptional regulation of the orc2 gene. The results, taken together, demonstrate that the orc2 gene is under the control of DREF pathway (Okudaira, 2005).

Chromatin signatures of the Drosophila replication program

DNA replication initiates from thousands of start sites throughout the Drosophila genome and must be coordinated with other ongoing nuclear processes such as transcription to ensure genetic and epigenetic inheritance. Considerable progress has been made toward understanding how chromatin modifications regulate the transcription program; in contrast, relatively little is known about the role of the chromatin landscape in defining how start sites of DNA replication are selected and regulated. This study describes the Drosophila replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. While the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. Chromatin features were identified that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. Accurate and predictive models were generated from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events (Eaton, 2011).

The chromatin landscape clearly impacts both the expression and the replication of the genome. For example, the transcriptionally active euchromatin typically replicates prior to the repressed heterochromatic sequences. Studies in yeast, Drosophila, and mammalian systems have shown that changes in histone acetylation are associated with changes in the replication program. However, a comprehensive view of the replication program in the context of chromatin modifications and DNA-binding proteins is lacking (Eaton, 2011).

The different modENCODE data types across multiple cell lines (The modENCODE Consortium 2010) allowed the definition of the chromatin and transcription landscape associated with features of the DNA replication program. For each replication data type (replication timing, early origins, and ORC binding), a 43 × 3 matrix was generated, with each column representing a specific cell line and each row representing the enrichment or correlations with chromatin marks, DNA-binding proteins, nucleosome density, histone variants, nucleosome turnover (CATCH-IT), and gene expression (RNA-seq). For the replication timing profiles where there are no discrete peak calls, the Spearman's correlation was calculated between each factor with the whole-genome replication timing profile. For early origins of replication and ORC-binding sites, the median log2 enrichment was calculated of each factor within all BrdU peaks and within 500 bp of ORC ChIP-seq peak centers, respectively (Eaton, 2011).

The selection and regulation of DNA replication origins was found to be associated with distinct sets of chromatin marks and DNA-binding proteins. Prior studies have associated early replication with active transcription and the presence of 'activating' chromatin modifications such as histone acetylation, whereas late replication is associated with 'repressive' chromatin marks such as those found in the heterochromatin. Indeed, this study found that gene expression is positively correlated with replication timing, as are generally euchromatic marks such as H3K4me1 and H3K18ac. In contrast, heterochromatic marks such as H3K27me3 and H3K9me2 are negatively correlated with replication timing. The sequences surrounding early origins were also enriched for activating chromatin marks as well as specific DNA-binding proteins, including chromatin remodeling factors (Eaton, 2011).

Because many of the ORC-binding sites colocalized with promoters of active genes, the ORC-binding sites were separated into those that are TSS proximal (within 1 kb of a TSS) and those that were not at a TSS (distal). Particular interest was placed on chromatin features that are shared between ORC-binding sites both proximal and distal to promoters. Additionally, marks that are specific to ORC sites distal from a promoter will be of interest, as these marks may be required for ORC binding or function in the absence of a promoter (Eaton, 2011).

ORC-binding sites proximal to TSSs were enriched for chromatin remodelers such as the NURF complex (NURF301 [also known as E(BX)], ISWI) as well as other DNA-binding proteins such as GAF, RNA Pol II, and CHRO. These TSS-associated ORC sites were also enriched for H3K9ac, H3K27ac, H3K4me2, and H3K4me3 -- marks frequently found at promoters. Interestingly, those ORC sites that did not overlap with a TSS (distal) were also enriched for chromatin remodelers ISWI and NURF301, as well as GAF, which has also been implicated in chromatin remodeling. Consistent with the idea of ORC localizing to dynamic and active chromatin, an enrichment was found for CATCH-IT and H3.3 at ORC sites both proximal and distal to TSSs, as well as a reduction in bulk nucleosome occupancy. ORC sites not located at promoters were enriched for many of the same histone marks as those at promoters, with a few notable exceptions. A decrease in H3K4me3 was found at ORC sites distal from a promoter, as well as an increase in H3K18ac and H3K4me1 (Eaton, 2011).

Chromatin features specific to transcription start sites such as RNA Pol II and H2Av were decreased at ORC-binding sites distal to promoter elements. A small amount of RNA Pol II signal remained in the TSS distal ORC-binding sites; however, in comparison to the local enrichment of ISWI and GAF, there was a clear reduction in local signal. The remaining signal may be due to unannotated transcription start sites (Eaton, 2011).

Chromatin marks that are associated with active transcription through gene bodies (e.g., H3K79me1, H3K36me1, and H3K36me3) were not found above background levels at any ORC-binding sites. However, H3K36me1 was found specifically flanking those ORC-binding sites that did not coincide with a TSS. ORC has been shown to facilitate the formation of heterochromatin and HP1 binding; however, ORC sites were depleted for heterochromatic histone modifications such as H3K27me3 and H3K9me2/3 and were only slightly enriched for HP1. This may be due, in part, to the inability to map distinct ORC-binding sites in repetitive sequences, a current limitation of high-throughput sequencing approaches (Eaton, 2011).

The chromatin signatures were examined of promoter elements with and without ORC associated to determine whether there were unique chromatin signatures specific for ORC associated promoters. Since those promoters with proximal ORC binding tend to be far more actively transcribed than those without ORC, the comparison was limited to active promoter elements only. It was found that ORC-associated promoters had modestly increased chromatin remodeling activities, decreased nucleosome occupancy, and greater evidence of nucleosome turn-over relative to other active promoters not associated with ORC. In summary, these results indicate that dynamic chromatin environments may contribute to ORC localization and the subsequent activation of replication origins (Eaton, 2011).

Integrative analysis of gene amplification in Drosophila follicle cells: parameters of origin activation and repression

In metazoans, how replication origins are specified and subsequently activated is not well understood. Drosophila amplicons in follicle cells (DAFCs) are genomic regions that undergo rereplication to increase DNA copy number. All DAFCs were identified by comparative genomic hybridization, uncovering two new amplicons in addition to four known previously. The complete identification of all DAFCs enabled investigation of these in vivo replicons with respect to parameters of transcription, localization of the origin recognition complex (ORC), and histone acetylation, yielding important insights into gene amplification as a metazoan replication model. Significantly, ORC is bound across domains spanning 10 or more kilobases at the DAFC rather than at a specific site. Additionally, ORC is bound at many regions that do not undergo amplification, and, in contrast to cell culture, these regions do not correlate with high gene expression. As a developmental strategy, gene amplification is not the predominant means of achieving high expression levels, even in cells capable of amplification. Intriguingly, it was found that, in some strains, a new amplicon, DAFC-22B, does not amplify, a consequence of distant repression of ORC binding and origin activation. This repression is alleviated when a fragment containing the origin is placed in different genomic contexts (Kim, 2011; full text of article).

Protein Interactions

Assembly of functionally active Drosophila origin recognition complex from recombinant proteins

In eukaryotes the sites for the initiation of chromosomal DNA replication are believed to be determined in part by the binding of a heteromeric origin recognition complex (ORC) to DNA. The genes encoding the subunits of the Drosophila ORC were cloned. Each of the genes is unique and can be mapped to discrete chromosomal locations implying that the pattern and developmental regulation of origin usage in Drosophila is not regulated solely by a large family of different ORC proteins. The six-subunit ORC can be reconstituted with recombinant proteins into a complex that restores DNA replication in ORC-depleted Drosophila or Xenopus egg extracts (Chesnokov, 1999).

With complete cDNAs for each of the Drosophila ORC subunits available it was of interest to determine if coexpression of the genes from baculovirus vectors would be sufficient for complex formation. Each of the genes was expressed individually; only ORC2 and ORC6 were found to be readily soluble proteins. However, upon coinfection of all six viral vectors, each of which carried a unique ORC subunit gene, all other proteins (i.e., ORC1, ORC3-ORC5) remained soluble and readily formed a complex. A His-tagged version of ORC1 was used to simplify purification. The six subunits cosediment as does the native material. (Chesnokov, 1999).

ORC1 appears to be critical for establishing a tight ternary complex betweeen ORC, DNA and chromatin

The origin recognition complex (ORC) is the DNA replication initiator protein in eukaryotes. A functional recombinant Drosophila ORC has been reconstituted and activities of the wild-type and several mutant ORC variants have been compared. Drosophila ORC is an ATPase, and the ORC1 subunit is essential for ATP hydrolysis and for ATP-dependent DNA binding. Moreover, DNA binding by ORC reduces its ATP hydrolysis activity. In vitro, ORC binds to chromatin in an ATP-dependent manner, and this process depends on the functional AAA+ nucleotide-binding domain of ORC1. Mutations in the ATP-binding domain of ORC1 are unable to support cell-free DNA replication. However, mutations in the putative ATP-binding domain of either the ORC4 or ORC5 subunits do not affect either of these functions. Evidence is provided that the Drosophila ORC6 subunit is directly required for all of these activities and that a large pool of ORC6 is present in the cytoplasm, cytologically proximal to the cell membrane. Studies reported here provide the first functional dissection of a metazoan initiator and highlight the basic conserved and divergent features between Drosophila and budding yeast ORC complexes (Chesnokov, 2001).

Six different mutant complexes and wild-type recombinant ORC were prepared. For each case, simultaneous expression of the wild-type or mutant genes in a baculovirus expression system results in complexes that could be purified to homogeneity through four chromatographic steps, and the mutant complexes assemble and exhibite no chromatographic differences during the purification. In a final step, the pooled peak fractions were subjected to glycerol-gradient sedimentation (Chesnokov, 2001).

The best understood functions of the yeast ORC are its DNA-binding and ATP hydrolysis functions. The bulk of recombinant (or purified embryonic) Drosophila ORC DNA binding activity is nonspecific and ATP-independent. However, this ATP-independent DNA binding activity can be titrated away with sufficient amount of carrier DNA when the carrier DNA is in a range 50-100 molar excess to the probe DNA. At physiologically relevant ATP concentrations (10 microM to 1 mM), the wild-type ORC binds to DNA 10-50-fold better than either the ORC1A or ORC1B mutant complex. Mutations in either the Walker A or B motif of ORC4 or the Walker A motif of ORC5 have no effect on the formation of ATP-dependent DNA-protein complex. These experiments support the idea that the recombinant Drosophila ORC, like the recombinant S. cerevisiae homolog, requires only the ORC1 component of the complex to bind ATP for tight DNA interactions. However, the complex missing the ORC6 subunit does not form an ATP-dependent DNA-protein complex (Chesnokov, 2001).

Kinetic analysis of ATP hydrolysis with multiple independent wild-type (wt) ORC preparations shows a Km of 1.92 µM and a Vmax of 0.4 mol ATP hydrolyzed per min per mol of complex. Binding to DNA has a small (2-fold) but measurable effect on slowing the rate of ATP hydrolysis by ORC. In these experiments, ATP was not limiting: the mutant ORC complexed to DNA was titrated to its maximal effect. In the absence of any carrier DNA, the saturation is reached at an approximate 2.5-fold molar excess of DNA to ORC. Complexes harboring similar mutations in either ORC4 or ORC5 hydrolyze ATP with equivalent kinetics to wild type, all displaying Km values and Vmax within the experimental error range of wild type. Consistent with the DNA-binding experiments, the ATP-hydrolysis rate for these mutant complexes is slowed by DNA similar to the effect observed for the wild-type ORC. In contrast, ORC1A or ORC1B mutants have severely crippled enzymatic activity, too close to background to measure any kinetic parameters. The ORC-6 complex is able to hydrolyze ATP at reduced levels, but this activity is unaffected by DNA, consistent with the finding that ORC6 is critical for formation of an ATP-dependent ternary complex (Chesnokov, 2001).

Chromatin binding assays were performed by using both mutant and wt ORC in extracts depleted of membranes. For these experiments Drosophila preblastula embryo extracts were immunodepleted of ORC by using antibody raised against ORC2 and ORC6. The effectiveness of immunodepletion was verified by immunoblotting. Demembranated sperm chromatin was added to the depleted extracts, and the binding activities of mutant and wild-type recombinant DmORC were compared with the endogenous Drosophila ORC. Treatment of the extracts with Apyrase abolishes ORC-chromatin binding, thus it is inferred that the binding process requires ATP. Endogenous ATP levels (which are estimated to be at 30-50 µM) were relied upon to mediate tight chromatin binding. Proteins associated with the chromosomes are separated from the unbound proteins by sedimentation. The results obtained via this assay parallel those obtained by the gel-shift experiments. Recombinant wt ORC, ORC4A, ORC4B, and ORC5A complexes associate with the chromatin with apparently the same efficiency as does endogenous protein, whereas the ORC1A, ORC1B, and ORC 6 complexes are severely crippled (Chesnokov, 2001).

Two independent measures of DNA replication competence were used for accessing the abilities of the mutant complexes to restore activity to depleted extracts. In the first assay, labeled precursor incorporation into high molecular DNA was detected by autoradiography of gels after electrophoresis or in a second assay after CsCl density gradient separation of DNA that was replicated in extracts with the density label precursor BrdUrd. As anticipated from the DNA and chromatin binding results, the ORC1A, ORC1B, and ORCdelta6 complexes were essentially inactive by at least 10-20-fold below the activity of wt recombinant ORC in restoring replication to the extracts. The ORC4A, ORC4B, and ORC5A mutants were effective in reconstitution but were in multiple experiments between 50% and 90% of wild-type complex (Chesnokov, 2001).

It has been concluded that the bulk of the subunits of the Drosophila ORC biochemically behave as a complex. ORC2 antibodies were used to track ORC in fractions from 0-12-h embryo extracts after gel-filtration chromatography. Two broad zones containing ORC were found. The highest apparent molecular weight fractions containing all ORC subunits were pooled and purified. A smaller complex was also detected that was apparently without ORC-1. However, when following ORC6 using ORC6-specific antibodies, a pool of ORC6 was devoid of other ORC subunits is detected. No other ORC subunits were found in a form unassociated with other ORC proteins. It is estimated that this free pool is at least one-half of the total ORC6 protein present in these extracts. Given the important role that Drosophila ORC6 plays in cell-free replication and the other activities of ORC, it was of interest to ask whether this separate pool of ORC6 is localized with the other ORC subunits in the cell (Chesnokov, 2001).

Transient ectopic expression of ORC1 or ORC2 GFP-fusion proteins in cultured cells shows a distinct nuclear localization; in unexpected contrast, the GFP-ORC6 fusion protein was found both in the nucleus and cytoplasm. The ORC6 cytoplasmic signal seems to be closely associated, in various focal planes, with the cytoplasmic membranes. These experiments rely on overexpression: this issue was probed further by direct immunofluorescence of endogenous levels of the ORC proteins in Drosophila embryos. Before the onset of cellularization, ORC6 protein localizes only with ORC2 in the nuclear space of both interphase and mitotic cells. However, after cellularization, ORC6 seems to localize in the cytoplasm and nucleus. The signals for ORC6 can be blocked by preincubating the affinity-purified antibodies with recombinant ORC6 proteins and are clearly distinct from the ORC2 pattern. Further work will be required to judge whether the cytoplasmic pool of ORC6 is truly membrane associated, but it is worth noting that the carboxyl terminus of Drosophila ORC6 contains a predicted leucine-zipper region that could be involved in mediating multiple heterologous protein-protein interactions (Chesnokov, 2001).

An important finding of this study is that the Drosophila ORC complex likely uses mechanisms for binding DNA that are similar to those reported for the budding yeast homolog. Of the three potential ATP binding proteins in ORC, only ORC1 seems to be critical for establishing a tight ternary complex with DNA and for binding to chromatin. Similarly, only mutations in the ATP binding domains of ORC1 critically affect a single round of DNA replication in cell-free extracts. Additional experimentation needs to be done to test the roles of the conserved domains in ORC4 and ORC5. Particularly intriguing is the wide conservation of the GKT (Walker A motif) and D (D/EE) (Walker B motif) in the ORC4 subunit. Such domains may be critical for recycling ORC for subsequent rounds of replication or for other activities of the complex in heterochromatin formation or putative check-point control. Drosophila ORC is an ATPase, and again ORC1 seems to play the critical role for ATP hydrolysis, since mutants in the putative ATPase domains of ORC4 and ORC5 do not affect the kinetic parameters of the mutant complex. Nevertheless, it is possible that in the presence of other bound factors, ATP binding or hydrolysis by the other subunits plays some critical role. ATP hydrolysis by any subunit does not seem important for DNA-binding activity. ADP could not mediate such a DNA-protein complex, and ATPgammaS is better at forming a ternary complex than ATP. X-ray crystallographic structure models for several AAA+ proteins have been solved, and a common fold has been observed. The crystal structure model of an archael Cdc6 ortholog was used as a guide for the ATP-binding structures of ORC1. In the nucleotide-binding domain of this protein family, both the GKT and the DE motifs contribute to nucleotide affinity. In fact, similar mutants in the amino-part of the Walker B motif of the S. cerevisiae ORC1 are defective for ATP binding, in contrast to mutations at the carboxyl end of the B motif that are competent for such activity. Moreover, the solvent-exposed surfaces present in these parts of the ORC1 protein may influence interactions with other partners, yielding a mutant complex with altered functions. These studies of the ATPase activity of DmORC indicate that turnover is slower when ORC is bound to DNA, but the effect is significantly less than that observed for the budding yeast complex. Divergence in the way in which these proteins interact with DNA is also highlighted by the critical role that the Drosophila ORC6 protein plays in ATP-dependent DNA binding. Perhaps, given the lack of amino acid homologies found between the ScORC6 and DmORC6 proteins, it is dangerous to consider each to be homologs (Chesnokov, 2001).

Overexpression of ORC1 trans-genes in Drosophila can alter DNA replication patterns. This overexpression leads to detectable levels of BrdUrd incorporation in normally quiescent cells or increased levels of replication in follicle cells normally amplifying the chorion genes. Similar ectopic expression of an ORC1A mutant (ORC1K604E) has no phenotype. The biochemical results with the ORC1A mutant K604A predict that their mutation might have a dominant negative effect on DNA replication in vivo. It is possible that the mutant gene would not be antimorphic by virtue of its not being able to compete with a wild-type ORC1 protein for assembly into complex. Leaving this point aside, one idea favored is that ORC1 is limiting for replication in some cellular environments and, for example, complexes containing solely ORC2-6 wait for ORC1 for activation. These pools may or may not be bound to chromosomal DNA. Recent work in mammalian systems indicates that ORC1 may be more loosely associated with chromatin than is ORC2. ORC2, presumably with some of the subunits, can be pelleted with the chromosomes. The results reveal that intact ORC needs ATP and functional ORC1 to bind tightly to chromatin. Are all of these data compatible, assuming a conservation in basic binding properties for ORC between mammals and Drosophila? Perhaps, in the absence of ORC1 other subunits mediate another sort of chromatin association. More complex notions are possible, including the interaction of unknown chromatin binding proteins that serve to tether a complex lacking ORC1 to the origin sites (Chesnokov, 2001).

It is suggested that ORC6 is another subunit that may play important and perhaps dynamic roles in regulating replication activity. The data show that ORC6 is an essential component of the complex per se and may be directly involved in DNA binding and other replication functions or needed for proper ORC assembly. In H. sapiens extracts, ORC6 is not found associated with other ORC subunits, but when expressed in the baculovirus system with the other ORC genes, the protein does join a six-subunit complex. The high levels of free ORC6 in embryonic and cultured cell extracts is intriguing. A considerable fraction of this pool as judged by cytological methods is cytoplasmic, and the protein is perhaps associated with or proximal to the cytoplasmic membranes. It is possible that this localization enables ORC6 to participate in functions unrelated to DNA replication per se, as has been suggested for the 'latheo' gene product, which is ORC3. Latheo seems to be involved in ion transport at neuromuscular junctions. Data now exist for both the budding yeast and for the Drosophila ORC, which directly indicate that all of the subunits are critical for DNA replication function, and complex models involving traffic of subsets of ORC subunits can be the subject of future work (Chesnokov, 2001).

Association of the highly conserved heterochromatin protein, HP1, with the specialized chromatin of centromeres and telomeres requires binding to a specific histone H3 modification of methylation on lysine 9. This modification is catalyzed by the Drosophila Su(var)3-9 gene product and its homologues. Specific DNA binding activities are also likely to be required for targeting this activity along with HP1 to specific chromosomal regions. The Drosophila HOAP protein is a DNA-binding protein that was identified as a component of a multiprotein complex of HP1 containing Drosophila origin recognition complex (ORC) subunits in the early Drosophila embryo. Direct physical interactions are demonstrated between the HOAP protein and HP1 and specific ORC subunits, including Orc1, Orc3 and Orc6. Two additional HP1-like proteins (HP1b and HP1c) were recently identified in Drosophila, and the unique chromosomal distribution of each isoform is determined by two independently acting HP1 domains (hinge and chromoshadow domain). Heterochromatin protein 1/origin recognition complex-associated protein (HOAP) is found to interact specifically with the originally described predominantly heterochromatic HP1a protein. Both the hinge and chromoshadow domains of HP1a are required for its interaction with HOAP, and a novel peptide repeat located in the carboxyl terminus of the HOAP protein is required for the interaction with the HP1 hinge domain. Peptides that interfere with HP1a/HOAP interactions in co-precipitation experiments also displace HP1 from the heterochromatic chromocenter of polytene chromosomes in larval salivary glands. A mutant for the HOAP protein also suppresses centric heterochromatin-induced silencing, supporting a role for HOAP in centric heterochromatin (Badugu, 2003).

Regulated Proteolysis of ORC1: Degradation during G1

The kinetics of ORC1 expression in cells is consistent with the idea that ORC1 accumulates at the G1/S boundary immediately after a pulse of E2F-dependent transcription and persists until its catastrophic destruction at the M/G1 boundary. In this scenario, cell cycle-modulated transcription and proteolysis both contribute to setting the level of ORC1. This idea was tested directly by uncoupling the expression of orc1 mRNA from its normal transcriptional signals. To this end, constitutive expression of ORC1-GFP was driven using the GMR promoter, which is turned on in all cells posterior to the morphogenetic furrow in the eye disc, and the distribution of green fluorescence was visualized both in situ and in FACS experiments (Araki, 2003).

Surprisingly, the accumulation of ORC1-GFP is essentially the same whether transcription is driven transiently by the orc1 promoter or constitutively by the GMR promoter. Protein levels first rise and then fall during the synchronous cycle in the eye disc as many of the cells go through mitosis. The majority of the cells are then in G1, with neither detectable CycB nor ORC1-GFP, despite persistent transcription in the case of the ORC1-GFP transgene. A minor population of cells in the posterior region of the disc reside in a prolonged G2 arrest upon emerging from the morphogenetic furrow, since they bear high levels of CycB. Unlike the neighboring G1-arrested cells, these have persistent ORC1-GFP. Taken together, these observations: (1) confirm the stability of ORC1-GFP in G2; and (2) imply that cell cycle-mediated destruction of ORC1-GFP occurs not only at the M/G1 transition but continues into G1, even though the endogenous ORC1 substrate is normally present at negligible levels during this period due to inactivity of the ORC1 promoter. As a control, expression of GFP bearing a nuclear localization sequence (NLS) results in accumulation of protein to essentially the same level in all cells posterior to the furrow, regardless of their cell cycle phase. Thus, cell cycle-dependent proteolysis of ORC1-GFP overrides constitutive transcription (Araki, 2003).

The results of FACS analysis of eye antennal discs that constitutively express ORC1-GFP under GMR control support the view that ORC1 is degraded from the M/G1 boundary throughout G1. The stable control protein, GFP, is found primarily in the G1 cells that predominate in the region of the disc where the GMR promoter is active. Despite this preponderance of G1 cells, ORC1-GFP is found almost entirely in the minor population of G2/M cells. Because the eye disc cells posterior to the morphogenetic furrow constitute a developmentally unusual population, it was of interest to ascertain the behavior of ORC1-GFP upon constitutive expression in a more typical proliferating population of imaginal disc cells. To this end, expression of ORC1-GFP (and, as a control, GFP) were driven with the engrailed (en) promoter, which is constitutively active in cells of the posterior compartment during the third larval instar. ORC1-GFP is essentially absent from G1 cells in these discs, whereas GFP is stable throughout the cell cycle. Thus, the instability of ORC1-GFP during G1 appears to be a general property of imaginal disc cells (Araki, 2003).

Several additional experiments demonstrate that the behavior of ORC1-GFP is exceptional. (1) ORC2-GFP behaves like the GFP control protein when driven by the GMR promoter, accumulating uniformly in every cell posterior to the furrow. (2) Constitutive transcription from transgenes encoding other cell cycle-regulated proteins is not masked by proteolysis. In particular, expression of CycE-GFP, E2F-GFP or untagged versions of the same proteins under GMR transcriptional control results in uniform protein accumulation throughout the posterior of the eye disc. In summary, the system responsible for degrading endogenous ORC1 at the end of M phase apparently is vigorous, active throughout G1 and relatively specific for ORC1 (Araki, 2003).

When expressed under GMR promoter control, the ORC1-GFP fusion is detectable until somewhat later in M phase than is endogenous ORC1. Early in M phase ORC1-GFP co-localizes with CycB off the chromatin following nuclear envelope breakdown. Subsequently, CycB drops beneath the level of detection but ORC1-GFP appears to reassociate briefly with the DNA when it forms pairs of tightly condensed spheres that presumably correspond to late telophase nuclei. This residual ORC1-GFP is then abruptly degraded as the cells divide and enter G1. Accumulation of ORC1-GFP in presumptive telophase nuclei is also apparent when transcription is driven by the ORC1 promoter. It is not known whether this minor difference in the behavior of endogenous ORC1 and ORC1-GFP is due to the ability to detect lower levels of ORC1-GFP, to enhanced transcription or to (modestly) enhanced stability of ORC1 by attachment of the GFP moiety. In any case, by using GMR to drive ORC1 expression, it is found that ORC1-GFP levels fall precipitously upon exit from mitosis and that protein newly synthesized in G1 is also rapidly degraded and thus fails to accumulate to an appreciable extent (Araki, 2003).

ORC1 is a member of a superfamily of ATP-binding proteins involved in DNA replication control that also includes ORC4, ORC5 and CDC6. Homology among these proteins is restricted to the C-terminal portion of ORC1, which bears the Walker A and B motifs. The role of the the N-terminal ORC1 domain is not known for any experimental system, although, in the case of human ORC1, it has been suggested that the N-terminal domain harbors signals that mediate its degradation during S phase (Mendez, 2002). Because Drosophila ORC1 is degraded at a different time in the cell cycle, it was of interest to determine whether the relevant signals also reside in the N-terminal region of the protein or whether they are embedded in the conserved C-terminal domain (Araki, 2003).

To map the degradation signals in Drosophila ORC1, transgenes that encode either the N- or C-terminal domain of ORC1 fused to GFP were prepared and transcription was driven in eye imaginal discs using the GMR promoter. The stability of these derivatives was monitored both by examination of GFP fluorescence in situ and by FACS analysis of dissociated disc cells. Both assays reveal that ORC1N is regulated in a manner essentially indistinguishable from the full-length protein, degraded at the M/G1 boundary as well as throughout G1. In contrast, ORC1C is stable throughout the cell cycle, although it is predominantly cytoplasmic, presumably because it lacks a functional NLS. To rule out the possibility that ORC1C is protected by virtue of its nuclear exclusion, sequences that encode the SV40 NLS were appended to the appropriate transgene and accumulation of the encoded protein throughout the cell cycle was monitored. ORC1C-NLS is stable throughout the cell cycle when targeted to the nucleus. Analysis of the level of protein and mRNA for each ORC1-GFP derivative supports the idea that the difference in steady-state accumulation of proteins bearing the ORC1 N-terminus is generated post-transcriptionally (Araki, 2003).

In summary, the signals that mediate degradation of both human and Drosophila ORC1 appear to reside in the N-terminal domain of each protein, even though they are degraded at different stages of the cell cycle (Araki, 2003).

In cultured cells, human ORC1 is degraded during S phase by Skp2-dependent SCF activity (2002). In contrast, the timing of ORC1 degradation in Drosophila strongly implies degradation by the APC, which degrades mitotic cyclins and securin to promote passage through and exit from mitosis. The APC is generally thought to be activated in succession, first by Fizzy (Fzy)/Cdc20, which promotes passage out of metaphase, and subsequently by Fizzy-related (Fzr)/Cdh1, which promotes exit from mitosis and suppresses CycB accumulation into G1. The ultimate consequence of APC activity is proteasome-dependent degradation of targeted substrates. The degradation of ectopically expressed ORC1 in G1 suggests the involvement of Fzr (Araki, 2003).

To examine the role of Fzr, the behavior of ORC1 was examined in mutant animals lacking Fzr activity. fzr mutants die in late embryogenesis, long before the imaginal discs can be studied; it also seems unlikely that fzr mutant somatic clones would proliferate and survive, precluding analysis in mosaic imaginal discs. Therefore, epithelial cells in stage 12-13 embryos were examined as they exited from M phase of division cycle 16 into G1 of cycle 17 (Araki, 2003). Whether ORC1 and ORC1-GFP behave in a similar manner in these embryonic cells as in proliferating imaginal disc cells was examined. In wild-type stage 12-13 embryos, most of the dorsal epithelial cells have entered a prolonged G1 arrest, with only a few cells still proliferating. These laggards have both detectable ORC1 and CycB, and thus are in S or G2 of cycle 16, whereas most of their neighbours have entered the prolonged G1 of cycle 17 and have neither protein. When expressed under the transcriptional control of the strong, constitutive actin5C promoter, ORC1-GFP accumulates to high levels only in the few epithelial cells still in S or G2, whereas the protein is destabilized in the remaining (G1) cells. The selective degradation of ORC1-GFP in G1 is mediated by N-terminal signals, as is the case in imaginal disc cells. Analysis of Western and Northern blots of embryonic samples is consistent with the idea that regulation of ORC1 accumulation is post-transcriptional. Thus, the stability of both the endogenous ORC1 and ectopically expressed ORC1-GFP is regulated in essentially the same fashion in the embryo and the imaginal disc (Araki, 2003).

Next, the dependence of ORC1 and CycB degradation on Fzr was examined, comparing the accumulation of each protein in sibling wild-type and fzr mutant embryos. Essentially every epithelial cell in fzr mutant embryos has appreciable levels of both ORC1 and CycB. This observation is consistent with the idea that ORC1 degradation is dependent on Fzr. However, loss of Fzr activity perturbs the cell cycle, promoting epithelial cells into an extra division. The accumulation of ORC1 might simply correlate with the progression of these cells into S and G2, where ORC1 has been shown to be stabilized (Araki, 2003).

To better test the role of Fzr in regulating ORC1 stability in vivo, it was desirable to perturb its activity without causing attendant dramatic changes in the cell cycle profile. To this end, either ORC1-GFP or the stable ORC1C-NLS-GFP derivative (as a control) were co-expressed in the eye imaginal disc with either Fzr or Rca1, a specific inhibitor of Fzr. Overexpression of Fzr causes a dramatic destabilization of ORC1-GFP, but not ORC1C-NLS-GFP. Conversely, co-expression of Rca1 modestly stabilizes ORC1-GFP but has no effect on ORC1C-NLS-GFP. Importantly, the effect of ectopic Fzr and Rca1 cannot readily be explained by primary effects on cell cycle progression with subordinate effects on ORC1 stability. Misexpression of neither Fzr nor Rca1 promotes significant S phase entry in the eye disc cells. Misexpression of Rca1 has no apparent effect on cell cycle progression analyzed by FACS, and misexpression of Fzr actually leads to accumulation of additional cells in G2 (where ORC1 is normally stable). In similar experiments, it was found that co-expression of Fzy has no apparent effect on ORC1-GFP stability, suggesting that ORC1 is preferentially targeted by Fzr. The simplest interpretation of these findings is that ORC1 is normally degraded upon exit from mitosis by Fzr-dependent APC activity (Araki, 2003).

To test the idea that Fzr acts directly to target ORC1 for degradation, it was asked whether Drosophila ORC1 is an APC substrate in vitro using a heterologous purified system. Ubiquitylation of a positive control, human polo-like kinase (Plk1), is stimulated by addition of APC activators from humans and flies, including Drosophila Fzr. Ubiquitinylation of ORC1N is also stimulated by Drosophila Fzr (and the human homolog, Cdh1, whereas none of the APC activators tested significantly stimulates ubiquitylation of ORC1C. These observations strongly support the idea that Drosophila ORC1 is targeted for degradation by Fzr by a mechanism that is conserved between vertebrates and flies (Araki, 2003).

DNA replication control through interaction of E2F-RB and the origin recognition complex

The E2F transcription factor and retinoblastoma protein control cell-cycle progression and DNA replication during S phase. Mutations in the Drosophila E2f1 and DP genes affect the origin recognition complex (DmORC) and initiation of replication at the chorion gene replication origin. Mutants of Rbf (an retinoblastoma protein homolog) fail to limit DNA replication. DP, E2f1 and Rbf proteins are located in a complex with ORC, and E2f1 and ORC are bound to the chorion origin of replication in vivo. These results indicate that E2f and Rbf function together at replication origins to limit DNA replication through interactions with ORC (Bosco, 2001).

To explore the possibility that E2f-Rbf is directly involved in controlling ORC activity, a test was performed to see whether a female-sterile mutant of Rbf (Rbf120a) has DNA replication and gene amplification defects in follicle cells of the Drosophila ovary. The Rbf120a mutation is due to a P-element insertion that causes reduced levels of wild-type Rbf protein, and Rbf14 is a null mutant. Ovaries from mutant Rbf120a/Rbf14 and heterozygous Rbf14/+ females were double labelled with 5-bromodeoxyuridine (BrdU) and anti-ORC2. Wild-type Drosophila follicle cells undergo endoreduplication cycles (endo cycles), reaching 16n ploidy by stage 9 or 10A of egg-chamber development. In stage 10B follicle cells, endo cycles have ceased, ORC has been cleared from the nucleus, and ORC is localized to discrete genomic regions undergoing amplification. Amplification is detected by BrdU incorporation at ORC localized foci. By contrast, the Rbf120a/Rbf14 mutant egg-chambers have a mosaic of follicle cells exhibiting striking replication defects: (1) some mutant follicle cells have inappropriate total nuclear ORC2 staining and continued endo cycles instead of amplification; (2) some follicle cells with specific ORC2 localization to replication origins have undergone gene amplification, and (3) some cells perform both amplification and genomic replication. Staining ovaries with anti-Rbf antibodies reveals a uniform absence of Rbf protein, and thus the mosaic phenotype cannot be explained by stochastic differences in Rbf protein levels (Bosco, 2001).

The Rbf120a/Rbf14 follicle cells undergoing gene amplification have large BrdU foci relative to sibling controls. Quantitation has confirmed that Amplification control element ACE3 DNA is amplified ~26-fold in mutant stage 13 egg-chambers, compared with ~16-fold in heterozygous egg-chambers. This phenotype in the Rbf120a/Rbf14 mutant is similar to the overamplification observed in the E2fi2 truncation mutant in which ACE3 is amplified 32-fold. Thus both Rbf and E2f are negative regulators of gene amplification (Bosco, 2001).

There is also a cell-cycle defect in the Rbf120a/Rbf14 mutant follicle cells. Inappropriate genomic replication seen in the mutant follicle cells results from the continuation of S/G endo cycles beyond the developmental stage at which they would normally cease. This predicts the presence of mutant follicle cells with greater DNA content than the wild-type 16n DNA. Fluorescence-activated cell sorting (FACS) analysis was carried out on purified ovarian nuclei, and heterozygous Rbf14/+ ovaries gave follicle cells with 2n, 4n, 8n and 16n DNA content. Rbf120a/Rbf14 ovaries, however, had cells with 32n DNA content, indicating that they had undergone at least one extra S-phase. It is concluded that stage 10B Rbf120a/Rbf14 mutant follicle cells undergo an ectopic S phase, and genomic replication in stage 10B cells is not due to a developmental delay. This result parallels that obtained with mutations in dDP (Bosco, 2001).

DNA replication also persists in later stages of mutant follicle cells. Wild-type stage 13 egg-chambers have no detectable ORC localization and little or no BrdU incorporation. In contrast, Rbf120a/Rbf14 stage 13 egg-chambers continue to undergo amplification and genomic replication that is consistent with persistent nuclear ORC staining. Some stage 13 cells exhibit characteristics of G1 cells, with nuclear ORC but no BrdU staining. This observation also supports the conclusion that Rbf120a/Rbf14 follicle cells continue bona fide G/S endo cycles (Bosco, 2001).

Tests were performed to see whether misregulation of important E2f target genes might account for the replication defects observed in the Rbf mutant follicle cells. Four important E2f target genes, Cyclin E, PCNA, RNR2 and ORC1, as well as ORC2 transcripts, are not normally induced in wild-type stage 10 follicle cells, and their transcripts are not elevated in the truncation E2fi2 mutant follicle cells. However, because overexpression of ORC1 is sufficient for initiating an ectopic endo cycle in stage 10 follicle cells, ORC1 and ORC2 transcripts were analyzed by in situ hybridization in Rbf mutant follicle cells. No significant differences were found in the amount of messenger RNA levels for either gene in Rbf120a/Rbf14 stage 9, 10 or 13 egg-chambers, as compared with Rbf14/+ sibling controls (Bosco, 2001).

Transcription of the reaper gene is highly induced in the follicle cells of wild-type stage 9 and 10 egg-chambers, and thus reaper levels were used as a measure of general transcriptional activity in an experiment in which attempts were made to block transcription of all genes. Egg-chambers were cultured in vitro for up to 6 h with or without alpha-amanitin. Rbf120a/Rbf14 egg-chambers cultured in the presence of alpha-amanitin abolish visible transcript levels of reaper in stage 10B follicle cells, whereas the Rbf120a/Rbf14 controls induce reaper normally. However, alpha-amanitin does not change the pattern of BrdU labelling in Rbf120a/Rbf14 stage 10 or 13 egg-chambers. Thus, the Rbf mutant replication defects persist even when general transcription is inhibited in follicle cells. It is possible that the in situ analysis of transcript levels or the inhibition of transcription by alpha-amanitin fail to uncover an effect of the Rbf mutant. Taken together, however, these data suggest that the gene amplification phenotype seen in Rbf120a/Rbf14 or E2fi2 follicle cells is not due to a misregulation of E2f target gene transcription in stage 10 egg-chambers (Bosco, 2001).

Whether E2f-Rbf complexes execute an S-phase function through a direct interaction with ORC was tested. Immunoprecipitations were carried out on ovary extracts; immunoblots of the pellets show that E2f and Rbf co-immunoprecipitate with Drosophila ORC when either anti-ORC2 or anti-ORC1 antibodies were used. The E2f-Rbf-ORC interaction could also be detected when immunoprecipitation reactions were performed with anti-E2f polyclonal or anti-DP monoclonal antibodies. This complex could be specifically immunoprecipitated from ovary extracts with five different antibodies. It is possible that in extracts the dDP-E2f-Rbf and ORC interaction might be due to dDP-E2f and ORC binding next to each other on DNA fragments. Therefore, immunoprecipitation reactions were carried out in the presence of ethidium bromide or micrococcal nuclease to disrupt protein-DNA interactions or cleave DNA fragments. Treatment of immunoprecipitation reactions with either reagent failed to disrupt the E2f-Rbf-ORC interaction. Furthermore, a mutation in DP predicted to reduce the DNA-binding activity of E2f did not abolish the E2f-Rbf-ORC interaction. It is therefore concluded that E2f and Rbf can co-immunoprecipitate with ORC through interactions that are independent of their respective DNA-binding activities (Bosco, 2001).

What is the functional relevance of this E2f-Rbf-ORC complex? One possible mechanism is that E2f-Rbf helps localize ORC to E2f-binding sites near the chorion replication origin. Another possibility is that ORC localization to the chorion replication origin is independent of its interaction with E2f-Rbf, and instead E2f-Rbf when bound next to an origin regulates replication initiation through its interaction with ORC. ORC binds the critical amplification control element ACE3 in vivo at a specific time in follicle cell development (stages 10A and 10B). Using anti-ORC2 antiserum, ACE3 has been specifically enriched relative to a control locus that does not bind ORC and is not amplified by using chromatin immunoprecipitation (CHIP). Using CHIP it was asked whether E2f also could be shown to localize specifically to ACE3 in vivo. Stabilization of protein-DNA interactions in live tissue is achieved by formaldehyde crosslinking. Subsequent CHIP enriches for specific trans-factors that are bound to genomic loci. The relative amounts of these loci are quantified by polymerase chain reaction (PCR). Sequence analysis reveals that there are several potential E2f-binding sites within 2.5 kilobases (kb) of ACE3. Using anti-E2f antibodies, it has been shown that ACE3 DNA is enriched ~15-fold relative to the rosy locus in stage 10 egg-chambers. Similarly, anti-ORC2 antibodies also enriched ACE3 DNA ~20-fold relative to the rosy locus. Thus, both E2f and ORC localize to ACE3 when amplification is occurring, and E2f binding is limited to sequences immediately adjacent to ACE3. This observation is consistent with E2f-Rbf functioning at replication origins and possibly controlling ORC activity (Bosco, 2001).

Since transactivation and RB-binding activities are known to be located in the C-terminal domain of mammalian E2F, a truncated form of Drosophila E2f predicted to lack the C-terminal transactivation and Rbf-binding domains was characterized. The E2fi2 mutation produces a stable, truncated E2fi2 protein that can still interact with DP. Truncated E2fi2 does not bind Rbf, as it does not co-immunoprecipitate, even when more than 10% of the total Rbf protein is immunoprecipitated. This failure to pellet the truncated E2fi2 protein is not due to low Rbf levels in mutant extracts because comparable amounts of Rbf in wild-type extracts can immunoprecipitate full-length E2f. Failure to detect this interaction is not due to low levels of truncated E2fi2 protein, because the amount of truncated E2fi2 in the supernatant represents 10% of total E2fi2 in the immunoprecipitation reaction and is comparable to full-length E2f that does interact with Rbf (Bosco, 2001).

Previous work has shown that mutant follicle cells producing this truncated E2fi2 protein specifically localize ORC to the amplification regions as in wild type, but that such cells have elevated levels of ACE3 amplification. This elevated level of amplification is probably due to extra rounds of origin initiation events, suggesting that both E2f and Rbf have a negative regulatory function in origin firing during amplification. The DNA-binding domain of the truncated E2fi2 protein might be sufficient to localize ORC, if it could still interact with ORC. Therefore, whether or not the truncated E2fi2 protein complexes with ORC was tested. Immunoprecipitation experiments show that truncated E2fi2 does not interact with ORC. This means that the C-terminal domain of E2f is necessary for its interaction with ORC, and possibly requires Rbf to mediate this interaction. In contrast to the stated hypothesis, however, localization of ORC to the amplification region does not require a physical complex with E2f (Bosco, 2001).

Thus, the Drosophila E2f-Rbf complex functions during S phase, specifically to regulate DNA replication initiation at origins. It is thought that DP-E2f-Rbf are bound near ORC at the amplification origin and regulate initiation by forming a complex with ORC. Although E2f does not direct ORC binding, it restricts its activity through Rbf. Five lines of evidence form the basis for this model: (1) reduced levels of Rbf result in increased gene amplification levels and genomic replication without measurable effects on transcription of E2f target genes; (2) a complex of dDP-E2f-Rbf-ORC is present in ovary extracts; (3) this complex is independent of DNA binding; (4) truncation of the C terminus of E2f eliminates this complex, and (5) in this truncation mutant, ORC is localized but increased amplification occurs. The mechanism by which the dDP-E2f-Rbf complex limits replication initiation at the chorion locus remains to be determined. It is possible that the dDP-E2f-Rbf proteins inhibit the activity of the ORC subunits through a physical interaction. Alternatively, E2f-Rbf might inhibit loading of other replication factors at origins, such as MCM proteins. Finally, Rbf might alter the local chromatin configuration, for example by histone deacetylation, and thereby affect origin firing. Although ORC does not need to be in the E2f-Rbf complex to bind specifically to the chorion replication origin, a mutation in the DNA-binding domain of E2f does result in loss of ORC localization in the follicle cells. This observation needs to be evaluated in the context of the result that ORC is localized in the E2fi2 mutant, in which the truncated E2f protein is able to bind DNA but does not complex with ORC. Thus, DNA binding by E2f seems to be a prerequisite for ORC localization, but ORC localization does not require complex formation with E2f. This may be because when E2f is not bound to the chorion region, E2f2 can bind to sites at ACE3 normally occupied by E2f, and E2f2-Rbf may repel ORC and preclude localization or antagonize ORC binding activity (Bosco, 2001).

The Rbf mutant provides insights into the controls leading to the cessation of the endo cell-cycle during follicle cell development. Both the female-sterile Rbf mutant and the dDP female-sterile mutant show inappropriate continuation of the endo cell cycle beyond stage 10 of egg-chamber development. In contrast, an ectopic S phase does not occur in either of the female-sterile E2f mutants. Like the dDPa1 mutant, the Rbf120a/Rbf14 mutant is expected to have effects on both E2f-Rbf and E2f2-Rbf complexes. Thus, it seems that DP-E2f2-Rbf is needed to exit endo cycles, whereas DP-E2f-Rbf is involved more directly in regulating ORC and gene amplification. Identification of mutations in E2f2 will permit direct analysis of the roles of E2f2 in the endo cell cycle and amplification. Although it has not been shown whether any other specific replication origins may be regulated in this manner, the E2f-Rbf-ORC complex has been found in embryonic extracts, indicating that E2f-Rbf may be a general repressor of replication origins in embryonic tissues. Notably, a region between the DmPolalpha and E2f genes, containing several known E2f-binding sites, has been identified as a replication initiation region. Human RB (and associated HDACs) co-immunolocalize to BrdU foci in early S phase of primary cells, suggesting that RB may have a role in replication initiation. This observation is consistent with the model that suggests that Drosophila E2F1-Rbf localizes to replication origins and regulates ORC activity through a direct protein-protein interaction. It will be of great value to determine whether mammalian E2F-RB complexes can interact with ORC. Such an interaction would allow for a better understanding of how E2F and RB function to regulate DNA replication and cell proliferation during tumor progression (Bosco, 2001).

HP1 associates with ORC2

There is a striking preferential but not exclusive association of Drosophila ORC2 with heterochromatin on interphase and mitotic chromosomes. DmORC is found on chromatin at all cell cycle stages of the embryonic syncytium in a diffuse, granular pattern throughout the DNA but is highly concentrated at foci along the apical surface of the interphase nuclei, consistent with the known orientation of pericentric heterochromatin. No differences in DmORC distribution are apparent in embryos after cellularization. HP1, a heterochromatin-localized protein required for position effect variegation (PEV), colocalizes with DmORC2 at these sites. Consistent with this localization, intact DmORC and HP1 are found in physical complex. DmORC2, 5 and 6 are also found in this complex. Neither DmORC2 nor 6 show reproducible interactions with HP1. The association of ORC1 with HP1 is shown biochemically to require the chromodomain and shadow domains of HP1. Amino acid residues 161-319 of DmORC1 are likely to carry multiple sites of contact with HP1. The amino terminus of DmORC1 contains a strong HP1-binding site, mirroring an interaction found independently in Xenopus by a yeast two-hybrid screen. Heterozygous DmORC2 recessive lethal mutations result in a suppression of PEV. These results indicate that ORC may play a widespread role in packaging chromosomal domains through interactions with heterochromatin-organizing factors (Pak, 1997).

In S. cerevisiae, the amino terminus of Orc1p appears to contain a specialized domain, perhaps dedicated to silencing (Bell, 1995). Although the amino termini of various metazoan Orc1 homologs are not well-conserved, it seemed possible that this domain of the protein might be involved in nonreplication functions of ORC, with the well-conserved carboxyl terminus dedicated to replication (Bell, 1995; Gavin, 1995; Muzi-Falconi, 1995). Therefore, it was of interest to determine whether Drosophila ORC1 interacts with HP1 and whether other Drosophila ORC subunit(s) might be contributing to the interaction. Coupled in vitro transcription/translation reactions using [35S]methionine were performed for each of the six Drosophia ORC subunits and the translation products were shown to comigrate in SDS-PAGE gels with ORC proteins purified from Drosophila embryos. ORC proteins were incubated with wild-type Flag-HP1, individually as well as together, to assess whether HP1 interacts with ORC subunits differently in the presence of the entire complex. Immunoprecipitations were conducted with anti-Flag monoclonal antibody-coupled beads, and the precipitated material was analyzed by SDS-PAGE and autoradiography. The results demonstrate that ORC1 binds to HP1 with the highest recovery. Lower and more variable levels of interaction were seen for the other subunits, but ORC3 and ORC4 also exhibited clearly greater levels of binding than the other subunits. Notably, when all six subunits are present, all ORC subunits can be coimmunoprecipitated with Flag-HP1. In particular, the in vitro translated products of ORC2 and ORC6, which individually show no reproducible interactions with HP1, are coprecipitated when the entire repertoire of ORC subunits was used (Pak, 1997).

To analyze further the strong ORC1-HP1 interaction, it was of interest to determine the region of ORC1 that binds to HP1. Various truncations of ORC1 were labeled with [35S]methionine by in vitro translation. These lysates were incubated individually with Flag-tagged full-length HP1 purified from Escherichia coli and immunoprecipitated with anti-Flag resin. A 160-amino-acid domain in the amino terminus of ORC1 spanning residues 161-319 was found to be necessary and sufficient for the interaction with HP1. However, a ORC1 fragment from 160-501 binds to HP1 very weakly, which may be due to improper folding or stability of that construct. A derivative of ORC1 (1-238) carrying a subfragment of the 161-319 region demonstrates intermediate levels of binding to HP1, suggesting that multiple contacts within this domain may be involved (Pak, 1997).

Independent confirmation of this observation and evidence that it also occurs in vertebrates came from a two-hybrid screen for proteins that bind to the Xenopus homolog of Orc1p (XOrc1) fused to the LexA DNA-binding domain. Three classes of positives were obtained. One class represented two subtypes of Xenopus HP1 homologs, HP1alpha and HP1gamma. Strikingly, the amino terminus of XOrc1 was also required for this interaction, paralleling that seen in Drosophila. The highly conserved carboxyl terminus of XOrc1 did not recognize either XHP1 species nor did the LexA domain alone or a panel of five negative control fusions. To confirm the specificity of this result, the fusion domains between XOrc1 and XHP1 homologs were exchanged. XOrc1 fused to LexA binds VP16HP1-alpha and VP16HP1-gamma; conversely, XOrc1 fused to the VP16 transcriptional activation domain interacts with the lexHP1-alpha and lexHP1-gamma fusions. Thus, the association between XOrc1 and XHP1 homologs in yeast was not dependent on the fusion domain in two-hybrid constructs (Pak, 1997).

Present models concerning the DNA replication cycle emphasize the point that ORC is continuously associated with chromosomes and that distinct steps toward building a replication complex occur at different stages of the cell cycle. This cyclical, stepwise assembly and disassembly would presumably safeguard inappropriate DNA replication origin initiation. However, recent genetic studies have shown that many temperature-sensitive orc5 mutants arrest in early M phase, before the metaphase-to-anaphase transition point and prior to the assembly of prereplicative initiation complexes late in mitosis. The continuous association of ORC with chromosomes may therefore be important because information is contained in the pattern of ORC binding, and/or ORC performs positive activities in the chromosome cycle outside of S phase and separate from a direct replication function. One such role of ORC may be in heterochromatin formation, which may itself underlie other evolutionarily conserved and equally critical requirements in chromosome folding. This would not only be a parsimonious use of the multiprotein complex but also could help coordinate multiple aspects of DNA dynamics. Thus, ORC may serve to monitor the state of chromatin, coupling the end of DNA replication to the beginning of chromosome condensation. Along these lines, certain ORC-binding sites may help regulate whether given regions decondense or not in early G1 phase. Involvement of ORC in chromatin remodeling for gene silencing may therefore be an example of an evolutionary variation on a general housekeeping function (Pak, 1997).

ORC associates with Myb and binds elements important for amplification of the chorion gene cluster

There is considerable interest in the developmental, temporal and tissue-specific patterns of DNA replication in metazoans. Site-specific DNA replication at the chorion loci in Drosophila follicle cells leads to extensive gene amplification, and the organization of the cis-acting DNA elements that regulate this process may provide a model for how such regulation is achieved. Two elements important for amplification of the third chromosome chorion gene cluster, ACE3 and Ori-ß, are directly bound by Orc (origin recognition complex), and two-dimensional gel analysis has revealed that the primary origin used is Ori-ß. The Drosophila homolog of the Myb (Myeloblastosis) oncoprotein family is tightly associated with four additional proteins, and the complex binds site-specifically to these regulatory DNA elements. Drosophila Myb is required in trans for gene amplification, showing that a Myb protein is directly involved in DNA replication. A Drosophila Myb binding site, as well as the binding site for another Myb complex member (p120), is necessary in cis for replication of reporter transgenes. Chromatin immunoprecipitation experiments localize both proteins to the chorion loci in vivo. These data provide evidence that specific protein complexes bound to replication enhancer elements work together with the general replication machinery for site-specific origin utilization during replication (Beall, 2002).

To identify proteins that bind to either ACE3 or Ori-ß, Drosophila tissue culture nuclear extracts were fractionated. DNase I protection was used to assay site-specific ACE3- and Ori-ß-binding proteins, and to follow their purification. The final glycerol gradient fractions were found to contain five polypeptides that co-elute with binding activity for both DNAs in multiple independent fractionation schemes from either Schneider L2 or Kc cell lines. Utilizing peptide sequences from proteolysed purified protein, database searches identified Drosophila Myb (p85) and Caf1 p55 proteins, as well as three new Drosophila proteins (p40, p120, p130; Berkeley Drosophila Genome Project CG15119, CG6061 and CG3480) (Beall, 2002).

Drosophila Myb recognizes a highly conserved DNA sequence, but the specific binding properties of the glycerol gradient fractions might be more complex than those of Myb alone. Therefore, whether any of the other proteins in the footprinting fractions might interact site-specifically with DNA was tested. Each protein was produced individually and purified to homogeneity as either a (His)6- or Flag-tagged protein using the baculovirus system. Only recombinant (r) Myb and rp120 bind to ACE3. rMyb protects nucleotides -471 to -511, and at higher concentrations protected -525 to -541, whereas rp120 protects -413 to -445, -525 to -541, and -575 to -603. However, the protection from the glycerol gradient fractions was more complex than the simple sums of the protections observed for these two purified proteins. Moreover, rMyb on its own does not bind to Ori-ß, whereas p120 does bind (Beall, 2002).

All five proteins co-immunoprecipitate together when any of the five antibodies are used. This association was not mediated through DNA, because ethidium bromide does not disrupt the interactions. Identical results were obtained using either 0-12-h embryo or ovary nuclear extracts. Myb co-fractionates with only the four other complex members. No indication of free or other Myb forms was found. It is therefore concluded that most of the Myb in these extracts is in a tight complex with the four additional proteins (Beall, 2002).

Since the Drosophila Myb complex binds to both ACE3 and Ori-ß in vitro, whether the Myb complex directly interacts with Orc was tested. Immunoprecipitation from Kc cell nuclear extracts shows that anti-Orc1 or anti-Orc2 antibodies co-immunoprecipitate Orc1, 2 and 6. Immunoprecipitations with anti-Myb antibodies co-immunoprecipitate Orc. Reciprocal experiments have shown that anti-Orc2 antibodies co-immunoprecipitate the Myb complex (Beall, 2002).

As a first step in exploring the role of the Drosophila Myb complex in vivo, chromatin immunoprecipitation assays were performed on whole ovaries dissected from females that were aged to maximize the number of stage-10 egg chambers. Antibodies against Myb, Orc2 and p120 specifically precipitate ACE3-containing chromatin. Thus, Myb, p120 and Orc are bound to ACE3 in ovaries enriched for egg chambers undergoing chorion gene amplification. E2F-containing complexes can bind to Orc and are associated with ACE3 in ovaries, but the location of this E2F cis-element is unknown. The interactions between the Myb complex, Orc and E2F proteins in sculpting the properties of the ACE3 element will be critical to understanding how this element functions as a replication enhancer (Beall, 2002).

Small P element transgenes containing ACE3 and Ori-ß amplify efficiently at ectopic genomic sites only when both elements are present. A minimal replication reporter was used to assess the role of protein binding sites within ACE3 to minimize the complications of redundant cis-elements. 'Suppressor of hairy wing binding sites' (SHWBS) were used to insulate the transgenes from chromosomal position effects. Such reporters allow for investigation, at various chromosomal positions, whether the binding sites in ACE3 for Myb and p120 are important cis-acting elements for amplification. Transgenes were constructed that contain deletions of each of the binding sites identified by DNase I protection and several transgenic lines were generated for each deletion. Mutations abolished DNA binding of both recombinant Myb and the entire complex to the regulatory sequences in electrophoretic mobility shift assay (EMSA) experiments. Deletion of either the Myb (-471 to -511) or one of the p120 binding sites (-413 to -445) results in severely reduced amplification in stage-13 egg chambers. Deletion of the other two p120 binding sites results in reduced levels of amplification, but to a less severe degree (Beall, 2002).

The mature somatic follicle cells that surround the developing oocyte derive from a series of mitotic cell divisions followed by genome-wide endocycles. At stage 10B, global DNA replication shuts down and the chorion loci on the X and third chromosomes (and two additional unidentified loci) begin locus-specific amplification. Amplification can be visualized by the incorporation of bromodeoxyuridine (BrdU) at four sub-nuclear foci using immunofluorescence microscopy, where the two largest foci represent incorporation on the X and third chromosomes (Beall, 2002).

Since Orc2 also localizes to these foci, ovaries were stained with either anti-Myb or anti-Orc2 antibodies. It was found that Myb is diffusely nuclear and not localized to the distinct sub-nuclear foci that contain Orc2. Identical results were observed with the four other complex members (Beall, 2002).

Drosophila Myb has been suggested to have a general role in S phase in several different tissues. However, a direct role for Drosophila Myb in replication at a specific location has not been demonstrated. To test the need for Drosophila Myb in trans for replication at the chorion loci, a directed mosaic system was used to generate green fluorescent protein (GFP)-negative, homozygous Drosophila Myb mutant clones in a heterozygous ovary. In the absence of Drosophila Myb, members of the pre-replication-complex (RC) are present at the sub-nuclear foci but are unable to initiate detectable replication. One prediction from these results is that in late-stage mosaic egg chambers with Myb clones, patches of thin, fragile eggshell and thin dorsal appendages should result from reduced chorion gene expression. Myb mutant patches in the regions that are normally responsible for dorsal appendage formation results in greatly reduced appendages. As egg chambers age, the Myb mutant nuclei appear more compact. There was no underlying chorion beneath the follicle cells in mutant patches (Beall, 2002).

Studies of Myb family members have largely focused on their functions as transcription factors, though important targets for gene activation that might explain the role of these proteins in the cell cycle remain unclear. Drosophila Myb has been shown in this study to play a direct role in DNA replication. These biochemical experiments imply that this protein functions in tight complex with four other proteins. Recent studies suggest that in a variety of tissues, but not all, Drosophila Myb seems to be important for S-phase progression. These findings support a role for Drosophila Myb in tissue-specific and temporally defined DNA replication, much as enhancer proteins define site-specific transcription (Beall, 2002).

The genetic studies and, in particular, the mosaic analyses indicate that Drosophila Myb probably functions at a late step in the replication process, since Orc2 and Cdt1 were both localized at discrete foci within Drosophila Myb mutant stage-10B follicle cells. It is inferred that Orc and other general replication proteins are localized at ACE3. However, the time in the developmental process when Orc appears at ACE3 with regard to Drosophila Myb loss is known. Thus, Drosophila Myb perdurance after genomic deletion could certainly complicate any conclusions about the role of Drosophila Myb in establishing a pre-RC at the amplification foci. In any case, Myb family members interact with both acetylases and deacetylases; thus, it is intriguing to consider the potential roles of this modification in either early or late steps of DNA replication initiation (Beall, 2002).

Visualization of replication initiation and elongation in Drosophila

Chorion gene amplification in the ovaries of Drosophila is a powerful system for the study of metazoan DNA replication in vivo. Using a combination of high-resolution confocal and deconvolution microscopy and quantitative realtime PCR, it was found that initiation and elongation occur during separate developmental stages, thus permitting analysis of these two phases of replication in vivo. Bromodeoxyuridine, origin recognition complex, and the elongation factors minichromosome maintenance proteins (MCM)2-7 and proliferating cell nuclear antigen were precisely localized, and the DNA copy number along the third chromosome chorion amplicon was quantified during multiple developmental stages. These studies revealed that initiation takes place during stages 10B and 11 of egg chamber development, whereas only elongation of existing replication forks occurs during egg chamber stages 12 and 13. The ability to distinguish initiation from elongation makes this an outstanding model to decipher the roles of various replication factors during metazoan DNA replication. This system was used to demonstrate that the pre-replication complex component, Double-parked protein/Cdt1, is not only necessary for proper MCM2-7 localization, but, unexpectedly, is present during elongation (Claycomb, 2002).

Three independent lines of evidence are presented that initiation and the bulk of elongation at a chorion amplicon occur during two separate developmental periods. (1) Deconvolution microscopy shows that ORC and BrdU initially colocalize at origins and then diverge, since ORC is lost in stage 11 and BrdU resolves into a double bar structure. (2) Elongation factors PCNA and MCM2-7 follow the same pattern as BrdU, resolving from foci early in amplification to a double bar structure by stage 12 to 13. (3) Quantitative realtime PCR shows a peak increase in DNA copy number at the origins by stage 11, with increases in flanking sequences becoming substantial in stages 12 and 13. Thus initiation ends by stage 11, and during stages 12 and 13 only the existing forks progress outward. Furthermore, these observations led to the unanticipated conclusion that DUP/Cdt1 travels with replication forks (Claycomb, 2002).

The realtime PCR and immunofluorescence data are remarkably consistent. (1) Both methods restrict initiation to stages 10B and 11 of oogenesis, and elongation to stages 12 and 13. Between stages 10B and 11, the maximum fold amplification was detected at amplification control element (ACE) on third chromosome (ACE3) by realtime PCR, ORC localized to origins, and the deconvolution showed a maximum increase in bar length. During stages 12 and 13, increases in fold amplification were detected only proximal and distal to ACE3, and ORC no longer localized to origins, whereas BrdU incorporation resolved into the double bar structure. (2) The distances of fork movement are consistent. Deconvolution measurements predicted that forks were maximally 30 +/- 3 kb apart in stage 10B, and this correlates with the 40-kb span of peak copy number detected by realtime PCR. In stage 11, forks were measured to have progressed across a 55 +/- 13-kb region by deconvolution and across a 45-kb region by realtime PCR. By stage 13, deconvolution showed that replication forks were maximally separated by 74 +/- 7 kb, whereas realtime PCR measured a 75-kb span (Claycomb, 2002).

The quantitative analysis of the amplification gradient provides insight into mechanisms affecting fork movement and termination and suggests that an onionskin structure impedes fork movement. The maximal rate of fork movement during amplification has been calculated to be 90 bp/min on average. In comparison, replication forks in the polytene larval salivary glands travel at ~300 bp/min (Steinemann, 1981), whereas rates of fork movement in both diploid Drosophila cell culture and embryo syncytial divisions are ~2.6 kb/min. From these rates, it seems that polyteny hinders replication fork movement, an effect even more pronounced in amplification, given that the chorion cluster has a rate of fork movement three times less than polytene salivary glands. The fact that by stage 13 there is a gradient of copy number, and not a plateau, further demonstrates the inefficiency of fork movement along the chorion cluster (Claycomb, 2002).

There do not seem to be specific termination sites to stop forks either along or at the ends of the chorion region, but fork movement may display some sequence or chromatin preference. The gradient of decreasing copy number implies that forks stop at a range of sites, because the presence of specific termination points along the region would be expected to cause steep drops in copy number. Despite this lack of specific termination sites, during stages 12 and 13 a greater increase is seen in copy number to one side of ACE3, and it was often observe by immunofluorescence that one of the two bars is shorter. This suggests that the sequence or chromatin structure to the other side of ACE3 hinders fork movement, and as fewer forks move out, less BrdU incorporation occurs and a shorter bar results (Claycomb, 2002).

These studies highlight the complex regulation of chorion gene amplification. How are the number of origin firings restricted to the proper developmental time? It is known that the number of rounds of origin firing at the chorion amplicons is limited by the action of Rb, E2F1, and DP. Perhaps Dup and MCM2-7 are also a part of this regulation, with origins firing only when MCM2-7 are properly loaded. It will also be interesting to decipher the regulation of Dup/Cdt1 during amplification. Recent studies have demonstrated that a Drosophila homologue of the metazoan re-replication inhibitor, Geminin, exists and interacts biochemically and genetically with Dup/Cdt1. Female-sterile mutations in geminin result in increased BrdU incorporation during amplification, raising the possibility that Geminin acts on DUP/Cdt1 at the chorion loci to limit origin firing. In addition to permitting the delineation of the regulatory circuitry controlling origin firing, the ability to developmentally distinguish initiation from elongation provides a powerful tool for the analysis of the properties of metazoan replication factors in vivo (Claycomb, 2002).

A novel motif governs APC-dependent degradation of Drosophila ORC1 in vivo

Regulated degradation plays a key role in setting the level of many factors that govern cell cycle progression. In Drosophila, the largest subunit of the origin recognition complex protein 1 (ORC1) is degraded at the end of M phase and throughout much of G1 by anaphase-promoting complexes (APC) activated by Fzr/Cdh1. None of the previously identified APC motifs targets ORC1 for degradation. Instead, a novel sequence, the O-box, is necessary and sufficient to direct Fzr/Cdh1-dependent polyubiquitylation in vitro and degradation in vivo. The O-box is similar to but distinct from the well characterized D-box. Finally, O-box motifs in two other proteins, Drosophila Abnormal Spindle and Schizosaccharomyces pombe Cut2, contribute to Cdh1-dependent polyubiquitylation in vitro, suggesting that the O-box may mediate degradation of a variety of cell cycle factors (Araki, 2005).

The O-box is an addition to the family of motifs that mediate APC-dependent degradation. Like the D-box, the O-box is a short portable motif that is sufficient to direct polyubiquitylation in vitro and cell cycle-dependent degradation in vivo. The O- and D-box signals compete at some currently unknown level in ubiquitylation reactions, demonstrating that, although they are structurally and functionally distinct, both motifs converge on at least one common component of the degradation machinery. Given the low sequence complexity of the motif and the current definition of essential residues solely by alanine scanning, it seems likely that O-boxes exist in other proteins (Araki, 2005).

A direct interaction occurs between Cdh1 and both KEN- and D-box peptides. Given the functional homology among the various destruction motifs, it was asked whether whether detect interactions could be demonstrated between O-box-containing peptides and Fzr in GST-pulldown, coimmuniprecipitation (co-IP), and yeast two-hybrid experiments. Specific binding of the O-box to Fzr/Cdh1 was not detected; perhaps more sensitive assays either using purified components or cross-linkable peptide substrates will be necessary to detect an inherently weak and transient interaction (Araki, 2005).

The N-terminal 554 amino acids of ORC1 contain three degradation motifs: one O-box, one KEN-box, and three D-boxes. What roles do these play? The O-box is primarily responsible for regulating cell cycle-coupled degradation of full-length ORC1. The results also clearly indicate that the KEN- and D-boxes, at best, play only minor roles in directing ubiquitylation in vitro and degradation in vivo. The most likely explanation for the inactivity of these motifs is that they are situated in an unfavorable context. When substituted for the O-box at positions 291-301 of ORC1, both D- and KEN-motifs direct very efficient polyubiquitylation in vitro (Araki, 2005).

It is assumed that degradation of ORC1 is integral to the process of resetting origins of replication after the initiation of S phase. In human cells, ORC1 is degraded during S phase by the SCF. Negative regulators of pre-RC formation, such as Geminin and Cyclin B, are degraded by the APC at the G2/M transition, and the pre-RC can then reform in G1 after ORC1 is resynthesized. In Drosophila, ORC1, Cyclin B, and presumably Geminin are all degraded by the APC; and it is thus unclear when the pre-RCs are re-established. One possibility is that ORC1 acts during the narrow window between activation of Fzy (leading to the destruction of Cyclin B and Geminin) and Fzr (leading to the destruction of ORC1) during the G2/M transition. Another possibility is that in Drosophila pre-RC formation does not take place until late in G1 when Fzr activity drops and ORC1 is resynthesized under the direction of E2F (Araki, 2005).

Expression of the stable O-box mutant derivative of ORC1 fused to GFP caused no significant cellular or organismal phenotypes using a variety of GAL4 drivers. However, expression of either tagged or untagged wild-type ORC1 using the same constellation of GAL4 drivers fails to phenocopy the organismal and cellular phenotypes that are associated with low-level constitutive expression of ORC1. It is assumed that, as is the case for many other cell cycle regulatory factors (e.g., APC), ORC1 activity is likely regulated by redundant mechanisms. If so, a more sensitive test of the role of ORC1 degradation would be to ask whether the stable O-box mutant form of the protein can substitute for all of the known or suspected roles of the wild-type protein, including not only replication control but also heterochromatin-associated transcriptional silencing, chromosome condensation, and the control of synaptic plasticity. Such experiments await the isolation and characterization of an ORC1- mutant (Araki, 2005).

Origin recognition complex subunit 1: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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