BIOLOGICAL OVERVIEW

Mutagen sensitive 209 (Mus209), more often referred to as Proliferating cell nuclear antigen (PCNA) is a DNA damage-inducible protein that performs essential functions in normal DNA replication, including the resynthesis during nucleotide excision repair of damaged DNA, as an auxiliary factor for DNA polymerases delta and epsilon. DNA polymerase delta is the functional DNA polymerase on the leading strand of the eucaryotic DNA replication fork. The high speed and processivity of replicative DNA polymerases reside in the processivity factor known as PCNA, which has been shown to be a ring-shaped protein. This protein, a sliding clamp, encircles DNA and tethers the DNA polymerase catalytic unit to the DNA template. Mammalian PCNA is loaded onto DNA by a special protein complex known as Replication factor C (RFC). RFC carries out multiple functions: these include the ability to recognize and bind to a DNA primer end and load the ring-shaped PCNA onto DNA in an ATP-dependent reaction. PCNA then tethers the polymerase to the template, allowing processive DNA chain elongation.

Perhaps the most intriguing property of temperature sensitive mutations in the mus209 gene that codes for Drosophila PCNA is suppression of the gene inactivation phenomenon known as position effect variagation (PEV). PEV in essence refers to a chromosomal rearrangement; the placing of a gene near heterochromatin, which leads to the inactivation of that gene in a capricious manner. Heterochromatin is the genetically silent area of a chromosome, a quiet haven in what is otherwise a noisy workzone. Variegation produces a quixotic gene expression; the gene is now turned on, now turned off. Variation in expression is found from fly to fly, and even between different cells in the same fly. This either on or off gene status is passed along from a cell to that cell's offspring. mus209 suppresses the PEV caused by the rearrangement of indicator genes to positions closer to the heterochromatin (Henderson, 1994).

The compaction model of PEV posits that gene inactivation occurs as the variegating gene becomes packaged as heterochromatin. In PEV, chromatin componenets normally restricted to heterochromatin are assumed to spread beyond the euchromatin-heterochromatin boundary, resulting in heterochromatinization of euchromatin and transcriptional inactivation of the variegating gene. To rationalize this model with the experimental results, requires that PCNA be either a component of heterochromatin or be involved somehow in chromatin assembly. It is suggested that suppression of variegation by mus209 mutants could be explained by postulating that an interaction between mutant PCNA and the Drosophila histone assembly machinery is impaired, thereby altering nucleosome positioning at euchromatin-heterochromatin boundaries. Alternatively, PCNA might be involved in regulating the timing of replicon firing or the rate of DNA polymerase progression during S phase, which may influence the transcriptional state of the gene. Thus, in mus209 mutants, a variegating gene might be expressed simply as a consequence of euchromatin-heterochromatin junctions undergoing early replication or altered chromatin assembly (Henderson, 1994 and references).

Recent evidence ties PCNA directly to a Polycomb group protein Cramped (Crm). Polycomb group proteins are involved in global gene silencing in Drosophila. The S-phase-specific nuclear localization of Cramped is reminiscent of the same localization in PCNA. During the first 13 nuclear division cycles, PCNA is present in all interphase nuclei and absent from metaphase chromosomes. Double immunostaining of Crm and PCNA reveals that the appearance and disappearance of the nuclear signals are identically timed during the preblastoderm cycles; by gastrulation, both proteins show overlapping patterns of expression. These two proteins are clearly observed in polytene tissues, and their staining pattens overlap. There is a genetic interaction between crm and mus209, the Drosophila gene encoding PCNA. Thus Crm may be the link between PCNA and Polycomb proteins regulating position effect variagation (Yamamoto, 1997).

Of special interest for an understanding PCNA function are the extensive analyses of the Drosophila mus209 promoter by Matsukage, Yamaguchi and their colleagues. The mus209 promoter contains at least three transcriptional regulatory elements: the URE (upstream regulatory element), DRE (DNA replication-related element), and E2F recognition sites. Three DREs are found in the Drosophila DNA polymerase alpha gene (Yamaguchi, 1996), two in the Drosophila raf gene (Ryu, 1997), and one each is found in the Cyclin A gene (Ohno, 1996) and mus209 (Yamaguchi, 1996). While the role of E2F in cell cycle regulation is fairly well documented , the role of DREF is only now beginning to be undersood. DREF is a novel transcription factor with a basic DNA binding domain and acidic and proline rich domains (Hirose, 1996).

Zerknullt (Zen) has been shown to repress mus209, but this effect is likely to be indirect (Yamaguchi, 1991b). The expression of of the Drosophila DNA polymerase alpha gene is also repressed by zerknullt. The expression of the zen results in reduction of the abundance of mRNA expression directed by mus209 and DNA polymerase alpha promoter fragments and also mRNAs for both mus209 and DNA polymerase alpha. Deletions of the DNA replication-related element (DRE) reveal that the DRE sequences are responsible for repression by Zen protein. zen expressing cells contain lesser amounts of the DRE-binding factor (DREF) than do untransfected or mutant zen-transfected cells. These results suggest that the Zen protein represses expression of DNA replication-related genes by reducing DREF, although the detailed mechanism of the repression remains to be elucidated (Hirose, 1994).

The view of PCNA as a mechanical clamp belies the complexity of PCNA protein interactions and the importance of its regulatory roles. Mammalian PCNA interacts with p21 (Drosophila homolog: Dacapo), an inhibitor of cyclin dependent kinases, suggesting that p21 plays a dual role, affecting both cell cycle regulators and the DNA synthetic machinery. PCNA physically interacts with nucleases involved in DNA repair. PCNA interacts with Gadd45, a protein that stimulates DNA excision repair and inhibits entry of cells into S phase. Thus GADD45 provides a link between the p53-dependent cell cycle checkpoint and PCNA involvement in DNA repair. Transcription of PCNA is also regulated by p53, and PCNA transcription is regulated by E2F downstream of Interleukin 2. Thus the wider view of PCNA regulation and protein interactions is a window into the complex regulatory networks involved in gene silencing, cell cycle progression, DNA synthesis, and repair of DNA damage.

Intrinsic negative cell cycle regulation provided by PIP box- and Cul4Cdt2-mediated destruction of E2f1 during S phase

E2F transcription factors are key regulators of cell proliferation that are inhibited by pRb family tumor suppressors. pRb-independent modes of E2F inhibition have also been described, but their contribution to animal development and tumor suppression is unclear. This study shows that S phase-specific destruction of Drosophila E2f1 provides a novel mechanism for cell cycle regulation. E2f1 destruction is mediated by a PCNA-interacting-protein (PIP) motif in E2f1 and the Cul4^Cdt2 E3 ubiquitin ligase and requires the Dp dimerization partner but not direct Cdk phosphorylation or Rbf1 binding. E2f1 lacking a functional PIP motif accumulates inappropriately during S phase and is more potent than wild-type E2f1 at accelerating cell cycle progression and inducing apoptosis. Thus, S phase-coupled destruction is a key negative regulator of E2f1 activity. It is proposed that pRb-independent inhibition of E2F during S phase is an evolutionarily conserved feature of the metazoan cell cycle that is necessary for development (Shibutani, 2008).

This study describes a novel mechanism for inhibiting activator E2F function. The destruction of Drosophila E2f1 during S phase requires PCNA and a Cul4^Cdt2 E3 ubiquitin ligase. A region was identified in E2f1 that when mutated stabilizes E2f1 during S phase, resulting in cell cycle acceleration, apoptosis, and aberrant development. These data suggest that replication-coupled degradation provides important, pRb-independent negative regulation of E2f1 activity during normal development (Shibutani, 2008).

The mechanism of E2f1 destruction during S phase is similar to that recently described for the pre-RC component, Cdt1, which interacts with chromatin-bound PCNA via a PIP box (Arias, 2006). This PCNA-Cdt1 interaction recruits Cul4^Ddb1-Cdt2, leading to the ubiquitylation and subsequent destruction of Cdt1, particularly after DNA damage. While it was not determined whether E2f1 binds PCNA directly or is ubiquitylated on chromatin, Dp, which is necessary for E2f1 to bind DNA as an E2f1/Dp dimer, is required for E2f1 destruction during S phase. Replication fork movement could bring PCNA to E2f1/Dp that is bound to specific sites throughout the genome. However, stalling replication forks with chemical inhibitors of DNA synthesis did not affect the kinetics of E2f1 destruction. Therefore, a model is favored where the nucleoplasmic pool of E2f1/Dp, in equilibrium with the DNA-bound pool, is the relevant Cul4^Cdt2 substrate and is recruited to PCNA bound at replication forks once S phase begins. Drosophila Cdt1 also contains a PIP box and is destroyed during S phase in a replication-dependent manner. Therefore, the Cul4/PIP box mechanism is conserved and has been coopted by different proteins during Drosophila evolution to couple destruction with ongoing DNA synthesis (Shibutani, 2008).

Genetic depletion of Drosophila Cul1^Slmb E3 ligase activity has been reported to stabilize E2f1 during S phase. Cul1 and Cul4 act redundantly to trigger Cdt1 destruction in human S phase cells (Nishitani, 2006). By analogy, multiple Cullin complexes may target E2f1. These experiments did not reveal a major role for a Cul1-based E3 ligase in S phase destruction of E2f1, but neither did they exclude the possibility that Cul1 regulates E2f1 levels at other times in the cell cycle. Perhaps Cul1 restrains E2f1 accumulation during G1, such that reduction of Cul1 function results in elevated levels of E2f1 prior to S phase, and this excess E2f1 cannot be depleted as rapidly as in wild-type cells once S phase begins (Shibutani, 2008).

There is not an obvious PIP box in mammalian activator E2Fs, and human E2F1 is targeted by a Cul1 E3 ubiquitin ligase. In addition, human E2F1 stability is modulated by interaction with pRb, whereas the current data indicate that the regulation of E2f1 protein accumulation during the cell cycle is independent of Rbf1. Thus, the mechanism for ubiquitin-mediated activator E2F destruction evolved differently in Drosophila than in mammals (Shibutani, 2008).

What appears to be evolutionarily conserved is a requirement to inhibit activator E2Fs during S phase independently of pRb family proteins. This can be achieved by different mechanisms. Furthermore, the failure of this inhibition results in apoptosis. In mammals, the phosphorylation of E2F1-bound-DP via Cyclin A/Cdk2, which interacts with the NH₂ terminus of E2F1, blocks DNA binding of E2F1/DP. Drosophila achieves the same effect by rapidly destroying E2f1 during S phase. Much like the current E2f1^PIP-3A results, the expression of an E2F1 allele that cannot bind Cyclin A results in an increase in the S phase population and apoptosis. Dp mutant wing imaginal discs do not display elevated apoptosis, suggesting that any free E2f1 that accumulates during S phase in this situation is not detrimental. Thus, cells may possess an S phase-specific sensing mechanism to detect chromatin-bound E2f1/Dp and trigger apoptosis (Shibutani, 2008).

What functions of activator E2Fs might necessitate their inhibition, or more specifically their removal from chromatin, during S phase? One possibility is that this provides a means to downregulate E2F transcriptional targets in S/G2. Consistent with this, the simultaneous mutation of the mouse E2F7 and E2F8 repressors, which lack a pRb interaction domain, results in a failure to downregulate the E2F1 and CDC6 genes in S/G2 in embryonic fibroblasts and causes widespread apoptosis in embryos (Li, 2008). E2F also controls the expression of genes at the G2/M transition in flies and mammals. Perhaps the precocious activation of G2/M targets because of persistent E2F activity during S phase prevents the normal coordination of events needed to progress from interphase to mitosis, contributing to the accumulation of S/G2 cells that was observed. Additionally, the interplay between activator and repressor E2Fs may be disrupted when chromatin-bound E2f1 persists during S phase. E2f1 prevents E2f2-mediated repression in Drosophila, likely by blocking access of E2f2 to specific DNA binding sites. Consequently, excess chromatin-bound E2f1 during S phase may antagonize the function of dREAM/MMB, a recently described E2f2-containing complex that regulates the expression of many genes that control both the cell cycle and development (Dimova, 2003; Georlette, 2007; Korenjak, 2004; Lewis, 2004; Stevaux, 2005). An analysis of whether E2f1 transcriptional activity is required for the cell cycle defects caused by stabilized E2f1 and a description of what transcriptional changes occur will be necessary to explore these questions (Shibutani, 2008).

Is replication-coupled destruction of E2f1 necessary for normal fly development? Because the current experiments involve ectopic overexpression of E2f1^PIP mutants and not replacement of endogenous E2f1, this question cannot be definitively answered. However, E2f1^PIP-3A expression in the larval salivary gland blocks endocycle progression, suggesting that at least in some tissues this regulatory mechanism is necessary. It cannot be unambiguously determined whether phenotypes caused by E2f1^PIP-3A result from changes in the timing (i.e., present in S phase) or total amount of E2f1 accumulation. In either case, coupling destruction of E2f1 to replication provides a possible explanation for previous data indicating that Cyclin E/Cdk2 activity is inversely correlated with E2f1 accumulation. This negative regulatory relationship is at the heart of a mechanism that maintains overall cell cycle timing. Cyclin E/Cdk2 may indirectly reduce E2f1 protein by triggering DNA replication. In this way, E2f1 destruction during each S phase would keep E2f1/Dp activity “in check” during the cell cycle by counteracting the positive feedback loop that occurs during the G1-to-S transition, in which E2f1 induces Cyclin E transcription and Cyclin E/Cdk2 phosphorylates and inhibits Rbf1, resulting in more E2f1 activity. Without replication-coupled destruction of E2f1 to break or dampen this loop, stable E2f1 may gradually accumulate over multiple cycles, thereby inappropriately accelerating the cell cycle in a proliferating cell population. Such cell cycle acceleration is incompatible with Drosophila development and may constitute a form of “oncogenic stress” in mammals that contributes to the onset of cancer (Shibutani, 2008).

This model may also explain a prior observation that Drosophila E2f1 actually accumulates during S phase in the blastoderm embryo. How E2f1 avoids destruction during these very earliest S phases of development is not known. At this stage of development, there is no zygotic transcription and no G1 phase. Consequently, positive feedback amplification between Rbf1, Cyclin E/Cdk2, and E2f1-induced transcription is not needed for cell cycle progression. Thus, replication-coupled E2f1 destruction is not necessary for S phase per se, but may rather provide an intrinsic rheostat to dampen the positive feedback loop that is necessary to trigger the G1-to-S transition in canonical G1-S-G2-M cell cycles (Shibutani, 2008).

The Enok acetyltransferase complex interacts with Elg1 and negatively regulates PCNA unloading to promote the G1/S transition

KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and are involved in cell cycle regulation. However, information regarding their roles in regulating cell cycle progression is limited. This study reports the identification of subunits of the Drosophila Enok complex and demonstrates that all subunits are important for its HAT activity. A novel interaction is reported between the Enok complex and the Elg1 proliferating cell nuclear antigen (PCNA)-unloader complex. Depletion of Enok in S2 cells resulted in a G1/S cell cycle block, and this block can be partially relieved by depleting Elg1. Furthermore, depletion of Enok reduced the chromatin-bound levels of PCNA in both S2 cells and early embryos, suggesting that the Enok complex may interact with the Elg1 complex and down-regulate its PCNA-unloading function to promote the G1/S transition. Supporting this hypothesis, depletion of Enok also partially rescued the endoreplication defects in Elg1-depleted nurse cells. Taken together, this study provides novel insights into the roles of KAT6 HATs in cell cycle regulation through modulating PCNA levels on chromatin (Huang, 2016).

This study reports that Enok forms a complex homologous to the human MOZ complex and that all four subunits contribute to its HAT function in vivo. Notably, in addition to stimulating the HAT activity of Enok toward H3K23, Br140 also expanded its substrate specificity to include H3K14 in vitro (Huang, 2014). This result suggests that Br140 plays a role in regulating the enzymatic specificity of the Enok complex, which is consistent with the recent study showing that the human homolog of Br140, BRPF1, switches the substrate specificity of the HBO1 HAT complex to histone H3 (Lalonde et al. 2013). However, although BRPF1 interacts with both MOZ and HBO1 in human cells, the Drosophila homolog of HBO1, Chameau, was not detected in Br140 purification, indicating that Br140 may be an Enok complex-specific component in flies (Huang, 2016).

This study has also revealed a novel physical and functional interaction between the Enok HAT complex and the Elg1 PCNA-unloader complex, suggesting a role for Enok in modulating PCNA levels on chromatin during cell cycle progression. The physical interaction between Enok and Elg1 complexes is also supported by a recent large-scale study on protein-protein interactions. This study reported the interacting partners of 459 Drosophila transcription-related factors, and four subunits of the Elg1 complex (Elg1, Rfc4, Rfc38, and Rfc3) were identified by affinity purification using Br140 as the bait. Interestingly, instead of Elg1, the largest component of the PCNA-loader complex (Rfc1) copurified with the yeast Sas3-containing NuA3 complex using Pdp3 as the bait protein. This difference in interacting partners between the Enok and Sas3 complexes may be one of the reasons that Enok-depleted S2 cells accumulate at the G1 phase but that populations with a ploidy ≥2C (G2/M) accumulate when SAS3 is deleted in gcn5Δ yeast cells. These results also raise the possibility that the roles of KAT6 HATs in regulating PCNA levels on the chromatin may be evolutionarily adapted by switching their interacting partners between different RFC/RFC-like complexes. The human MOZ complex has been implicated in playing a role in DNA replication via interacting with the MCM helicase and has been shown to regulate cell cycle arrest at the G1 phase by promoting p21 expression. Given that MOZ is a critical regulator of proliferation of hematopoietic precursors and is involved in leukemia, it may advance knowledge of hematopoiesis to investigate whether the MOZ complex also interacts with an RFC/RFC-like complex and regulates PCNA loading/unloading (Huang, 2016).

Reducing enok expression levels by dsRNA increased the rate of G2/M progression. While this faster G2/M progression is not dependent on Elg1, an ∼40% increase was also detected in the mRNA levels of the Drosophila CDC25 phosphatase that activates the mitotic kinase Cdk1, string (stg), in Enok-depleted cells. As it has been reported previously that Enok plays a positive role in transcriptional activation by acetylating H3K23 (Huang, 2014), Enok may promote transcription of some repressor genes that down-regulate stg expression. Alternatively, Enok might repress transcription at a subset of gene loci, including stg, in a context-dependent manner, and, last, the possibility cannot be excluded that Enok may directly interact with other protein machinery to regulate G2/M progression (Huang, 2016).

Depletion of Enok also resulted in a block at the G1/S transition that is partially dependent on Elg1. This partial Elg1 dependence indicates that Enok has other roles in regulating the G1/S transition. While it is conceivable that Enok may regulate the expression of genes involved in cell cycle regulation, no significant changes were detected in the mRNA levels of Cyclin A, Cyclin B, Cyclin D, Cyclin E, Cyclin G, Cdk2, E2f1, Rbf (Rb), dap (p21/p27), Dp, or Myc in Enok-depleted S2 cells as compared with cells treated with control LacZ dsRNA. Nevertheless, further genome-wide analysis of gene expression levels in Enok-depleted cells may provide more information on the transcriptional roles of Enok in cell cycle regulation (Huang, 2016).

The model proposed in this paper that the Enok complex interacts with Elg1 via Br140 and down-regulates the PCNA-unloading function of Elg1. This hypothesis is supported by the findings that Br140 interacts with Elg1 in vivo and in vitro and that knocking down enok decreased the PCNA levels on chromatin. The Elg1 dependence of the G1/S block in Enok-depleted S2 cells and the genetic interaction between enok and elg1 in germline cells also agree well with the model, further supporting the negative role of the Enok complex in regulating Elg1 activity. Interestingly, small decreases were often observed in the Elg1 protein levels in Enok-depleted S2 cells or germline cells compared with the controls, while the elg1 mRNA levels remained largely unaffected by Enok depletion. This observation suggests that, in addition to regulating the PCNA-unloading function of the Elg1 complex, Enok may be involved in maintaining Elg1 protein levels or that the protein level and the PCNA-unloading activity of Elg1 may be inversely coregulated. Taken together, the physical and functional interactions between Enok and Elg1 provide a novel insight into the mechanisms underlying regulation of cell proliferation by KAT6 HATs (Huang, 2016).

GENE STRUCTURE

Immediately adjacent to, but distrinct from the PCNA gene, is plutonium (plu). The PLU gene product controls DNA replication early in Drosophila development. plu mutant females lay unfertilized eggs that have undergone extensive DNA synthesis. In fertilized embryos from plu mutant mothers, S-phase is uncoupled from mitosis. The gene is expressed only in ovaries and embryos; null alleles are strict maternal effect mutations, and the phenotype of inappropriate DNA replication is the consequence of loss-of-gene function. plu therefore negatively regulates S-phase at a time in early development when commitment to S-phase does not depend on cyclic transcription. plu encodes a protein with two ankyrin-like repeats, including a domain for protein-protein interaction (Axton, 1994).

Bases in 5' UTR - 89

Exons - 2

Bases in 3' UTR - 374

PROTEIN STRUCTURE

Amino Acids - 260

Structural Domains

A protein with an apparent mass of 36 kDa was purified from Drosophila melanogaster embryos using a protocol developed for the purification of proliferating cell nuclear antigen (PCNA) from human 293 cells. The Drosophila protein comigrates with human PCNA on one-dimensional sodium dodecyl sulfate-polyacrylamide gels and cross-reacts with monoclonal anti-rabbit PCNA antibodies. NH2-terminal amino acid sequence analysis reveals that the putative Drosophila PCNA is highly homologous to human PCNA. Of the first 22 amino acids, 16 are found to be identical between the two species, and four of the remaining six are changed conservatively. Results of total amino acid analysis are also consistent with a high degree of similarity between Drosophila PCNA and human PCNA. Functional analysis using the reconstituted simian virus 40 in vitro DNA replication system demonstrates that Drosophila PCNA can substitute, albeit with reduced efficiency, for human PCNA in stimulating simian virus 40 DNA synthesis. Affinity-purified anti-Drosophila PCNA antibodies cross-react with human PCNA and are able to recognize specifically Drosophila PCNA both on crude homogenate immunoblots and by indirect immunofluorescence analysis of proliferating cells in larval tissues in situ (Ng, 1990).

The genomic and cDNA clones for a Drosophila proliferating cell nuclear antigen (PCNA) were isolated and sequenced. The coding sequence for a 260-amino-acid residue polypeptide is interrupted by a single short intron of 60 base pairs (bp); about 70% of the deduced amino acid sequence of the Drosophila PCNA is identical to the rat and human PCNA polypeptides, with conserved unique repeats of leucine in the C-terminal region. The highly conserved sequence between residues 61 and 80 shows an alpha-helix-turn-alpha-helix motif: this is a putative DNA-binding domain found in several proteins. Genomic Southern blot hybridization analysis indicates the presence of a single gene for PCNA per genome (Yamaguchi, 1990).

date revised: 1 Nov 97

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