Proliferating cell nuclear antigen


Promoter Structure

The major transcription initiation site (cap site) of PCNA is localized 89 bp upstream from the ATG codon. Neither a TATA box nor a CAAT box is found within the 600-bp region upstream of the cap site. Clusters of 10 bp of sequence, similar to the binding sites for Drosophila proteins containing homeodomains, were found in the region from -127 to -413. DNase I footprint analysis reveals that the Drosophila homeodomain proteins coded by even-skipped and zerknullt genes can specifically bind to these sites. There are two sequences, starting at -52 and -39, of which 8 and 7 (respectively) of 10 nucleotides match the consensus sequence for HeLa cell transcription factor Sp 1 binding. These results suggest that the expression of the PCNA gene is under the control of genes coding for homeodomain proteins (Yamaguchi, 1990).

The proliferating-cell nuclear antigen (PCNA) promoter function resides within a 192-bp region (-168 to +24 with respect to the transcription initiation site). Cotransfection with a zerknullt (zen)-expressing plasmid specifically represses CAT expression. However, cotransfection with expression plasmids for either a nonfunctional zen mutation, even-skipped, or bicoid shows no significant effect on CAT expression. RNase protection analysis reveals that the repression by Zen is at the transcription step. The target sequence of Zen maps within the 34-bp region (-119 to -86) of the PCNA gene promoter, even though it lacked Zen protein-binding sites. Transgenic flies were established carrying the PCNA gene regulatory region (-607 to +137 or -168 to +137) fused with lacZ. When these flies were crossed with the zen mutant, ectopic expression of lacZ was observed in the dorsal region of gastrulating embryos carrying the transgene with either construct. These results indicate that Zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter (Yamaguchi, 1991a).

The regulatory region of Drosophila proliferating cell nuclear antigen (PCNA) gene consists of a promoter region (-168 to +24 with respect to the transcription initiation site) and an upstream region containing three homeodomain protein binding sites (HDB) (-357 to -165). The PCNA gene regulatory regions with HDB (-607 to +137) or without HDB (-168 to +137) were fused with the lacZ and transgenic flies were established by P-element-mediated transformation. Expression of the lacZ was high in embryos, first and second instar larvae, and adult females, and low at other stages of development. Only a marginal difference in expression was observed between flies carrying the homeodomain protein binding region and those not carrying it. In genetic crossing experiments of transgenic flies with those carrying mutation in homeobox genes, no significant change in the lacZ expression pattern was observed. However, when male transgenic flies were crossed with female flies homozygous for a torso gain-of-function allele, repression of the lacZ expression was observed in the central region of the embryo. Because these local changes in the lacZ expression depend on the homeodomain protein binding region, unidentified homeodomain proteins are probably involved. These results suggest that the promoter region is practically sufficient for expression of the PCNA gene and that the homeodomain protein binding region functions as a silencer when torso is activated ectopically (Yamaguchi, 1995a).

Promoter regions of the PCNA gene and the DNA polymerase alpha 180-kDa catalytic subunit gene contain a common 8 base pair (bp) promoter element: 5'-TATCGATA (DRE, Drosophila DNA replication-related element). The DRE of the PCNA gene is found at nucleotide positions -93 to -100 with respect to the transcription initiation site. The 10-bp sequence from positions -91 to -100, essential for complex formation with DREF (DRE-binding factor), is also essential for activation of the promoter in cultured cells. The 8-bp sequence is also important for DRE function during development, but the 8-bp sequence requirement for DRE function is less stringent in living flies than in the cultured cell (Yamaguchi, M., 1995b).

In transgenic experiments the PCNA gene promoter seems to function in a manner independent of cell cycle progression or of functions of the string gene (Yamaguchi, 1995c).

Sequences similar to the transcription factor E2F recognition site have been found within the Drosophila proliferating cell nuclear antigen (PCNA) gene promoter. These sequences are located at positions -43 to -36 (site1) and -56 to -49 (site 2), with respect to the cap site. E2F and DP proteins cooperate and bind to the potential E2F sites in the PCNA promoter in vitro. A binding factor(s) to these sequences with similar binding specificity to that of E2F has been detected in nuclear extracts of Drosophila Kc cells. Transient expression of the target site for the activation coincides with the E2F sites. These results indicate that the PCNA gene is a likely target gene of E2F. Examination of mutations in either or both of two E2F sites introduced into flies by germ line transformation reveals that site 2 plays a major role in the PCNA promoter activity during embryogenesis and larval development, although both sites are required for optimal promoter activity. However, for maternal expression in ovaries, either one of the two sites is essentially sufficient to direct optimal promoter activity. These results demonstrate, for the first time, an essential role for E2F sites in regulation of PCNA promoter activity during development of a multicellular organism (Yamaguchi, 1995d).

The DNA replication-related element (DRE) consisting of an 8-bp palindrome, TATCGATA, and not neighboring sequences, is responsible for activating promoters of the Drosophila melanogaster (Dm) PCNA (proliferating cell nuclear antigen)- and DNA polymerase alpha-encoding genes in both cultured cell and transgenic fly systems. 153 copies of DRE have so far been found in the Dm gene database. 73 of them are concentrated within the 600-bp upstream regions from the transcription start points of 61 genes. Interestingly, many of these genes are involved in either DNA replication, transcription, translation, signal transduction, cell cycle or other putative regulatory functions, and are possibly related to cell proliferation. It seems likely that DRE is an element common to the regulation of cell-proliferation-related genes, although their expression patterns may be different depending on which regulatory elements other than the DRE are combined (Matsukage, 1995).

Promoter regions of the Drosophila proliferating cell nuclear antigen (PCNA) gene and the DNA polymerase alpha gene contain a common 8-base pair promoter element (DRE: DNA replication-related element). The examination of a common expression mechanism for DNA replication-related genes, which is regulated positively by growth signals and negatively by differentiation signals would be of interest. PCNA-LacZ fusion genes were generated in which the 5'-flanking sequence of the PCNA gene was mutated. An examination of the expression of these fusion genes, introduced into flies by germ-line transformation, has led to the identification of another distinct regulatory element, URE (upstream regulatory element), within the region from -168 to -119 with respect to the transcription initiation site. During embryogenesis, the region containing the DRE sequence (-108 to -91) greatly stimulates the PCNA gene minimal promoter (-86 to +130), when it is placed upstream of the promoter in both normal and reverse orientations. Addition of the URE sequence further stimulates the promoter activity twofold. During larval stages, both DRE and URE are indispensable to the promoter activity, since neither of the sequences alone activate the minimal promoter. URE plays an essential role in various larval tissues such as salivary gland and imaginal disc. While the minimal promoter region alone directes maternal expression of lacZ in ovaries of adult females, both DRE and URE further stimulate promoter activity. These results show several elements of the PCNA gene promoter play roles during Drosophila development (Yamaguchi, 1996).

The Drosophila proliferating cell nuclear antigen (PCNA) gene promoter contains at least three transcriptional regulatory elements, the URE (upstream regulatory element), DRE (DNA replication-related element), and E2F recognition sites. In nuclear extracts of Drosophila Kc cells, a novel protein factor(s) has been identified [CFDD (common regulatory factor for DNA replication and DREF genes)] that appears to recognize two unique nucleotide sequences (5'-CGATA and 5'-CAATCA) and binds to three sites in the PCNA gene promoter. These sites are located at positions -84 to -77 (site 1), -100 to -93 (site 2) and -134 to -127 (site 3) with respect to the transcription initiation sites. Sites 2 and 3 overlap with DRE and URE, respectively, and the 5'-CGATA matches with the reported recognition sequence of BEAF-32 (boundary element-associated factor of 32 kDa). Detailed analyses of CFDD recognition sequences and experiments with specific antibodies to DREF (DRE-binding factor) and BEAF-32 suggest that CFDD is different from DREF, UREF (URE-binding factor) and BEAF-32. Polypeptides of approximately 76 kDa in the nuclear extract interact directly with the CFDD site 1 sequence. CFDD site 1 plays an important role for the promoter activity both in cultured cells and in living flies. In addition to the PCNA gene, multiple CFDD sites were found in promoters of the DNA polymerase alpha and DREF genes; CFDD binding to the DREF promoter has been confirmed. Therefore, CFDD may play important roles in regulation of Drosophila DNA replication-related genes (Hayashi, 1997).

The Drosophila Proliferating cell nuclear antigen promoter contains multiple transcriptional regulatory elements, including the upstream regulatory element (URE), a DNA replication-related element, and E2F recognition sites. In addition to DRE and E2F sites, the PCNA promoter contains three CFDD (common regulatory factor for DNA replication and DREF genes recognition) sites. Among these three, at least site 1 could be demonstrated to play an important role in promoter activity in both cultured cells and living flies. In addition to the PCNA gene, multiple CFDD sites have been found in promoters of the DNA polymerase and DREF genes. In nuclear extracts of Drosophila embryos, a protein factor, the URE-binding factor (UREF), has been detected that recognizes the nucleotide sequence 5'-AAACCAGTTGGCA located within URE. Analyses in Drosophila Kc cells and transgenic flies reveal that the UREF-binding site plays an important role in promoter activity both in cultured cells and in living flies. A yeast one-hybrid screen using URE as a bait allows isolation of a cDNA encoding a transcription factor, Grainyhead/nuclear transcription factor-1 (Grh/NTF-1). The nucleotide sequence required for binding to Grh is indistinguishable from that for UREF detected in embryo nuclear extracts. Furthermore, a specific antibody to Grh reacts with UREF in embryo nuclear extracts. From these results it is concluded that GRH is identical to UREF. Although Grh has been thought to be involved in regulation of differentiation-related genes, this study demonstrates for the first time the involvement of a Grh-binding site in the regulation of the DNA replication-related Proliferating cell nuclear antigen gene (Hayashi, 1999).

E2F proteins control cell cycle progression by predominantly acting as either activators or repressors of transcription. How the antagonizing activities of different E2Fs are integrated by cis-acting control regions into a final transcriptional output in an intact animal is not well understood. E2F function is required for normal development in many species, but it is not completely clear for which genes E2F-regulated transcription provides an essential biological function. To address these questions, the control region of the Drosophila PCNA gene has been characterized. A single E2F binding site within a 100-bp enhancer is necessary and sufficient to direct the correct spatiotemporal program of G1-S-regulated PCNA expression during development. This dynamic program requires both E2F-mediated transcriptional activation and repression, which, in Drosophila, are thought to be carried out by two distinct E2F proteins. The data suggest that functional antagonism between these different E2F proteins can occur in vivo by competition for the same binding site. An engineered PCNA gene with mutated E2F binding sites supports a low level of expression that can partially rescue the lethality of PCNA null mutants. Thus, E2F regulation of PCNA is dispensable for viability, but is nonetheless important for normal Drosophila development (Thacker, 2003).

Two sequences upstream of the PCNA transcription start site are capable of binding E2F in vitro, and chromatin IP experiments have demonstrated that this region is occupied by both dE2F1 and dE2F2 in cultured SL2 cells. The two E2F binding sites have been reported to be necessary for PCNA expression in vivo, as determined by measuring the amount of β-Gal activity in whole-animal extracts from PCNA-lacZ transgenes. However, this approach does not assess the contribution E2F binding sites make to endogenous patterns of gene expression that represent the complex spatiotemporal program of PCNA expression during development. To determine this, transgenic flies were engineered carrying PCNA-GFP fusion constructs and in situ hybridization of embryos carrying these transgenes was performed with a GFP probe. The results indicate that a 100-bp sequence containing the two E2F binding sites reproduces the entire complex embryonic profile of cell cycle-correlated PCNA expression (Thacker, 2003).

PCNA-GFP fusion protein expression was analyzed in several larval tissues by confocal microscopy. The final synchronous mitotic cycle prior to cell differentiation (called the 'second mitotic wave') in third instar eye imaginal discs can be easily visualized as a stripe of cells that incorporates BrdU and that also expresses PCNA in an E2F-dependent fashion. The accumulation of PCNA-GFP fusion protein from the reporter transgene faithfully reproduces this pattern of cell cycle control. In the optic lobe of the larval brain, PCNA-GFP expression correlates with the inner and outer proliferative zones, which are separated by a field of quiescent cells. PCNA-GFP expression also accurately reports patterns of proliferation in the wing imaginal disc. Expression is absent in the zone of nonproliferating cells (ZNC) located at the juxtaposition of dorsal and ventral compartments in which E2F is thought to be inactive, and it is present in the surrounding cells in a pattern consistent with known proliferation patterns in this part of the disc. Thus, the 100-bp enhancer element containing two E2F binding sites accurately reproduces S phase-associated, E2F-dependent PCNA expression at many stages of Drosophila development. These transgenes should prove useful as tools to mark replicating cells in situ by using methods other than BrdU labeling (Thacker, 2003).

To determine the binding sites through which dE2F2 acts, the activity of each PCNA reporter construct was observed in dE2F2 mutant eye discs by in situ hybridization. The expression of PCNA-GFP and PCNA-Δ site I-GFP in dE2F2 mutant eye discs was very similar to wild-type, suggesting that dE2F1 is sufficient to provide both activating and repressing activities necessary to generate the pattern of PCNA expression. PCNA-Δ site II-GFP was expressed within the morphogenetic furrow of dE2F2 mutant eye discs, which is in contrast to the lack of expression of this reporter in wild-type eye discs. This ectopic expression appears to require dE2F1, because PCNA-Δ site I&II-GFP was not expressed in dE2F2 mutant eye discs. Thus, dE2F1 and dE2F2 can compete for site I when site II is absent, suggesting that activating and repressing influences on the PCNA gene can act through the same E2F binding site. However, the relevance of this observation to endogenous PCNA regulation is unclear, since E2F binding site II alone is sufficient to drive the spatiotemporal pattern of PCNA expression. Perhaps dE2F2 modulates the overall level of output of PCNA transcription by binding to site I, rather than whether the gene is fully repressed or not (Thacker, 2003).

RT-PCR was used to directly measure whether loss of E2F binding sites could support some expression below the level detectable by cytological methods. Whereas GFP transcripts were undetectable in wild-type embryos, they were detected at similar levels in both PCNA-GFP and PCNA-Δ site I-GFP lines. GFP transcripts were also reproducibly detected in Δ site II and Δ site I&II lines compared to controls. In the Δ site I and Δ site I&II lines, the amount of GFP mRNA detected was lower than with PCNA-GFP or PCNA-Δ site I-GFP lines, although there was enough line to line variability within a single construct that conclusive quantitative comparisons could not be made between constructs. Nevertheless, these data indicate that the PCNA enhancer can support transcription in the absence of functional E2F binding sites. Similarly, simultaneous loss of dE2F1 and dE2F2 reduces, but does not eliminate, endogenous PCNA expression in the eye disc (Thacker, 2003).

Chronic derepression in the absence of E2F proteins might provide sufficient expression to permit cell cycle progression. Thus, the essential function of the PCNA gene could possibly be provided in the absence of E2F-regulated transcription. To test this, PCNA minigenes were constructed that lack one or both of the E2F binding sites (by fusing the site mutants used above to the PCNA coding region instead of GFP) and it was asked if these transgenes could complement the lethality of PCNA null mutants. As expected, both a wild-type control transgene and two independent Δ site I-PCNA transgenes fully complement the lethality of PCNA null mutant animals. The two independent Δ site I&II-PCNA and one Δ site II-PCNA transgenes tested in this assay also complemented PCNA lethality, although less efficiently than wild-type or Δ site I transgenes. The Δ site II-PCNA rescue was the least efficient (7% of expected for each of two PCNA alleles), although, since only a single line was tested, an inhibitory effect from insertion position cannot be excluded. Nevertheless, it is possible that E2F2-mediated inhibition through site I further inhibits expression from this transgene relative to the Δ site I&II-PCNA construct. In sum, while E2F regulation is not absolutely essential for PCNA gene function, it appears to provide a level of PCNA expression necessary for normal Drosophila development (Thacker, 2003).

Loss of dE2F1 function is lethal, and replication in cells that lack dE2F1 is impaired relative to that in wild-type cells. Because dE2F1 is also required for the expression of genes encoding essential replication factors, dE2F1-mediated activation appears to be necessary for normal cell cycle progression. Nevertheless, sufficient PCNA function is provided in the absence of E2F regulation to support development. This expression may result from the absence of E2F/RBF-dependent repression and/or the presence of other factors (e.g., DREF) that bind within the 100-bp enhancer and contribute to PCNA activation. S. cerevisiae cells can proliferate in the absence of cell cycle-regulated activation of transcription of replication factors. Similarly, DNA synthesis in the second mitotic wave appears rather normal in eye imaginal discs lacking both dE2F1 and dE2F2. Here, direct control of cyclin E transcription by the Ci transcription factor in response to Hh signaling appears to control the onset of S phase (Thacker, 2003).

If sufficient biosynthesis of essential replication factors like PCNA can be achieved in the absence of E2F, then why is E2F essential? The data indicate that the absence of E2F-regulated PCNA expression is not tolerated very well. Thus, the optimal level of PCNA gene function requires E2F input. Since PCNA is not expected to provide a cell cycle regulatory role, per se, it is suspected that the lack of cell cycle regulation and the consequent low level of ubiquitous expression is not the problem. Rather, the lack of high-level expression achieved during S phase may attenuate DNA synthesis and inhibit normal development. This may be true for many E2F target genes, such that the coordinated loss of entire E2F-directed programs of gene expression is much more detrimental than the loss of E2F regulation of a single gene like PCNA (Thacker, 2003).

The gene promoter of Drosophila PCNA contains several transcriptional regulatory elements, such as upstream regulatory element (URE), DNA replication-related element (DRE, 5'-TATCGATA), and E2F recognition sites. In the present study, a yeast one-hybrid screen using three tandem repeats of DRE in PCNA promoter was used as the bait allowed isolation of a cDNA encoding Cut, a Drosophila homolog of mammalian CCAAT-displacement protein (CDP)/Cux. Electrophoretic mobility shift assays showed that Cut binds to both DRE and the sequence 5'-AATCAAAC in URE, with much higher affinity to the former. Measurement of PCNA promoter activity by transient luciferase expression assays in Drosophila S2 cells after an RNA interference for Cut or DREF showed DREF activates the PCNA promoter while Cut functions as a repressor. Chromatin immunoprecipitation assays in the presence or absence of 20-hydroxyecdysone further showed both DREF and Cut proteins to be localized in the genomic region containing the PCNA promoter in S2 cells, especially in the Cut case upon induction of differentiation. These results indicate that Cut functions as a transcriptional repressor of PCNA gene by binding to the promoter region in the differentiated state, while DREF binds to DRE to promote expression of PCNA during cell proliferation (Seto, 2006).

Transcriptional Regulation

A coordinate program of transcription of S-phase genes (DNA polymerase alpha, PCNA and the two ribonucleotide reductase subunits) can be induced in Drosophila embryos by the G1 cyclin, cyclin E. This program drives an intricate spatial and temporal pattern of gene expression that perfectly parallels the embryonic program of S-phase control. string mutation does not disrupt this dynamic pattern of expression. Thus, the transcriptional program is not a secondary consequence of cell cycle progression. It seems that developmental signals control this transcriptional program and the activation of this program either directly or indirectly drives transition from G1 to S phase in the stereotyped embryonic pattern (Duronio, 1994).

The expression of the 1152-base pair 5'-flanking region (-1107 to +45 nucleotide positions with respect to the major transcription initiation site) of the Drosophila DNA polymerase alpha gene is repressed by zerknullt (zen) as previously observed with the proliferating cell nuclear antigen gene promoter. The zen expression results in reduction of the abundance of mRNA expression directed by these promoter fragments and also mRNAs for both DNA polymerase alpha and PCNA. Various deletion of the DNA replication-related element (DRE), a positive cis-acting element found in both DNA polymerase alpha and PCNA genes, reveals 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).

DREF, a transcription regulatory factor that specifically binds to the promoter-activating element DRE (DNA replication-related element) of DNA replication-related genes, was purified to homogeneity from nuclear extracts of Drosophila Kc cells. DREF is a polypeptide of 701 amino acid residues that contains three characteristic regions: one rich in basic amino acids, another rich in proline, and the third in acidic amino acids. A part of the N-terminal basic amino acid region (16-115 amino acids) is responsible for the specific binding to DRE. Antibodies against DREF specifically inhibit the transcription of the DNA polymerase alpha promoter in vitro. Overproduction of DREF protein overcomes the repression of the proliferating cell nuclear antigen gene promoter by the zerknüllt gene product. DREF is a trans-activating factor for DNA replication-related genes. DREF polypeptide is present in nuclei after the eighth nuclear division cycle, suggesting that nuclear accumulation of DREF is important for the coordinate zygotic expression of DNA replication-related genes carrying DRE sequences (Hirose, 1996).

The E2F transcription factor, a heterodimer of E2F and DP subunits, is capable of driving the G1-S transition of the cell cycle. However, mice in which the E2F-1 gene has been disrupted develop tumors, suggesting a negative role for E2F in controlling cell proliferation in some tissues. The consequences of disrupting the DP genes have not been reported. A screen was carried out for mutations that disrupt G1-S transcription late in Drosophila embryogenesis and five mutations in the dDP gene were identified. Sequencing of dDP reveals the presence of several important motifs, including the DNA-binding region, the DEF box that is predicted to be required for DP/E2F heterodimerization, and three other highly homologous regions named DP-conserved box 1 (DCB1), DCB2, and negatively charged box (NCB). Although mutations in dDP or dE2F nearly eliminate E2F-dependent G1-S transcription, S-phase still occurs. Cyclin E has been shown to be essential for S-phase in late embryogenesis, but in dDP and dE2F mutants the peaks of G1-S transcription of cyclin E are missing. Thus, greatly reduced levels of cyclin E transcript suffice for DNA replication until late in development. Both dDP and dE2F are necessary for viability, and mutations in the genes cause lethality at the late larval/pupal stage. The mutant phenotypes reveal that both genes promote progression of the cell cycle (Royzman, 1997).

The striking observation from the Drosophila dDP and dE2F mutants is that although cyclic transcription of cyclin E, PCNA, and ribonucleotide reductase 2 (RNR2) is not detectable, S phase still occurs. Although the possibility that cyclic transcription of these genes occurs at a low level driven by maternal pools of dDP and dE2F cannot be excluded, the bursts of transcription that normally precede S phase are not essential for the G1-S transition. In these mutants the cell cycle may be driven by basal levels of transcripts and post-transcriptional regulation. The maternal pools of components of the replication machinery can persist until late in development, as evidenced by the fact that mutations in PCNA and MCM2 cause late larval lethality (Royzman, 1997 and references).

Suppression of the rbf null mutants by a de2f1 allele that lacks transactivation domain

E2F positively regulates many of the genes required for initiation of S phase (the DNA synthetic phase). In mammals, the tumor suppressor RB interacts with, and negatively regulates, E2F, but it is not clear whether the function of pRB is solely mediated by E2F. In addition, E2F has been shown to mediate both transcription activation and repression; it remains to be tested which function of E2F is critical for normal development. Drosophila homologs of the RB and E2F family of proteins Rbf and E2f have been identified. The genetic interactions between Rbf and E2f were analyzed during Drosophila development, and the results show that Rbf is required at multiple stages of development. Unexpectedly, Rbf null mutants can develop until late pupae stage when the activity of E2f is experimentally reduced, and can develop into viable adults with normal adult appendages in the presence of an E2f mutation that retains the DNA binding domain but lacks the transactivation domain. These results indicate that most, if not all, of the function of Rbf during development is mediated through E2f. In turn, the genetic interactions shown here also suggest that E2f functions primarily as a transcription activator rather than a co-repressor of Rbf during Drosophila development. Analysis of the expression of an E2F target gene Pcna in eye discs shows that the expression of PCNA is activated by E2f in the second mitotic wave and repressed in the morphogenetic furrow and posterior to the second mitotic wave by Rbf. Interestingly, reducing the level of Rbf restores the normal pattern of cell proliferation in E2f mutant eye discs but not the expression of E2f target genes, suggesting that the coordinated transcription of E2f target genes does not significantly affect the pattern of cell proliferation (Du, 2000).

Interestingly, lowering the activity of Rbf can partially suppress the E2f null phenotypes. There are at least two possible explanations for this observation. One possibility is that Rbf has a function downstream of E2f; the other possibility is that Rbf can affect the E2f mutant phenotypes through a parallel pathway (for example Rbf may be able to regulate the expression of E2f target genes through another target such as dE2F2). The second explanation seems to be more likely for the following reasons: (1) in the suppressed E2f null mutants, the expression of E2f target genes is not restored, and the larvae growth is still greatly retarded, suggesting that reducing the level of Rbf bypasses rather than restores the function lost by E2f mutation; (2) the rbf null mutant phenotype is fully suppressed by an E2f mutant that lacks transcription activation and an Rbf binding domain, demonstrating that Rbf functions upstream of E2f. In summary, these results do not point to a role for Rb downstream of E2f. In contrast, loss of Rbf indeed causes deregulation of the expression of PCNA even in the absence of transcription activation by E2f, supporting the notion that Rbf can regulate the expression of PCNA by targets other than E2f (Du, 2000).

Control of endoreduplication domains in the Drosophila gut by the knirps and knirps-related genes

Endoreduplication cycles that lead to an increase of DNA ploidy and cell size occur in distinct spatial and temporal patterns during Drosophila development. Only little is known about the regulation of these modified cell cycles. Fore- and hind-gut development have been investigated and evidence is presented that the knirps and knirps-related genes are key components to spatially restrict endoreduplication domains. Lack and gain-of-function experiments show that knirps and knirps-related, which both encode nuclear orphan receptors, transcriptionally repress S-phase genes of the cell cycle required for DNA replication and that this down-regulation is crucial for gut morphogenesis. Furthermore, both genes are activated in overlapping expression domains in the fore- and hind-gut in response to Wingless and Hedgehog activities emanating from epithelial signaling centers that control the regionalization of the gut tube. These results provide a novel link between morphogen-dependent positional information and the spatio-temporal regulation of cell cycle activity in the gut (Fuss, 2001).

The development of the gut epithelium is accompanied by a stereotyped pattern of cell cycle regulation. The fore- and hind-gut primordia undergo a fixed number of postblastodermal cell divisions until late stage 10/early stage 11. Endoreduplication cycles have been described to occur at stages 13/14 in the hindgut. BrdU incorporation studies have shown that the hindgut epithelium displays a subdivision into replicating tissues (such as the developing large intestine) and quiescent tissues (such as the developing small intestine) and the rectum from stage 11 onwards. The replicative activity is reflected by a specific BrdU incorporation pattern in the hindgut: no incorporation is observed in the small intestine and rectum and but high incorporation is observed in the large intestine primordia in between. Notably, the kni/knrl expression pattern in the hindgut of wild-type embryos is complementary to the BrdU incorporation pattern. This complementarity also applies to the foregut in which kni/knrl are ubiquitously expressed. Endocycles have not been described for the developing foregut and BrdU is not incorporated from stage 11 onwards (Fuss, 2001).

In Df(3L)riXT1 mutant embryos, the analysis of the BrdU incorporation pattern reveals a tissue and time specific defect of cell cycle activity in the hindgut epithelium. An ectopic domain of DNA replication in the rectum and a slight expansion of DNA replication into the small intestine is detectable using the BrdU incorporation assay in stage 13 mutant embryos. The appearance of a G1 phase in the endoreduplicative cycle and the transition from G1 to S phase is accompanied by a molecular network controlling the coordinate transcription of cycE. CycE in turn regulates the activity of the S-phase genes Polalpha, PCNA and RNR2. Since cycE is only weakly expressed in the hindgut whether its expression is changed in Df(3L)riXT1 mutant embryos could not be analyzed (both kni and knrl are unchanged in cycE mutants). On the contrary, the Polalpha, PCNA and RNR2 genes which are activated in response to CycE activity, indeed do have a strong expression pattern in the hindgut that parallels the BrdU incorporation pattern in wild-type embryos. In line with the BrdU experiments, loss of kni/knrl function in Df(3L)riXT1 mutant embryos leads to an ectopic expression of RNR2, PCNA and Polalpha in the rectum and an upregulation of these genes in the small intestine prior to the upcoming defect in these gut regions. To further investigate this, gain-of-function experiments were performed using the UAS-Gal4 system. Ectopic expression of either kni or knrl in the entire hindgut using the 14-3fkh-Gal4 driver and the UAS-Kni or UAS-Knrl effector lines merely leads to a mild reduction of the BrdU incorporation domain in the large intestine. Ectopic expression of both kni and knrl has a strong effect on DNA replication in the hindgut. The BrdU domain is completely abolished, suggesting a combinatorial function of both genes in the suppression of endoreduplication cycles. The expression of various cell cycle components was analyzed. Ectopic kni and knrl activities in the entire hindgut are able to completely repress the transcription of RNR2, PCNA and Polalpha in the large intestine. Notably, cycE mutants in which no endoreduplication occurs in the large intestine, display a mutant phenotype that is similar to the one obtained when kni and knrl are ubiquitously expressed in the hindgut (Fuss, 2001).

Functional antagonism between E2F family members

E2F is a heterogenous transcription factor and its role in cell cycle control results from the integrated activities of many different E2F family members. Unlike mammalian cells, which have a large number of E2F-related genes, the Drosophila genome encodes just two E2F genes, de2f1 and E2F transcription factor 2 (de2f2). de2f1 and de2f2 provide different elements of E2F regulation, and they have opposing functions during Drosophila< development. dE2F1 and dE2F2 both heterodimerize with dDP and bind to the promoters of E2F-regulated genes in vivo. dE2F1 is a potent activator of transcription, and the loss of de2f1 results in the reduced expression of E2F-regulated genes. In contrast, dE2F2 represses the transcription of E2F reporters and the loss of de2f2 function results in increased and expanded patterns of gene expression. The loss of de2f1 function has previously been reported to compromise cell proliferation. de2f1 mutant embryos have reduced expression of E2F-regulated genes, low levels of DNA synthesis, and hatch to give slow-growing larvae. These defects are due in large part to the unchecked activity of dE2F2, since they can be suppressed by mutation of de2f2. Examination of eye discs from de2f1;de2f2 double-mutant animals reveals that relatively normal patterns of DNA synthesis can occur in the absence of both E2F proteins. This study shows how repressor and activator E2Fs are used to pattern transcription and how the net effect of E2F on cell proliferation results from the interplay between two types of E2F complexes that have antagonistic functions (Frolov, 2001).

The relatively normal patterns of cell proliferation in de2f1;de2f2 mutants are, at first glance, difficult to reconcile with the idea that E2F is a critical regulator of gene expression and cell proliferation. The expression of de2f1-regulated genes was examined in de2f2 and de2f1; de2f2-mutant animals. Third instar eye discs were chosen for this analysis to avoid the possible contribution of maternally supplied products. Initially expression of PCNA, one of the best-characterized E2F-regulated genes, was monitored by in situ hybridization. In wild-type eye discs, the pattern of PCNA expression is tightly coupled to the pattern of DNA replication. High levels of PCNA transcripts are present anterior to the furrow and within a narrow stripe that overlaps with the second mitotic wave. No PCNA expression is present in the furrow and in the cells just anterior to the furrow. In de2f1;de2f2 double-mutant eye discs, PCNA expression is abnormal. In these animals, a weak staining is observed in mutant eye discs without any specific pattern. PCNA transcripts appear to be present at a low level in the anterior portion of the disc, including the morphogenetic furrow and the second mitotic wave. In de2f2 mutant animals, PCNA expression is not downregulated as cells enter the furrow, and this results in an expanded region of PCNA transcription that encompasses both the first and second mitotic waves and the morphogenetic furrow (Frolov, 2001).

To compare the overall levels of E2F-target genes expression, Northern blot analysis was performed using total RNA from third instar eye imaginal discs dissected from de2f2 single and de2f1;de2f2 double-mutant larvae. The steady state level of PCNA transcripts is elevated in the de2f2 mutant and declines in the double mutant to a level that is lower than the wild-type control. These results suggest that dE2F2 represses PCNA expression and that the elevated level of PCNA expression in the de2f2 mutant is due to the activity of dE2F1. The expression of MCM3, another proposed target of dE2F1, increases in the de2f2 single mutant and this effect is suppressed in the double mutant, but in this case the total amount of MCM3 RNA in the double-mutant discs is indistinguishable from wild type. Even though the pattern of RNR2 expression has been shown to be dependent on dE2F1, little change was seen in the total level of RNR2 transcripts in de2f1 mutant larvae or in de2f2, or de2f1;de2f2 mutant eye discs (Frolov, 2001).

It is concluded that both de2f1 and de2f2 are required for the normal pattern of PCNA expression. de2f2 is needed to repress PCNA expression in the morphogenetic furrow, whereas de2f1 is required for the high levels of expression in the first and second mitotic waves. Although the pattern of PCNA, MCM3, and RNR2 transcripts depends on de2f1 and/or de2f2, significant levels of each of these transcripts can be detected in the absence of both de2f1 and de2f2 (Frolov, 2001).

Distinct mechanisms of E2F regulation by Drosophila RBF1 and RBF2

RBF1, a Drosophila pRB family homolog, is required for cell cycle arrest and the regulation of E2F-dependent transcription. RBF2, a second family member, represses E2F transcription and is present at E2F-regulated promoters. Analysis of in vivo protein complexes reveals that RBF1 and RBF2 interact with different subsets of E2F proteins. E2F1, a potent transcriptional activator, is regulated specifically by RBF1. In contrast, RBF2 binds exclusively to E2F2, a form of E2F that functions as a transcriptional repressor. RBF2-mediated repression requires E2F2. Moreover, RBF2 and E2F2 act synergistically to antagonize E2F1-mediated activation, and they co-operate to block S phase progression in transgenic animals. The network of interactions between RBF1 or RBF2 and E2F1 or E2F2 reveals how the activities of these proteins are integrated. These results suggest that there is a remarkable degree of symmetry in the arrangement of E2F and RB family members in mammalian cells and in Drosophila (Stevaux, 2002).

Repression of E2F transcription is a hallmark of RB family proteins. To determine whether RBF2 regulates E2F-dependent transcription, its ability to repress E2F-regulated reporter constructs in SL2 cells was assayed. RBF1 and RBF2 expression constructs were generated and transfected into SL2 cells together with a PCNA reporter that had been previously used to measure the activity of dE2F1 and dE2F2. In order to monitor the expression levels from both constructs, RBF1 and RBF2 were HA-tagged on their N-terminal ends. Titration experiments showed that RBF2 represses transcription from the wild-type PCNA promoter but has no effect on the mutant PCNA reporter construct lacking E2F-binding sites. The repression properties of RBF1 and RBF2 were examined. RBF2 represses transcription from the PCNA promoter, as well as the MCM3 and DNA Polalpha promoters, two other E2F-regulated genes. In these reporter assays, RBF1 proved a more effective repressor than RBF2, when expressed at the same level. The reason why RBF1 and RBF2 expression plasmids give different levels of protein expression is not known. This difference may reflect a property of the endogenous proteins, since quantitative blots show that SL2 cells contain ~30 times more RBF1 than RBF2. It is concluded that RBF2 can repress E2F-dependent transcription, but in a less efficient manner than RBF1 (Stevaux, 2002).

To determine whether RBF1 and RBF2 are present at these promoters in vivo under physiological conditions, a chromatin immunoprecipitation (ChIP) assay was used with specific RBF1 or RBF2 antibodies. DNA sequences from the PCNA and the DNA Polalpha promoters are selectively enriched in the RBF1 and RBF2 immunoprecipitations. It is concluded that RBF1 and RBF2 are both able to repress transcription from E2F-regulated promoters, and that the endogenous RBF1 and RBF2 proteins are normally found at these promoters in vivo. The presence of both Drosophila pocket proteins at E2F promoters suggests that RBF1 and RBF2 may have overlapping functions in the regulation of E2F targets genes (Stevaux, 2002).

Since the overexpression of RBF2 is able to repress E2F-dependent transcription, it seemed likely that RBF2 would repress dE2F1-mediated activation in a manner similar to that previously shown for RBF1. To test this possibility, RBF2 and dE2F1 expression constructs were co-transfected together with a PCNA reporter plasmid in SL2 cells. To ensure that dE2F1 was not saturating, small quantities of the dE2F1 expression plasmid were used that resulted in a 4.5-fold activation of the PCNA reporter. Co-transfection of HA-RBF1 completely blocked this dE2F1-induced transcriptional response, and a significant degree of repression was observed when a low amount of HA-RBF1 was transfected (Stevaux, 2002).

Previous studies have revealed that dE2F1 and dE2F2 have distinct biochemical and functional properties. Both proteins can be found at endogenous E2F-regulated promoters, but dE2F1 functions primarily as an activator of transcription, whereas dE2F2 is a transcriptional repressor. Studies of de2f1, de2f2 and de2f2;de2f1 double mutants demonstrate that the normal expression patterns of E2F-target genes depends on the integrated activities of both dE2F1 and dE2F2. Thus far, it has been observed that (1) RBF2 interacts specifically with dE2F2, (2) dE2F2 is able to antagonize dE2F1 in a manner dependent upon its interaction with RBF proteins, and (3) RBF2-mediated repression requires dE2F2. These results suggest that transcriptional repression by RBF2 is not mediated via dE2F1. Rather, RBF2 appears to form an RBF2-dE2F2 complex that antagonizes dE2F1 indirectly, by altering the balance between the transcriptional activities of dE2F1 and dE2F2 (Stevaux, 2002).

This idea was tested directly in SL2 cells, where constant levels of transfected dE2F1 were challenged with increasing amounts of transfected dE2F2. The overall level of transcription generated by both dE2Fs at the PCNA promoter were compared with and without co-transfected RBF2. The combined expression of dE2F2 and RBF2 is able to reduce dE2F1-mediated activation of the PCNA reporter far more effectively than dE2F2 alone. Thus, in SL2 cells, RBF2 and dE2F2 cooperate to antagonize the transcriptional activity of dE2F1 (Stevaux, 2002).

TRF2 associates with DREF and directs promoter-selective gene expression in Drosophila

Drosophila TATA-box-binding protein (TBP)-related factor 2 (TRF2) is a member of a family of TBP-related factors present in metazoan organisms. Recent evidence suggests that TRF2s are required for proper embryonic development and differentiation. However, true target promoters and the mechanisms by which TRF2 operates to control transcription remain elusive. A Drosophila TRF2-containing complex has been purified by antibody affinity; this complex contains components of the nucleosome remodelling factor (NURF) chromatin remodelling complex as well as the DNA replication-related element (DRE)-binding factor DREF. This latter finding leads to potential target genes containing TRF2-responsive promoters. A combination of in vitro and in vivo assays has been used to show that the DREF-containing TRF2 complex directs core promoter recognition of the proliferating cell nuclear antigen (PCNA) gene. Additional TRF2-responsive target genes involved in DNA replication and cell proliferation have also been identified. These data suggest that TRF2 functions as a core promoter-selectivity factor responsible for coordinating transcription of a subset of genes in Drosophila (Hochheimer, 2002).

An 80K protein associated with TRF2 is identical to DREF1. DREF and its corresponding response element DRE have been well documented to be important for the regulation of cell-cycle and cell-proliferation genes in Drosophila (that is, genes for PCNA and the 180K and 73K subunits of DNA polymerase). The identification of the promoter-selective DNA-binding protein DREF was intriguing because Drosophila TRF2 thus far had failed to bind to canonical TATA-box elements, which suggests that TRF2 may cooperate with DREF to execute promoter specificity and perhaps operate like a metazoan sigma factor (Hochheimer, 2002).

The 140K protein associated with TRF2 is identical to Drosophila ISWI, which is the catalytic ATPase subunit of NURF, ACF and CHRAC chromatin remodelling complexes. Moreover, the 55K and 38K proteins associated with TRF2 turned out to be NURF-55/CAF-1 and NURF-38/inorganic pyrophosphatase, respectively. Notably, the peptide sequences obtained from the three largest (300K, 250K and 230K) proteins associated with TRF2 do not match NURF-301, suggesting that the presence of some NURF subunits is not merely a result of contaminating NURF in the TRF2 complex. However, analysis of the cDNAs encoding the 190K and 160K proteins associated with TRF2 revealed that both proteins contain conserved sequence motifs for 11 and 5 zinc finger motifs (C2H2), respectively; these smaller proteins thus resemble factors like the CCCTC-binding factor CTCF that has been implicated in mediating chromatin-dependent processes such as the regulation of insulator function. It is therefore possible that the TRF2 complex encompasses both promoter-selectivity functions and NURF-like components as well as other activities with distinct subunits and specificity (Hochheimer, 2002).

Having identified DREF as a tightly associated component of the TRF2 complex, it was next asked whether TRF2 can function as a true core promoter recognition factor and selectively initiate transcription at a promoter that is documented to be stimulated by the DRE/DREF system. The DREF-responsive PCNA promoter, which contains at least three promoter-proximal regulatory elements including an upstream regulatory element URE, DRE and two E2F recognition sites located within 200 bp upstream of the start site, was chosen (Hochheimer, 2002).

To test the responsiveness of the PCNA promoter in vitro and to map the transcription start site(s), a -580 PCNA (-580 to +56) promoter fragment, which contains all known regulatory elements, and a -64 PCNA (-64 to +56) promoter fragment, which lacks all regulatory elements except for the E2F-binding sites were used as DNA templates for in vitro transcription. Increasing amounts of a partially purified Drosophila embryo nuclear extract (H.4) that contains all the necessary basal factors were added, as well as both the TRF2 complex and limiting amounts of TFIID to the transcription reaction. Using the -580 PCNA template two distinct transcription start sites separated by 63 nucleotides were detected. Promoter 1 (with start site at position +1) was stimulated with increasing amounts of H.4 supplemented with TFIID whereas promoter 2 (with start site at position -63) was detected only with the lowest amounts of H.4 added. Using the truncated -64 PCNA, template, transcription from promoter 2 was essentially abolished, whereas a weak activity could be detected from promoter 1 by adding the maximum amount of H.4 + TFIID. In vitro and in vivo results suggest that promoter 2 might be TRF2- and DRE-dependent, whereas promoter 1 appears to be mediated by TFIID (Hochheimer, 2002).

It was next asked whether TRF2 can contribute to the enhancer-dependent activated transcription of the PCNA promoters by E2F and DP, which cooperate in DNA-binding and transcriptional activation. The co-expression of just the trancriptional activators E2F and DP in the absence of exogenous TRF2/DREF results in a substantial transcriptional activation of the PCNA reporter. It is likely that this activation by E2F/DP is mediated by endogenous TRF2. This activation is abolished with the -64 PCNA reporter, which lacks the DRE-binding sites but still contains the E2F-binding sites. As expected, inducing the co-expression of all three promoter recognition factors -- TRF2, DREF and E2F -- results in a strong synergistic activation of the PCNA promoters (80-fold) in a DRE-dependent fashion. These results suggest that in SL2 cells TRF2 and DREF can work together to stimulate the PCNA reporter in a DRE-dependent fashion. This is consistent with the finding in vitro that the TRF2 complex can selectively initiate transcription from promoter 2 of the PCNA gene in a DRE-dependent manner (Hochheimer, 2002).

To investigate whether TRF2 is involved in the coordinate regulation of other DNA replication and cell cycle genes in the Drosophila genome, oligonucleotide-based microarrays representing 13,500 Drosophila genes were hybridized with RNA probes isolated at different time points after induction of TRF2 expression in SL2 cells. The microarray analysis revealed that only 1.9% of all genes analysed were upregulated more than 2-fold and only 1.6% downregulated by more than 2-fold. These biochemical studies and cell based assays suggested that TRF2 functions as a core promoter-selectivity factor that collaborates with DREF. It was therefore asked whether there are other TRF2-responsive genes that also contain a promoter-proximal DRE. An analysis of the distribution of DREs in the Drosophila genome revealed that about 100 genes bear a consensus DRE within 1 kb of the predicted promoter region. Microarray analysis revealed that 38 of these DRE-containing genes were also responsive to TRF2 overexpression. For example, genes encoding PCNA, the 180K subunit of DNA polymerase, the a-subunit of mitochondrial DNA polymerase, and E2F were all found to be upregulated 2-5-fold by TRF2 in the microarray analysis and confirmed by RNase protection assays. Three additional DRE-regulated genes encoding TBP, the 73K subunit of DNA polymerase, and the 50K subunit of DNA polymerase were found to be downregulated (Hochheimer, 2002).

Because these studies have relied largely on 'gain of function' assays double stranded RNA interference (dsRNAi) was imployed in flies and SL2 cells25 to determine the consequences of ablating TRF2/DREF on transcription of putative target genes such as PCNA and DNApol 180 in cultured cells. SL2 cells were treated with in vitro synthesized dsRNA and the depletion of TRF2 and DREF proteins was monitored by immunoblot analysis. After 2 days of incubation the specifically targeted TRF2 and DREF proteins were severely depleted. After 48 h of dsRNA treatment, a significant number of cells sloughed off the plate and died. This is in accordance with previous finding that TRF2 RNAi in Drosophila embryos is lethal for embryos (Hochheimer, 2002).

However, between 24 and 48 h of dsRNAi treatment it was possible to reproducably measure the activity of transiently transfected luciferase reporters fused to either the PCNA or DNApol 180 promoters and compare them to an internal control reporter gene. The activities from both the PCNA (-580 to +56) and DNApol 180 (-620 to +20) promoters were significantly reduced in TRF2-depleted cells (4.2-fold and 3-fold, respectively) relative to a Renilla luciferase control reporter driven by the HSV TK promoter. Likewise, these two target gene promoters were downregulated in DREF-depleted cells (3.5-fold and 5-fold, respectively). These depletion experiments using dsRNAi thus support the findings that TRF2 and DREF participate in directing transcription of a select subset of genes that include PCNA and DNApol 180 (Hochheimer, 2002).

Although the dsRNAi studies provide an independent line of evidence to support the notion that TRF2/DREF play a role in promoter selectivity in vivo, they fail to provide a direct mechanistic link between TRF2/DREF and the PCNA and DNApol 180 promoters. Chromatin immunoprecipitation (ChIP) experiments were carried to determine the occupancy of TRF2/DREF at the PCNA and DNApol 180 promoters in formaldehyde-treated SL2 cells using antibodies raised against TRF2, DREF and TBP. The precipitated DNA fragments with an average length of 500-1,000 base pairs (bp) were analysed directly by polymerase chain reaction (PCR). The PCNA promoter region was specifically precipitated by anti-TRF2 and anti-DREF and to a lesser extent by anti-TBP; this is consistent with previous findings and confirms that TRF2, DREF and the TFIID subunit TBP can co-localize and directly interact with the PCNA promoter region in living cells. As further evidence for the targeting of specific promoters by TRF2 and DREF, an analysis was carried out of the DRE-containing DNApol 180 promoter region, which is selectively precipitated by the TRF2-, DREF- and TBP-specific antibodies. These ChIP experiments strongly support the in vitro transcription results as well as the cell-based dsRNAi transcription assays in establishing a core promoter selectivity for the TRF2/DREF complex (Hochheimer, 2002).

The E2F cell cycle regulator is required for Drosophila nurse cell DNA replication and apoptosis

During Drosophila oogenesis nurse cells become polyploid, enabling them to provide the developing oocyte with vast amounts of maternal messages and products. The nurse cells then die by apoptosis. In nurse cells, as in many other polyploid or polytene tissues, replication is differentially controlled and the heterochromatin is underreplicated. The nurse cell chromosomes also undergo developmentally induced morphological changes -- from being polytene, with tightly associated sister chromatids, to polyploid, with dispersed sister chromatids. Female-sterile dE2F1 and dDP mutants have been used to assess the role of the E2F cell cycle regulator in oogenesis and the relative contributions of transcriptional activation versus repression during nurse cell development. E2F1 transcriptional activity in nurse cells is essential for the robust synthesis of S-phase transcripts that are deposited into the oocyte. dE2F1 and dDP are needed to limit the replication of heterochromatin in nurse cells. In dE2F1 mutants the nurse cell chromosomes do not properly undergo the transition from polyteny to polyploidy. It is also found that dDP and dE2F1 are needed for nurse cell apoptosis, implicating transcriptional activation of E2F target genes in this process (Royzman, 2002).

Whether the activity of the E2F transcription factor is needed for transcription of known E2F target genes in the nurse cells was tested by examining transcript levels of PCNA, ORC1, and RNR2 by in situ hybridization. All three of these transcripts are present at background levels until stage 9 of egg chamber development. The transcripts are induced at high levels in stage 10. Staining of egg chambers with antibodies against dE2F1 protein show that the level of protein increases at the same time that E2F targets are expressed. The dE2F1i1, dE2F1i2, and dDPa1 mutations all caused a significant reduction, although not elimination, of these transcripts. To compare the transcript levels more accurately in the mutants, the in situ hybridizations and histochemical staining were done for ORC1 with dDPa1/CyO controls and dDPa1/Df mutant ovaries in the same tube. The mutant ovaries were distinguished from sibling controls by their smaller size and by the dumpless phenotype. In this experiment it was clear that the level of ORC1 transcript is significantly reduced in dDPa1 mutants. The mutations in dDP and dE2F1 do not generally block transcription at stages 9 and 10 of egg chamber development, because the level of cyclin E transcript is not detectably altered by these mutations. A reduction in the level of RNR2 transcript is observed in germline clones of dDP mutants (Royzman, 2002).

Sunspot, a link between Wingless signaling and endoreplication in Drosophila

The Wingless (Wg)/Wnt signaling pathway is highly conserved throughout many multicellular organisms. It directs the development of diverse tissues and organs by regulating important processes such as proliferation, polarity and the specification of cell fates. Upon activation of the Wg/Wnt signaling pathway, Armadillo (Arm)/beta-catenin is stabilized and interacts with the TCF family of transcription factors, which in turn activate Wnt target genes. This study shows that Arm interacts with a novel BED (BEAF and Dref) finger protein that has been termed Sunspot (Ssp). Ssp transactivates Drosophila E2F-1 (dE2F-1) and PCNA expression, and positively regulates the proliferation of imaginal disc cells and the endoreplication of salivary gland cells. Wg negatively regulates the function of Ssp by changing its subcellular localization in the salivary gland. In addition, Ssp was found not to be involved in the signaling pathway mediated by Arm associated with dTCF. These findings indicate that Arm controls development in part by regulating the function of Ssp (Taniue, 2010).

Arm is composed of 12 imperfect protein interaction repeats (Armadillo repeat domain) flanked by unique N and C termini. In an attempt to identify novel Arm-binding proteins, a yeast two-hybrid screen of a Drosophila embryo cDNA library was performed using the Armadillo repeat domain of Arm as bait. Positive clones containing the same insert of a novel gene (CG17153) were isolated that were named sunspot (ssp; named after the phenotype of mutant flies). Sequence analysis of the full-length cDNA revealed that it encodes a protein of 368 amino acids. A region near its N terminus (amino acids 34 to 98) shows similarity to the BED (BEAF and Dref) finger domain, which is predicted to form a zinc finger and to bind DNA (Taniue, 2010).

To confirm the interaction between Ssp and Arm, whether Ssp produced by in vitro translation could interact with the Armadillo repeat domain of Arm fused to glutathione S-transferase (GST) was tested. Ssp specifically interacted with the Armadillo repeat domain of Arm (amino acids 140 to 713), but failed to interact with Pendulin (Pen), a Drosophila homolog of importin α, which also possesses the Armadillo repeat domain. Pull-down assays with a series of deletion fragments of Ssp showed that a fragment of Ssp containing amino acids 235 to 307 (termed the ABR, the Arm-binding region) binds to Arm in vitro. Also, it was found that Armadillo repeats 2-8 of Arm are responsible for binding to Ssp. Although TCF is known to bind to Armadillo repeats 3-10 of Arm, Ssp did not compete with TCF for binding to Arm (Taniue, 2010).

Next, whether Ssp is associated with Arm in living cells was examined. Drosophila Schneider-2 (S2) cells were transfected with Arm along with GFP-Ssp, GFP-SspδC (amino acids 1 to 217; a mutant lacking the ABR) or GFP-SspABR (amino acids 235 to 342; a fragment containing the ABR). GFP-fusion proteins were immunoprecipitated from S2 cell lysates and subjected to immunoblotting with anti-GFP and anti-Arm antibodies. For immunoprecipitation of GFP-fusion proteins, a 13-kDa GFP-binding fragment was used derived from a llama single chain antibody, which was covalently immobilized to magnetic beads (GFP-Trap-M), as the molecular weight of GFP-SspδC is the same as that of IgG. It was found that Arm is associated with GFP-Ssp and GFP-SspABR. By contrast, Arm barely co-immunoprecipitated with GFP-SspδC. In addition, pull-down assays were also performed with a mixture of lysates of S2 cells transfected with Arm alone and GFP-Ssp alone, respectively. It was found that Ssp and Arm co-precipitate only when both proteins are co-expressed in S2 cells, excluding the possibility that Ssp and Arm associate after cells are lysed. Taken together, these results suggest that Ssp interacts via its ABR with Arm not only in vitro but also in vivo (Taniue, 2010).

One lethal P-element insertion line, l(3)j2D3j2D3, was found in which a P-element had been inserted into the gene adjacent to ssp, CG6801, which is located about 250 bp upstream of the 5' end of ssp. RT-PCR analysis revealed that the expression level and size of the CG6801 transcript were not changed compared with in wild-type larvae, which is consistent with the P-element being inserted into an intron in CG6801. To generate mutants that have a deletion in ssp but have intact CG6801, a local hop and imprecise excision approach was used. l(3)j2D3j2D3 was used in a local hop to generate a P-element insertion line, sunspotP, that completely complemented the lethality of l(3)j2D3j2D3. Then ssp mutants were generated by imprecise excision of the P-element from sunspotP. One allele was found that has a deletion of about 600 bp, and this was designated as ssp598. Sequence analysis showed that the deletion extends from a position 60 bp downstream of the presumptive ssp transcription start site to the ssp gene locus. Because this deletion removes the start codon and the BED finger domain of ssp, it is presumed that ssp598 represents a null allele for ssp. RT-PCR analysis revealed that ssp598 generates a truncated transcript. The truncated transcript encodes a peptide consisting of 13 amino acids, which is unrelated to Ssp. By contrast, RT-PCR analysis revealed that the intact CG6801 transcript is expressed in ssp598 mutant larvae, and that the expression level of CG6801 is unchanged in ssp598 mutant larvae compared with that in wild-type larvae. Furthermore, ssp598 fully complemented the phenotype of l(3)j2D3j2D3, indicating that this mutant contains intact CG6801 (Taniue, 2010).

The imaginal discs, salivary glands and central nervous system of larvae homozygous for ssp598 were smaller than those of their normal counterparts. ssp598 homozygotes reached the third instar stage, but failed to reach the pupal stage and died between 10 and 20 days after egg laying (AEL). Furthermore, melanotic pseudotumors were formed in ssp598 mutant larvae. Melanotic pseudotumors are groups of cells within the larvae that are recognized by the immune system and encapsulated within a melanized cuticle. One or more small melanotic pseudotumors first appeared in the ssp mutants at 6 days AEL, and the number and size of these melanotic pseudotumors increased during the development of the larvae. Similar phenotypes were observed with hemizygotes for ssp598 and Df(3L)BK9, which has a deletion larger than that of ssp598 and lacks ssp. In situ hybridization analysis of imaginal discs using the coding region of the ssp cDNA as a probe revealed that ssp transcripts are expressed ubiquitously. Therefore whether ubiquitous expression of ssp restores the phenotypes of ssp598 homozygous animals was examined. It was found that ubiquitous expression of the full-length ssp cDNA with the Gal4-UAS system rescued the lethality and other phenotypes associated with ssp598 homozygous animals. Taken together, these results suggest that the phenotypes of ssp598 homozygotes are caused by the loss of ssp function, and that ssp is required for cell proliferation and morphogenesis of the imaginal disc and central nervous system (Taniue, 2010).

Arm is a key transducer of Wg signaling and many of the Arm-binding proteins are known to function as a component of the Wg signal transduction pathway. To explore the possibility that Ssp is related to the Wg signal transduction pathway, the effect of Wg on the distribution of GFP-Ssp was examined. Because imaginal disc cells are too small for detailed study, focus for this analysis was placed on the third instar salivary glands, and whether the subcellular localization of GFP-Ssp is linked to Wg signaling was studied. The larval salivary gland mainly consists of secretory gland cells and imaginal ring cells. Gland cells are large polyploid epithelial cells. Small imaginal ring cells reside at the proximal end of the secretory gland. Immunostaining with anti-Wg antibody revealed that Wg is expressed in imaginal ring cells. Furthermore, Drosophila frizzled 3 (dfz3)-lacZ, a target gene of Wg signaling, was found to be expressed in imaginal ring cells and proximal gland cells, which reside within several cell diameters of the Wg-expressing cells. These results suggest that Wg signaling is active in the proximal region in the third instar salivary gland. When GFP-Ssp was expressed ubiquitously under the control of dpp-Gal4 in the larval salivary gland, GFP-Ssp was found to be localized predominantly at the nuclear envelope in proximal gland cells. In addition, GFP-Ssp was detected as aggregates in the nucleus in the distal region of the salivary gland. To examine whether this region-specific subcellular localization of GFP-Ssp is related to Wg signaling, Wg or Axin, a negative regulator of Wg signaling, was overexpressed in the salivary gland under the control of dpp-Gal4. It was found that expression of Wg along with GFP-Ssp resulted in the accumulation of a certain population of GFP-Ssp at the nuclear envelope in both the distal and proximal regions. Again, a significant amount of GFP-Ssp was localized in nuclear aggregates in both distal and proximal cells, suggesting that ectopic expression of Wg can also change the subnuclear localization of Ssp in proximal cells, from the nuclear periphery to nuclear aggregates. This result also suggests that ectopic expression of Wg in distal cells is not sufficient to change the subnuclear localization of all GFP-Ssp protein, from nuclear foci to the nuclear periphery. By contrast, when Axin was expressed along with GFP-Ssp, GFP-Ssp was detected as nuclear aggregates, not only in the distal region but also in the proximal region, but was no longer detected at the nuclear envelope. These results suggest that the subcellular localization of Ssp is regulated at least in part by Wg signaling in the third instar salivary gland (Taniue, 2010).

To examine whether the effect of Wg signaling on Ssp localization is mediated by the direct interaction between Arm and Ssp, Ssp localization was studied in larvae expressing an RNAi targeting Arm. It was found that Ssp was localized in nuclear aggregates and that Wg overexpression did not alter its localization when the expression of Arm was suppressed by RNAi. Thus, Arm is required for Wg-induced Ssp relocalization. Ssp localization was also examined in cells expressing δArm, a mutant of Arm that localizes at the plasma membrane. It was found that overexpression of δArm under the control of dpp-Gal4 results in the localization of GFP-Ssp at the plasma membrane throughout the salivary gland. Next the subcellular localization of SspδC, a mutant that lacks the ABR and is unable to interact with Arm, was examined. When GFP-SspδC was expressed ubiquitously, it was found to localize homogenously in the nucleus of both distal and proximal cells. This result indicates that the localization of Ssp to nuclear aggregates requires the ABR and suggests that Ssp requires a direct interaction with Arm to localize to its target sites in the nucleus. Furthermore, it was found that the localization of GFP-SspδC was not changed by coexpression with Wg, or δArm. Taken together, these results suggest that the direct interaction between Arm and Ssp is required for the regulation of Ssp localization by Wg signaling (Taniue, 2010).

The N-terminal region of Ssp contains a BED finger domain. This presumptive DNA-binding domain is known to be contained in several Drosophila proteins, such as Dref and BEAF-32. Dref regulates the transcription of genes involved in DNA replication and cell proliferation, including dE2F-1 and PCNA, the promoters of which contain BED finger-binding elements (BBEs). To clarify whether Ssp regulates the transcription of these genes, the expression levels of dE2F-1 and PCNA were examined. For this purpose, the P-element (lacZ) insertion lines E2F07172 and PCNA02248 were used. dE2F-1-lacZ and PCNA-lacZ expression were found to be high in distal cells compared with proximal cells in the larval salivary gland. When ssp was ectopically expressed in the salivary gland, dE2F-1-lacZ expression was markedly elevated in distal cells, whereas it was only slightly elevated in proximal cells. However, PCNA-lacZ expression was markedly elevated throughout the salivary gland. By contrast, dE2F-1-lacZ and PCNA-lacZ expression were not elevated in distal cells of ssp mutant salivary glands compared with in wild-type salivary glands, and dfz3-lacZ expression in ssp mutant and Ssp-overexpressing salivary glands was not changed compared with in wild-type salivary glands, suggesting that Ssp is not involved in Arm-dTCF-mediated transactivation of Wg target genes. In addition, overexpression of Wg resulted in a decrease in the expression levels of dE2F-1-lacZ and PCNA-lacZ in distal cells. Thus, Ssp is active in the distal region where Wg signaling is not active, and Ssp is aggregated in the nucleus. Conversely, Ssp is not very active in the proximal region where Wg signaling is active, and Ssp is accumulated in the nuclear envelope (Taniue, 2010).

The expression of dE2F-1, PCNA and dfz3 was examined in the wing disc. Clones of cells lacking Ssp function were generated by FLP/FRT-mediated somatic recombination. Clones of ssp mutant cells underwent only a few divisions after they were generated in the presumptive wing blade: the mutant cells proliferated slowly and either died or were actively eliminated from the disc epithelium. Therefore, a Minute mutation, M(3)65F, was used to confer a growth advantage upon cells homozygous for ssp. When mitotic recombination was induced in a M(3)65F background using enhancer trap lines, ssp mutant cells exhibited reduced levels of dE2F-1-lacZ and PCNA-lacZ expression but did not show any change in the levels of dfz3-lacZ and Arm expression. These results suggest that ssp regulates the expression of dE2F-1 and PCNA, but is not involved in Arm-dTCF-mediated Wg signaling (Taniue, 2010).

To confirm these results, endogenous expression of dE2F-1 and PCNA was examined by quantitative real-time RT-PCR analysis using RNA from late third instar larvae. Flies carrying heat-shock-inducible Gal4 (hs-Gal4) were crossed with transgenic flies carrying UAS-GFP, UAS-ssp or UAS-wg. Consistent with the above results, overexpression of ssp resulted in elevated steady state levels of dE2F-1 and PCNA transcripts. Furthermore, overexpression of Wg induced decreases in the numbers of dE2F-1 and PCNA transcripts. These results suggest that dE2F-1 and PCNA expression is regulated positively by Ssp and negatively by Wg (Taniue, 2010).

Also whether Ssp regulates the expression of dE2F-1 by binding directly to its promoter region was examined. Electrophoretic mobility-shift assays (EMSA) showed that GST-Ssp, but not GST, bound to a 40-mer oligonucleotide corresponding to a region in the dE2F-1 promoter that contains three BBEs. By contrast, GST-Ssp barely bound to a mutated probe in which CG in each BBE had been replaced with AA. Binding of Ssp to the wild-type probe was inhibited in the presence of an excess amount of unlabeled wild-type probe, whereas the mutated probe did not inhibit the interaction significantly. When anti-Ssp antibody was included in the reaction mixture, the Ssp band was not detected. Furthermore, it was found that GST-SspδBFD, a mutant Ssp lacking the BED finger domain, did not bind to the wild-type probe. These results suggest that Ssp regulates dE2F-1 expression by binding directly to the BBEs in the dE2F-1 promoter region via its BED finger domain (Taniue, 2010).

To further elucidate the function of Ssp and Wg, the third instar salivary glands of ssp and wg mutants were examined. In the third instar salivary gland, the distal region undergoes greater endoreplication than does the proximal region. As a result, the nuclear size of distal gland cells is markedly larger than that of proximal gland cells. However, the nuclear size of ssp mutant distal cells was found to be smaller than that of wild-type distal cells. By contrast, the nuclear size of wg mutant proximal cells was larger than that of wild-type proximal cells. Thus, the difference in nuclear size between proximal and distal cells was also small in the salivary glands of wg mutants (Taniue, 2010).

To confirm these results, the effects were examined of Ssp and/or Wg overexpression on the nuclear size of salivary gland cells. When Ssp was overexpressed, the nuclear size of both proximal and distal cells was heterogenous. Overexpression of Wg decreased the nuclear size of distal cells: the difference in nuclear size between Wg-overexpressing proximal and distal cells was small. However, when Wg was overexpressed along with Ssp, the effect of Ssp was suppressed and the heterogeneity of nuclear size was not observed. Furthermore, to confirm that Ssp and Wg play important roles in the regulation of endoreplication, δArm-expressing clones were generated using the flip-out technique. It was found that the nuclear size of δArm-expressing cells is much smaller than that of surrounding cells. This result suggests that δArm mislocalizes Ssp to the plasma membrane, thereby negatively regulating Ssp activity for endoreplication (Taniue, 2010).

To directly show that ssp mutant cells undergo fewer endoreplications than do wild-type cells, BrdU-labeling experiments were performed. When wild-type salivary glands were labeled with BrdU, distal cells efficiently incorporated BrdU, indicating that they underwent at least one round of DNA replication during the labeling period. By contrast, very few nuclei of ssp mutant cells and Wg-overexpressing cells were labeled with BrdU (Taniue, 2010).

dMyc has also been reported to be required for the endoreplication of salivary gland cells. It is therefore interesting to examine the relationship between dMyc, Wg and Ssp in endoreplication. It was found that dMyc expression was unchanged in both ssp mutant and Ssp-overexpressing salivary glands. Thus, Ssp might not be involved in the regulation of dMyc (Taniue, 2010).

Taken together, these results suggest that Ssp and Wg play important roles in the regulation of endoreplication in the third instar salivary gland, and that Wg might exert its effect by negatively regulating the function of Ssp. It is interesting to speculate that Ssp plays a general role for endoreplication in all larval endocycling tissues (Taniue, 2010).

It is believed that Wg/Wnt target genes are transactivated by Arm/β-catenin associated with TCF. However, expression of some human genes is transactivated by β-catenin that is associated with proteins other than TCF. For example, β-catenin interacts with the androgen receptor in an androgen-dependent manner and enhances androgen-mediated transactivation. In the present study, it was shown that Arm interacts with Ssp and negatively regulates its function. Ssp transactivates dE2F-1 and PCNA expression, and positively regulates the endoreplication of salivary gland cells. Furthermore, the Wg signal represses the function of Ssp by altering the subcellular localization of Ssp in the salivary gland: the Wg signal induces the accumulation of Ssp at the nuclear envelope. Interestingly, recent studies indicate that the nuclear membrane provides a platform for sequestering transcription factors away from their target genes. For example, it has been shown that the tethering of transcription factors such as c-Fos and R-Smads to the nuclear envelope prevents transcription of their target genes. The results appear to be consistent with these findings. Although the precise mechanism remains to be investigated, the interaction between Arm and Ssp appears to be required for the regulation of Ssp localization by Wg signaling. It remains to be investigated whether the mechanisms identified in the salivary gland are applicable to other tissues (Taniue, 2010).

Protein Interactions

Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and repair. It is a molecular matchmaker required for loading of proliferating cell nuclear antigen (PCNA) onto double-stranded DNA and, thus, for PCNA-dependent DNA elongation by DNA polymerases delta and epsilon. To elucidate the mode of RF-C binding to the PCNA clamp, modified forms of human PCNA were used that could be 32P-labeled in vitro either at the C or the N terminus. Using a kinase protection assay, it was shown that the heteropentameric calf thymus RF-C is able to protect the C-terminal region but not the N-terminal region of human PCNA from phosphorylation, suggesting that RF-C interacts with the PCNA face at which the C termini are located (C-side). A similar protection profile was obtained with the recently identified PCNA binding region (residues 478-712), but not with the DNA binding region (residues 366-477) of the human RF-C large subunit. RF-C 36 kDa subunit of human RF-C can interact independently with the C-side of PCNA. The RF-C large subunit from a third species, namely Drosophila melanogaster, interacts similarly with the modified human PCNA, indicating that the interaction between RF-C and PCNA is conserved through eukaryotic evolution (Mossi, 1997).

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).

Proliferating cell nuclear antigen: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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