Transcriptional Regulation

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).

Targets of Activity

DNA POL alpha (the alpha subunit of DNA polymerase), ribonucleotide reductase large (DmRNR1) and small (DmRNR2), and Proliferating cell nuclear antigen are coordinately transcribed in transient pulses that parallel and slightly precede DNA synthesis in cells entering S phase from quiescence (Duronio, 1994). Both DmRNR2 and PCNA require DE2F function for transcription (Duronio, 1995a).

E2F recognition sequences have been identified in the promoter of Drosophila DNA polymerase alpha gene (Ohtani, 1994).

Cyclin E expression at G1-S requires E2F, activation of E2F without cyclin E is not sufficient for S phase. Early in G1, ectopic expression of cyclin E alone can bypass E2F and induce S phase. Cyclin E is the downstream gene that couples E2F activity to G1 control. However, not all embryonic cycles are similarly coupled to E2F activation. The rapidly proliferating CNS cells that exhibit no obvious G1 express cyclin E constitutively and independently of E2F. Instead, cyclin E expression activates E2F in the CNS. Thus, this tissue-specific E2F-independent transcription of cyclin E reverses the hierarchical relationship between cyclin E and E2F. Both hierarchies activate expression of the full complement of replication functions controlled by E2F; however, whereas inactivation of E2F can produce a G1 when cyclin E is downstream of E2F (as in cells with a delayed S period), an E2F-independent source of cyclin E (neuroblasts) eliminates G1 (Duronio, 1995b).

Cyclin B expression is not detected in E2F-deficient clones of the eye disc, while elav is expressed normally in E2F-deficient clones (Brook, 1996)

Simultaneous overexpression of both E2F subunits, E2F and DP, stimulates the expression of multiple E2F-target genes including cyclin E, and also causes the initiation of S phase. Mutation of cyclin E prevents the initiation of S phase after overexpression of E2F/DP without affecting induction of target gene expression. Thus, E2F-directed transcription cannot bypass loss of cyclin E in Drosophila embryos. It is concluded that Cyclin E has an essential in vivo role in S phase induction other than induction of E2F activity (in mammals accomplished by phosphorylation and inactivation of Rb). Reduction of cyclin E expression blocks E2F-induced S phase in epidermis, but not in the midgut. These results suggest that different cell types have different sensitivity thresholds to cyclin E expression, and that as little as a two fold change in Cyclin E levels can have dramatic effects of cell cycle (Duronio, 1996).

Sequences similar to the transcription factor E2F recognition site are found within the Drosophila proliferating cell nuclear antigen (PCNA) gene promoter. These sequences are located at positions -43 to -36 (site I). and -56 to -49 (site II) with respect to the cap site. E2F and DP cooperate and bind to the potential E2F sites in the PCNA promoter in vitro. Thus the PCNA gene is a likely target gene of E2F. Site II 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 an essential role for E2F sites in regulation of PCNA promoter activity during development (Yamaguchi, 1995).

E2F mediates developmental and cell cycle regulation of ORC1 in Drosophila

Throughout the cell cycle of Saccharomyces cerevisiae, the level of origin recognition complex (ORC) is constant and ORCs are bound constitutively to replication origins (See Drosophila Orc2 for more information on the ORC). Replication is regulated by the recruitment of additional factors such as CDC6. ORC components are widely conserved, and it generally has been assumed that they are also stable factors bound to origins throughout the cell cycle. In this report, it is shown that the level of the Origin recognition complex subunit 1 (ORC1) subunit changes dramatically throughout Drosophila development. The accumulation of ORC1 is regulated by E2F-dependent transcription. In embryos, ORC1 accumulates preferentially in proliferating cells. In the eye imaginal disc, ORC1 accumulation is cell cycle regulated, with high levels in late G1 and S phase. In the ovary, the sub-nuclear distribution of ORC1 shifts during a developmentally regulated switch from endoreplication of the entire genome to amplification of the chorion gene clusters. Furthermore, it has been found that overexpression of ORC1 alters the pattern of DNA synthesis in the eye disc and the ovary. Thus, replication origin activity appears to be governed in part by the level of ORC1 in Drosophila (Asano, 1999)

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

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

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

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

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

Drosophila Double parked: a conserved, essential replication protein that colocalizes with the origin recognition complex and links DNA replication with mitosis and the down-regulation of S phase transcripts

To test if the cell cycle transcription of double parked (dup) is dependent on E2F, embryos homozygous for a null allele of the dE2F1 subunit, dE2F91 were collected and hybridized with dup riboprobes. The levels of dup transcript are decreased in the endoreplicating gut and appear to be slightly decreased in the CNS. A similar effect on dup transcript is seen in embryos that are homozygous mutant for the other subunit of the E2F transcription factor, of the genotype dDPa2. Thus, dup is a downstream target of the E2F transcription factor. Interestingly, yeast cdt1 transcription is also cell-cycle regulated. Expression of cdt1 is controlled by the G1-S transcription factor Cdc10 that, like E2F, regulates transcription of many genes required for S phase (Hofmann, 1994). This suggests that cell cycle control of dup may be conserved, and Dup may prove to be an important downstream target of E2F in mammalian cells (Whittaker, 2000).

Cyclin E is required to regulate positively the transcription of S phase genes in the nervous system and to downregulate these transcripts in endo cycling cells. The cyclinEl(2)305 and cyclin EPZ5 mutations and a cyclin E deficiency, Df(2L)TE35D1, have similar effects on double parked transcripts. In these embryos, dup is not downregulated properly in the endoreplicating gut such that dup transcripts persist at higher levels than wild type in the anterior midgut, central midgut, and posterior midgut in later embryonic stages. In cyclin E mutant embryos dup transcripts are reduced in the CNS, although not to as great an extent as other S phase genes. Thus, dup expression also is regulated by cyclin E (Whittaker, 2000).

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).

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).

The contribution of E2F-regulated transcription to Drosophila PCNA gene function

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).

Involvement of an SCFSlmb complex in timely elimination of E2F upon initiation of DNA replication in Drosophila

Cul1 is a core component of the evolutionarily conserved SCF-type ubiquitin ligases that target specific proteins for destruction. SCF action contributes to cell cycle progression but few of the key targets of its action have been identified. This study found that expression of the mouse Cul1 (mCul1) in the larval wing disc has a dominant negative effect. It reduces, but does not eliminate, the function of SCF complexes, promotes accumulation of Cubitus interruptus (a target of SCF action), triggers apoptosis, and causes a small wing phenotype. A screen for mutations that dominantly modify this phenotype showed effective suppression upon reduction of E2F function, suggesting that compromised downregulation of E2F contributes to the phenotype. Partial inactivation of Cul1 delayed the abrupt loss of E2F immunofluorescence beyond its normal point of downregulation at the onset of S phase. Additional screens showed that mild reduction in function of the F-box encoding gene slimb enhanced the mCul1 overexpression phenotype. Cell cycle modulation of E2F levels is virtually absent in slimb mutant cells in which slimb function is severely reduced. This implicates Slimb, a known targeting subunit of SCF, in E2F downregulation. In addition, Slimb and E2F interacted in vitro in a phosphorylation-dependent manner. This study used genetic and physical interactions to identify the G1/S transcription factor E2F as an SCFSlmb target in Drosophila. These results argue that the SCFSlmb ubiquitin ligase directs E2F destruction in S phase (Heriche, 2003; full text of article).

Cell cycle genes regulate vestigial and scalloped to ensure normal proliferation in the wing disc of Drosophila melanogaster

In Drosophila, the Vestigial-Scalloped (VG-SD) dimeric transcription factor is required for wing cell identity and proliferation. Previous results have shown that VG-SD controls expression of the cell cycle positive regulator dE2F1 during wing development. Since wing disc growth is a homeostatic process, the possibility was investigated that genes involved in cell cycle progression regulate vg and sd expression in feedback loops. The experiments focused on two major regulators of cell cycle progression: dE2F1 and the antagonist Dacapo (Dap). The results reinforce the idea that VG/SD stoichiometry is critical for correct development and that an excess in SD over VG disrupts wing growth. Transcriptional activity of VG-SD and the VG/SD ratio are both modulated by down-expression of cell cycle genes. A dap-induced sd up-regulation was detected that disrupts wing growth. Moreover, a rescue was observed of a vg hypomorphic mutant phenotype by dE2F1 that is concomitant with vg and sd induction. This regulation of the VG-SD activity by dE2F1 is dependent on the vg genetic background. The results support the hypothesis that cell cycle genes fine-tune wing growth and cell proliferation, in part, through control of the VG/SD stoichiometry and activity. This points to a homeostatic feedback regulation between proliferation regulators and the VG-SD wing selector (Legent, 2006).

Cell proliferation relies on the tight control of cell cycle genes, and, in the wing pouch, VG-SD is also critically required. Accordingly, vg up-regulates dE2F1 expression and antagonizes the CKI dap. This study investigated the effects of these two antagonistic proliferation regulators in the wing pouch of the disc, and tested the hypothesis that cell cycle genes fine-tune proliferation, through regulation of the respective expressions of vg and sd and VG-SD dimer activity, thereby providing a feedback control (Legent, 2006).

Combined loss and gain of function experiments has ascertained the requirement of a precise VG/SD ratio for normal wing development and has shown that an excess in SD disrupts VG-SD function in wing growth, and probably acts as a dominant-negative through titration of functional VG-SD dimers. Therefore, sd induction may efficiently restrain VG-SD function in vivo, and a similar effect may also be physiologically achieved down-regulating vg. Moreover, since SD DNA target selectivity is modified upon binding of VG to SD in vitro, the hypothesis cannot be discarded that, in vivo too, VG-SD targets might be different from the targets of SD alone. This could explain to some extent the phenotypes observed when sd is induced (Legent, 2006).

The results show that the CKI member DAP, homogeneously expressed in the wing disc, regulates VG-SD function. dap heterozygotes display a wild type wing phenotype, reduced levels of both vg and sd transcripts, but an almost normal vg/sd ratio, thus VG-SD activity is normal. Consistently, no abnormal wing phenotype could be detected. Therefore, the relative vg/sd stoichiometry, rather than absolute vg and sd expression levels, determines wing growth. Interestingly, it had been observed that dap homozygous mutant adult escapers display duplication of the wing margin, indicating a role of DAP at the D/V boundary. This phenotype could be linked to an enhanced proliferation due to the absence of CKI function. Moreover, D/V-specific over-expression of dap alters wing margin structures. This dap over-expression triggers both ectopic expression of sd and subsequent impairment of VG-SD activity associated with a proliferation decrease.The associated wing phenotype is clearly enhanced in vg heterozygous flies, providing evidence that dap over-expression affects VG/SD stoichiometry and represses VG-SD activity in wing development. This reveals a model in which, in the wing pouch, cell proliferation down-regulation through cyclin/CDK inhibition by DAP, may be enhanced by an additive reduction of VG-SD proliferation function. Such a mechanism probably participates in vivo in the control of balanced wing growth (Legent, 2006).

The results also demonstrate that dE2F1-DP regulates VG-SD: the dE2F1 heterozygote displays a reduced vg/sd ratio due to a decrease in vg and an increase in sd transcripts, associated with reduced dimer activity, comparable to the vgnull/+ context. Thus, dE2F1 is required for vg normal expression. This supports the hypothesis that the slower proliferation observed in these contexts is linked to an imbalance in the dimer ratio (Legent, 2006).

Conversely, over-expressing dE2F1-DP-P35, in a vg83b27 hypomorphic mutant context, rescues expression of both vg and sd and normal VG-SD function, wing appendage specification and growth. This is not observed in vgnull flies implying the necessity for vg sequences, but the second intron, missing in the vg83b27 mutant. In addition, it was ascertained that not all the genes triggering cell cycle progression or cell proliferation can induce vg expression. Neither ectopic expression of CYC E, which promotes dE2F1-induced G1/S cell cycle transition, nor the growth regulator Insulin receptor (InR) is sufficient to elicit VG expression and wing growth in the vg83b27 mutant. These results demonstrate that vg induction is a prerequisite for vg83b27 wing pouch growth in response to dE2F1 activity (Legent, 2006).

In a vg+ genetic background, dE2F1 over-expression induces only sd, disrupting VG/SD stoichiometry. Consistently, at the D/V boundary, wing notching was observed. Therefore, although dE2F1 basically displays a positive role in proliferation, this sd induction in response to dE2F1 over-expression is clearly associated with wing growth impairment. This effect is significantly weaker in a vg heterozygote background, and a rescue of the wing phenotype was observed, supporting the hypothesis that VG/SD stoichiometry is restored. Therefore, sd induction by dE2F1 depends on the vg genetic context. This indicates that the effects of over-expressing dE2F1 differ depending on the growth-state of the wing pouch, which is tightly linked with the vg genotype (Legent, 2006).

Clearly, feedback regulations rule the growth of the wing disc. Regulation has been noted in three different vg genetic contexts that can be analyzed in the light of a homeostasis hypothesis. In the vg83b27under-proliferative wing pouch, ectopic dE2F1 expression coordinately increases vg and sd expression in a positive feedback loop. This triggers VG-SD activity, and induces both cell proliferation and wing specification in the mutant. Conversely, no such crosstalk occurs in a correctly grown vg+ disc, where over-growth should be prevented. In this latter case, sd induction (VG/SD decrease) probably restrains the proliferation function of dE2F1. Consistently, wings were not overgrown, but notches were observed. This phenotype was partially suppressed in a vg heterozygote background. As a whole, these results support the hypothesis that VG-SD/dE2F1 coordination tends to ensure normal wing growth and that the dimer does not trigger unrestricted cell proliferation in a vg+ context, since an excess in dE2F1 attenuates VG-SD function in a negative feedback loop. Thus, molecular interactions between dE2F1, vg and sd, display a clear plasticity depending on the vg genetic context (Legent, 2006).

Establishing and maintaining homeostasis is critical during development. This is achieved in part through a balance between cell proliferation and death. In mammals E2F1 and p21, the dacapo homolog, play a key role in this process. In the wing disc compensatory proliferation induced by cell death has been observed. However, the role of cell cycle genes in this process has not yet been established. How patterns of cell proliferation are generated during development is still unclear. It seems nevertheless likely that the gene responsible for regulating differentiation also regulates proliferation and growth. For instance, Hedgehog (HH) induces the expression of Cyclins D and E. This mediates the ability of HH to drive growth and proliferation. In the same way, other data support a direct regulation of dE2F1 by the Caudal homeodomain protein required for anterio-posterior axis formation and gut development. Wingless (WG) also displays both patterning and a cell cycle regulator function during Drosophila development (Legent, 2006).

Growth control in the wing pouch seems to be achieved through both positive and negative feedback regulations linking dE2F1 and VG-SD, but also via additive impairment of VG-SD by DAP. In fact, in a vg+ background, over-expression of both dap and dE2F1 induces sd, impairs VG-SD and alters wing development. Nevertheless, clear opposite behaviors are observed in vgnull/+ flies where dap-induced nicks are enhanced, while those of dE2F1 are partially rescued. This highlights the functional discrepancy between these two types of feedback regulation. It is suggested that dap expression inhibits cell proliferation through a process involving both Cyclin-CDK inhibition and VG-SD impairment in the wing pouch. In contrast, it is proposed that dE2F1 over-expression triggers a homeostatic response. It will either induce vg and sd to ensure proliferation (in a vg83b27 genotype), or decrease the VG/SD ratio in a vg+ context. In this latter genotype, down-regulation probably counteracts fundamental proliferative properties of dE2F1 and governs homeostatic wing disc growth (Legent, 2006).

At late third instar, wing discs display a Zone of Non-proliferating Cells (ZNC) along the wing pouch D/V boundary. It has been shown that, although dE2F1-DP is expressed in this area, its proliferative function is inactivated late, because of RBF1-induced G1 arrest. Accordingly, although expression of vg and sd presents a peak at the D/V boundary, in late third instar, VG-SD activity is decreased in D/V cells, and it was suggested to result from an excess of SD. Therefore, the ZNC setting may also reflect a VG-SD/dE2F1 coordinated dialogue that triggers a decrease in proliferation signals in this area (Legent, 2006).

Previous studies of homeostatic control of cell proliferation in the wing reported that, to some extent, over-expression of positive or negative cell cycle regulators only weakly affects the overall division rate. For instance, although dap over-expression alters dE2F1 function in G1-S cell cycle transition, it also promotes dE2F1 expression and function in G2-M transition, preventing a decrease in the overall rate of cell division. Strikingly, the cells seemed able to monitor each phase length and maintain cell cycle duration and normal proliferation in the wing pouch of the disc. Therefore, dE2F1 is a central component that enables cells to ensure normal proliferation in the wing disc and prevents imbalance in the process. The fact that dE2F1 triggers quite different or opposite responses in vg+ or vg hypomorphic contexts suggests that the VG-SD/dE2F1 crosstalk plays a role in the same sort of homeostatic process that ensures entire wing growth (Legent, 2006).

Such regulations are likely to reveal a precise physiological fine-tuning of vg and sd by cell cycle effectors, promoting an exquisite control of wing growth. Feedback loops between the developmental selector VG-SD and cell cycle effectors may stand for a control mechanism to guarantee that the tissue can sustain balanced growth and a reproducible size. Such a subtle mechanism, on a local scale, would correct the alterations in cell proliferation that may occur during development (Legent, 2006).

A gradient of epidermal growth factor receptor signaling determines the sensitivity of rbf1 mutant cells to E2F-dependent apoptosis

Retinoblastoma (Rb) family proteins control E2F-dependent transcription and restrict cell proliferation. In the early G1 phase of the cell cycle, Rb family proteins bind to E2F family members, inhibiting their ability to activate transcription and recruiting repressor complexes to DNA. In late G1 to S phase, cyclin-dependent kinases (CDK) phosphorylate Rb family proteins, liberating E2F and activating E2F-dependent transcription. One of the least-well-understood aspects of in vivo studies of Rb function is the fact that the inactivation of Rb often sensitizes cells to apoptosis. The extent of apoptosis caused by the inactivation of Rb is highly cell type and tissue specific, but the underlying reasons for this variation are poorly understood. This study characterizes specific time and place during Drosophila development where rbf1 mutant cells are exquisitely sensitive to apoptosis. During the third larval instar, many rbf1 mutant cells undergo E2F-dependent cell death in the morphogenetic furrow. Surprisingly, this pattern of apoptosis is not caused by inappropriate cell cycle progression but instead involves the action of Argos, a secreted protein that negatively regulates Drosophila epidermal growth factor receptor (EGFR [DER]) activity. Apoptosis of rbf1 mutant cells is suppressed by the activation of DER, ras, or raf or by the inactivation of argos, sprouty, or gap1, and inhibition of DER strongly enhances apoptosis in rbf1 mutant discs. RBF1 and a DER/ras/raf signaling pathway cooperate in vivo to suppress E2F-dependent apoptosis and the loss of RBF1 alters a normal program of cell death that is controlled by Argos and DER. These results demonstrate that a gradient of DER/ras/raf signaling that occurs naturally during development provides the contextual signals that determine when and where the inactivation of rbf1 results in dE2F1-dependent apoptosis (Moon, 2006).

This study takes advantage of the observation that the inactivation of rbf1 in the Drosophila eye results in a distinctive pattern of apoptosis that is tightly linked to eye development, and this model system was used to define a cellular context in which RBF1 is needed to protect cells against dE2F1-dependent cell death. The results show that the cellular response to the inactivation of rbf1 involves a combination of signals. Deregulated dE2F1 provides one function that is required for apoptosis. However, in most situations, deregulation of the endogenous dE2F1 is not sufficient to induce apoptosis. In addition, a second condition, the down-regulation of an EGFR/Ras/Raf signaling pathway, is also necessary. In the eye imaginal disc, the EGFR/Ras/Raf signaling pathway is down-regulated at the region immediately anterior to the 'intermediate group' (IG) of cells, from which the R8 founder cell will be selected. When rbf1 mutant cells pass through this gradient, they become highly sensitive to dE2F1-dependent apoptosis. Elevation of the level of DER/Ras/Raf signaling by a variety of means suppresses apoptosis in rbf1 mutant cells. Conversely, expression of a dominant-negative mutant of DER strongly synergized with mutation of rbf1 to induce apoptosis (Moon, 2006).

Before starting this work, several different ways were considered in which the inactivation of RBF1 might result in apoptosis. If differentiated/differentiating cells try to reenter the cell cycle following the inactivation of RBF1, then an abnormal or inappropriate S-phase entry might cause apoptosis. Alternatively, one could argue that rapidly proliferating cells contain the highest levels of E2F transcriptional activity, and hence these cells ought to be most sensitive to E2F-induced apoptosis when RBF is removed. Although both models were plausible, in fact, neither explanation fits the data. rbf1 mutant eye discs display little apoptosis in either the population of differentiated cells or in actively cycling cells. Instead, rbf1 mutant cells are sensitive to apoptosis in the MF, at a time when some cells exit the cell cycle and initiate a differentiation program. This apoptosis was not accompanied by inappropriate cell cycle progression. Indeed, when rbf1 mutant cells were rescued from apoptosis, they showed no indication of S-phase entry. Hence rbf1 mutant cells were not dying because they were inappropriately progressing through the cell cycle. Instead, apoptosis was dependent on a specific developmental context. This need for the correct context may be particularly significant when designing cell culture-based screens for treatments that are synthetic lethal with the inactivation of Rb (Moon, 2006).

Several studies have shown that E2F complexes regulate the expression of proapoptotic genes, but why would the effects of losing RBF1 be sensitive to EGFR signaling? While it seems likely that deregulated dE2F1 activates transcription of several proapoptotic targets, the results indicate that an important part of the explanation lies in the regulation of the proapoptotic gene hid. hid transcripts are up-regulated in rbf1 mutant eye discs and halving the gene dosage of hid dramatically reduced apoptosis. Previous studies have shown that HID-induced apoptosis is highly sensitive to EGFR/Ras/Raf signaling. Signaling through this pathway suppresses transcription of hid and is thought to induce an inhibitory phosphorylation on the HID protein. This provides a simple model, in which the loss of RBF1 results in the elevated expression of a proapoptotic protein, which is then held in check by EGFR/Ras/Raf-mediated signaling. Apoptosis would then occur when EGFR signals are reduced. Consistent with this model, it was found that the region of the eye disc that is most sensitive to loss of RBF1 is also highly sensitive to low levels of ectopic hid expression (Moon, 2006).

Why does this pattern of apoptosis occur? It is likely that several different factors are needed to establish the gradient of DER activity. Important regulators of DER activity in the eye include Gap1, Sprouty, and Argos. In this particular context, the ability of Argos to diffuse and act at a distance from the p-Erk-positive cells appears to be important. It is suggested that the pattern of Argos expression in the developing eye disc generates a zone in which cells that have failed to exit the cell cycle and inappropriately inactivate RBF1 become prone to undergo apoptosis. In essence, this could be viewed as a developmental failsafe mechanism against inappropriate proliferation. In support of this, it is noted that E2F1 levels are transiently elevated in G1 phase cells in the MF, even though E2F regulation is not needed for S phase entry in the second mitotic wave. Consistent with the idea that this region of the disc may be more sensitive to apoptosis, it was found that a transient pulse of cyclin E expression, which drives ectopic S phases throughout much of the disc, generates a similar stripe of apoptosis in the MF. It is curious that this sensitivity occurs at the time when the role of EGFR is apparently changing from being needed for cell proliferation in the anterior part of the disc to being required for differentiation in the posterior part of the disc. It will be interesting to discover whether similarly sensitive regions exist in other discs (Moon, 2006).

As seen with rbf1, the effects of mutating Rb in the mouse are most evident at points in development when cells attempt to exit the cell cycle and differentiate. Rb-null mouse retinas show increased cell death during the transition from proliferation to differentiation. Whether this is due to an analogous interaction between Rb/E2F and EGFR/Ras signaling has not been tested but is an interesting possibility. It is also tempting to speculate that some of the different cellular responses to the inactivation of Rb in the mouse retina may be caused by differences in EGFR/Ras-mediated differentiation signals (Moon, 2006).

There are several indications that the general phenomenon described in this study is likely to be conserved in mammalian cells. For example, recent studies have shown that apoptosis in cultured fibroblasts lacking Rb family proteins (TKO) can be suppressed by activation of Ras/Raf. Interestingly, a functional homologue of Hid, Smac/Diablo, was recently shown to be a direct target of E2F1 in mammalian cells, raising the possibility that mammalian cells may contain a regulatory loop that directly parallels the regulation of Hid. However, a connection between the proapoptotic function of Smac/Diablo and EGFR pathway has yet to be described (Moon, 2006).

The molecular events underlying the convergence of EGFR signaling and Rb/E2F may be different between flies and humans. It is noted that Akt activation suppresses E2F1-induced apoptosis in mammalian tissue culture cells, while neither the overexpression of dAKT1 nor the mutation of dPTEN is sufficient to prevent cell death in rbf1 mutant eye discs. This may reflect a difference between an in vivo analysis and tissue culture conditions, or it may reflect species-specific differences in the regulation of apoptosis. It is known, for example, that caspase activation is regulated differently between species. In vertebrates, cytochrome c release from mitochondria is a key step in the promotion of caspase activation, while in Drosophila, this step is largely dispensable. It is possible that EGFR activity converges on E2F-dependent cell death through a previously identified E2F target whose activity is regulated by Raf/Erk- and/or AKT-mediated signals, such as Bim. In order to define this circuitry, it is first necessary to identify the appropriate in vivo context in mice or humans in which Rb/E2F and EGFR activity cooperate to regulate cell survival. Once the appropriate context is found, then it may be possible to identify the molecular mechanism linking E2F-dependent cell death to survival signals (Moon, 2006).

Both EGFR family and Rb pathways are often altered in cancer. Given that developmentally controlled fluctuations in EGFR signaling have dramatic effects on the sensitivity of rbf1 mutant cells to apoptosis, it is speculated that therapeutic cancer drugs that target EGFR family proteins may induce cell death most efficiently in tumor cells that have the highest levels of E2F1 activity. One of the curious features of human retinoblastoma is that, unlike many other cancers, these tumors rarely contain mutations in p53, suggesting that either these cells do not need to mutate p53 or that they find a more effective way to suppress apoptosis. Identification of the critical components that protect premaligant Rb mutant cells from apoptosis may lead to new ways to target these cells for treatment (Moon, 2006).

A double-assurance mechanism controls cell cycle exit upon terminal differentiation in Drosophila

Terminal differentiation is often coupled with permanent exit from the cell cycle, yet it is unclear how cell proliferation is blocked in differentiated tissues. The process of cell cycle exit was examined in Drosophila wings and eyes; cell cycle exit can be prevented or even reversed in terminally differentiating cells by the simultaneous activation of E2F1 and either Cyclin E/Cdk2 or Cyclin D/Cdk4. Enforcing both E2F and Cyclin/Cdk activities is required to bypass exit because feedback between E2F and Cyclin E/Cdk2 is inhibited after cells differentiate, ensuring that cell cycle exit is robust. In some differentiating cell types (e.g., neurons), known inhibitors including the retinoblastoma homolog Rbf and the p27 homolog Dacapo contribute to parallel repression of E2F and Cyclin E/Cdk2. In other cell types, however (e.g., wing epithelial cells), unknown mechanisms inhibit E2F and Cyclin/Cdk activity in parallel to enforce permanent cell cycle exit upon terminal differentiation (Buttitta, 2007).

Current models for cell cycle exit invoke repression of Cyclin/Cdk activity by CKIs or repression of E2F-mediated transcription by RBs as the proximal mechanisms by which cell cycle progression is arrested. Since these models include the potential for positive feedback between E2F and CycE/Cdk2, they predict that the induction of either E2F or a G1 Cyclin/Cdk complex should be sufficient to maintain the activity of the other and thereby sustain the proliferative state. However, in differentiating Drosophila tissues, both E2F and G1 Cyclin/Cdk activities had to be simultaneously upregulated to bypass or reverse cell cycle exit. An explanation for this resides in two observations. First, the ability of Cyclin/Cdk activity to promote E2F-dependent transcription is lost or reduced in the wing and eye after terminal differentiation. Second, increased E2F cannot sustain functional levels of CycE/Cdk2 activity after terminal differentiation, despite an increase in cycE and cdk2 mRNA to levels higher than those observed in proliferative-stage wings. Thus, crosstalk between E2F and Cyclin/Cdk activity appears to be limited, in both directions, as a consequence of differentiation (Buttitta, 2007).

How are these two regulatory interactions altered? One possibility is that Rbf2- or E2F2-dependent repression prevents ectopic Cyclin/Cdk activity from promoting E2F-dependent transcription after prolonged exit. While mRNA expression data and the existing genetic data on E2F2 and Rbf2 do not support this possibility, the roles of Rbf2 or E2F2 have not been tested in the presence of continued Cyclin/Cdk activity. Therefore, transcriptional repression of E2F targets by Rbf2 or E2F2 remains an important issue to address in future experiments (Buttitta, 2007).

More enigmatic is the inability of the ectopic CycE/Cdk2 provided by overexpressed E2F to promote cell cycle progression. One plausible explanation for this is that novel inhibitors of CycE are expressed with the onset of differentiation, and that these raise the threshold of Cyclin/Cdk activity required to promote cell cycle progression. Such inhibitors might make the critical substrates of CycE/Cdk2, which reside on chromatin in DNA-replication and -transcription initiation complexes, less accessible or otherwise recalcitrant to activation. The notion of an increased Cdk threshold is consistent with the observation that the >10-fold increase in CycE/Cdk2 provided by direct overexpression of the kinase bypassed cell cycle exit in conjunction with E2F, while the ~4-fold increase provided indirectly by ectopic E2F is insufficient to drive the cell cycle. Although a >10-fold increase in Cdk activity as applied in these experiments is far above the normal physiological range, such dramatic deregulation of cell cycle genes may be physiologically relevant to cancers, in which gene expression can be greatly amplified (Buttitta, 2007).

Recent studies of cycle exit in larval Drosophila eyes have concluded that Rbf1 and Dap are required to inhibit E2F and CycE/Cdk2 in differentiating photoreceptors. Other studies document the roles of Ago/Fbw7 and components of the Hippo/Warts-signaling pathway in downregulating CycE for cell cycle exit in nonneural cells in the eye. Although the data are consistent with these studies in the eye, Ago and the Hippo/Warts pathway are dispensable for cell cycle exit in the wing. Furthermore, deletion of Rbf1 did not prevent cell cycle exit in the epithelial wing, even when high levels of CycE/Cdk2 were provided. Conversely, deletion of Dap was not sufficient to keep wing cells cycling, even when excessive E2F activity was provided. These observations suggest that unknown inhibitors of E2F and Cyclin/Cdk activity mediate cell cycle exit in specific contexts, such as the wing (Buttitta, 2007).

In attempts to identify upstream factors regulating cell cycle exit, a variety of growth and patterning signals were manipulated in the pupal wing and eye, and their effects on cell cycle exit were examined. Surprisingly, signals that act as potent inducers of proliferation in wings and eyes at earlier stages did not prevent or even delay cell cycle exit upon terminal differentiation. Thus, an important focus for future studies will be the nature of the signals upstream of E2F and CycE that mediate cell cycle exit. These could be novel signals, or combinations of known signals delivered in unappreciated ways (Buttitta, 2007).

How general is double assurance? Studies of cell cycle exit in mammals do not offer a consistent answer to this question. S phase re-entry can be achieved in differentiated cells by activating E2F, CycE/Cdk2, or CycD/Cdk4 alone, but this does not lead to cell division or continued proliferation. Several studies with mammalian cells in vivo have shown that neither increased E2F nor Cyclin/Cdk activity alone is sufficient to fully reverse differentiation-associated quiescence, consistent with the double-assurance model propose in this study. Also consistent with this model is the ability of proteins from DNA tumor viruses, such as adenovirus E1A, SV40 LargeT, and HPV E6 and E7, to fully reverse differentiation-associated cell cycle exit in many cell types. These viral onco-proteins stimulate cell cycle progression by targeting multiple cell cycle factors, which ultimately increase both E2F and G1 Cyclin/Cdk activities simultaneously. For example, LargeT and E1A inhibit both RBs and CKIs, such as p21Cip1 and p27Kip1 (Buttitta, 2007).

There are some instances, however, in which differentiation-associated cell cycle exit has been bypassed, not just delayed, by the deletion of CKIs or RBs. In one such case, p19Ink4d and p27Kip1 were knocked out in the mouse brain, and ectopic mitoses were documented in neuronal cells weeks after they normally become quiescent. Similar results have been obtained with hair and support cells in the mouse inner ear, where deletion of p19Ink4d, p27Kip1, or pRB can bypass developmentally programmed cell cycle exit. In light of these findings, it is interesting to speculate that certain differentiated tissues may retain some ability to repair or regenerate by maintaining the capacity for positive feedback between E2F and CycE/Cdk2 activity. Inner-ear hair cells may be such an example, since in many vertebrates they are capable of regeneration, although this ability has been lost in mammals. Although the mammalian brain has a very limited capacity for regeneration, the cell cycle can be reactivated in the brains of other vertebrates, such as fish, in response to injury. Thus, the retention of crosstalk between E2F and Cyclin/Cdk activities in the evolutionary descendents of regeneration-competent cells might explain some of the tissue-specific sensitivities to loss of CKIs or RBs observed in mammals (Buttitta, 2007).

Rbf1-independent termination of E2f1-target gene expression during early Drosophila embryogenesis

The initiation and maintenance of G1 cell cycle arrest is a key feature of animal development. In the Drosophila ectoderm, G1 arrest first appears during the seventeenth embryonic cell cycle. The initiation of G117 arrest requires the developmentally-induced expression of Dacapo, a p27-like Cyclin E-Cdk2 inhibitor. The maintenance of G117 arrest requires Rbf1-dependent repression of E2f1-regulated replication factor genes, which are expressed continuously during cycles 1-16 when S phase immediately follows mitosis. The mechanisms that trigger Rbf1 repressor function and mediate G117 maintenance are unknown. This study shows that the initial downregulation of expression of the E2f1-target gene Ribonucleoside diphosphate reductase small subunit (RnrS), which occurs during cycles 15 and 16 prior to entry into G117, does not require Rbf1 or p27Dap. This suggests a mechanism for Rbf1-independent control of E2f1 during early development. E2f1 protein is destroyed in a cell cycle-dependent manner during S phase of cycles 15 and 16. E2f1 is destroyed during early S phase, and requires ongoing DNA replication. E2f1 protein reaccumulates in epidermal cells arrested in G117, and in these cells the induction of p27Dap activates Rbf1 to repress E2f1-target genes to maintain a stable G1 arrest (Shibutani, 2007).

The finding that p27Dap expression is not necessary for the downregulation of E2f1 targets was unexpected, based on the known regulatory circuitry of the pRb-E2F pathway. This result led to the hypothesis that mechanisms in addition to Rbf1 binding are used to control E2f1 activity in the early embryo. It was found that E2f1 is destroyed during S phase of the post-blastoderm divisions in the embryonic epidermis, has been reported for cells in wing and eye imaginal discs. E2f1 destruction first occurs during S15, at the same time that E2f1-regulated transcripts such as RnrS begin to decline. Because E2f1 functions as a transcriptional activator, and because Rbf1 is not required for the initial decline in RnrS transcripts, it is proposed that the loss of E2f1 protein contributes to the initial termination of replication factor gene expression. Rbf1 is first required during development for the maintenance of G117 arrest and the continued repression of E2f1-target genes. The data suggest that Rbf1 is converted to a repressor after the developmentally-induced expression of Dap, most likely because the consequent inhibition of CycE-Cdk2 results in the accumulation of hypophosphorylated Rbf1. Dap expression accompanies the downregulation of Cyclin E transcription, and each of these mechanisms of inhibition of CycE-Cdk2 contributes to G1 arrest (Shibutani, 2007).

The high level of E2f1 protein in G117 epidermal cells may permit the formation of E2f1-Rbf1 complexes necessary to actively and stably repress replication factor genes during G1 arrest, and also provides a simple explanation for why the loss of Rbf1 function results in the ectopic expression of E2f1 targets. After hatching, and in response to the first instar larvae beginning to feed, the epidermal cells start to endoreduplicate. Thus, the accumulation of Rbf1-E2f1 complexes during G1 arrest may prepare cells for rapid production of replication factors and efficient re-entry into the cell cycle upon activation of G1 Cyclin-Cdk complexes after growth stimulation (Shibutani, 2007).

RnrS expression is lost in E2f1 zygotic mutant embryos, but not until cell cycle 17. One interpretation of this result is that maternal stores of E2f1 are sufficient for the early induction of replication gene expression in the post-blastoderm divisions. Consistent with this, maternal E2f1 protein persists into cycle 14, coincident with the commencement of zygotic transcription of E2f1 targets such as RnrS. In addition, mutation of the E2f1-binding sites in the regulatory region of the Pcna gene (mus209 - Flybase) is sufficient to abolish zygotic Pcna expression. However, the data do not demonstrate a requirement for E2f1 for early zygotic RnrS expression, and E2f1 may be only one of several factors necessary for early zygotic expression of genes encoding replication factors. For instance, the transcription of Cyclin E requires E2f1 in embryonic endocycles, but also occurs independently of E2f1 via tissue-specific enhancer elements such as those operating in the CNS. Thus, any control of replication factor gene expression by E2f1 abundance may be modulated by other transcription factors, or bypassed entirely in certain cell types by E2f1-independent modes of expression (Shibutani, 2007).

The data suggest that E2f1 destruction is coupled to DNA synthesis. CycE-Cdk2 has been suggested as a possible cell cycle input for E2f1 destruction in imaginal cells because it is activated at the G1-S transition when E2f1 is destroyed. However, CycE-Cdk2 is continuously active during the embryonic post-blastoderm cell cycles, whereas E2f1 is destroyed only during S phase. Thus, CycE-Cdk2 is unlikely to be the only signal, and actively replicating DNA may provide a necessary input into E2f1 destruction. This model is consistent with the observation that E2f1 destruction occurs after DNA synthesis begins, resulting in cells that are positive for both E2f1 and BrdU incorporation in early interphase (Shibutani, 2007).

Previous studies have suggested that mammalian E2f1 is degraded by the ubiquitin-proteasome pathway. In this pathway, E3 ubiquitin ligases bind to and mediate the ubiquitylation of specific proteins. The SCF class of cullin-dependent E3 ligases has been implicated in E2F1 destruction. In Drosophila, evidence from genetic and cell biology studies suggest that SCFSLMB mediates E2f1 destruction at the G1-S transition in wing imaginal disc cells (Heriche, 2003). Although there is no evidence implicating a specific E3 ligase in the destruction of embryonic E2f1, there are interesting parallels with recent experiments describing the destruction of Cdt1/Dup. Like E2f1, Cdt1/Double parked is degraded at the G1-S transition and cannot be detected during S phase. In vertebrates, Cdt1 destruction is mediated by two independent and apparently redundant mechanisms: direct Cdk2 phosphorylation that targets Cdt1 to SCFSKP2, and binding of PCNA to the Cdt1/Dup amino-terminus that targets Cdt1 to Cul4DDB1. This latter result is consistent with a recent study indicating that Drosophila Dup hyperaccumulates in cells where DNA synthesis is attenuated. Thus, more than one E3 ubiquitin ligase may participate in E2f1 destruction. Determining the molecular mechanism of E2f1 destruction should permit a direct test of whether prevention of E2f1 destruction would affect replication factor gene expression in the embryo (Shibutani, 2007).

E2F is necessary for the development of worms, flies and mice. Remarkably, however, pRb is not needed for the entirety of mouse embryonic development. This could in part be due to redundancy with other pRb family members, such as p107 and p130. Alternatively, a pRb-independent mechanism of regulating E2F activity may control S phase gene expression and cell cycle progression during early mammalian development. This idea is supported by experiments modeling the cell cycles of early vertebrate development in cell culture using murine embryonic stem cells. These pluripotent cells have a cell cycle composed mostly of S phase that is characterized by ubiquitous Cdk activity and the absence of CKIs. As in the Drosophila embryo, E2F-regulated transcripts are also ubiquitous even though pRb family members are expressed. Differentiation requires the lengthening of G1 and the negative regulation of Cdk2 activity, which is accomplished both by increases in the level of CKIs and by the downregulation of Cyclin E1 expression via inhibition of E2F. Thus, evolutionarily conserved regulatory mechanisms operating in early development may mediate the conversion from rapid cell cycles driven by intrinsic cues to slower, more highly regulated cycles that are influenced by extrinsic developmental and environmental cues (Shibutani, 2007).

E2F1 and E2F2 have opposite effects on radiation-induced p53-independent apoptosis in Drosophila

The ability of ionizing radiation (IR) to induce apoptosis independent of p53 is crucial for successful therapy of cancers bearing p53 mutations. p53-independent apoptosis, however, remains poorly understood relative to p53-dependent apoptosis. IR induces both p53-dependent and p53-independent apoptoses in Drosophila, making studies of both modes of cell death possible in a genetically tractable model. Previous studies have found that Drosophila E2F proteins are generally pro-death or neutral with regard to p53-dependent apoptosis. This study reports that dE2F1 promotes IR-induced p53-independent apoptosis in larval imaginal discs. Using transcriptional reporters, evidence is provided that, when p53 is mutated, dE2F1 becomes necessary for the transcriptional induction of the pro-apoptotic gene hid after irradiation. In contrast, the second E2F homolog, dE2F2, as well as the net E2F activity, which can be depleted by mutating the common cofactor, dDp, is inhibitory for p53-independent apoptosis. It is concluded that p53-dependent and p53-independent apoptoses show differential reliance on E2F activity in Drosophila (Wichmann, 2010).

This study has taken advantage of the relative simplicity of Drosophila E2F and p53 families to study the role of E2Fs in p53-independent apoptosis. The results indicate that Drosophila E2F homologs play opposing roles in regulating p53-independent apoptosis in response to IR. dE2F1, a homolog of the mammalian 'activator' E2Fs, is required for Chk2-/p53-independent apoptosis, while dE2F2, a homolog of the mammalian 'repressor' E2Fs, limits p53-independent apoptosis. The net E2F activity in the cell, reduced by mutations in dDP, is inhibitory towards p53-independent apoptosis (Wichmann, 2010).

One surprising finding from these studies is that 2 kb of hid promoter confers IR-induced transcriptional activation in a p53-dependent manner. This is surprising because in embryos, transcriptional activation of hid by IR in a p53-dependent manner requires the IRER (irradiation responsive enhancer region) that lies next to rpr, ~ 200 kb away from hid, and is regulated epigenetically by histone modification (Zhang, 2008). Yet, as shown previously, 2 kb of hid promoter is enough to allow IR-induced GFP expression in eye and wing imaginal discs (Tanaka-Matakatsu, 2009). This study shows that this induction is p53-dependent. Clearly, regulation of hid by IR is different between embryos and larval discs (Wichmann, 2010).

Mammalian p53 consensus is a tandem repeat of 10 nucleotides with the sequence RRRCWWGYYY where R = G/A, W = A/T and Y = T/C and invariant C and G are shown in bold. Drosophila p53 binds to a DNA damage response element at the rpr locus that differs from the mammalian consensus at one position shown in lower case; tGACATGTTT GAACAAGTCg. Manual examination of 2 kb of hid promoter fragment that responds to p53 status shows a potential binding sequence at −2006 from the start of hid transcription that deviates from the mammalian consensus at two positions, and another at −1667 that deviates at three positions. These are ttGCATGCTC GctCATGTTC and GtGCAAGagT GtGCTTGaat respectively. Since the consensus for Drosophila p53 has not been determined, it is possible that either or both of these are responsible for the effect of p53 on hid-driven GFP reporter (Wichmann, 2010).

The 2 kb hid enhancer includes E2F consensus sequences. Rb has been shown to repress the expression of hid-driven GFP reporter when E2F binding sequences are intact but not when these are mutated (Tanaka-Matakatsu, 2009). That is, E2F binding sites allow for repression of hid by Rb although which E2F mediates this repression is not known. In any case, the finding that net E2F activity is inhibitory towards apoptosis would be consistent with the published result that Rb inhibits hid expression via E2F consensus sites. It is not known if dE2F1 plays a permissive role (e.g. by allowing elevated basal expression of pro-apoptotic genes) or an instructive role (e.g. by allowing for induction of pro-apoptotic genes by IR), or both. The results with hid-driven GFP reporter are consistent with an instructive role but do not rule out a permissive role (Wichmann, 2010).

In the absence of p53, dE2F1 is needed for the transcriptional induction of hid> GFP reporter by IR. This can explain two published results: that hid is necessary for IR-induced p53-independent apoptosis (McNamee, 2009), and that hid is transcriptionally induced in p53 mutants after a time delay. Human E2F1 can bind to the promoter of a hid ortholog, Smac/DIABLO, and can, when ectopically expressed, activate the transcription of the latter in vivo. The role of p53 status in this process or the significance of this mode of regulation was not investigated. It is speculated that the role of E2F1 in IR-induced, p53-independent transcriptional activation of Smac/DIABLO genes may be conserved in mammals (Wichmann, 2010).

Previous work has shown that dE2F1 and dE2F2 exhibit antagonistic functions, with dE2F1 activating and dE2F2 repressing the transcription of a reporter containing canonical E2F sites from the PCNA promoter. dE2F1 and dE2F2 occupy the PCNA promoter and the ratio of the two E2F proteins influenced the degree of transcriptional activation or repression. In wild type, PCNA expression is tightly coupled to the pattern of S phase, in eye imaginal discs for example. In de2f2 mutants, PCNA is no longer down-regulated outside pattern of the S phase. The loss of all E2F activities, either in de2f1, de2f2 double mutants or in dDP single mutants, results in de-repression of PCNA such that a low but significant level is expressed throughout the cell cycle. Thus the net result of opposing E2F activities is the cyclical expression of PCNA in concert with DNA replication (Wichmann, 2010).

The paradigm of E2F-dependent regulation of PCNA aids in understanding the role of E2F proteins in p53-independent apoptosis. dE2F1 and dE2F2 might similarly influence p53-independent apoptosis by regulating pro-apoptotic gene(s) such as hid. According to this model, dE2F2 (with dDp) provides a net repressive activity that inhibits IR-induced apoptosis. This activity must be operative only in the absence of p53;dE2F2 mutations that have no effect on apoptosis when p53 is present. In p53 mutants, dE2F1 counteracts dE2F2 after irradiation and thus promotes apoptosis. Disabling transcriptional activation by dE2F1, which is what the allelic combination de2f1i2/de2f17172 is predicted to cause, would result in the failure to overcome dE2F2/Dp. Removal of dE2F2 with null alleles, would result in increased gene expression and more apoptosis. Reducing the ability of dDP to interact with dE2Fs, which is what the allelic combination dDPa1/dDPa2 is predicted to cause, would reduce dE2F1 and dE2F2 activities simultaneously. Since this results in more apoptosis, the net E2F activity is inhibitory on apoptosis when p53 is absent (Wichmann, 2010).

This study and published studies in wing imaginal discs reveal significant differences in the effect of E2F/DP mutations on p53-dependent apoptosis (typically assayed at 4–6 h post irradiation) and p53-independent apoptosis (18–24 h after irradiation in p53 mutants). The clearest difference is that dE2F2 null mutations have little or no effect on p53-dependent apoptosis, but increase the level of p53-independent apoptosis. dDP loss-of-function mutations decrease p53-dependent apoptosis throughout eye imaginal discs and in most cells of the wing pouch, whereas they increase the level of p53-independent apoptosis. In contrast, loss-of-function mutations in dE2F1 reduced both p53-dependent apoptosis in most cells of the wing imaginal disc and p53-independent apoptosis in the wing imaginal disc. These differences raise the question ‘how does p53 status alter the role of dE2F2 and dDP in IR-induced apoptosis?’ In the presence of p53, dE2F2 has little effect and dDP is stimulatory. In the absence of p53, dE2F2 and dDP play inhibitory roles. Perhaps the occupancy of transcriptional factors at the target loci such as the hid promoter is sensitive to p53 status (Wichmann, 2010).

In the eye imaginal disc, mutations in ago, a ubiquitin ligase, result in elevated apoptosis. This mode of cell death occurs via elevated E2F1 activity, increased expression of hid and rpr and is independent of apoptosis. Thus, the role of dE2F1 in promoting p53-independent apoptosis is conserved in another tissue of the larvae (Wichmann, 2010).

Previous studies found that the role of Drosophila E2F transcription factors in apoptosis is context-dependent and is influenced by, for example, whether the cells are in the wing pouch or at the dorsal/ventral margin of the wing disc and whether apoptosis is induced by radiation or by the loss of a tumor suppressor homolog, Rb. The current study addresses the role of E2F family members in IR-induced p53-independent apoptosis. The most significant finding in this study is that reducing E2F activity, as in the case of dDP mutants, allows p53-null cells to die following IR exposure. This is in clear contrast to the finding that a similar reduction of E2F activity prevents p53 wild type cells from dying following IR exposure. Several E2F antagonists are being considered in cancer therapy. The current results from Drosophila studies would caution that p53 status must be considered when using such therapies in conjunction with radiation treatment. If the findings in Drosophila apply to human cancers, an E2F antagonist would help kill p53-deficient cancer cells following radiation treatment, but would help p53-wild type cancer cells survive. In addition, an E2F antagonist may be particularly suitable for combination therapy with radiation to eradicate p53-deficient tumors because it may sensitize p53-deficient cancer cells to radiation while protecting p53-wild type somatic cells from the cell-killing effects of IR (Wichmann, 2010).

Cooperation between dE2F1 and Yki/Sd defines a distinct transcriptional program necessary to bypass cell cycle exit

The Hippo signaling pathway regulates organ size homeostasis, while its inactivation leads to severe hyperplasia in flies and mammals. The transcriptional coactivator Yorkie (Yki) mediates transcriptional output of the Hippo signaling. Yki lacks a DNA-binding domain and is recruited to its target promoters as a complex with DNA-binding proteins such as Scalloped (Sd). In spite of recent progress, an open question in the field is the mechanism through which the Yki/Sd transcriptional signature is defined. This study reports that Yki/Sd synergizes with and requires the transcription factor dE2F1 to induce a specific transcriptional program necessary to bypass the cell cycle exit. Yki/Sd and dE2F1 bind directly to the promoters of the Yki/Sd-dE2F1 shared target genes and activate their expression in a strong cooperative manner. Consistently, RBF, a negative regulator of dE2F1, negates this synergy and limits the overall level of expression of the Yki/Sd-dE2F1 target genes. Significantly, dE2F1 is needed for Yki/Sd-dependent full activation of these target genes, and a e2f1 mutation strongly blocks yki-induced proliferation in vivo. Thus, the Yki transcriptional program is determined through functional interactions with other transcription factors directly at target promoters. It is suggested that such functional interactions would influence Yki activity and help diversify the transcriptional output of the Hippo pathway (Nicolay, 2011).

While recent work has provided insight into how the regulation of Yki occurs via the location within the cell through protein-protein interactions, less is known about how Yki-mediated transcription is regulated. The results presented in this study suggest that Yki may rely on a combinatorial network of transcription factors to modulate transcriptional output in response to Hippo pathway signaling. One such transcription factor is dE2F1, which is required for the full activation of specific target genes by Yki/Sd (Nicolay, 2011).

These studies were prompted by the strong enhancement of the wts mutant phenotype by an rbf mutation. Both the pRB and Hippo pathways are negative regulators of cell proliferation. In flies, RBF functions to limit the activity of the transcriptional activator dE2F1, while the Wts kinase inhibits the transcriptional coactivator Yki. Therefore, one possibility is that, in rbf wts double mutants, dE2F1 and Yki are left unchecked to independently induce genes that promote cell proliferation. However, the data do not support such a trivial explanation. Microarray profiling followed by gene ontology analysis demonstrated that the rbf wts double mutant gene expression signature was distinct from that of either rbf or wts single mutants. Importantly, the rbf wts double mutant signature contained a significant number of up-regulated genes involved in cell cycle progression and cell proliferation that were not present in the rbf or wts single mutant signatures. Thus, an alternative explanation, one that is favored, is that, in rbf wts double mutants, hyperactivated dE2F1 and Yki synergistically up-regulate a novel set of genes and establish the distinct gene expression signature needed to overcome terminal cell cycle exit upon differentiation. Importantly, the synergy results from a direct binding and cooperation between the two factors on the target promoters, since both can be detected by ChIP on dE2F1-Yki/Sd coregulated genes. Consistently, inhibition of dE2F1 by RBF, which is also present on the same set of promoters, is sufficient to limit this synergistic activation by dE2F1 and Yki/Sd (Nicolay, 2011).

Previous studies demonstrated that, in the absence of de2f1, Yki fails to drive inappropriate proliferation, indicating that Yki alone is not sufficient to induce the transcriptional program to prevent cell cycle exit. Importantly, Yki is still active and capable of inducing other Yki-dependent target genes, such as dIAP1. Thus, it appears that the interplay between Yki/Sd and dE2F1 is highly specific to the activation of a distinct set of target genes and is not simply a reflection of a Yki transcription program gone awry. It is suggested that Yki requires an assist from dE2F1 to up-regulate some, if not all, of the dE2F1-Yki/Sd target genes. This assist is critical, since, in the absence of dE2F1, Yki is unable to fully activate these genes to a level sufficient to bypass the cell cycle exit and undergo inappropriate proliferation. Such an interpretation is supported by the transcriptional reporter assays demonstrating that the activation potential of Yki/Sd is reduced in dE2F1-depleted cells. It is noteed that the dE2F1-Yki/Sd target genes are regulated primarily through activation. It remains unclear why RBF/dE2F2 complexes are bound at promoters that are regulated by dE2F1, yet these genes remain insensitive to RBF/dE2F2-mediated repression. Interestingly, two of the dE2F1-Yki/Sd target genes, dDP and cdc2c, were isolated in a genome-wide RNAi screen for factors that are required for Yki to activate a synthetic reporter (Ribeiro, 2010). Given that de2f1 is a transcriptional target of Yki activity as well, it is tempting to speculate that a positively reinforcing signaling loop occurs between Yki/Sd and dE2F1 (Nicolay, 2011).

Yki is a potent oncogene and can elicit a dramatic effect on cell proliferation and apoptosis. Therefore Yki is tightly regulated at multiple levels, including its transcriptional activity, nuclear localization, and degradation. Additionally, it appears that Yki target gene specificity is determined by the transcription factors that interact with Yki and tether it to DNA. For example, Yki partners with Sd and Hth transcription factors. Notably, Hth/Yki transcriptional complexes appear to be important for promoting cell proliferation and survival within the anterior compartment of the eye disc, while in the posterior of the eye disc, Yki switches to partner with Sd to regulate a different set of target genes. The ability of Yki to partner with different DNA-binding proteins in different contexts is thought to provide a basis for altering the transcriptional output of the Hippo pathway. The current results exemplify how, under oncogenic conditions, another transcription factor, such as dE2F1, helps to set up a specific Yki/Sd gene expression signature that is needed to overcome the cell cycle exit. Thus, one conclusion drawn from these results is that the Yki transcriptional program is determined not only by DNA binding proteins that recruit Yki to its target genes, but additionally through interactions with other transcription factors directly at specific target genes. Such functional interactions would influence Yki activity and essentially help to further shape the transcriptional output of the Hippo pathway (Nicolay, 2011).

Another implication of the results is that not only does dE2F1 help to engage a Yki/Sd transcriptional program, but, conversely, a hyperactive Yki/Sd complex contributes to the deregulation of E2F transcription in rbf wts double mutant cells. Given that E2F-dependent transcription is often deregulated in tumor cells, this is an important point. Thus, depending on the identity of other cooperating mutations in pRB-deficient tumor cells, E2F can potentially synergize with a distinct repertoire of transcription factors to engage in transcriptional programs unique to tumor cells of different origins (Nicolay, 2011).

Although initially Yki-induced ectopic proliferation was characterized by an up-regulation in the expression of cyclin E, cyclin A, and cyclin B in flies, this mechanism does not appear to be conserved. In mammals, the up-regulation of cyclin D1 by YAP (the Yki mammalian homolog) is thought to be more critical in promoting inappropriate cell divisions. Thus, it is possible that, in mammals, YAP relies on a different network of transcription factors to promote cell cycle progression than Yki does in flies. Indeed, although YAP has been shown to partner with the Sd homologs TEAD1-4 in mammals, it is also known to interact with other transcription partners (SMAD1 and p73) under specific contexts. Thus, it appears that, similar to Yki, YAP may rely on a distinct repertoire of transcription factors to relay the response to various cellular stimuli (Nicolay, 2011).

Intriguingly, it has been demonstrated that the pRB and Hippo pathways are functionally integrated in human cells. However, the precise mechanism of interaction has seemingly evolved, as it has been shown that inactivation of the Wts homolog LATS2 interferes with the formation of the p130/DREAM repressor complex at E2F target promoters. The inability to repress E2F targets in the absence of LATS2 prevents pRB-induced senescence in human cells . In contrast, the Drosophila dREAM complex appears to be functional in wts mutants (data not shown), and instead the cross-talk between the two pathways occurs at the level of cooperation between Yki and dE2F1. Nonetheless, although the mechanistic paths taken may have diverged between flies and humans, the end point is the same: limit E2F transcriptional activity to prevent inappropriate proliferation (Nicolay, 2011).

To date, the most well-defined oncogenic role for YAP, in the context of Hippo pathway signaling, is in the formation of hepatocellular carcinoma (HCC). However, YAP is also capable of transforming immortalized human mammary epithelial cells, which appears to be through an interaction with the EGFR signaling pathway. In the future, it will be interesting to determine how many other signaling networks oncogenic YAP activity is dependent on, and with what degree these interactions are tissue- or cell type-specific. Finally, these findings support a conserved function of the pRB and Hippo pathways and suggest that a complex coordination of gene expression by these two pathways may underlie a key mechanism during oncogenic proliferation (Nicolay, 2011).

Post-transcriptional Regulation Interactions

mir-11 limits the proapoptotic function of its host gene, dE2f1

The E2F family of transcription factors regulates the expression of both genes associated with cell proliferation and genes that regulate cell death. The net outcome is dependent on cellular context and tissue environment. The mir-11 gene is located in the last intron of the Drosophila E2F1 homolog gene dE2f1, and its expression parallels that of dE2f1. This study investigated the role of miR-11 and found that miR-11 specifically modulates the proapoptotic function of its host gene, dE2f1. A mir-11 mutant was highly sensitive to dE2F1-dependent, DNA damage-induced apoptosis. Consistently, coexpression of miR-11 in transgenic animals suppressed dE2F1-induced apoptosis in multiple tissues, while exerting no effect on dE2F1-driven cell proliferation. Importantly, miR-11 repressed the expression of the proapoptotic genes reaper (rpr) and head involution defective (hid), which are directly regulated by dE2F1 upon DNA damage. In addition to rpr and hid, a novel set of cell death genes was identified that was also directly regulated by dE2F1 and miR-11. Thus, these data support a model in which the coexpression of miR-11 limits the proapoptotic function of its host gene, dE2f1, upon DNA damage by directly modulating a dE2F1-dependent apoptotic transcriptional program (Truscott, 2011).

Coupled expression of a microRNA and its host transcript creates a unique situation where the microRNA can modulate the function of its host. This study found a novel relationship in which an embedded microRNA directly repressed targets that were directly regulated by the host gene transcription factor itself. MiR-11 specifically down-regulated the expression of rpr and hid, which were also directly targeted by dE2F1. However, dE2F1 also activates the expression of genes associated with cell proliferation, and this function was not modulated by miR-11. Therefore, a selective, or partial, negative feed-forward loop was identified in which one of the functions of dE2F1 is modulated by miR-11 (Truscott, 2011).

Target regulation by microRNAs has been shown to follow an incoherent feed-forward loop in which microRNAs buffer against the stochastic fluctuation of expression of their targets, and this facilitates the robustness of changes of expression in response to different cues. For example, miR-7 modulates the expression of its targets in response to environmental fluctuation, while miR-9a modulates proneural signaling to ensure proper specification of sensory organ precursors. However, miR-11 and dE2F1 are part of a somewhat unusual incoherent feed-forward loop in which the expression of both miR-11 and dE2F1 can be induced by the same signal, after which miR-11 negatively regulates only a subset of dE2F1 targets. In this sense, miR-11 imparts robustness to one of the functions of dE2F1, the regulation of expression of proapoptotic targets, while not influencing the function of dE2F1 in modulating the cell cycle. In doing so, miR-11 prevents an apoptotic response due to dE2F1 activity unless it is warranted, such as in the case of response to irradiation-induced DNA damage. Under these circumstances, dE2F1 would be recruited to proapoptotic gene promoters, and miR-11-mediated repression would be released, which could accelerate the apoptotic response. Therefore, these results are consistent with the view that microRNAs function in the canalization of development and response to environmental cues, and demonstrate that the proapoptotic function of the dE2F1 transcription factor is intrinsically modulated by the miR-11 gene, which is embedded in the dE2f1 locus (Truscott, 2011).

The critical role of dE2F1 in irradiation-induced apoptosis is well established and is conserved between flies and mammals. Elimination of dE2F1 activity by a dDP mutation fully blocks DNA damage-induced cell death. Conversely, rbf mutant discs are sensitized to irradiation-induced apoptosis, and this sensitivity is due to elevated activity of dE2F1 (Moon, 2008). Consistent with the idea that miR-11 limits the proapoptotic function of dE2F1, the response of mir-11 mutant cells to irradiation closely parallels the response of rbf-deficient cells. Like rbf mutants (Moon, 2008), mir-11 mutant cells undergo apoptosis more quickly following irradiation than wild-type cells, and the stripe of cells with caspase activity anterior to the MF is expanded in both rbf and mir-11 mutants. Such a strikingly similar phenotype is likely due to abnormally high levels of two key mediators of DNA damage-induced apoptosis, rpr and hid, in both mutants. It is noted, however, that mir-11 and rbf mutations do not fully phenocopy each other. Unirradiated rbf mutant cells are prone to apoptosis within the MF during larval eye disc development, which was attributed to a requirement of EGFR signaling for survival of rbf mutant cells. In contrast, unirradiated mir-11 mutant cells did not undergo apoptosis in the MF, suggesting that miR-11 is not required to limit the proapoptotic function of dE2F1 in this developmental context (Truscott, 2011).

It is well established that dE2F1 induces proliferation and apoptosis, and that the balance between these two opposing activities must be tightly regulated in a context-dependent manner. However, the precise mechanism that determines the net outcome has remained elusive. E2Fs cooperate with different transcription factors in the binding and regulation of different gene promoters, and this was suggested to impart specificity to E2F-mediated transcription. However, E2F has been shown to activate cell death genes in proliferating cells; therefore, post-transcriptional regulation must be in place to limit the function of these cell death genes. The data suggest that such regulation could be also mediated by microRNAs, and in Drosophila, mir-11 would play this role. Although there are other microRNAs that inhibit apoptosis, the coexpression of mir-11 with dE2f1 puts miR-11 in a special position, since it would be expressed precisely where and when dE2F1 is expressed. This is thought to be the first example of an imbedded intronic microRNA regulating direct gene targets of its transcription factor host (Truscott, 2011).

Intriguingly, the ability of miR-11 to regulate dE2F1-specific proapoptotic targets extends beyond rpr and hid. This study has identified a novel set of genes involved in apoptosis that could be directly regulated by both dE2F1 and miR-11. While the described functions of some of these genes are not linked to determining whether or not a cell should die, their deregulation has been associated with cell death. Therefore, fluctuations in the expression levels of genes in this set of common targets could stress the cells, and it is proposed that the presence of miR-11 buffers against such fluctuations, thereby contributing to the maintenance of the overall stability of the cell, while still permitting normal cellular activities (Truscott, 2011).

These data suggest that, in normal cells, while the cell death targets of dE2F1 may be transcribed and poised for translation, miR-11 would prevent the accumulation of an excess of such transcripts. The presence of such transcripts is not sufficient to trigger cell death, as an elevated level of apoptosis is not found in mir-11 mutants during normal development. However, it appears that the loss of mir-11 sensitizes cells to apoptotic signals such as irradiation. Given that miR-11 does not seem to regulate cell death at the level of the core executioners, the caspases, and that at least one target shared by miR-11 and dE2F1, pink1, carries anti-apoptotic functions, cells that express miR-11 would not be unable to die. Indeed, overexpression of miR-11 was not as effective as p35 at blocking dE2F1-induced apoptosis, indicating that some level of apoptosis can be induced even in the presence of a high level of miR-11. One could extend this idea and speculate that, in the case of replicative stress resulting from deregulated dE2F1 activity, for example, miR-11 itself would be subject to negative regulation, thus permitting high expression of the proapoptotic dE2F1 targets, and if necessary, cell death would follow (Truscott, 2011).

While there is no mammalian homolog of miR-11, E2F-induced cell death can be inhibited by microRNAs in mammalian cells. However, the precise mechanism appears to be different. In humans, E2F induces the expression of let-7a-d, let-7i, mir-15b-16-2, and mir-106b-25 at the G1-to-S-phase transition. These microRNAs down-regulate critical cell cycle regulators, which are also targets of E2Fs and also directly down-regulate E2F1 and E2F2. In addition, E2Fs induce the expression of the mir-17-92 polycistronic cluster of microRNAs. mir-17-5-p and mir-20a, in turn, target E2F factors, thereby limiting E2F activity at the level of E2F expression levels. By inducing these microRNAs, E2F initiates a negative feedback mechanism that limits the activation of E2F cell cycle gene targets directly and indirectly, thereby preventing replicative stress-induced cell death. Therefore, excessive E2F activity could be limited by coexpressed microRNAs in flies and mammals (Truscott, 2011).

In summary, these data suggest a model whereby coexpression of miR-11 with dE2F1 from the same locus permits transcriptional activation of cell cycle genes by dE2F1 in the absence of apoptosis. In this scenario, miR-11 initiates a partial negative feed-forward loop in which miR-11 specifically limits the dE2F1 proapoptotic transcriptional program. Intriguingly, although this regulation occurs in normal proliferating cells and leads to up-regulation of dE2F1 proapoptotic targets rpr and hid, the loss of mir-11 was not sufficient to trigger spontaneous apoptosis. Instead, mir-11 becomes important in specific settings, such as protecting cells from dE2F1-dependent DNA damage-induced apoptosis. Therefore, miR-11 buffers against apoptosis, in part by directly modulating the proapoptotic function of its host gene, dE2f1 (Truscott, 2011).

Characterization of null and hypomorphic alleles of the Drosophila l(2)dtl/cdt2 gene: Larval lethality and male fertility

The Drosophila lethal(2)denticleless (l(2)dtl) gene was originally reported as essential for embryogenesis and formation of the rows of tiny hairs on the larval ventral cuticle known as denticle belts. It is now well-established that l(2)dtl (also called cdt2) encodes a subunit of a Cullin 4-based E3 ubiquitin ligase complex that targets a number of key cell cycle regulatory proteins, including p21, Cdt1, E2F1 and Set8, to prevent replication defects and maintain cell cycle control. To investigate the role of l(2)dtl/cdt2 during development, existing l(2)dtl/cdt2 mutants were characterized and new deletion alleles were generated, using P-element excision mutagenesis. Surprisingly, homozygous l(2)dtl/cdt2 mutant embryos developed beyond embryogenesis, had intact denticle belts, and lacked an observable embryonic replication defect. These mutants died during larval stages, affirming that loss of l(2)dtl/cdt2 function is lethal. The data show that L(2)dtl/Cdt2 is maternally deposited, remains nuclear throughout the cell cycle, and has a previously unreported, elevated expression in the developing gonads. E2f1 was found to regulate l(2)dtl/cdt2 expression during embryogenesis, possibly via several highly conserved putative E2f1 binding sites near the l(2)dtl/cdt2 promoter. Finally, hypomorphic allele combinations of the l(2)dtl/cdt2 gene result in a novel phenotype: viable, low-fertility males. It is concluded that 'denticleless' is a misnomer, but that l(2)dtl/cdt2 is an essential gene for Drosophila development (Sloan, 2012).

RBF binding to both canonical E2F targets and noncanonical targets depends on functional dE2F/dDP complexes

The retinoblastoma (RB) family of proteins regulate transcription. These proteins lack intrinsic DNA-binding activity but are recruited to specific genomic locations through interactions with sequence-specific DNA-binding factors. The best-known target of RB protein (pRB) is the E2F transcription factor; however, many other chromatin-associated proteins have been described that may allow RB family members to act at additional sites. To gain a perspective on the scale of E2F-dependent and E2F-independent functions, genome-wide binding profiles of RBF1 and dE2F proteins were generated in Drosophila larvae. RBF1 and dE2F2 associate with a large number of binding sites at genes with diverse biological functions. In contrast, dE2F1 was detected at a smaller set of promoters, suggesting that it overrides repression by RBF1/dE2F2 at a specific subset of targets. Approximately 15% of RBF1-bound regions lacked consensus E2F-binding motifs. To test whether RBF1 action at these sites is E2F independent, dDP mutant larvae were examined that lack any functional dE2F/dDP heterodimers. As measured by chromatin immunoprecipitation-microarray analysis (ChIP-chip), ChIP-quantitative PCR (qPCR), and cell fractionation, the stable association of RBF1 with chromatin was eliminated in dDP mutants. This requirement for dDP was seen at classic E2F-regulated promoters and at promoters that lacked canonical E2F-binding sites. These results suggest that E2F/DP complexes are essential for all genomic targeting of RBF1 (Korenjak, 2012).

The Drosophila RBF and E2F families lack the complexity of their mammalian counterparts and the distribution of RBF1- and dE2F-binding sites described in this study gives a snapshot of the Rb-E2F network in an in vivo context. The results show that RBF1 acts at > 2000 sites in the Drosophila genome and, in accordance with the idea that RB-family proteins regulate gene expression, most of these binding sites are close to promoter regions (Korenjak, 2012).

RBF1-bound regions overlap extensively with binding sites for dE2F2. Additionally, RBF1 is a component of dREAM/Myb-MuvB complexes and a large percentage (74%) of RBF1-bound regions correspond to dREAM/Myb-MuvB binding sites. In contrast, only a relatively small proportion of the regions that were bound by RBF1 were also bound by dE2F1. These findings are consistent with the idea that dE2F2-RBF1 repressor complexes are present throughout the genome, with their effects being transiently reversed by dE2F1 at a small subset of these targets in proliferating cells. One of the implications of this model is that different subsets of RBF1-repressed genes may be activated in different cellular contexts. In support of this it is noted that GO classification of RBF1-bound promoters showed enrichment for several functional categories that were absent from the lists of dE2F1-bound genes, and potentially these targets may represent groups of RBF1 repressed genes that may only be activated by dE2F1 under specific circumstances, or that may be activated by factors that are different from dE2F1. Since E2F-mediated activation can result from the transient presence of dE2F1, a caveat to this interpretation is the likelihood that RBF1 proteins that are components of a stable repressor complex will be more easily detected by ChIP than a transiently bound dE2F1-activator and it is probable that the list of dE2F1-bound promoters underestimates the overall number of dE2F1 targets. Interestingly it found that dE2F1 target genes with functions in mitosis were conspicuously underrepresented in the lists of genes bound by dE2F1 in larvae, even though they were bound by RBF1 and dE2F2, and they were bound by dE2F1 when chromatin prepared from S2 tissue culture cells was profiled. This difference is consistent with the fact that many larval cells grow via endocycles and do not need to express mitotic genes, but it also suggests that dE2F1 binds to different sets of promoters in mitotically-dividing cells and endoreduplicating tissue. Although dE2F1 is thought to be a ubiquitous driver of cell proliferation, these results suggest that different programs of dE2F1-activated transcription are used in different cell types (Korenjak, 2012).

This experimental system was used to address a key issue in E2F/RB research: how much of the RB-protein that is found on chromatin is bound via E2F, and how much is bound in an E2F-independent manner and might potentially have E2F independent functions? This has been a long-standing question, in part because pRB has been shown to associate with many different chromatin-associated proteins, and in part because mammalian cells express many different E2F complexes and it is difficult to dissect the relative contributions of these complexes on pRB chromatin association. The genome wide binding studies for pRB family members that have been carried out in mammalian cells show an enrichment of E2F consensus sequences in the regions bound by pRB, p107 and p130, but it has been unclear if these motifs are essential for the pocket proteins to associate with chromatin, and whether E2F-binding sites represent either the sole genomic location, the main location, or one of many potential locations for the set of pRB-family proteins. Previous studies have examined the distribution of pRB506 family members in cultured cells and it is unclear how accurately the distributions seen in cell lines reflect the distribution of these proteins in normal tissues (Korenjak, 2012).

In the experiments described in this study, the ability to detect chromatin-associated peaks of RBF1 by ChIP was absolutely dependent on dDP. Consistent with this, most RBF1- bound regions contained a sequence corresponding to a 'consensus' E2F-binding site, but this dependency on dDP was also seen in regions that lacked an obvious E2F-binding motif. In agreement with the ChIP results, in cell fractionation experiments no full length RBF1 was found tightly associated with chromatin in nuclear extracts from dDP mutant larvae. The simplest interpretation of these data is that the stable association of RBF1 with chromatin in Drosophila larvae is completely dependent on its recruitment by dE2F/dDP complexes (Korenjak, 2012).

This finding is surprising given the number of reports that have proposed that RB family proteins have E2F independent functions. There are several potential explanations for this paradox. First, the experiments described here provide a picture of the general properties of RBF1 in larval chromatin, but do not exclude the possibility that RBF1 has E2F-independent targets in specific subsets of cells, or at other stages of animal development. It is noted that loss of RBF1 causes defects in chromosome compaction in neuroblasts that were not seen in de2f1, de2f2 or dDP mutants. This role might not require a stable association of RBF1 with chromatin, or alternatively there may be types of RBF1-DNA complexes that were not detected using the ChIP conditions and were disrupted by the fractionation procedure (Korenjak, 2012).

A second possibility is that E2F proteins may play a larger role in the differentiation-specific functions of pRB than has been appreciated. For example, the fact that pRB and Runx2 co-localize at osteoblast-specific promoters during osteogenic differentiation, and the fact that Runx2 is required for pRB to be recruited to these promoters in Saos-2 cells does not exclude the possibility that E2F proteins might also be involved in this process. A recent study demonstrated that the osteogenesis defects resulting from the conditional deletion of murine Rb can be suppressed by the combined inactivation of E2F1. An alternative possibility is that some of the differentiation defects seen when pRB-family members are inactivated are due primarily to problems in establishing permanent cell cycle exit rather than to a direct role for pRB in differentiation (Korenjak, 2012).

A third scenario is suggested by the observation that pRB may act in some contexts by blocking the function of inhibitors of differentiation, such as EID-1, RBP2 and Id2. Id2 is targeted for degradation by the anaphase promoting complex (APC/C) upon cell cycle exit. Interestingly, during cell cycle exit pRB promotes a physical interaction between the APC/C and Skp2 that results in the targeted degradation of Skp2. Although such activities are not E2F-dependent, it is possible that they normally occur in the vicinity of E2F-recruited complexes. Alternatively, they might not occur in the context of chromatin at all (Korenjak, 2012).

Fourth, almost all of the E2F-independent functions of Rb-family proteins have been discovered using mammalian cells and it is possible that there is a basic difference in the roles of these proteins between flies and mammals. Perhaps, during evolution, mammalian pRB has acquired an ability to interact with additional proteins, while the Drosophila orthologs have remained specific partners of dE2Fs (Korenjak, 2012).

These results provide a cautionary note to the use of 'consensus' E2F-binding motifs. 'Consensus' E2F-binding sequences are widely used to predict sites of E2F regulation. However, human E2F proteins bind to a large number of promoter regions and many of these regions do not harbor classical E2F consensus sequences. Approximately 15% of RBF1-binding sites lacked a clear E2F-binding motif. Further analysis of examples of these promoters showed not only that dE2F/dDP proteins could be detected at these regions, but also the analysis of dDP mutant larvae showed that that these complexes were essential for the recruitment of RBF1 (Korenjak, 2012).

These observations raise the question whether additional sequence motifs (and binding proteins) mediate or assist the binding of E2F complexes to DNA. As a component of dREAM/Myb-MuvB complexes, RBF1 is associated with proteins such as dMyb and Mip120 that have known DNA binding activites, and recent work suggests that several dREAM/Myb-MuvB subunits may assist the binding of dE2F2 and RBF1 to E2F target genes. In contrast to a previous study, which identified a consensus DNA binding site for Myb in a small subset of dREAM/Myb-MuvB binding sites, this study failed to detect an enrichment of this motif in dE2F/RBF bound regions. However a sequence motif with similarity to the CHR element was significantly enriched at dE2F/RBF binding sites. CDE/CHR elements have recently been linked to the sequence-specific DNA binding of the human and C. elegans Lin54 proteins, which are the orthologs of Mip120. Moreover, human DREAM binds the cyclin B2 promoter preferentially through the CHR, but not the CDE element. Given that the majority of E2F binding in Drosophila appears to be in the context of dREAM/Myb- MuvB, CHR elements may play an important role in dE2F DNA binding and be useful in predicting functional E2F binding sites (Korenjak, 2012).

Interestingly, dE2F/RBF bound promoters that lack an E2F consensus motif show a strong enrichment for a sequence resembling the DNA replication element (DRE). DRE's are often found in core promoter sequences in Drosophila and are bound by the DNA replication element binding factor (DREF). dE2F and DREF regulate the expression of an overlapping set of genes involved in DNA replication and both proteins are required for cell proliferation in vivo. DREF forms a complex with TATA-box-binding protein-related factor 2 (TRF2) that regulates the expression of several E2F target genes, including PCNA. In the PCNA promoter the DRE was found to be required, in addition to an E2F-binding site, for dE2F1/dDP-dependent transcriptional activation. It seems likely therefore that, at some promoters, DRE/DREF facilitates the binding and action of dE2F/RBF1 complexes (Korenjak, 2012).

Despite the presence of these additional motifs and their potential role in dE2F/dDP DNA binding, the complete lack of RBF1 binding to chromatin in dDP mutants shows that no additional proteins can compensate for the absence of dE2F/dDP in Drosophila larvae and allow the recruitment of RBF1 to chromatin. This suggests that RBF1 and dE2F/dDP function are intimately linked in Drosophila. In support of this, larval lethality due to the homozygous inactivation of rbf1 can be rescued by a mutation in dE2F1 that deletes sequences in the RBF1-binding and transactivation domains. The binding of Mip130, another dREAM/Myb-MuvB subunit, to polytene chromosomes was only marginally affected in dDP animals, suggesting that submodules of dREAM/Myb-MuvB complexes are recruited to chromatin independent of one another. The results are consistent with the idea that protein/protein interactions with dREAM/Myb-MuvB subunits and DREF may help to recruit dE2F/dDP/RBF complexes to promoters, and this may be especially relevant at promoters that lack a consensus E2F motif, but even in this situation the presence of RBF1 at these promoters is completely dependent on dE2F/dDP complexes. When it becomes possible to completely inactivate all of the different classes of E2F complexes in mammalian cells, it will be interesting to learn how this finding compares with the recruitment of pRb, p107 and p130 to chromatin in humans (Korenjak, 2012).

dLin52 is crucial for dE2F and dRBF mediated transcriptional regulation of pro-apoptotic gene hid

Drosophila lin52 (dlin52) is a member of Myb transcription regulator complex and it shows a dynamic pattern of expression in all Drosophila tissues. Myb complex functions to activate or repress transcription in a site-specific manner; however, the detailed mechanism is yet to be clearly understood. Members of the Drosophila melanogaster Myb-MuvB/dREAM complex have been known to regulate expression of a wide range of genes including those involved in regulating apoptosis. E2F and its corepressor RBF also belong to this complex and together they regulate expression of genes involved in cell cycle progression, apoptosis, differentiation, and development. This study examined whether the depletion of dlin52 in developing photoreceptor neurons results in enhanced apoptosis and disorganisation of the ommatidia. Strikingly, it was found that dLin52 is essential for transcriptional repression of the pro-apoptotic gene, hid; decrease in dlin52 levels led to dramatic induction of hid and apoptosis in eye-antennal discs. Reduction of Rpd3 (HDAC1), another member of the dREAM complex, also led to marginal upregulation of Hid. In addition, it was demonstrated that an optimum level of dLin52 is needed for dE2F1/2 activity on the hid promoter. dlin52 cooperates with dRBF and dE2F1/2 for recruitment of repressor complex on the hid promoter. Preliminary data indicates that Rpd3/HDAC1 also contributes to hid repression. Based on the findings, it is concluded that dLin52 functions as a co-factor and modulates activity of members of dMyb/dREAM complex at hid promoter, thus, regulating apoptosis by repressing this pro-apoptotic gene in the developing Drosophila eye (Bhaskar, 2014).

Regulated progression through the cell cycle is essential for ordered cell proliferation. Changes in the balance between cell cycle-driving proto-oncogene-dependent pathways and inhibiting signals from tumour suppressors are a common cause for cancer. One of the best characterised tumour suppressors is the retinoblastoma protein pRB, the first cloned tumour suppressor. This gene was first described as a susceptibility gene for retinoblastoma, an eye tumour in children; it is now known to be mutated in many cancers. Currently, more than 120 proteins have been reported to be associated with pRb, and a wide assortment of chromatin-modifying and binding complexes have been implicated in pRB mediated repression. The association of these complexes with pRb has broadened its range of functions. In humans, one of the complexes known as the DREAM complex is required to arrest expression of genes essential for cell cycle progression. A similar complex is also found in C.elegans (synMuvB complementation group or DRM complex) as well as in Drosophila (dREAM complex). The function of this complex seems to be conserved across taxa and has definitive control over normal cell development. In the past few years, the function and composition of this complex has been unravelled. The role of the dREAM complex has now been extended to development, differentiation, and apoptosis. Its composition has also been reported to vary according to the function and site of the action (Bhaskar, 2014).

The smallest member of dREAM/MMB complex, dLin52 is found in Drosophila. The dynamic pattern of expression of dLin52 in various tissues has been reported and a strong conservation was found of this protein across taxa (Bhaskar, 2012). Hence, this research was extended to explore the functional characterisation of dlin52. GMR-GAL4 and UAS-dlin52-RNAi were used to deplete dLin52 in the developing eye and examine its role in the developing photoreceptors (Bhaskar, 2014).

A recent study, Lewis (2012), has shown that dLin52 is needed for viability, adult eye development and embryogenesis via its maternal effect. The mutant phenotypes could be rescued by heterozygous deletion of mip120 or loss of function allele of mip130. It was concluded that Lin-52 and Myb proteins counteract against the repressive activities of the other members of the MMB/dREAM complex at specific genomic loci in a developmentally controlled manner (Bhaskar, 2014).

The findings of the current study showed that down regulation of dlin52 leads to rough eye phenotype, which is primarily caused by enhanced apoptosis. this observation is in accordance with a study of mammalian cells where depletion of LIN52 sensitised gastrointestinal stromal cells to imatinib induced apoptosis, suggesting a similar mechanism for regulation of apoptosis by LIN52 in higher organisms (Boichuk, 2013). A connection between E2Fs, regulation of hid, and apoptosis has also been found in Drosophila. While dE2F1 activates, dE2F2 has been found to represses hid in wing discs. However, it has been reported that the depletion of dE2F1 in S2 cells leads to activation of hid, showing for the first time the ability of dE2F1 to repress or negatively regulate transcription (Bhaskar, 2014).

The present study established that apoptosis due to dlin52 down regulation is cell autonomous. The components of Drosophila cell death regulatory pathway are conserved in higher organisms. This includes inhibitor of apoptosis proteins (IAPs) which bind to caspases and pro-apoptotic proteins. This study observed that over-expression of DIAPI rescued dlin52-RNAi phenotype. Further, only loss of function alleles of hid suppressed dlin52 rough eye phenotype but not rpr or grim alleles. Similarly, depletion of dLin52 also led to increase in hid transcript levels but not that of rpr or grim. The pattern of hid activation caused by dlin52-RNAi is in agreement with the earlier studies which reported the loss of dRBF and dE2F1; similar to those of previous studies, the cells immediately posterior to the MF were more sensitised to dLin52 downregulation in the eye-antennal discs. Therefore, these findings, supported by the previous observations with loss of RBF and E2F1, suggest that the reduction of dLin52 results in apoptosis is mediated via upregulation of Hid. Additionally, dlin52 also showed robust genetic interaction with Rbf, E2f, and E2f2alleles. It is evident that dLin52 works synergistically with dRBF and dE2F1/2 to mediate transcriptional repression of hid. Previous studies revealed that loss of dRBF and dE2F1/2 activates hid and increases apoptosis, while this tudy identified a third important component, dLin52, vital for hid regulation (Bhaskar, 2014).

Although Lin52 itself is not DNA binding, in humans, Lin52 has been shown to be essential for the formation of transcription repressor complex. Thiss tudy demonstrated that ablated dLin52 levels diminish both dE2F1 and dE2F2 recruitment on the hid promoter. As mutated dE2F binding site on the hid promoter fails to enhance dlin52-RNAi phenotype, it indicates that dE2F1 mediates repression of hid, aided by dLin52 and dRBF1. It was observed that recruitment of dE2F2 was also affected with loss of dLin52; it is likely that both E2F1 and E2F2 function to repress hid, and the stoichiometry of both E2F1 and E2F2 are important for their regulatory function. Taken together these data suggest dLin52 as the essential factor for dRBF, dE2F1/2 mediated repression of hid expression. It was also found that transcriptional regulation of hid is mediated via the dE2F binding site present in the 5' UTR of hid. Previous studies showed that dE2F1 not only binds to a unique site in promoter region of hid but also regulates its transcription in presence of dRBF. Therefore, it is concluded that optimum level of dLin52 is needed for recruitment of dE2F1/2 to the hid promoter; reducing dLin52 dramatically compromises with the dE2F1/2 binding to the cis-acting sequences on the hid promoter (Bhaskar, 2014).

Histone deacetylase1 (HDAC1) has been the most thoroughly studied HDAC at the biochemical and functional levels. HDACs are known to promote heterochromatin silencing by deacetylating H3. They target hundreds of genes in the genome and play a major role as a direct transcriptional repressor. HDAC1 controls segmentation genes through interaction with the Groucho corepressor and has also been linked to silencing by Polycomb repressors. HDAC3 and HDAC1 mutants together can suppress position-effect variegation (PEV), indicating the ability of both the HDACs in mediating heterochromatin silencing while working together. Recently, Zhu, found Drosophila HDAC3 regulating the wing imaginal disc size through suppression of apoptosis, while HDAC3 mutants shows ectopic Hid levels in wing discs. Mammalian HDAC1 mutant also shows increased apoptosis. Rpd3, the Drosophila homologue of HDAC1/2, has been purified along with dMyb/MMB/dREAM complex. Function of both HDAC3 and Rpd3 appears to overlap in regulating silencing and chromatin modification. In this study, it was observed that down-regulation of Rpd3 alone in eye-antennal discs led to a rough eye phenotype and induced marginal increase of Hid; however, ectopic expression was not limited to a few rows of cells posterior to the MF but traversed the differentiating photoreceptors, implying that Rpd3 mediated hid regulation is not limiting to cells specifically regulated by dLin52, dRbF and dE2F1/2 but expands to a much broader area in the developing photoreceptors. Similarly, lowering of both dlin52 and Rpd3 at the same time led to substantial increase in Hid levels with enhanced disorganisation of the ommatidia. Although Rpd3 in Drosophila lacks LXCXE motif, it might be recruited at the repressed sites by other members of the complex. Hence, it may be concluded that both dLin52 and Rpd3 (HDAC1) have a major role to play in negative regulation of hid expression. However, this is only a preliminary report suggesting that dLin52 and Rpd3 can work in parallel to regulate hid expression. Further experiments like biochemical purification of dREAM complex in the presence and absence of dLin52 would help in elaborating function of Rpd3 in regulating apoptosis (Bhaskar, 2014).

This study is in agreement with the earlier findings that Lin52 plays an important role in forming dREAM complex. Human Lin52 phosphorylation is needed for assembling of the dREAM complex. Earlier, it was reported that the Serine-28 residue in the human Lin52 is conserved in dLin52. This study proposes that limiting dLin52 might actually be equivalent to un-phosphorylated dLin52 which is non-functional, resulting in assembling defects of the dREAM complex. Therefore, it is hypothesised that dLin52 is a vital survival signal, needed for suppressing hid transcription and apoptosis and conclude that dLin52 is a crucial cofactor essential for assembling members of dREAM/MMB complex (dRBF, dE2F1/2). In addition, this study has also presented preliminary data indicating that Rpd3 functions together with dLin52, dRBF, and dE2F1/2 for mediating transcriptional repression of hid (Bhaskar, 2014).

A proposed model shows that dLin52 and Rpd3 (HDAC1) together with dRBF and dE2Fs are part of a repressor complex, repressing hid activation. When dLin52 becomes limiting, E2F1/2 cannot be recruited to the consensus E2F1/2 binding site on the hid promoter, resulting in the inability of the repressor complex to assemble. This leads to derepression and activation of hid, followed by increased apoptosis (Bhaskar, 2014).

Chemical inhibitors that block HDAC activity are of considerable interest in cancer research because of their ability to induce tumour cell killing by activating cell death pathway leading to apoptosis. Hence, it is proposed that Lin52 may also be selectively inhibited in inducing apoptosis in tumour cells (Bhaskar, 2014).

This study established the important role of dLin52 in repressing apoptosis. This leads to the belief that dLin52 is needed for maintenance of proper development, differentiation, normal physiology, and homeostasis in Drosophila (Bhaskar, 2014).

This study has found down regulation of dlin52 resulting in a rough eye phenotype. Based on these findings, it is suggested that the rough eye phenotype is due to increase in apoptosis. This study established through genetic analysis that downregulation of dlin52 increases hid expression. As dLin52 by itself is not DNA binding, it is predicted that it may be regulating hid along with the members of dREAM complex. Based on the observations, it can be concluded that dRBF, dE2F1, dE2F2, and Rpd3 work together with dLin52 for repressing hid activation, because the loss of function alleles of these genes led to strong enhancement of dlin52-RNAi eye phenotype. ChIP experiment demonstrated that reduced dlin52 levels also affect dE2F1 and dE2F2 binding to its consensus binding site on hid promoter (Bhaskar, 2014).

Furthermore, it can be concluded that dLin52 regulates apoptosis in eye-antennal discs by repressing hid transcription; loss of dLin52 induces hid expression. This suggests that dLin52 is a co-factor, needed for repression of hid along with dE2Fs and dRBF. Additionally, loss of dLin52 also affected binding of dE2F1 and dE2F2 to their consensus sequence, which means like DP, dLin52 can also affect DNA binding capacity of RBF and E2Fs. Taken together these data suggest that dLin52, dRBF, dE2F1, dE2F2, and dRpd3 cooperate to negatively regulate hid transcription and apoptosis. Further studies in understanding the role of Drosophila lin52 in apoptosis will shed light on the role of LIN52 in higher organisms (Bhaskar, 2014).

An intronic microRNA links Rb/E2F and EGFR signaling

The importance of microRNAs in the regulation of various aspects of biology and disease is well recognized. However, what remains largely unappreciated is that a significant number of miRNAs are embedded within and are often co-expressed with protein-coding host genes. Such a configuration raises the possibility of a functional interaction between a miRNA and the gene it resides in. This is exemplified by the Drosophila melanogaster dE2f1 gene that harbors two miRNAs, mir-11 and mir-998, within its last intron. miR-11 was demonstrated to limit the proapoptotic function of dE2F1 by repressing cell death genes that are directly regulated by dE2F1, however the biological role of miR-998 was unknown. This study shows that one of the functions of miR-998 is to suppress dE2F1-dependent cell death specifically in rbf mutants by elevating EGFR signaling. Mechanistically, miR-998 operates by repressing dCbl, a negative regulator of EGFR signaling. Significantly, dCbl is a critical target of miR-998 since dCbl phenocopies the effects of miR-998 on dE2f1-dependent apoptosis in rbf mutants. Importantly, this regulation is conserved, as the miR-998 seed family member miR-29 repressed c-Cbl, and enhanced MAPK activity and wound healing in mammalian cells. Therefore, the two intronic miRNAs embedded in the dE2f1 gene limit the apoptotic function of dE2f1, but operate in different contexts and act through distinct mechanisms. These results also illustrate that examining an intronic miRNA in the context of its host's function can be valuable in elucidating the biological function of the miRNA, and provide new information about the regulation of the host gene itself (Truscott, 2014. PubMed ID: 25058496).

Protein Interactions

E2F is really a heterodimer, consisting of E2F itself and DP protein. A Drosophila DP protein which can interact co-operatively with E2F proteins is a physiological DNA binding component of Drosophila E2F. Some aspects of the mechanisms which integrate early cell cycle progression with the transcription apparatus are thus conserved between Drosophila and mammalian cells. The distinct expression patterns of Drosophila DP and E2F suggest that the formation of DP/E2F heterodimers, and hence E2F, is subject to complex regulatory cues (Hao, 1995).

Drosophila DP and human DP-1 proteins share 61% identity over a region between residues 91 and 315 that includes the putative DNA-binding domain. Drosophila DP binds to Drosophila E2F fusion proteins containing amino acids 225-805 or 1-432, but fails to bind to a fusion protein that includes only aa 47-343. Thus a region of Drosophila E2F between aa225 and 432 may be required for interaction with Drosophila DP. This region of the protein is highly conserved. Drosophila DP has a weak nonspecific DNA-binding activity, while Drosophila E2F alone is unable to bind DNA. When the two proteins are mixed, E2F acquires DNA-binding activity, and the affinity of DP for DNA is dramatically enhanced. The enhanced binding is site-specific (Dynlacht, 1994).

Whereas the ectopic expression of Cyclin E activates dE2F-dependent transcription, it has been proposed that Cyclin E does not act directly on E2F but targets a negative regulator of E2F activity. Such a regulator might be analogous to the family of RB-related proteins (pRB, p107, and p130) that associate with E2F in humans. Drosophila Retinoblastoma-family protein) combines several of the structural features of pRB, p107, and p130, suggesting that it may have evolved from a common ancestor to the three human genes. RBF associates with Drosophila E2F and DP in vivo and is a stoichiometric component of E2F DNA-binding complexes. RBF specifically repressed E2F-dependent transcription and suppressed the phenotype generated by ectopic expression of dE2F and dDP in the developing Drosophila eye. RBF is phosphorylated by a cyclin E-associated kinase in vitro, and loss-of-function cyclin E mutations enhanced an RBF overexpression phenotype, consistent with the idea that the biological activity of RBF is negatively regulated by endogenous cyclin E. The properties of RBF suggest that it is the intermediary factor that was proposed to allow cyclin E induction of E2F activity. These findings indicate that RBF plays a critical role in the regulation of cell proliferation in Drosophila (Du, 1996a).

A role for the DP subunit of the E2F transcription factor in axis determination during Drosophila oogenesis

The E2F family of transcription factors contributes to cell cycle control by regulating the transcription of DNA replication factors. Functional 'E2F' is a DNA-binding heterodimer composed of E2F and DP proteins. Drosophila contains two E2F genes (E2F and E2F2) and one DP gene. Mutation of either E2F or DP eliminates G1-S transcription of known replication factors during embryogenesis and compromises DNA replication. However, the analysis of these mutant phenotypes is complicated by the perdurance of maternally supplied gene function. To address this and to further analyze the role of E2F transcription factors in development mitotic clones of DP, mutant cells in the female germline have been phenotypically characterized. DP has been shown to be required for several essential processes during oogenesis. In a fraction of the mutant egg chambers the germ cells execute one extra round of mitosis, suggesting that in this tissue DP is uniquely utilized for cell cycle arrest rather than cell cycle progression. Mutation of DP in the germline also prevents nurse cell cytoplasm transfer to the oocyte, resulting in a 'dumpless' phenotype that blocks oocyte development. This phenotype likely results from both disruption of the actin cytoskeleton and a failure of nurse cell apoptosis, each of which is required for normal cytoplasmic transfer. DP is required for the establishment of the dorsal-ventral axis, since loss of DP function prevents the localized expression of the Egfr ligand Gurken in the oocyte: Gurken initiates dorsal-ventral polarity in the egg chamber. Thus new functions for E2F transcription factors during development have been uncovered, including an unexpected role in pattern formation (Myster, 2000).

Drosophila oogenesis relies on communication between the nurse cells and the oocyte; this includes transfer of nurse cell cytoplasm to the oocyte. There are two successive phases of cytoplasmic transport during oogenesis. Early transport is slow and highly selective. Later in oogenesis a rapid phase of cytoplasmic transfer occurs as nurse cells contract to squeeze their contents into the oocyte. DP mutant egg chambers are defective in this rapid transport. They are also defective in nurse cell apoptosis. How might dDP affect both nurse cell cytoplasmic transport and apoptosis? Recent evidence reveals that entry into apoptosis may be coupled to rapid cytoplasm transport. In wild-type egg chambers several events indicate an apparent initiation of a nurse cell apoptotic pathway just prior to rapid cytoplasm transfer. The nurse cells undergo actin cytoskeleton rearrangements and their nuclear membranes become permeabilized. Delays in apoptosis seen in dumpless mutants that disrupt the actin cytoskeleton, such as chickadee (profilin) and kelch (a ring canal component), suggest that an apoptotic pathway and a rapid cytoplasmic transport pathway are interconnected. Germline mutant clones of Drosophila dcp-1, a CED-3-related caspase, display defects in actin bundle assembly, nuclear envelope permeabilization and cytoplasmic transport. Thus, it has been suggested that activation of an apoptotic pathway in nurse cells may lead to the formation of the actin bundles and subsequent nurse cell to oocyte cytoplasmic transfer. This model places a signal to enter apoptosis at the start of the pathway, followed by dcp-1 function, followed by chickadee, quail (villin) and singed (fascin) functions in bundling actin, and ending with cytoplasm transfer. In the context of this model, DP could act either by directly regulating genes involved in actin cytoskeleton function (e.g., chickadee) or an apoptotic pathway (e.g., dcp-1). The well-documented ability of both mammalian and Drosophila E2F to induce apoptosis suggests the latter hypothesis, i.e. that dDP is required for the initiation of nurse cell apoptosis, which triggers subsequent cytoplasmic dumping (Myster, 2000).

Surprisingly, loss of dDP function significantly inhibits the establishment of dorsal-ventral polarity during oogenesis. Germline cells mutant for either of two alleles of dDP prevent the normal accumulation of Grk protein. In DP oocytes, Grk protein fails to accumulate to wild-type levels. Grk protein is detected at higher levels in DP mutants than DP mutants, but fails to properly localize to the oocyte plasma membrane. These data suggest that both Grk accumulation and intracellular localization are disrupted by mutation of DP. Because Grk is required for an oocyte-follicle cell signaling system that initiates dorsal follicle cell fate, the DP mutants displayed a ventralized eggshell. Defects in Gurken accumulation may result from disruption of the cell cycle caused by loss of DP. There is already evidence that Gurken expression is linked to cell cycle control. Mutation of the 'spindle' class genes spnB and okra prevent normal Grk accumulation and establishment of the dorsal-ventral axis. spnB and okra encode proteins with sequence similarity to yeast genes required for repair of double strand breaks (DSBs). The failure to accumulate Grk in spnB and okra mutants is a downstream developmental consequence of a meiotic checkpoint activated in response to the persistence of DSB generated during meiotic recombination. Perhaps loss of DP function alters replication sufficiently to activate a checkpoint pathway. Alternatively, the dorsal-ventral defect in DP mutants may reflect a DP requirement for expression of spnB and okra during meiosis (Myster, 2000).

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

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

To explore the possibility that E2f-Rbf is directly involved in controlling ORC activity, a test was performed to see whether a female-sterile mutant of Rbf (Rbf120a) has DNA replication and gene amplification defects in follicle cells of the Drosophila ovary. TheRbf120a mutation is due to a P-element insertion that causes reduced levels of wild-type Rbf protein, and Rbf14 is a null mutant. Ovaries from mutant Rbf120a/Rbf14 and heterozygous Rbf14/+ females were double labelled with 5-bromodeoxyuridine (BrdU) and anti-ORC2. Wild-type Drosophila follicle cells undergo endoreduplication cycles (endo cycles), reaching 16n ploidy by stage 9 or 10A of egg-chamber development. In stage 10B follicle cells, endo cycles have ceased, ORC has been cleared from the nucleus, and ORC is localized to discrete genomic regions undergoing amplification. Amplification is detected by BrdU incorporation at ORC localized foci. By contrast, the Rbf120a/Rbf14 mutant egg-chambers have a mosaic of follicle cells exhibiting striking replication defects: (1) some mutant follicle cells have inappropriate total nuclear ORC2 staining and continued endo cycles instead of amplification; (2) some follicle cells with specific ORC2 localization to replication origins have undergone gene amplification; and (3) some cells perform both amplification and genomic replication. Staining ovaries with anti-Rbf antibodies reveals a uniform absence of Rbf protein, and thus the mosaic phenotype cannot be explained by stochastic differences in Rbf protein levels (Bosco, 2001).

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

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

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

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

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

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

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

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

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

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

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

The role of RBF in developmentally regulated cell proliferation in the eye disc and in Cyclin D/Cdk4 induced cellular growth

During Drosophila eye development, cell proliferation is coordinated with differentiation. Immediately posterior to the morphogenetic furrow, cells enter a synchronous round of S phase called second mitotic wave. This study examines the role of RBF, the Drosophila RB family homolog, in cell cycle progression in the second mitotic wave. RBF-280, a mutant form of RBF that has four putative cdk phosphorylation sites mutated, can no longer be regulated by Cyclin D or Cyclin E. RBF-280 retains the wild-type RBF ability to inhibit transactivation by E2F1. Expression of RBF-280 in the developing eye reveals that RBF-280 does not inhibit G1/S transition in the second mitotic wave; rather, it delays the completion of S phase and leads to abnormal eye development. These observations suggest that RB and E2F control the rate of S-phase progression instead of G1/S transition in the second mitotic wave. Characterization of the role of RBF in Cyclin D/Cdk4-mediated cellular growth shows that RBF-280 blocks Cyclin D/Cdk4 induced cellular growth in the proliferating wing disc cells but not in the non-dividing eye disc cells. By contrast, RBF-280 does not block activated Ras-induced cellular growth. These results suggest that the ability of Cyclin D/Cdk4 to drive growth in the proliferating wing cells is distinct from that in the non-dividing eye cells or the ability of activated Ras to induce growth, and that RBF may have a role in regulating growth in the proliferating wing discs (Xin, 2002).

e2f1 null mutant eye discs undergo a second mitotic wave when the level of RBF is reduced, indicating that transcription activation by E2F1 is not required for S-phase entry in the second mitotic wave. There are two possible explanations for this observation: one possibility is that derepressed basal E2F target gene expression in the second mitotic wave is sufficient to drive S-phase entry. Alternatively, it is possible that cells in the second mitotic wave are driven into S phase through an E2F-independent mechanism. The results presented in this report suggest that S-phase entry in the second mitotic wave is probably driven by an E2F-independent mechanism, since overexpression of a non-regulated RBF inhibits E2F target gene expression but does not inhibit S phase there. These results are consistent with the observation that Hh signaling is required for S phase entry in the second mitotic wave through direct induction of Cyclin E. Since Hh signal is known for its role in neuronal differentiation and in pattern formation of the developing eye, it appears that developmentally regulated G1/S transition in the second mitotic wave is controlled by the same signal that also controls differentiation and pattern formation to coordinate the cell proliferation with differentiation. Although RB/E2F does not control the S-phase entry in this case, RB and E2F appear to be important for the rapid progression through S phase in the second mitotic wave. The observation that RBF-280 delays S-phase completion in the second mitotic wave and severely disrupts normal eye development indicates the importance of coordinating the rate of cell proliferation and differentiation in the developing eye (Xin, 2002).

Besides the observation that RB and E2F play important roles regulating S-phase progression in the developing eye, RB/E2F has also been shown to affect S-phase progression in developing embryos and wing discs. Thus, RB and E2F appear to regulate S-phase progression in multiple developmental settings. Similarly, RB and E2F have also been shown to regulate G1/S transition in a number of other developmental settings. The question is when do RB and E2F regulate G1/S transition and when do they regulate S phase progression? It appears that RB and E2F often play important roles in the G1 arrested cells (such as the G1 arrested cells in the embryos and in the eye discs) to prevent ectopic S phase entry. In these cases, Rb and E2F probably function through inhibiting the expression of Cyclin E. By contrast, developmentally regulated cell proliferation (G1/S transition) appears to be tightly linked to the developmentally regulated transcription of cyclin E, which is controlled by a large cis-regulatory region containing tissue- and stage-specific components. Temporal and tissue specific Cyclin E expression will drive cells into S phase and lead to the inactivation of RB and the coordinated E2F target gene expression, which might be required for the timely progression through S phase. The observation that RBF-280 expression inhibits E2F target gene expression and delays the completion of S phase supports a role for RB/E2F in S phase progression in the second mitotic wave (Xin, 2002).

There are at least two possible mechanisms that may contribute to the function of RBF in regulating S-phase progression. One mechanism is through the inhibition of E2F target gene expression besides cyclin E. Because several E2F target genes such as PCNA, RNR2, Orc1 and DNA polalpha are components of the DNA replication machinery, inhibition of E2F target gene expression may result in an insufficient amount of DNA replication machinery, which may delay the completion of S phase. A second possibility is that RBF may regulate DNA replication directly. Recently, it has been shown that the E2F1/RBF complex is localized to the DNA replication origin, and interacts with ORC proteins directly (Bosco, 2001). In addition, mammalian RB can interact with MCM7 and regulate DNA replication directly. Thus, it is possible that RBF can regulate S-phase progression directly by controlling firing at replication origins (Xin, 2002).

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).

Surprisingly, however, HA-RBF2 had no effect on dE2F1-activated transcription. RBF2 also failed to repress the dE2F1 activation of the DNA Pola reporter. In keeping with these transfection results, it was noted that in transgenic animals, contrary to RBF1, RBF2 overexpression fails to suppress phenotypes caused by elevated levels of dE2F1 in various tissues. Since overexpression of RBF2 does not inhibit dE2F1-driven transcription and does not suppress dE2F1-induced phenotypes in vivo, it appears that RBF1 and RBF2 regulate E2F-dependent transcription in a distinct manner (Stevaux, 2002).

To understand the relationship between RBF1, RBF2, and the E2F proteins, the pattern of dE2F-RBF protein interactions that exist in Drosophila SL2 cells was examined. Specific antibodies for dDP, dE2F2, dE2F1 were used to immunoprecipitate protein complexes from SL2 extracts. These immune complexes were analyzed by Western blotting with monoclonal antibodies specific for RBF1 or RBF2. A single 85 kDa band was detected by an anti-RBF2 monoclonal antibody in dDP and dE2F2 immunoprecipitates, but not in the dE2F1 or the control immunoprecipitates. The blot was stripped and re-probed with an anti-RBF1 monoclonal antibody and, as expected, RBF1 was detected in each of the test lanes. In the reciprocal experiment, dE2F1 was detected in RBF1 immune complexes, but not in RBF2 immune complexes, whereas dE2F2 was found in both RBF1 and RBF2 immune complexes. These results indicate that, under physiological conditions, RBF1 forms complexes with dE2F1 or dE2F2. RBF2, however, does not bind dE2F1, the activator Drosophila E2F, but associates exclusively with the repressor Drosophila E2F, dE2F2 (Stevaux, 2002).

This pattern of interactions could explain, in a very simple way, why RBF2 is unable to block dE2F1-mediated activation. This arrangement also predicts that the effects of RBF2 on E2F-regulated transcription are likely to be mediated via dE2F2. To test this hypothesis, the ability of RBF2 to repress transcription in cells depleted for dE2F2 was assessed by RNA-mediated interference (RNAi). Cells were treated with control or dE2F2-specific double-stranded (ds) RNA and subsequently transfected with an E2F reporter construct and RBF1 or RBF2 expression plasmids. Western blots of the dsRNA-treated cells showed that the level of dE2F2 was substantially reduced by the RNAi treatment after 4 days. The ability of RBF2 to repress transcription from the MCM3 promoter is completely inhibited in cells treated with dE2F2 dsRNA. In contrast with RBF2, the ability of RBF1 to repress transcription is unaffected by the depletion of dE2F2, presumably because of the presence of dE2F1, the other E2F partner of RBF1. This experiment indicates that dE2F2 is necessary for the proper repression of E2F target genes by RBF2. To test whether dE2F2 is required for the detection of RBF2 at E2F-regulated promoters, ChIP assays was performed on cells treated with dE2F2 dsRNA. The depletion of dE2F2 from SL2 cells eliminates the binding of dE2F2 and RBF2 to the DNA Pola promoter, while leaving dE2F1 binding unaffected (Stevaux, 2002).

These experiments show that RBF2 can no longer be localized to E2F-regulated promoters or repress transcription in the absence of the DNA-binding activity provided by dE2F2. While the levels of HA-RBF2 expressed in transient transefction assays were unaffected by depletion of dE2F2, it was noticed that levels of endogenous RBF2 protein were slightly reduced when dE2F2 was depleted by RNAi. Interestingly, Western blots of larval extracts prepared from wild-type and de2f2 mutant larvae show that a long-term consequence of removing dE2F2 is that RBF2 becomes barely detectable. The reduction of RBF2 protein is due to post-transcriptional effects, since rbf2 transcripts are readily detectable in total RNA preparations from de2f2 mutant larvae and, indeed, are present at elevated levels in the de2f2 mutants. The most likely explanation is that RBF2 becomes less stable in the absence of its binding partner. At present, the formal possibility that dE2F2 influences the synthesis of RBF2 cannot be excluded. Nevertheless, these observations all support the notion that the function of RBF2 depends on the presence of dE2F2 (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).

The combined expression of dE2F2 and RBF2 generates a small rough eye phenotype. This small eye phenotype is similar to, but weaker than, what is observed with an ey-Gal4/UAS-rbf1 transgenic line. Wing discs overexpressing RBF2 and dE2F2 were examined to determine the physiological basis of the observed phenotype. The co-expression of RBF2 and dE2F2 causes a significant decrease in DNA synthesis. Overexpression of RBF2 and dE2F2 gives a phenotypic range that varies from discs that are shrunken and abnormal to those discs that have relatively normal morphology but display reduced BrdU incorporation. The defects do not appear until sufficient amounts of dE2F2 and RBF2 have accumulated in sensitive tissues (Stevaux, 2002).

To further assess the effects of RBF2-E2F2 expression on the cell cycle, clones of dE2F2-RBF2-overexpressing cells were generated in the wing discs and marked with GFP. The cell cycle profile of these cells was determined by FACS analysis and compared with wild-type cells from the same discs. The overexpression of dE2F2 or RBF2 alone has no effect on cell cycle distribution. However, the co-expression of dE2F2 and RBF2 causes a significant increase in the population of cells with a G1 DNA content, and a decrease of S phase and G2 cells. Taken together, these experiments demonstrate that, when overexpressed, dE2F2 and RBF2 act synergistically to antagonize dE2F1-mediated transcriptional activation and to block S phase entry in vivo (Stevaux, 2002).

It is concluded that although RBF1 and RBF2 are both able to repress E2F-dependent transcription, they appear to act in markedly different ways. RBF1 was originally identified by virtue of its ability to physically interact with the transcriptional activation domain of dE2F1. RBF1 is a potent inhibitor of dE2F1-mediated activation and it readily suppresses dE2F1-induced phenotypes in vivo. Unlike RBF1, RBF2 does not associate with dE2F1 in vivo, and it is unable to suppress the effects of overexpressed dE2F1. In vivo, RBF2 associates specifically with dE2F2. The recruitment of RBF2 to E2F-regulated promoters, and its ability to repress transcription, requires dE2F2. In support of the idea that RBF2 acts in a stable complex with dE2F2, these proteins act synergistically when overexpressed in SL2 cells or in transgenic animals, and that RBF2 levels are strongly reduced in de2f2 mutant larvae (Stevaux, 2002).

Of the four proteins, only dE2F1 activates transcription. It is suggested that RBF proteins modulate dE2F1-mediated activation in two distinct ways. (1) dE2F1 can be directly regulated through a physical interaction with RBF1. (2) dE2F1-mediated activation can be antagonized by the presence of RBF2-dE2F2 and RBF1-dE2F2 repressor complexes at the promoter of E2F-regulated genes. Because E2F-regulated promoters often contain multiple E2F-binding sites, it is unclear whether these complexes compete for the same binding element or whether they act antagonistically through adjacent sites. The results described here suggest that there is a hierarchy of effects, with RBF1 being a stronger antagonist of dE2F1 than either of the dE2F2-containing complexes. Nevertheless, the level of dE2F1-dependent transcription is influenced by dE2F2 and RBF2, and changing the levels of RBF2 alters the balance between dE2F1-mediated activation and dE2F2-mediated repression. One of the implications of this arrangement is that a pocket protein does not need to bind directly to an E2F subunit in order to influence its activity (Stevaux, 2002).

Strikingly, a similar arrangement of E2F/pocket proteins exists in mammalian cells. Although mammalian cells contain multiple E2F and pRB family members, recent studies have suggested that the different forms of E2F can be subdivided into two groups depending on whether they appear to be primarily involved in activation or repression. Intriguingly, pRB, like RBF1, interacts with both sets of E2Fs, whereas p107 and p130, like RBF2, interact specifically with the co-repressor E2Fs. Consistent with this, the loss of RBF1 function induces phenotypes that are remarkably similar to the effects of mutating pRB (deregulation of E2F, ectopic S phases, increased apoptosis), and the mutation of dE2F1 gives phenotypes that are very similar to those recently described for the combined mutation of the murine E2F1, E2F2 and E2F3 genes (G1 arrest and loss of E2F-dependent transcription). Furthermore, the genetic interactions observed between RB and E2f1 or RB and E2f3 alleles in mice are reminiscent of the genetic interactions between rbf and de2f1 in flies. The logical extension of this homology is that the roles of RBF2-E2F2 in Drosophila may be similar to the repressor complexes formed by p107/p130 and E2F4/E2F5 in mammalian cells. Despite the attractions of this analogy, it is noted that the phylogenetic tree of Retinoblastoma-related proteins shows that RBF1 and RBF2 are more closely related to one another than they are to any mammalian protein. This suggests that RBF1 and RBF2 were generated by a gene duplication event, most likely from an ancestral protein that resembled p107 or p130. Consequently, similarities between the arrangement of Drosophila and mammalian RB-E2F complexes are more likely to result from a convergent evolutionary process, rather than the conservation of functional differences between pocket proteins (Stevaux, 2002).

What is the advantage of such an arrangement of complexes, and why might it be selected? The distinction between activator and repressor E2Fs is most important if one considers the effects when the complexes are disrupted. Since dE2F2 appears to be unable to activate transcription, the release of RBF1 or RBF2 from a dE2F2 complex is predicted to de-repress E2F target genes. In contrast, the release of RBF1 from a dE2F1-containing complex would liberate a strong activator of transcription. These types of E2F complexes therefore allow three different types of E2F regulatory transitions: (1) from repression to de-repression (in which RBF2-dE2F2 or RBF1-dE2F2 complexes are disrupted and removed from the promoters); (2) from repression to activation (in which RBF1-dE2F1 complexes are disrupted by phosphorylation liberating dE2F1, a strong activator of transcription); and (3) from repression to de-repression to activation (in which RBF2-dE2F2 or RBF1-dE2F2 repressors are disrupted and replaced by dE2F1) (Stevaux, 2002).

The evolution of multiple E2F-RB complexes may offer several additional advantages. Individual complexes may preferentially regulate different subsets of targets. This specificity might be achieved by different DNA-binding subunits (e.g. E2F1-RBF1 versus E2F2-RBF1) or by different protein-protein interactions with adjacent factors at the promoter. Indeed, several studies have suggested that mammalian E2F/pocket proteins may target specific promoters. A second possibility is suggested by the fact that many chromatin-remodeling activities have been linked to pocket proteins, potentially allowing a wide variety of activities to be recruited to E2F-regulated promoters. Perhaps RBF1 and RBF2 provide a bridge to different types of complexes. A third possibility is that the pocket protein-E2F complexes may be differentially regulated. pRB appears to be uniquely required for DNA damage-induced cell cycle arrest. pRB is specifically required in TGFß and p16-induced cell cycle arrest. In a similar way, RBF2 may be required for cell cycle arrest at a specific stage of development or in particular tissues. The high levels of RBF2 protein in early embryos and in dissected ovaries may reflect specific roles in embryonic cell cycle and oogenesis. Alternatively, RBF1- and RBF2-containing complexes might occupy E2F sites during different phases of the cell cycle. E2F has recently been shown to control the expression of genes encoding mitotic functions whose transcription is induced later than the G1 to S transition. RBF2, like p107, is expressed at higher levels in actively cycling cells and is a likely E2F target gene. Potentially, this newly synthesized pocket protein may provide a repressor activity during S phase on mitosis-specific promoters or during G2 at S phase-specific promoters (Stevaux, 2002).

The answer to many of these questions will stem from a careful comparison between rbf1 mutants, rbf2 mutants and rbf1;rbf2 double mutants. This analysis will be needed to separate the specific functions of RBF1 and RBF2 in vivo and to uncover the functions that are redundant between the two Drosophila pocket proteins (Stevaux, 2002).

Nitric oxide pathway interacts with the RB pathway to control growth

Animal organ development requires that tissue patterning and differentiation is tightly coordinated with cell multiplication and cell cycle progression. Several variations of the cell cycle program are used by Drosophila cells at different stages during development. In imaginal discs of developing larvae, cell cycle progression is controlled by a modified version of the well-characterized mammalian retinoblastoma (Rb) pathway, which integrates signals from multiple effectors ranging from growth factors and receptors to small signaling molecules. Nitric oxide (NO), a multifunctional second messenger, can reversibly suppress DNA synthesis and cell division. In developing flies, the antiproliferative action of NO is essential for regulating the balance between cell proliferation and differentiation and, ultimately, the shape and size of adult structures in the fly. The mechanisms of the antiproliferative activity of NO in developing organisms are not known, however. Transgenic flies expressing the Drosophila nitric oxide synthase gene (dNOS1) and/or genes encoding components of the cell cycle regulatory pathways (the Rb-like protein RBF and the E2F transcription factor complex components dE2F and dDP) combined with NOS inhibitors were used to address this issue. Manipulations of endogenous or transgenic NOS activity during imaginal disc development can enhance or suppress the effects of RBF and E2F on development of the eye. These data suggest a role for NO in the developing imaginal eye disc via interaction with the Rb pathway (Kuzin, 2000).

To regulate ectopic production of NO during development, transgenic lines of Drosophila were generated in which the expression of dNOS1 cDNA was controlled either by the heat-shock-inducible hsp70 promoter (hs-dNOS1 flies) or by the eye-specific GMR promoter, which functions in all cells of the eye imaginal disc in, and posterior to, the morphogenetic furrow [19] (GMR-dNOS1 flies). Examination of scanning electron micrographs of the eyes of, and thin sections of the retinas of, different transgenic lines did not reveal obvious differences among eyes of wild-type flies, transgenic hs-dNOS1 flies with or without heat-shock and GMR-dNOS1 flies. This indicates that a moderate increase in NO production on its own does not noticeably affect eye development (Kuzin, 2000).

To investigate the relationship between NOS activity and cell cycle progression, NOS activity was manipulated in transgenic flies ectopically expressing genes of the Rb pathway in the developing eye. Drosophila RBF is structurally related to the mammalian proteins of the Rb family and, like the Rb proteins, RBF is a negative regulator of cell cycle progression. The RBF transgene was placed under control of the GMR promoter and flies with either two (GMR-RBF2) or four (GMR-RBF4) copies of the transgene were used in these experiments. The eyes of adult flies with two copies of the RBF transgenes (GMR-RBF2) appear normal, indicating that at this dosage the RBF transgene does not noticeably disturb cell division in the eye disc. When GMR-RBF2 flies are crossed to hs-dNOS1 flies and the progeny larvae are treated with heat shock before pupariation, however, the resulting adults have multiple defects in the eyes, including missing bristles and pigment cells. Pigment cells, which comprise the boundaries of each ommatidia, appear as a characteristic honeycomb pattern in thin sections of normal eyes. A lack of the regular number of pigment cells in GMR-RBF2 + hs-dNOS1 flies results in the appearance of many fused ommatidia and a rough eye phenotype. Thus, hs-dNOS1 and GMR-RBF2 flies, both of which do not affect the development of eye structure when overexpressed on their own, nevertheless yield eye defects when overexpressed together. This transgenic interaction suggests that NOS and RBF genes interact synergistically during the development of ommatidia. A similar effect was observed employing a different genetic strategy. This time, the overexpression of dNOS1 was restricted to the developing eye by crossing GMR-RBF2 flies with GMR-dNOS1 flies. These double-transgenic flies also display eye defects similar to those of heat-shocked GMR-RBF2 + hs-dNOS1 flies — missing pigment and bristle cells and fused ommatidia. Thus, regardless of the promoter that drives the expression of the dNOS1 transgene, elevated levels of NO and RBF synergize to limit cell number in the developing eye, supporting the notion of interaction between dNOS1 and RBF genes (Kuzin, 2000).

Both RBF and NOS act to suppress cell division. If indeed NOS acts in concert with RBF during eye development, then inhibition of NOS might suppress RBF function and restore the normal number and shape of ommatidia to GMR-RBF4 flies. To test this, endogenous NOS activity was blocked in larvae of GMR-RBF4 flies using a specific NOS inhibitor L-nitroarginine methyl ester, L-NAME (which alone did not affect the eye morphology of the wild-type flies. Remarkably, the eyes of these drug-exposed transgenic flies have an almost normal phenotype as regards the number of photoreceptor and accessory cells and the number and shape of the ommatidia; only a few bristles were still missing. The antiproliferative activity of NO results from its ability to suppress DNA synthesis, as BrdU labeling of the eye imaginal discs showed that the number of cells in S phase is decreased after heat shock in flies carrying the hs-dNOS1 transgene and is increased upon inhibition of NOS activity in GMR-RBF4 flies. Thus, the inhibitory effect of RBF overexpression on cell proliferation is almost completely rescued when endogenous NOS activity is inhibited in the developing larvae (Kuzin, 2000).

In mammalian cells, Rb and Rb-related proteins bind to transcription factors of the E2F family and inhibit E2F-dependent transcription. When phosphorylated by cyclin-dependent kinases, Rb does not bind E2F and E2F-dependent transcription of several genes required for the synthesis of DNA and entry into S phase of the cell cycle is induced. Ectopic overexpression of E2F overcomes the Rb-mediated repression and induces quiescent cells to enter S phase. Similarly, in Drosophila cells, RBF is associated with the E2F transcription factor complex. In transgenic flies overexpressing dE2F and dDP under control of the GMR promoter (GMR-dE2FdDP flies), ommatidia form irregular rows and lack their regular hexagonal shape; in addition, many eye bristles are duplicated. This observation indicates that overexpression of RBF and of E2F have reciprocal effects on cell proliferation in the developing eye (Kuzin, 2000).

To determine whether the antiproliferative activity of NO can counteract excessive precursor cell proliferation caused by E2F overexpression, GMR-dE2FdDP flies were crossed with hs-dNOS1 flies. When progeny larvae are treated with heat shock, a normalized adult eye developed; in some cases a revertant (wild-type) pattern of ommatidial rows, regularly shaped ommatidia, and the usual number of bristles are seen. Similarly, progeny of a cross between GMR-dE2FdDP flies and GMR-dNOS1 flies developed more normal eyes, corroborating a specific dNOS1-E2F interaction. Thus, in contrast to GMR-RBF4 flies, in with which inhibition of NOS was needed to rescue the mutant phenotype (underproliferation of precursor cells), overexpression of dNOS1 is needed to rescue the phenotype of GMR-dE2FdDP flies (overproliferation of precursor cells). This reciprocal effect of NO levels on RBF and E2F function in cell cycle control adds considerable genetic strength to the idea that NO acts in concert with the Rb pathway to suppress cell division during eye development (Kuzin, 2000).

RBF blocks E2F-dependent transcription in cotransfection assays, in accordance with its ability to sequester E2F proteins. When expressed in the eye, GMR-RBF suppresses the rough-eye phenotype of the GMR-dE2FdDP transgenic flies. Thus, overexpression of RBF and E2F have opposing effects on the decision of precursor cells to enter the cell cycle. A test was performed to see whether NO modulates the effects of GMR-RBF2 on E2F function by inhibiting NOS activity in GMR-RBF2 + GMR-dE2FdDP flies. Ectopic expression of E2F in the developing eye increases both cell proliferation and programmed cell death; the net effect is the appearance of more cells in the eye, however. To minimize the E2F-induced augmentation of cell death, an effective inhibitor of apoptosis, the baculoviral p35 gene, under control of the GMR promoter, was used. Whereas the combination of GMR-dE2FdDP and GMR-p35 transgenes produce an even more severe phenotype than the GMR-dE2FdDP transgene alone, the GMR-RBF2 + GMR-dE2FdDP + GMR-p35 flies have a normal eye phenotype, confirming that, in the absence of programmed cell death, RBF suppresses the consequences of E2F over-expression and rescues the E2F phenotype. In contrast, inhibition of NOS activity in these GMR-RBF2 + GMR-dE2FdDP + GMR-p35 larvae prevents RBF from rescuing the E2F phenotype. In particular, when endogenous NO production is suppressed, the arrangement of ommatidia is still abnormal, and many additional bristles and pigment cells are still observed. This suggests that the RBF-E2F interaction involves NOS and that RBF requires NO to antagonize the E2F activity (Kuzin, 2000).

This study of the developing Drosophila eye presents a series of reciprocal genetic interactions that consistently suggest that NO modulates a signaling pathway involved with cell cycle control. Specifically, increased production of NO in the developing eye acts as an antiproliferative signal, whereas inhibition of NOS activity promotes additional rounds of cell division. It is considered that the reciprocal effects of E2F and NOS and complementary effects of Rb and NOS are best explained by the hypothesis that NO affects the Rb signaling pathway, thereby regulating entry into the S phase of the cell cycle (Kuzin, 2000).

Native E2F/RBF complexes contain Myb-interacting proteins and repress transcription of developmentally controlled E2F target genes

The retinoblastoma tumor suppressor protein (pRb) regulates gene transcription by binding E2F transcription factors. pRb can recruit several repressor complexes to E2F bound promoters; however, native pRb repressor complexes have not been isolated. E2F/RBF repressor complexes have been isolated from Drosophila embryo extracts and their roles in E2F regulation have been characterized. These complexes contain RBF, E2F, and Myb-interacting proteins that have previously been shown to control developmentally regulated patterns of DNA replication in follicle cells. The complexes localize to transcriptionally silent sites on polytene chromosomes and mediate stable repression of a specific set of E2F targets that have sex- and differentiation-specific expression patterns. Strikingly, seven of eight complex subunits are structurally and functionally related to C. elegans synMuv class B genes, which cooperate to control vulval differentiation in the worm. These results reveal an extensive evolutionary conservation of specific pRb repressor complexes that physically combine subunits with established roles in the regulation of transcription, DNA replication, and chromatin structure (Korenjak, 2004).

The Drosophila genome encodes two pocket proteins, RBF1 and RBF2, and two E2F proteins, dE2F1 and dE2F2, that act in heterodimers with a common partner, dDP. It was reasoned that this streamlined version of the E2F/pRb network would greatly simplify the chromatographic separation of native complexes. Drosophila embryo nuclear extracts were subject to gel filtration to verify the presence of RBF complexes. RBF1 was detected by Western blot in many fractions ranging in apparent molecular weight from 66 kDa to 1.2 MDa. RBF2 was detected in a narrower peak with an apparent molecular weight of 669 kDa to 1.2 MDa. dDP was likewise detected in fractions ranging in molecular weight from 443 kDa to 1.2 MDa. These findings suggest that Drosophila embryos contain multisubunit dE2F/RBF complexes (Korenjak, 2004).

Next, extracts were subjected to ion exchange chromatography. This resolved three peaks of RBF1 activity. Peak I contained RBF1 but did not contain RBF2, dE2F, or dDP. When peak I was subjected to gel filtration, RBF1 eluted with an apparent molecular weight of 100 kDa, close to its theoretical molecular weight (91.8 kDa), suggesting that peak I contains monomeric RBF1. RBF1, dE2F1, and dDP coeluted in peak II. During subsequent gel filtration, these three proteins coeluted with an estimated molecular weight of 500 kDa. Analysis of peak III revealed the presence of RBF1, RBF2, dE2F2, and dDP. These four proteins coeluted during gel filtration with an apparent molecular weight of 669 kDa to 1.2 MDa (Korenjak, 2004).

Previous studies have shown that RBF1 associates with both dE2F1 and dE2F2, whereas RBF2 interacts exclusively with dE2F2, and these binding specificities are reflected in the elution profile. Interestingly, peak III fractions contain both RBF1 and RBF2 even though they do not interact with each other. This suggests that peak III contains two separate dE2F2/RBF1 and dE2F2/RBF2 complexes with similar subunit composition. The molecular weight of these complexes (669 kDa to 1.2 MDa) indicates that they contain additional subunits. Therefore dE2F2/RBF complexes present in peak III were purified (Korenjak, 2004).

dE2F2/RBF complexes were purified by classical chromatography. dE2F2, dDP, RBF1, and RBF2 coeluted from the final gel filtration column with an apparent molecular weight of 669 kDa to 1.2 MDa. Silver staining detected seven bands that perfectly coeluted with the Western signals. These polypeptides were present in similar stoichiometric amounts, with the exception of one (55 kDa) that was stained more intensely. Peptide mass fingerprinting revealed that the 55 kDa band comprised two distinct polypeptides (Korenjak, 2004).

Identification of copurifying polypeptides revealed Twilight (also known as Mip130; from here on referred to as Mip130/TWIT), RBF1, RBF2, dMyb, dDP, dE2F2, CAF1p55, and Mip40. The identity of these polypeptides was confirmed by Western blot. Intriguingly, Mip130/TWIT, dMyb, CAF1p55, and Mip40 have recently been identified as components of a dMyb complex that regulates chorion gene amplification in follicle cells. The fifth subunit of the dMyb complex, Mip120, was apparently absent from the final preparation as judged by silver staining. However, Western analysis with Mip120-specific antibody has demonstrated that Mip120 coelutes with other complex subunits throughout the fractionation. The Mip120 signal became progressively weaker during purification but was still detectable in fractions eluting from the final gel filtration column, suggesting that Mip120 might have been progressively lost or degraded. Indeed, several results presented below suggest that Mip120 is a bona fide complex subunit. Since these complexes are a composite of known transcriptional regulators, they go by the acronym dREAM (Drosophila RBF, E2F, and Myb-interacting proteins) (Korenjak, 2004).

The idea that E2F proteins have tissue-specific, developmentally regulated functions is supported by the identification of novel E2F regulated genes in human, mouse, and fly. In addition to cell cycle-related E2F targets, these studies reveal numerous genes that have developmental functions or display a strictly tissue-specific expression pattern. Analysis of the E2F transcriptional program in Drosophila indicates that there are at least two different types of E2F regulation (Dimova, 2003): expression of cell cycle-regulated E2F targets is primarily dependent on dE2F1/dDP-mediated activation and is repressed by RBF1 (A group genes). In contrast, other E2F targets are actively repressed in proliferating cells by dE2F2, dDP, and either RBF1 or RBF2, and these genes are expressed in developmentally regulated patterns (E group genes). These two types of regulation appear to be combined in differing proportions over the spectrum of E2F targets, generating a broad variety of E2F control (Korenjak, 2004).

dREAM repressors are required for a recently discovered aspect of dE2F transcriptional regulation. RNAi-mediated disruption of dREAM complexes by depletion of Mip130/TWIT and Mip120 specifically derepresses E group genes, genes that have previously been shown to be repressed in a cell cycle-independent manner by dE2F2, dDP, and a function that is redundant between RBF1 and RBF2. Although depletion of Mip130/TWIT and Mip120 has no effect on expression of A group genes, it is probable that dREAM complexes also repress cell cycle-related targets in other situations: ChIP experiments show that dE2F2, RBF1, and RBF2 are normally present at almost all dE2F-regulated genes, including A group genes; the distinction between A and E group genes lies, therefore, not in the binding of the repressor proteins but in the binding of dE2F1 (Dimova, 2003). Accordingly, in cells lacking dE2F1, dE2F2-mediated repression prevents the expression of both cell cycle-dependent and -independent targets (Dimova, 2003). The extensive colocalization of dE2F2, RBF1, Mip120, and Mip130/TWIT on polytene chromosomes suggests that dREAM complexes are present at most sites of dE2F action (Korenjak, 2004).

The fact that dMyb is a stoichiometric subunit of dREAM complexes hints at an extensive collaboration between dE2F and dMyb. However, depletion of dMyb has no effect on expression of the A and E group genes tested. It is clear, therefore, that dMyb is not required for all aspects of dREAM complex function. However, it is possible that dE2F and dMyb cooperate to regulate transcription of other genes that have not been investigated. Moreover, as will be discussed below, dE2F2 and dMyb appear to converge on the regulation of chorion gene amplification (Korenjak, 2004).

The mechanism of E2F regulation provided by dREAM appears to be highly conserved during evolution. Strikingly, with the exception of dMyb, all components of dREAM are either homologs of previously described C. elegans synMuv class B genes (mip130/twit/lin-9, rbf1 and rbf2/lin-35, de2f2/efl-1, ddp/dpl-1, and caf1p55/lin-53), contain regions of sequence conservation (Mip40/lin-37), or produce a synMuv phenotype when the corresponding C. elegans gene is inactivated (Mip120/JC8.6). Genetic studies have shown that synMuv class B genes are required for development of the worm's male and female reproductive systems, and it has been suggested that some encode subunits of a hypothetical complex that represses vulva-specific gene transcription; however, the precise transcriptional changes underlying the synMuv phenotype are unknown (Ceol, 2001). The discovery of dREAM complexes suggests an intriguing model for synMuv class B gene function: it is proposed that at least seven synMuv class B gene products physically associate to form a complex that, like its Drosophila counterpart, represses sex-related targets and that misexpression of these genes causes a change in cell fate. Given the vast differences between C. elegans and Drosophila embryogenesis, it is considered unlikely that REAM complexes will regulate the exact same set of genes in both species. However, it is proposed that, in both organisms, REAM complexes control transcriptional programs required for development of the reproductive system. In agreement with this model, dE2F2 has been shown to be needed to repress genes like vasa and spn-E that are important for Drosophila gametogenesis (Dimova, 2003) and that dE2F2 mutants have both male and female fertility defects (Korenjak, 2004).

Do mammalian cells contain similar complexes? Mammalian homologs exist for all dREAM subunits. Intriguingly, B-Myb associates with the N terminus of p107. RbAp48/p46, human orthologs of CAF1p55, were first isolated through their ability to bind a pRb-affinity column but are now known as components of several chromatin-associated complexes, including a putative pRb-histone deacetylase and the NuRD complex. Human homologs of Mip130/TWIT, Mip120, and Mip40 had not previously been linked to pRb. All three interact with pRb in vitro. Furthermore, endogenous hMip130/TWIT associates specifically with pRb, p107, and p130 fusion proteins. In agreement with these results, interaction has been demonstrated between pRb and Mip130/TWIT in human cells in vivo (S. Gaubatz, personal communication to Korenjak, 2004). Clearly, further studies are needed to define the properties and biological roles of pRb/hMip complexes. Nevertheless, these preliminary findings suggest that such complexes may well exist in mammalian cells, and, if studies in C. elegans and Drosophila are a guide, it might be expected that they function in developmentally regulated aspects of E2F/pRB function (Korenjak, 2004).

What is the biochemical function of dREAM complexes? dREAM complexes lack known chromatin-modifying enzymes. Studies of mammalian E2F targets show that activation and repression correlate with histone acteylation and deacetylation, respectively. The finding that dREAM complexes associate specifically with unmodified histone H4 tails but fail to bind hyperacetylated tails implies that they bind specifically to deacetylated histones that are characteristic of repressed chromatin. Consistent with this, dE2F2, RBF, Mip120, and Mip130/TWIT colocalize at chromosomal sites that are not actively transcribed. It is proposed that dREAM complexes bind deacteylated nucleosomes, protecting them from modification, and in doing so maintain a repressive state that is both stable and readily reversible (Korenjak, 2004).

One might predict that dREAM would act synergistically with histone deacetylases. Indeed, the C. elegans synMuv B class includes an ortholog of HDAC1, and the dRPD3 histone deacetylase coimmunoprecipitates with RBF from extracts of cell lines. However, dRPD3 is not a stoichiometric component of dREAM complexes. Furthermore, inhibition of histone deacetylases in SL2 cells, either by the depletion of dRPD3 or treatment with deacetylase inhibitors, does not derepress group E genes (Taylor-Harding, 2004). Thus, while histone deacetylation may be a prerequisite for histone binding by dREAM, deacetylases are not required to maintain repression of E group genes (Korenjak, 2004).

The discovery that E2F/RBF complexes contain five subunits of a recently described dMyb complex is particularly intriguing. This complex binds the ACE3 element of a chorion gene locus and has been suggested to regulate chorion gene amplification in ovary follicle cells. Amplification involves both cessation of general genomic replication and relicensing and firing of origins in a temporally and spatially restricted manner. Remarkably, dE2F2, dDP, RBF1, and Mip130/TWIT are all needed to shut off genomic replication in vivo. The discovery of dREAM complexes offers a mechanistic explanation for these genetic results and implies that dREAM complexes function to shut off genomic replication in this cell type. Interestingly, dRPD3 and histone deacetylation have been shown to counteract chorion origin firing (Aggarwal, 2004), lending further support to the idea that 'transcriptional' regulators can also influence DNA replication events (Korenjak, 2004).

Recent genetic studies have suggested that the effects of Mip130/TWIT are reversed at rereplicating sequences by dMyb, possibly following an activating modification of dMyb itself (Beall, 2004). Interestingly, dE2F1 and dMyb colocalize to amplifying foci, and dE2F1, like dMyb, is needed to promote rereplication. Since dE2F1 works at least in part by overriding dE2F2-mediated repression and dMyb has been proposed to selectively counteract Mip130/TWIT activity, the discovery that dE2F2 and Mip130/TWIT reside in the same complex suggests that dE2F1 and activated dMyb may collaborate at the ACE3 locus to reverse repressive effects of dREAM complexes. In this setting, dMyb and E2F appear to share a similar mechanism of action, relying on an activator (dE2F1) or an activating event (modification of dMyb) to relieve the effects of a common repressor (Korenjak, 2004).

It should be noted that mutant alleles of de2f2 and mip130/twit are not lethal but do suffer from reduced viability and fertility. Hence, dREAM complexes are not essential. Amplification of chorion loci in follicle cells represents a highly specialized case of DNA replication. The general patterns of DNA replication are unaffected by mutation in de2f2 and mip130/twit, arguing against a strict requirement for replication per se. Nevertheless, several studies of mammalian cells have linked pRb and E2F proteins to various aspects of DNA replication, but their precise roles in replication remain to be established (Korenjak, 2004).

dREAM complexes are the first native RBF repressor complexes to be purified, but it is noted that additional complexes likely exist. Fractionation reveals an additional complex containing RBF1, dE2F1, and dDP that might act at other E2F targets that were unaffected by the depletion of dREAM components. The results show that specific RBF-containing complexes are important at specific subsets of dE2F-regulated promoters. It is becoming clear that pRb/RBF tumor suppressors assemble distinct molecular machines to exert distinct functions. More work is needed to determine which complexes are needed for each of their ascribed functions. The striking parallels between studies of pRb and E2F orthologs in C. elegans and Drosophila indicate that their basic mechanisms of action are well conserved. Perhaps the most definitive picture will emerge by integrating information from each of the available model organisms (Korenjak, 2004).

The Drosophila Myb complex has roles in both activating and repressing developmentally regulated DNA replication. Drosophila Myb has been shown to form a stable complex with four additional proteins, Mip130, Mip120, Mip40, and Caf1/p55. This five-subunit complex was originally identified as an activity present in Drosophila extracts that specifically recognizes two critical control elements (ACE-3 and ori-beta) required for chorion gene DNA replication-mediated amplification in the follicle cells surrounding the developing oocyte. To further understand biochemically the functions of the Myb complex, Drosophila embryo extracts were fractionated, relying upon affinity chromatography. E2F2, DP, RBF1, RBF2, and the Drosophila homolog of LIN-52, a class B synthetic multivulva (synMuv) protein, copurify with the Myb complex components to form the Myb-MuvB complex. In addition, the transcriptional repressor protein, lethal (3) malignant brain tumor protein, L(3)MBT, and the histone deacetylase, Rpd3, both associate with the Myb-MuvB complex. Members of the Myb-MuvB complex were localized to promoters and were shown to corepress transcription of developmentally regulated genes. These and other data now link together the Myb and E2F2 complexes in higher-order assembly to specific chromosomal sites for the regulation of transcription (Lewis, 2004).

The discovery that the Myb complex proteins are needed to repress developmentally regulated genes raises the possibility that some of the phenotypes observed in mip130 mutant animals may be due in part to the inappropriate expression of differentiation factors. The number of such target genes regulated by the repressive Myb-MuvB complex identified here is likely quite large. Multiple site-specific DNA-binding proteins contained together in one complex (such as Myb, Mip120, and E2F2:DP), increase the potential diversity for DNA sites that may be bound. Thus, at certain enhancers, the E2F2 site in combination with Mip120 may target the assembly, while at other sites the Myb DNA-binding activity may be important. Furthermore, although the majority of the Myb complex subunits in embryo extracts are present in the Myb-MuvB complex, it seems likely that the Myb and E2F2 proteins function independently at some chromosomal positions. It is also posited that the Myb complex may be modified in such a way as to provide a signal for activation rather than repression. In work to be presented elsewhere, by using microarray analysis and genomic localization of the Myb complex, it has been found that a family of transcripts is indeed dependent upon the Myb complex for expression. Thus a network of chromosomal domains may be independently regulated by these factors. In the context of such a network, it is intriguing that as an activator of DNA amplification, it appears as if Myb plays an active role in targeting the Mips and associated activities to the ACE-3 site. In contrast to the transcriptional repression studied in cell culture, Myb does not seem important for such targeting. Clearly understanding the DNA sequence context and associated factors in activation may shed some light on this difference (Lewis, 2004).

The Drosophila repressor characterized in this work has been called the Myb-Muv B complex because of the striking resemblance of its protein composition to that encoded by the synMuv class B genes of C. elegans. Elegant genetic screens have defined a regulatory pathway essential for vulval development that is entirely consistent with a model in which these synMuv proteins are individual members of a large complex. Together with the known expression patterns of the proteins associated with the Drosophila Myb-MuvB complex and phenotypes of the mutants for many of the factors of the complex, the biochemical data argue for a general role for the repressor in many tissue types. Phenotypic differences between the putative nematode complex and the Drosophila counterpart may ultimately be ascribed to subunit composition or perhaps other more complex differences in the actual developmental programs between the two organisms. To highlight these in vivo differences, it is worthwhile to briefly review the SynMuv mutant phenotypes (Lewis, 2004).

Wild-type C. elegans hermaphrodites contain a single vulva organ, while synMuv mutants may posses multiple vulva. In the wild-type organism, the activity of the synMuv genes antagonize the effects of the basal activity of the RTK/Ras pathway by repressing transcription of vulval genes. The class B synMuv genes likely inhibit vulval induction by a conserved mechanism whereby the class B synMuv proteins form a repressive complex with the sequence-specific transcription factor EFL-1/E2F protein, and recruit corepressor proteins to inhibit the transcription of vulval specification genes via EFL-1/E2F-binding sites. As a result, those cells adopt the nonvulval fate. However, in the key vulval precursor cells, the antagonistic action of the synMuv genes is inactivated or can be overcome by the activated RTK/Ras pathway, thereby permitting downstream activation and transcription of keys genes for vulval fate. Some of the findings from studies on the biochemical properties of the components of the Drosophila Myb-MuvB complex may be relevant to a putative nematode complex. The Mip120 protein binds specifically to the ACE-3 and ori- and is probably involved in sequence-specific interactions for the Myb-MuvB complex. It is therefore possible that the C. elegans Mip120 homolog, LIN-54, is also a sequence-specific DNA-binding protein that helps direct the class B gene complex to specific promoters for repression of vulval genes (Lewis, 2004).

L(3)MBT, a homolog of LIN-61, is similar to the Drosophila polycomb group protein Sex Combs on Midleg (SCM), which is a member of the PRC1 complex. PRC1 is thought to primarily repress gene expression through blocking the nucleosome remodeling activity of SWI/SNF. As shown in this study, RNAi directed against L(3)MBT indicates that it is required for transcriptional repression at many of the sites coordinately repressed by E2F2 and the Myb-associated proteins. The L(3)MBT protein appears substoichiometric relative to core Myb-MuvB complex subunits. Like L(3)MBT in the Myb-MuvB complex, the SCM protein is present in substoichiometric quantities relative to the other subunits of the human and Drosophila hPRC-H and PRC1 complexes. Both L(3)MBT and LIN-61 contain multiple MBT repeats that are evolutionarily conserved domains found throughout metazoa. The X-ray crystal structure of the MBT repeats provide some hints as to how both LIN-61 and the Drosophila L(3)MBT protein may function. The MBT repeat consists of a five-stranded beta-barrel core domain that shares structural similarity to the Tudor and chromodomains. The Tudor domain interacts with methylated arginine residues, and chromodomains interact with methylated lysine residues of histone H3. Consistent with the speculation that this domain is critical for function of the MBT family and that the proteins bind modified histones, several hypomorphic mutations in Drosophila SCM map to residues within the putative ligand-binding pocket. The MBT domains in LIN-61 and L(3)MBT may maintain a repressed chromatin domain through interaction with histone tails methylated at specific lysine residues on neighboring nucleosomes, thus hindering the nucleosome mobility by chromatin remodeling factors. For maximum repression, genes regulated by the Myb-MuvB or the putative nematode complex may require additional mechanisms of repression such as histone modification and thus the association of the deacetylase Rpd3 (Lewis, 2004).

Like the Myb-MuvB complex the putative C. elegans complex may also play a wide role in repression in different tissues. For example, several C. elegans class B synMuv genes, including the homologs of Mip130, E2F2, DP, and RBF, have been shown to function independently of synMuv A genes for regulation of the G1/S transition (Lewis, 2004).

The particular genes regulated by the Myb-MuvB complex are likely determined in a tissue-specific and cell-type-specific manner. In Drosophila tissue culture cells, E2F2 appears to function primarily for repression of developmentally regulated genes, while E2F1/RBF1 complexes are involved in regulating genes involved in cell cycle progression. However, microarray studies performed in e2f2 and rbf1 mutant follicle cells indicate that both E2F2 and RBF1 are involved in the repression of several S-phase genes, including CDT1 and the ORC and MCM complex subunits. Therefore, it is likely that the set of genes regulated by the Myb-MuvB complex may change depending on the developmental context (Lewis, 2004).

In the embryo extracts that were fractionated, the repressive form of the Myb complex seems to predominate. However, other much less abundant complexes between the previously identified Myb complex and activators should also be found. Certainly at ACE3, E2F1 and the Myb complex cooperate for amplification and proper ORC localization, and it has been proposed that a switch between E2F2 and E2F1 at ACE3 may be epistatic to activation. Other RNAi studies using Drosophila cell lines indicate that E2F1 and E2F2 primarily occupy and regulate the expression of a non-overlapping set of genes, and the work presented here implies that this non-overlapping control may be dictated by other proteins associated with the well-studied E2F proteins. The Myb complex might assemble with either activators or repressors of the E2F family to regulate either transcription or DNA replication in response to appropriate developmental cues. In future work it will be important to understand how in a given cell type, the cis-acting DNA sites and chromosomal context determine a region for either repression or activation (Lewis, 2004).

Chromatin regulation mediated by Domino and PcG-like factors controls E2F activity and cell growth

Regulation of chromatin structure is critical in many fundamental cellular processes. Previous studies have suggested that the Rb tumor suppressor may recruit multiple chromatin regulatory proteins to repress E2F, a key regulator of cell proliferation and differentiation. Taking advantage of the evolutionary conservation of the E2F pathway, a genome-wide RNAi screen was conducted in cultured Drosophila cells for genes required for repression of E2F activity. Among the genes identified are components of the putative Domino chromatin remodeling complex, as well as the Polycomb Group (PcG) protein-like fly tumor suppressor, L3mbt, and the related Scm-related gene containing four mbt domains (CG16975/dSfmbt). These factors are recruited to E2F-responsive promoters through physical association with E2F and are required for repression of endogenous E2F target genes. Surprisingly, their inhibitory activities on E2F appear to be independent of Rb. In Drosophila, domino mutation enhances cell proliferation induced by E2F overexpression and suppresses a loss-of-function cyclin E mutation. These findings suggest that potential chromatin regulation mediated by Domino and PcG-like factors plays an important role in controlling E2F activity and cell growth (Lu, 2007).

This study identified the putative Dom/SWR1 chromatin remodeling complex and the PcG-like MBT domain-containing factors were identified as E2F repressors. These proteins are recruited to E2F target promoters through association with E2F and inhibit E2F in an apparently Rb-independent manner. Depletion of these genes resulted in derepression of some endogenous E2F target genes accompanied by changes in histone modification. More importantly, dom genetically interacts with the E2F pathway. These proteins show an extensive degree of evolutionary conservation, indicating the mechanism of E2F regulation provided by these factors may be well conserved (Lu, 2007).

Regulation of E2F is tightly linked to cell proliferation and differentiation. Existing evidence suggests that perturbation of the Dom and MBT proteins may cause dysregulation of these cellular processes. Apart from the fact that the heterozygous dom mutation modifies cell growth in an E2F-transgenic or a cycE hypomorphic background, fly mutants homozygous for several dom alleles show enlarged lymph glands apparently because of excessive proliferation of prehemocytes. In human, the Dom complex subunit YL1 possesses growth suppressive activity, and the Dom homolog p400 is an essential target for the viral oncoprotein E1A-mediated transformation. Indeed, overexpression of E1A disrupts the association of E2F with the Dom complex in mammalian cells. Furthermore, mutations in the fly tumor suppressor gene l3mbt result in overgrowth of the larval brain lobes and epithelial imaginal discs, and failure of neural differentiation (Wismar, 1995). This is intriguing, because in mammalian cells, many E2F-regulated genes are repressed during quiescence and differentiation, and mammalian MBT proteins are found in an inhibitory E2F complex purified from quiescent cells (Lu, 2007).

Although the mechanism of Rb-mediated repression on E2F is complex, these studies indicate that Dom and MBT possess Rb-independent activities. In support of this view, recent studies suggest that the C. elegans Dom and Rb homologs share redundant functions in vulva development, a process controlled by the E2F pathway (Ceol, 2004). In addition, these proteins may participate in distinct E2F complexes. Mammalian MBT orthologs have been identified from Rb-independent complexes, and they can associate with E2F forms lacking the Rb-binding motif, such as E2F6 and a C-terminal truncated E2F3 mutant. Interestingly, L3mbt is shown to interact with dREAM, a dE2F2-Rb complex, even though it is not a stoichiometric subunit. But unlike L3mbt, RNAi of dE2F2 and several other components of the core dREAM complex had no effect on the E2F reporter. This observation may hence indicate the existence of multiple L3mbt-containing complexes or hint at a potential collaboration among different E2F regulatory activities. So far, there is no evidence linking Dom and CG16975 to Rb. It is likely that both Rb-mediated and -independent chromatin modulations play critical roles in E2F regulation and cell proliferation. Future biochemical and genetic studies may shed light on these potentially independent and collaborative relations (Lu, 2007).

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 Cul4Cdt2 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 Cul4Cdt2 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 Cul4Ddb1-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 Cul4Cdt2 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 Cul1Slmb 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 E2f1PIP-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 E2f1PIP mutants and not replacement of endogenous E2f1, this question cannot be definitively answered. However, E2f1PIP-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 E2f1PIP-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).

Control of Drosophila endocycles by E2F and CRL4CDT2

Endocycles are variant cell cycles comprised of DNA synthesis (S)- and gap (G)-phases but lacking mitosis. Such cycles facilitate post-mitotic growth in many invertebrate and plant cells, and are so ubiquitous that they may account for up to half the world's biomass. DNA replication in endocycling Drosophila cells is triggered by cyclin E/cyclin dependent kinase 2 (CYCE/CDK2), but this kinase must be inactivated during each G-phase to allow the assembly of pre-Replication Complexes (preRCs) for the next S-phase. How CYCE/CDK2 is periodically silenced to allow re-replication has not been established. This study used genetic tests in parallel with computational modelling to show that the endocycles of Drosophila are driven by a molecular oscillator in which the E2F1 transcription factor promotes CycE expression and S-phase initiation, S-phase then activates the PCNA/replication fork-associated E3 ubiquitin ligase CRL4CDT2 (Cul-4), and this in turn mediates the destruction of E2F1 (Shibutani, 2008). It is proposed that the transient loss of E2F1 during S phases creates the window of low Cdk activity required for preRC formation. In support of this model overexpressed E2F1 accelerated endocycling, whereas a stabilized variant of E2F1 blocked endocycling by deregulating target genes, including CycE, as well as Cdk1 and mitotic cyclins. Moreover, it was found that altering cell growth by changing nutrition or target of rapamycin (TOR) signalling impacts E2F1 translation, thereby making endocycle progression growth-dependent. Many of the regulatory interactions essential to this novel cell cycle oscillator are conserved in animals and plants, indicating that elements of this mechanism act in most growth-dependent cell cycles (Zielke, 2011).

Altogether these results indicate that periodic E2F1 degradation is necessary for endocycling for three reasons: (1) it creates a window of low CYCE/CDK2 activity; (2) it promotes high APCFzr/Cdh1 activity and thereby suppresses geminin accumulation; and (3) it allows E2F2 to maintain repression of CDK1 and its cyclins. Each of these conditions is required for preRC assembly and endocycle progression. This cell cycle mechanism is fundamentally different from that used in mitotic cycles, wherein destruction of the M-phase cyclins by APCCdc20/Fzy, rather than of E2F1 by the CRL4CDT2, throws the switch that allows preRC assembly. Indeed it is noteworthy that the periodic degradation of E2F1 and depletion of CYCE are not required for mitotic cell cycles in Drosophila. CRL4CDT2 is required for endocycling in plants, indicating that this element of the endocycle oscillator is conserved (Zielke, 2011).

Finally, it was asked what factors control E2F production to regulate endocycle rates. Endocycle speed and number can be manipulated by altering cell growth through changes in dietary protein or growth-regulatory genes including Myc and insulin/PI3K/TOR signalling components. Hence larvae were starved of protein to suppress insulin/TOR signalling, reduce protein synthesis, and block cell growth. Starvation arrested the salivary endocycles within 24h and strongly depleted E2F1. E2f1 and Dp mRNA levels were not affected, but the E2F targets CycE, pcna and rnrS were reduced. To test whether this was responsible for starvation-induced endocycle arrest E2F1 was overexpressed in the salivary glands of starved animals. Although these glands failed to grow their nuclei incorporated BrdU and accrued approximately sevenfold more DNA than controls. Overexpression of RHEB, which activates the Target of rapamycin (TOR) kinase and increases ribosome biogenesis and cap-dependent translation, also restored cell growth, E2F1 protein, and endocycle progression in starved animals. Thus E2F1 appears to act as a 'growth sensor' that couples rates of endocycle progression to rates of cell growth. A likely mechanism for this, corroborated by modelling, involves increased translation of E2F1 in rapidly growing cells. Indeed, it was found that the association of E2F1 mRNA with polyribosomes was greatly reduced in protein-starved animals. Translational control of E2F is an attractive mechanism for coupling growth to G1/S progression not only in endocycling cells, but also in growth-dependent mitotic cells with extended G1 periods (Zielke, 2011).

Rbf1 degron dysfunction enhances cellular DNA replication

The E2F family of transcription factors contributes to oncogenesis through activation of multiple genes involved in cellular proliferation, a process that is opposed by the Retinoblastoma tumor suppressor protein (RB). RB also increases E2F1 stability by inhibiting its proteasome-mediated degradation, but the consequences of this post-translational regulation of E2F1 remain unknown. To better understand the mechanism of E2F stabilization and its physiological relevance, this study examined the streamlined Rbf1-dE2F1 network in Drosophila. During embryonic development, Rbf1 is insulated from ubiquitin-mediated turnover by the COP9 signalosome, a multi-protein complex that modulates E3 ubiquitin ligase activity. This study report that the COP9 signalosome also protects the Cullin4-E3 ligase that is responsible for dE2F1 proteasome-mediated destruction. This dual role of the COP9 signalosome may serve to buffer E2F levels, enhancing its turnover via Cul4 protection and its stabilization through protection of Rbf1. It was further shown that Rbf1-mediated stabilization of dE2F1 and repression of dE2F1 cell cycle-target genes are distinct properties. Removal of an evolutionarily conserved Rbf1 C terminal degron disabled Rbf1 repression without affecting dE2F1 stabilization. This mutant form of Rbf1 also enhanced G1-to-S phase progression when expressed in Rbf1-containing S2 embryonic cells, suggesting that such mutations may generate gain-of-function properties relevant to cellular transformation. Consistent with this idea, several studies have identified mutations in the homologous C terminal domains of RB and p130 in human cancer (Raj, 2012).

E2F: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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