Retinoblastoma-family protein


REGULATION

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.

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).

Repression of dMyc expression by Wingless promotes Rbf-induced G1 arrest in the presumptive Drosophila wing margin

Little is known about how patterns of cell proliferation and arrest are generated during development, a time when tight regulation of the cell cycle is necessary. In this study, the mechanism by which the developmental signaling molecule Wingless generates G1 arrest in the presumptive Drosophila wing margin is examined in detail. Wg signaling promotes activity of the Drosophila retinoblastoma family (Rbf) protein, which is required for G1 arrest in the presumptive wing margin. Wg promotes Rbf function by repressing expression of the G1-S regulator Drosophila myc (dmyc). Ectopic expression of dMyc induces expression of Cyclin E, Cyclin D, and Cdk4, which can inhibit Rbf and promote G1-S progression. Thus, G1 arrest in the presumptive wing margin depends on the presence of Rbf, which is maintained by the ability of Wg signaling to repress dmyc expression in these cells. In addition to advancing the understanding of how patterned cell-cycle arrest is generated by the Wg signaling molecule during development, this study indicates that components of the Rbf/E2f pathway are targets of dMyc in Drosophila. Although Rbf/E2f pathway components mediate the ability of dMyc to promote G1 progression, dMyc appears to regulate growth independently of the RBF/E2f pathway (Duman-Scheel, 2004).

This investigation examines the mechanism by which Wg signaling promotes G1 arrest in the presumptive Drosophila wing margin. It was postulated that Rbf might mediate the ability of Wg to induce G1 arrest, since loss of Wg signaling promotes expression of dE2f1 target genes. Overexpression of Rbf can block this induction of dE2f1 target gene expression. Strikingly, loss of Rbf in the zone of nonproliferating cells (ZNC) prevents G1 arrest, as evidenced by ectopic BrdUrd incorporation in Rbf mutant clones. This requirement for Rbf in the ZNC is notable. Surprisingly few developing fly tissues display such an absolute requirement for Rbf to promote G1 arrest. To date, Rbf has been shown to be required to limit DNA replication in the embryo and in the ovary. However, in many tissues, loss of Rbf does not result in ectopic S phase; a likely explanation for this finding is that in other developing tissues, Rbf may function as one of several redundant mechanisms that function to promote G1 arrest. Such redundancy would help to ensure that the cell cycle is regulated tightly during development (Duman-Scheel, 2004).

In an attempt to better understand the mechanism by which Wg promotes Rbf function, this investigation uncovered interactions between dMyc and components of the Rbf/E2f pathway. Wg signaling normally inhibits dMyc expression in the ZNC. Ectopic expression of dMyc in the ZNC can induce expression of dE2f1 target genes, which can be blocked by the addition of Rbf-280 (a constitutively active form of Rbf). Thus, overexpression of dMyc, which results from loss of Wg signaling in the ZNC, must somehow inactivate Rbf. These data indicate that inhibition of dMyc expression in the ZNC is critical for Rbf function (Duman-Scheel, 2004).

The results indicate why exclusion of dMyc from the ZNC is necessary for Rbf activity. Overexpression of dMyc leads to high levels of Cyclin E, Cyclin D, and Cdk4 transcripts. dMyc also regulates Cyclin E posttranscriptionally in Drosophila. G1-S Cyclins/Cdks function to phosphorylate and inhibit Rbf, suggesting that dMyc blocks Rbf activity through activation of G1-S Cyclins/Cdks. Thus, inhibition of dMyc by Wg helps to ensure that G1-S Cyclins/Cdks do not activate S phase. This idea is supported by the results that indicate that only a combination of both Dap and constitutively active Rbf (that cannot be regulated by Cyclins/Cdks) can restore G1 arrest when Wg signaling is blocked or when dMyc is expressed. These data suggest that either Cyclin D or Cyclin E activity can mediate the ability of dMyc to promote S phase in the ZNC. Coexpressing Dap alone with dMyc, which would block only Cyclin E/Cdk2 activity, does not restore G1 arrest. Furthermore, overexpression of dMyc in a cdk4 mutant background still results in ectopic S phases, suggesting that Cyclin E/Cdk2 also are sufficient to mediate dMyc's ability to promote G1 progression. Thus, either Cyclin D/Cdk4 or Cyclin E/Cdk2 is sufficient to mediate the ability of dMyc to promote G1 progression. The ability of Wg to inhibit dMyc expression is thus critical for RBF activation and G1 arrest in the ZNC. Still, it is possible that Wg promotes G1 arrest through other mechanisms that have not yet been uncovered. The observation that overexpression of a dominant-negative form of dTCF (dTCFDeltaN) with C96>Gal4 can promote S phase, even in a dmyc mutant background, supports this idea (Duman-Scheel, 2004).

It is likely that dMyc/dMax directly up-regulate transcription of Cyclin D and cdk4 in Drosophila. Myc/Max heterodimers regulate transcription by binding to various consensus sequences, such as the E box. Previous studies indicated that cMyc induces Cyclin D2 expression in mice by binding to two consensus E boxes in the Cyclin D2 promoter. cdk4 also was identified as a transcriptional target of c-Myc. Furthermore, it has been suggested that cdk4 is a transcriptional target of dMyc and Cyclin D is a transcriptional target of dMax. Although future studies should analyze the Drosophila Cyclin D and Cdk 4 regulatory regions in more detail, these results suggest that the observed ability of dMyc to induce Cyclin D and Cdk4 expression in the ZNC most likely occurs through transcriptional regulation of these proteins by dMyc/dMax. In contrast, Cyclin E was not identified as a target of dMyc or dMax. It is more likely that the ability of dMyc to induce growth in the wing indirectly leads to increased Cyclin E transcript levels (Duman-Scheel, 2004).

Recent studies indicate that both dMyc and Rbf can regulate cellular growth in the Drosophila wing. dMyc induces cellular growth, whereas Rbf inhibits cellular growth and proliferation. dMyc can promote cellular growth in the presence of constitutively active Rbf, suggesting that dMyc can induce growth independently of the Rbf/E2f pathway. Such results are consistent with previous studies that indicate that Ras, which can induce growth by increasing levels of dMyc protein, also is capable of inducing growth in the presence of Rbf. It is likely that dMyc regulates growth through induction of genes encoding regulators of protein synthesis, such as ribosomal proteins and the DEAD-box helicase Pitchoune, as well as other proteins that regulate cellular metabolism (Duman-Scheel, 2004).

Wnt signaling is generally associated with the stimulation of cell proliferation during development and in tumor cells. However, in the ZNC, Wnt/Wg signaling actually promotes cell-cycle arrest. Ironically, in the ZNC, Wg signaling suppresses expression of dmyc; however, a cMyc reporter was found to be directly up-regulated by Tcf4 in a colon carcinoma cell line. Thus, Wg appears to be able to up-regulate Myc expression in some tissues and to repress it in others (Duman-Scheel, 2004).

The ability of Wg signaling to either activate or repress the same target gene in different situations has been observed in other cases. For example, in the developing Drosophila midgut, low levels of Wg signaling, in conjunction with Dpp, stimulate expression of Ubx and lab; high levels of Wg signaling result in the repression of Ubx and lab by means of the transcriptional repressor Teashirt. Thus, expression of Wnt target genes can be turned on or off in response to the modulation of Wg levels as well as by the presence or absence of the various proteins that can regulate transcription in conjunction with, or in response to, Wg signaling. Such flexibility is advantageous to a developing organism (Duman-Scheel, 2004).

Wg signaling can be modulated to affect expression of the same target gene differently in various situations. Moreover, Wg signaling can be modulated to promote or inhibit the different, somewhat conflicting cellular processes of patterning, growth, proliferation, and differentiation. The same is true for Hh signaling, which also regulates all of these cellular processes. Thus, it seems, at least in the case of Hh and Wg, that one signaling molecule can regulate many different types of cellular and developmental events. In order for various cellular programs to be implemented and coordinated during development, the way that a particular cell type responds to Wg or Hh signaling at any given time must be tightly regulated. The delicate balance between various processes that can occur in response to Hh or Wg signaling is likely maintained through tight control of the temporal and spatial expression patterns of Hh and Wnt targets and the molecules that regulate them (Duman-Scheel, 2004).

Epigenetic silencers Lola and Pipsqueak collaborate with Notch to promote malignant tumours by Rb silencing

Cancer is both a genetic and an epigenetic disease. Inactivation of tumour-suppressor genes by epigenetic changes is frequently observed in human cancers, particularly as a result of the modifications of histones and DNA methylation. It is therefore important to understand how these damaging changes might come about. By studying tumorigenesis in the Drosophila eye, two Polycomb group epigenetic silencers, Pipsqueak and Lola, have been identified that participate in this process. When coupled with overexpression of Delta, deregulation of the expression of Pipsqueak and Lola induces the formation of metastatic tumours. This phenotype depends on the histone-modifying enzymes Rpd3 (a histone deacetylase), Su(var)3-9 and E(z), as well as on the chromodomain protein Polycomb. Expression of the gene Retinoblastoma-family protein (Rbf ) is downregulated in these tumours and, indeed, this downregulation is associated with DNA hypermethylation. Together, these results establish a mechanism that links the Notch-Delta pathway, epigenetic silencing pathways and cell-cycle control in the process of tumorigenesis (Ferres-Marco, 2006).

Correct organ formation depends on the balanced activation of conserved developmental signalling pathways (such as the Wnt, Hedgehog and Notch pathways). If insufficient signals are received, organ growth may be deficient. By contrast, excess signalling leads to an overproduction of progenitor cells and a propensity to develop tumours. When such hyperproliferation is associated with the capacity of cells to invade surrounding tissue and metastasis to distant organs, cancer develops. Indeed, activation of the Wnt, Hedgehog and Notch pathways is a common clinical occurrence in cancers. Curiously, activation of any of these pathways in animal models seems to be insufficient for cancer to develop, indicating that synergism with other genes is required for these pathways to produce cancer (Ferres-Marco, 2006).

Cellular memory or the epigenetic inheritance of transcription patterns has also been implicated in the control of cell proliferation during development, as well as in stem-cell renewal and cancer. Proteins of the Polycomb group (PcG) are part of the memory machinery and maintain transcriptional repression patterns. The upregulation of several PcG proteins has been associated with invasive cancers. Thus, increased amounts of EZH2, the human homologue of the Drosophila histone methyltransferase E(z), is associated with poorer prognoses of breast and prostate cancers (Ferres-Marco, 2006).

Another histone methyltransferase implicated both in gene silencing and in cancer is SUV39H1, a homologue of Drosophila Su(var)3-9. SUV39H1 and Su(var)3-9 methylate histone H3 on lysine 9 (H3K9me), and this epigenetic tag is characteristic of heterochromatin and DNA sequences that are constitutively methylated in normal cells. DNA methylation is another mechanism involved in cellular memory that actively contributes to cancer. Indeed, numerous tumour-suppressor genes, including the retinoblastoma gene RB, are silenced in cancer cells by DNA hypermethylation. Inactivation of the RB tumour-suppressor pathway is considered an important step towards malignancy; thus, it is important to understand how these damaging epigenetic changes are initiated in cells that become precursors of cancer. Moreover, it is equally important to determine the connection between these processes and the developmental pathways controlling proliferation (Ferres-Marco, 2006).

Forward genetic screening in suitable animals is a powerful tool with which to identify tumour-inducing genes and to reveal changes that precede neoplastic events in vivo. The developing eye of Drosophila melanogaster is a good model for such studies because it is a simple and genetically well-defined organ. The growth of the eye depends on Notch activation in the dorsal-ventral organizer by its ligands Delta (human counterparts, DLL-1, -3, -4) and Serrate (human counterparts, JAGGED-1, -2). This study used the 'large eye' phenotype, produced by overexpression of Delta, as a tool to screen for mutations that interact with the Notch pathway and convert tissue overgrowths into tumours. One mutation, eyeful, was isolated that combined with Delta induces metastatic tumours. eyeful forces the transcription of two hitherto unsuspected growth and epigenetic genes, lola and pipsqueak (psq). The identification of eyeful has been a starting point from which to unravel crosstalk between the Notch and epigenetic pathways in growth control and tumorigenesis. The fact that many epigenetic factors are involved in cancer suggests that these processes may be more generally involved in tumorigenesis than at first it might seem (Ferres-Marco, 2006).

To identify genes that interact with the Notch pathway and that influence growth and tumorigenesis, the Gene Search (GS) system was used to screen for genes that provoked tumours when coexpressed with Delta in the proliferating Drosophila eye. The ey-Gal4 line was used for both eye-specific and ubiquitous induction, resulting in the transactivation of UAS-linked genes throughout the proliferating eye discs. It was through such a screen that the GS88A8 line was isolated. Generalized overexpression of Delta by ey-Gal4 (hereafter termed ey-Gal4 > Dl) produces mild eye overgrowth. In most of the flies in which the GS88A8 line was coexpressed with Delta, tumours developed in the eyes. Moreover, in ~30% of the mutant flies, secondary eye growths were observed throughout the body, and in some flies the whole body filled up with eye tissue. These secondary eye growths had ragged borders, indicating invasion of the mutant tissue into the surrounding normal tissue. As a result, the GS88A8 line was named 'eyeful' (Ferres-Marco, 2006).

A developmental analysis of the tumours was undertaken. To facilitate analyses, a triple mutant strain was generated carrying the eyeful, UAS-Dl and ey-Gal4 transgenes all on the same chromosome (ey-Gal4 > eyeful > Dl. In this strain, mutant eye discs showed massive uncontrolled overgrowth (some discs were more than five times their normal size). In most discs, the epithelial cells had lost their apical-basal polarity, and some had a disrupted basement membrane and grew without differentiating (Ferres-Marco, 2006).

These results were extended to the wing disc. (1) dpp-Gal4 was used to direct coexpression of eyeful and Delta along the anterior-posterior boundary of the wing (perpendicular to the endogenous Delta domain along the dorsal-ventral boundary. In a normal wing disc, the dpp-Gal4 driver typically establishes a stripe of green fluorescent protein (GFP) expression with a sharp border at the boundary. Whereas wild-type (or single eyeful) cells expressing GFP conformed with this pattern, some of the eyeful and Delta cells were found outside this stripe, indicating that the mutant cells can disseminate and invade adjacent regions of the disc. (2) The MS1096-Gal4 line was used to direct expression in the dorsal wing disc compartment. Under these conditions, the wing tissue grew massively and aggressively, and the mutant tissue failed to differentiate. These observations suggest that, when coupled with Delta overexpression, an excess of the gene products flanking the eyeful insertion site induces the formation of tumours capable of metastasising (Ferres-Marco, 2006).

The genomic DNA flanking the eyeful P-element was isolated and sequenced. eyeful is inserted in an intron of the gene longitudinals lacking (lola), which is known to be a chief regulator of axon guidance. lola encodes 25 messenger RNAs that are produced by alternative splicing and that generate 19 different transcription factors. All of the different isoforms share four exons that encode a common amino terminus, which contains a BTB or POZ domain. In addition, all but one of these transcription factors are spliced to unique exons encoding one or a pair of zinc-finger motifs (Ferres-Marco, 2006).

The GS P-elements allow Gal4-dependent inducible expression of sequences flanking the insertion site in both directions. The nearest gene in the opposite direction to transcription of lola is the psq gene. This gene encodes nine variants produced by alternative splicing and alternative promoter use, generating four different proteins. Three of the psq isoforms contain a BTB or POZ domain in the N terminus, and a histidine- and glutamine-rich region downstream of this domain. Two of the BTB-containing isoforms and the isoform that lacks this domain contain four tandem copies of an evolutionarily conserved DNA-binding motif, the Psq helix-turn-helix (HTH) motif (Ferres-Marco, 2006).

psq was initially identified for its 'grandchildless' and posterior group defects and was subsequently shown to have a role in retinal cell fate determination. Psq is essential for sequence-specific targeting of a PcG complex that contains histone deacetylase (HDAC) activity. Psq binds to the GAGA sequence, which is present in many Hox genes and in hundreds of other chromosomal sites (Ferres-Marco, 2006).

Both polymerase chain reaction with reverse transcription (RT-PCR) and in situ hybridization experiments confirmed that transcription of lola and psq was influenced by eyeful in response to Gal4 activation (Ferres-Marco, 2006).

To determine whether lola and/or psq was responsible for the tumour phenotype, 11 enhancer promoter (EP) P-elements inserted into the lola and psq region were tested. In contrast to the GS lines, the EP lines allows Gal4-dependent inducible expression of sequences flanking only one end of the P-element. It was found that none of the EP lines induced tumours; thus, it was reasoned that the deregulation of both genes might be required to produce the tumours (Ferres-Marco, 2006).

The complexity of lola and psq loci, which together produce 23 proteins, hampers identification of the transcripts responsible for the eyeful phenotype by gain-of-expression mutants (that is, by expressing individual or combinations of isoforms). Therefore, this issue was resolved by isolating point mutations that reverted the phenotype caused by deregulated expression of lola and psq. In this analysis, the chemical mutagen ethyl-methane sulphonate (EMS) was used to induce preferentially single nucleotide changes (Ferres-Marco, 2006).

The parental eyeful GS line was viable in trans with deficiencies that removed both lola and psq. In contrast, a set of 14 EMS-induced mutations on the eyeful chromosome failed to complement these deficiencies and were found to be alleles of psq or lola. The EMS-induced mutations that best recovered a normal eye size were sequenced. Each individual mutation had a single base change or a small deletion that considerably altered the predicted Psq or Lola proteins (Ferres-Marco, 2006).

All psq- mutations induced on the eyeful chromosome prevented eyeful from producing eye tumours and metastases. Three alleles affected the BTB domain (psqrev2, psqrev7 and psqrev9), and three other alleles contained either a premature stop codon that would produce truncated proteins lacking the Psq HTH repeats (psqrev4 and psqrev14) or a missense mutation that would change a conserved amino acid in the third Psq HTH repeat (psqrev12). All lola- mutations induced on the eyeful chromosome, including the presumptive null allele (lolarev6), reduced eye tumour size but still permitted sporadic secondary growth (Ferres-Marco, 2006).

These data show an unequal contribution of Psq and Lola in this process, whereby Psq is the most important factor in the tumorigenic phenotype. The BTB subfamily of transcriptional repressors includes the human oncogenes BCL6 and PLZF. In these oncogenes, the BTB domain is crucial for oncogenesis through the recruitment of PcG and HDAC complexes. It is therefore speculated that deregulated Psq and Lola could lead to tumorigenesis by epigenetic processes and that Drosophila counterparts of HDACs and PcG proteins might be involved in the progression of these tumours. Indeed, genetic evidence was found that both Lola and Psq function as epigenetic silencers in vivo (Ferres-Marco, 2006).

Attempts were made to determine the specific epigenetic mechanisms through which deregulation of Psq and Lola might induce tumorigenesis in conjunction with Delta overexpression. Methylation of histone on lysine is a central modification in both epigenetic gene control and in large-scale chromatin structural organization. For example, trimethylation of histone H3 on K4 (H3K4me3) is associated with the active transcription of genes and open chromatin structure. By contrast, histone hypoacetylation and H3K9 and H3K27 methylation are characteristic of heterochromatin state and gene silencing. To determine whether any changes in these epigenetic markers might coincide with the induction of tumorigenesis, eye discs were immunolabelled with antibodies against specific histone H3 modifications. Because dorsal eye disc cells are refractory to Delta, the dorsal region of the discs provided an internal control for these studies (Ferres-Marco, 2006).

With the exception of some scattered cells, a prominent loss or strong reduction of H3K4me3 was observed in the ventral region of the mutant discs. Notably, although the loss of Notch in clones does not affect this epigenetic tag, overexpression of Delta caused a significant reduction in staining for H3K4me3. The H3K4me3 depletion was already apparent in discs showing moderate hyperplasia and thus preceded neoplasm formation. Changes in other epigenetic tags (such H3K9me3 and H3K27me2) could not be reproducibly resolved; perhaps more sensitive methods or antibodies might facilitate detection of such changes (Ferres-Marco, 2006).

H3K4 methylation is thought to be permissive for maintaining and propagating activated chromatin and is thought to neutralize repressor tags by precluding binding of the HDAC complex and impairing SUV39H1-mediated H3K9 methylation. Thus, H3K4me3 depletion may contribute to tumour formation by permitting aberrant chromatin silencing. It was found that a 50% reduction in dosage of the HDAC gene Rpd3 or of Su(var)3-9 decreased the tumour phenotype dominantly. In contrast, reducing the activity of the H3K4 histone methyltransferase genes Trx (known as ALL1 or MLL in humans) or Ash1, which would be expected to deplete the H3K4me3 tag further, did not visibly enhance the tumours (Ferres-Marco, 2006).

E(z) when complexed with the Extra sex combs (Esc) protein becomes a histone methyltransferase. The E(z)-Esc complex and its mammalian counterpart Ezh2-Eed show specificity for H3K27 but may also target H3K9. The complex also contains the HDAC Rpd3, and this association with Rpd3 is conserved in mammals. H3K27 methylation facilitates binding of the chromodomain protein Pc (HPC in humans), which then creates a repressive chromatin state that is a stable silencer of genes (Ferres-Marco, 2006).

Although loss of E(z) does not cause proliferation defects within discs, halving the E(z) gene dosage dominantly suppressed tumorigenesis, indicating that histone methylation by the E(z)-Esc complex is also a prerequisite for the excessive proliferation of these tumours. Accordingly, Esc- or Pc- mutations also notably reduced the tumours (Ferres-Marco, 2006).

Together, these findings suggest that the development of these tumours involves, at least in part, changes in the structure of chromatin brought about by covalent modifications of histones. These changes probably switch the target genes from the active H3K4me3 state to a deacetylated H3K9 and H3K27 methylation silent state (Ferres-Marco, 2006).

From this above data, it is considered that the tumours might form as a result of aberrant gene silencing. If so, then the expression of genes involved in cell-cycle control is likely to be altered in the mutant cells. The transcription of 12 tumour-related genes in the mutant and wild-type discs was compared. Transcription of the gene Rbf, a fly homologue of the RB/Rb family of genes, was strongly downregulated in this assay (and even in ey-Gal4 > Dl flies). A second Rb gene, Rbf2, remained unchanged in the different genetic backgrounds, highlighting the specificity of Rbf silencing (Ferres-Marco, 2006).

It was found that Rbf depletion seems to be intricately associated with tumorigenesis: (1) reducing Rbf gene dosage by 50% enhanced tumour growth; (2) re-establishing Rbf expression in the eye (using an UAS-Rbf transgene) consistently prevented eye tumours and occurrence of secondary growths (Ferres-Marco, 2006).

Inactivation of RB1 in retinoblastoma, a form of eye cancer in children, can occur through DNA hypermethylation of the promoter. Unlike in mammals, however, there is little cytosine methylation of the genome in Drosophila during developmental stages, and its potential role during tumorigenesis is unknown. DNA methylation seems to depend on one DNA methyltransferase, Dnmt2, that preferentially methylates cytosine at CpT or CpA sites. The fly genome also encodes one methyl-CpG DNA-binding MBD2/3 protein. Because there are no known Dnmt2 loss-of-function mutations, the role of this gene in tumorigenesis could not be tested (Ferres-Marco, 2006).

Nevertheless, whether the CpG islands that were observed in the Rbf gene were potential targets for repression by DNA methylation was tested by two methods. (1) Methylation-sensitive restriction enzymes analysis was used; this showed that the regions around the promoter and transcription start site of the Rbf gene are susceptible to methylation. This approach showed aberrant DNA hypermethylation of Rbf in eyeful and Delta eye discs and mild hypermethylation in Delta discs; however, at best only very mild methylation was detected in discs from wild-type flies or from flies with the control psq gene (Ferres-Marco, 2006).

(2) Direct bisulphite sequencing of genomic DNA was carried out from mutant discs. This approach confirmed the notable increase in methylated DNA in eyeful and Delta discs when compared with wild-type discs (and a moderate increase in methylated DNA in the Delta discs). Hypermethylation of the Rbf promoter was not simply the result of de novo transcription of Dnmt2 (ey-Gal4 > Dnmt2), indicating that activation of the Notch pathway is a crucial step in this de novo hypermethylation of Rbf (Ferres-Marco, 2006).

This study has used Drosophila genetics to search for genes that collaborate with the Notch pathway during tumorigenesis in vivo. Psq and Lola were identified as decisive factors to foment tumour growth and invasion when coactivated with the Notch pathway. These proteins are presumptive transcription repressors that contain a BTB domain and sequence-specific DNA-binding motifs and behave as epigenetic silencers in vivo (Ferres-Marco, 2006).

In addition, crosstalk between the Notch pathway and different epigenetic regulators was identified. It is likely that alterations in this crosstalk provoke the aberrant epigenetic repression (and perhaps also derepression) of genes that contribute to cellular transformation. The Rbf gene was identified as one target for this epigenetic regulation and Rbf depletion was shown to directly contribute to the tumours (Ferres-Marco, 2006).

It is proposed that the sequence of events that leads to these tumours commences with hyperactivation of the Notch pathway, which initiates gene repression. Subsequently, or at the same time as Notch, Psq-Lola could bind to the silenced genes and enforce silencing by recruitment of HDAC or PcG repressors. Given the conservation of the Psq-like HTH domains in Psq and of BTB domains, it seems likely that other transcriptional repressors containing such domains strongly influence the tumour-inducing capacities of HDACs and PcG repressors in human cancers (Ferres-Marco, 2006).

Finally, the collaboration between PcG-mediated cellular memory and the Notch pathway may have implications in other processes controlled by Notch, including the second mitotic wave in the Drosophila eye, and the organization of eye and wing growth. In these processes, the memory mechanism could ensure that cells keep a record of the Notch signals received at an earlier stage or when the progenitor cells were closer to the Delta source. In this way, they might remain proliferative without having to receive continuous instructions from Notch. Likewise, such a situation could be conceived for tumorigenesis. The oncogenic signals could opportunistically take advantage of the memory mechanism to fix and to maintain their instructions of continuous proliferation in progenitor or stem cells, thereby fostering tumour growth and metastasis (Ferres-Marco, 2006).

The H3K27me3 demethylase dUTX is a suppressor of Notch- and Rb-dependent tumors in Drosophila

Trimethylated lysine 27 of histone H3 (H3K27me3) is an epigenetic mark for gene silencing and can be demethylated by the JmjC domain of UTX. Excessive H3K27me3 levels can cause tumorigenesis, but little is known about the mechanisms leading to those cancers. Mutants of the Drosophila H3K27me3 demethylase dUTX display some characteristics of Trithorax group mutants and have increased H3K27me3 levels in vivo. Surprisingly, dUTX mutations also affect H3K4me1 levels in a JmjC-independent manner. A disruption of the JmjC domain of dUTX results in a growth advantage for mutant cells over adjacent wild-type tissue due to increased proliferation. The growth advantage of dUTX mutant tissue is caused, at least in part, by increased Notch activity, demonstrating that dUTX is a Notch antagonist. Furthermore, the inactivation of Retinoblastoma (Rbf in Drosophila) contributes to the growth advantage of dUTX mutant tissue. The excessive activation of Notch in dUTX mutant cells leads to tumor-like growth in an Rbf-dependent manner. In summary, these data suggest that dUTX is a suppressor of Notch- and Rbf-dependent tumors in Drosophila melanogaster and may provide a model for UTX-dependent tumorigenesis in humans (Herz, 2010).

Mammalian UTX, UTY, and JmjD3 and Drosophila UTX (dUTX) are histone demethylases that specifically demethylate di- and trimethylated lysine 27 on histone H3 (H3K27me2 and H3K27me3, respectively). The catalytic domain of this activity is the Jumonji C (JmjC) domain, located at the C terminus of these proteins. The N-terminal domains of UTX, UTY, and dUTX contain several tetratricopeptide repeats (TPRs) thought to be required for protein-protein interactions (Herz, 2010).

H3K27me3 is a histone mark for Polycomb (Pc)-mediated genomic silencing and transcriptional repression and is associated with animal body patterning, X-chromosome inactivation, genomic imprinting, and stem cell maintenance. H3K27 methylation is catalyzed by Polycomb repressive complex 2 (PRC2), which in Drosophila is composed of the catalytic subunit enhancer of zeste [E(z)] (EZH2 in mammals), extra sex combs (Esc), suppressor of zeste 12 [Su(z)12], and nucleosome remodeling factor 55 (Nurf55). H3K27me3 is recognized by the chromodomain of Pc, which is a component of a different silencing complex, called PRC1, which, in addition to Pc, contains Polyhomeotic (Ph), posterior sex combs (Psc), and dRING. The wild-type function of UTX is to demethylate H3K27me3 and, thus, to antagonize Polycomb-mediated silencing (Herz, 2010).

UTX is also a component of mixed-lineage leukemia complex 3 (MLL3) and MLL4. MLL complexes are histone methyltransferases for H3K4. The function of UTX in MLL3 and MLL4 is unknown. However, it appears that UTX is not required for the H3K4 methyltransferase activity of MLL3 and MLL4. The best-characterized targets of H3K27me3/Pc-mediated silencing are homeotic genes, which are critical regulators of animal patterning. However, many other genes are also enriched for H3K27 methylation and Pc binding. Furthermore, elevated H3K27me3 levels due to an increased activity of the methyltransferase EZH2 could be a leading cause of certain human cancers. Recently, mutations that inactivate UTX, and which are thus expected to cause increased H3K27me3 levels, have been linked to the development and progression of human cancer. However, the precise mechanisms by which this occurs are largely unknown (Herz, 2010).

Notch is the receptor of a highly conserved signaling pathway involved in many biological processes, including lateral inhibition, stem cell maintenance, and proliferation control. The binding of Delta or Serrate, the two ligands in Drosophila melanogaster, triggers the proteolytic processing of Notch, resulting in the release and translocation of the Notch intracellular domain (NICD) into the nucleus, where it regulates gene expression. Aberrant, oncogenic Notch signaling has been linked to tumor development in humans, including T-cell acute lymphoblastic leukemias (T-ALLs), pancreatic cancer, medulloblastoma, and mucoepidermoid carcinoma. Thus, an improved understanding of Notch signaling will have significant implications for human health (Herz, 2010).

In Drosophila, the Notch signaling pathway also controls the growth of the eye primordium and wing margin formation during development. Although the mechanistic details are unclear, one way by which Notch signaling controls proliferation during Drosophila eye development is through the negative regulation of the Retinoblastoma (Rb) family member Rbf. Rbf inactivation has also been implicated in Notch-induced eye tumors in Drosophila. Rb is a tumor suppressor that negatively regulates cell cycle progression through the inhibition of the transcription factor E2F. Rb binds directly to E2F and represses its transcriptional activity. The release of Rb activates E2F to induce the transcription of cell cycle regulators such as cyclin E and PCNA. Therefore, the inactivation of Rbf by increased Notch signaling can trigger increased proliferation, which may lead to cancerous growth (Herz, 2010).

This study genetically characterizes loss-of-function mutations of dUTX. dUTX mutants display some of the characteristics of Trithorax group mutants and have increased H3K27me3 levels in vivo. Surprisingly, dUTX mutations also affect H3K4me1 levels in a JmjC-independent manner. dUTX mutant tissue has an H3K27me3-dependent growth advantage over wild-type tissue due to increased proliferation in the developing eye. The growth advantage of dUTX mutant tissue is caused by increased Notch activity, demonstrating that dUTX is a Notch antagonist. The inactivation of Rbf contributes to the growth advantage of dUTX mutant tissue. Moreover, an excessive activation of Notch in dUTX mutant cells leads to tumor-like growth in an Rbf-dependent manner. In summary, these data suggest that dUTX is a suppressor of Notch- and Rbf-dependent tumors in Drosophila and may provide a model for UTX-dependent tumorigenesis in humans (Herz, 2010).

Based on the enzymatic activity of the JmjC catalytic domain as H3K27me3 demethylases, UTX proteins are predicted to counteract Polycomb function. Consistently, it was found that dUTX mutants display genetic characteristics of Trithorax group genes. In vitro studies have shown that dUTX and UTX demethylate H3K27me2 and H3K27me3. However, dUTX mutants affect the global levels of only H3K27me3 but not of H3K27me2. Nevertheless, this observation does not mean that dUTX does not demethylate H3K27me2 in vivo. There may be fewer genes regulated by dUTX at the H3K27me2 level such that the global levels are not detectably altered in dUTX mutants (Herz, 2010).

Interestingly, dUTX mutants also affect global levels of H3K4me1, which are significantly reduced in mutant tissue. Mammalian UTX is a component of the MLL3 and MLL4 methyltransferase complexes, and based on the reduction of H3K4me1 levels, it is predicted that dUTX is also a component of the Drosophila equivalent of the MLL3/MLL4 methyltransferase complex, which contains Trithorax-related (Trr) as a histone methyltransferase. The function of UTX in MLL3 and MLL4 complexes is currently unknown. It was suggested previously that UTX is not required for H3K4 methylation, but in these studies, only H3K4me2 and H3K4me3 were investigated. Consistently, the global levels of H3K4me2 and H3K4me3 are not affected in dUTX mutant clones. The data demonstrate that dUTX is required for the monomethylation of H3K4. Interestingly, the JmjC demethylase domain of dUTX is not required for H3K4me1 methylation, suggesting that other domains of dUTX, such as the TPR domains, may be necessary for mediating this function. The finding that the global levels of H3K4me2 and H3K4me3 are not affected in dUTX mutants is also quite interesting, as it implies that the monomethylation of H3K4 is not required for the di- or trimethylation of H3K4 (Herz, 2010).\

The epigenetic control of gene expression has been best studied for the control of homeotic gene expression, which is established during embryogenesis and maintained throughout animal life. However, not only homeotic genes are regulated through epigenetic modifications. Other genes in different developmental processes are also subject to epigenetic control. In this study, by analyzing the dUTX mutant phenotype, a role was establised of H3K27me3 levels in cell cycle control. The data suggest that increased H3K27me3 levels in dUTX clones cause the epigenetic silencing of several genes involved in Notch signaling. This includes both positive and negative regulators of Notch signaling activity as well as target genes that are either positively or negatively regulated by the Notch pathway. Such an incoherent control of gene expression by the Notch pathway has been reported previously, suggesting that the final outcome of Notch activity may be determined by the relative expression levels of positive or negative regulators. Because this study determined that the overrepresentation phenotype of dUTX clones is caused by elevated levels of Notch signaling, it appears that the silencing of Notch inhibitors is dominant over the silencing of Notch activators, resulting in a net increase of Notch activity. However, this increased Notch activity may be specific for the cell cycle phenotype of dUTX mutants, since increased Notch activity was not found for other Notch-dependent paradigms, such as E(spl)m8-lacZ. This is also consistent with the finding that E(spl) genes contain increased H3K27me3 levels in dUTX mutants. Thus, the wild-type function of dUTX is to restrict the cell cycle through the negative control of Notch. Therefore, the data link H3K27me3-dependent Notch activity with enhanced tissue growth, implying that dUTX is a Notch antagonist regarding the cell cycle and explaining the overrepresentation phenotype of dUTX mutant clones (Herz, 2010).

However, this phenotype is subtle compared to that of mutants in growth control pathways such as the Hippo pathway. Nevertheless, the overgrowth of dUTX clones is strongly potentiated by the additional activation of Notch. The expression of Delta in dUTX clones causes a strong tumor-like growth phenotype. Thus, dUTX functions as a suppressor of Notch-induced tumors under normal conditions. This synergistic interaction between the loss of dUTX and increased Notch activity is a clear example that tumor development requires several hits for progression (Herz, 2010).

The overrepresentation phenotype of dUTX clones can be dominantly enhanced by the genetic loss of Rbf, suggesting that the reduction of Rbf contributes to the overrepresentation phenotype. However, the reduction of Rbf activity in dUTX clones is not caused by direct epigenetic silencing at the Rbf locus. No increased H3K27me3 levels was found at the Rbf locus in dUTX mutants, and mRNA levels of Rbf were unchanged. Instead, Rbf is negatively regulated by the Notch pathway during eye growth. Thus, the increased activity of Notch in dUTX clones leads to a partial inactivation of Rbf and increased proliferation, causing the overrepresentation phenotype. Currently, it is unknown how Notch regulates Rbf (Herz, 2010).

The control of cell cycle progression by UTX proteins is likely conserved in mammals. A parallel study performed by Wang showed that the loss of mammalian UTX also results in elevated levels of proliferation (Wang, 2010). Consistent with the current work, that study also implicated the inactivation of Rb function in increased proliferation in response to UTX knockdown. Similar to the current study, Rb itself is not subject to increased H3K27m3 silencing, but the promoters of several genes in the Rb network were found to be occupied and likely controlled by UTX (Wang, 2010). Thus, although the mechanisms of Rb control by UTX proteins (Notch in this study and the Rb network in the study reported previously by Wang are distinct, both studies established the control of the Rb pathway as a common element of cell cycle control by UTX proteins. Wang also demonstrated a link between UTX and Rb during vulval development in Caenorhabditis elegans. Thus, these studies combined suggest a well-conserved function of UTX proteins for Rb control (Herz, 2010).

Although these studies establish a link between UTX genes and Rb for cell cycle control, it should be noted that the loss of dUTX (and likely mammalian UTX) affects many genes. While the deregulation of individual genes may not cause a significant phenotype on its own, the combined deregulation may disrupt gene regulatory networks, which accounts for the growth phenotype of dUTX mutants. Thus, while aberrant Notch signaling was identified as an important element of the overrepresentation phenotype of dUTX mutants, other genes and signal transduction pathways may also contribute to this phenotype. For example, this study also identified genes involved in growth control by the Hippo pathway (four-jointed [fj] and warts) associated with increased H3K27me3 levels in dUTX mutants and showed reduced transcript levels for fj. Thus, it is possible that the Hippo pathway and other genes contribute to the overrepresentation phenotype of dUTX mutants (Herz, 2010).

These observations have important implications for the initiation and development of human tumors. Increased levels of H3K27me3 due to the elevated activity of the H3K27me3 methyltransferase EZH2 have been associated with human cancer. Furthermore, mutations that inactivate UTX have been linked to human cancer, and low UTX activity correlates with poor patient prognosis. This study establishes that increased levels of H3K27me3 affect Notch activity, which in turn affects Rbf activity. Rb is a well-known tumor suppressor, the loss of which causes human tumors. Therefore, tumors associated with the loss of UTX and, thus, increased H3K27me3 levels may be caused by decreased Rb activity. It should also be noted that aberrant Notch signaling is the cause of several human cancers, including T-cell acute lymphoblastic leukemias (T-ALLs), pancreatic cancer, medulloblastoma, and mucoepidermoid carcinoma. In summary, these data demonstrate that the appropriate control of H3K27 methylation is critical for normal tissue homeostasis, and increased H3K27me3 levels may contribute to cancer through the inactivation of Rb (Herz, 2010).

Paradoxical instability-activity relationship defines a novel regulatory pathway for retinoblastoma proteins

The retinoblastoma (RB) transcriptional corepressor and related family of pocket proteins play central roles in cell cycle control and development, and the regulatory networks governed by these factors are frequently inactivated during tumorigenesis. During normal growth, these proteins are subject to tight control through at least two mechanisms. First, during cell cycle progression, repressor potential is down-regulated by Cdk-dependent phosphorylation, resulting in repressor dissociation from E2F family transcription factors. Second, RB proteins are subject to proteasome-mediated destruction during development. To better understand the mechanism for RB family protein instability, Rbf1 turnover and the protein motifs required for its destabilization were characterized in Drosophila. Specific point mutations in a conserved C-terminal instability element strongly stabilize Rbf1, but strikingly, these mutations also cripple repression activity. Rbf1 is destabilized specifically in actively proliferating tissues of the larva, indicating that controlled degradation of Rbf1 is linked to developmental signals. The positive linkage between Rbf1 activity and its destruction indicates that repressor function is governed in a manner similar to that described by the degron theory of transcriptional activation. Analogous mutations in the mammalian RB family member p107 similarly induce abnormal accumulation, indicating substantial conservation of this regulatory pathway (Acharya, 2010).

During Drosophila development, cell-cycle regulation deviates considerably from the classical four-stage G1/S/G2/M pattern, exhibiting rapid direct S-M cycling early in development, stepwise acquisition of G2 and G1 phases, and endoreplication. These alternative cycles involve a variety of regulatory features, including constitutive inactivation of Rbf proteins by phosphorylation, transcriptional regulation of the rbf1 and rbf2 genes, and regulated degradation of the E2F1 protein. This study provides evidence that this regulatory richness also includes a novel developmentally-triggered degradation of Rbf1 that paradoxically appears to be required for repression activity. This study indicates that Rbf1 lability is tightly linked to repression activity, both in a cellular as well as a whole organismal context. The instability element (IE) identified in the C terminus of this protein appears to be a complex domain with dual functions, so that even a few lysine to alanine mutations can dramatically enhance protein stability while inhibiting transcriptional activity, while other lesions enhance the protein's activity (Acharya, 2010).

Not only is the turnover of Rbf1 required for effective gene regulation, but it appears that this turnover can be developmentally cued, presumably to be coordinated with the engagement of Rbf1 with regulation of the cell cycle. Highly proliferative imaginal disc tissue appears to provide one such context, where levels of wild-type, but not an instability element mutant, Rbf1 protein decrease sharply, presumably in response to the engagement of this protein during cell cycling. In the eye imaginal disc, the Rbf1 protein levels drop sharply in the posterior, where cells are becoming terminally differentiated. Presumably, Rbf1 is activated and consumed in the coordinated cell divisions that occur in the two stripes flanking the morphogenetic furrow; the absence of any further transcription leads to global depletion of Rbf1. The Rbf1 protein lacking the IE accumulates inappropriately in differentiating cells (Acharya, 2010).

How might the repression activity of Rbf1 be linked to protein turnover? Protein lability has previously been found to underlie the action of some eukaryotic transcriptional activators. The activation domain of the VP16 protein was found to be subject to modification by ubiquitylation, enhancing the transcriptional potency of this factor as well as destabilizing it. This process is thought to affect other transcriptional activators as well. The exact mechanism by which ubiquitylation enhances transcriptional activation is poorly understood. The ubiquitin tag may serve a dual purpose of facilitating interactions with the transcriptional machinery as well as attracting the 26S proteasome. Alternatively, the proteasome itself, or portions of this multi-protein complex, may directly enhance transcription; chromatin immunoprecipitation experiments have placed the 'lid' of the proteasome on specific genomic locations (Acharya, 2010).

Until now, there have been no examples of a connection between transcriptional repression and turnover. If it is the modification of the protein with ubiquitin that potentiates Rbf1's repressor activity, this moiety may allow efficient interaction with the transcriptional machinery, similar to the manner in which SUMOylation of PPAR-γ enhances interaction with NCoR corepressors to silence inflammatory genes. Ubiquitylation would in this case attract the 26S proteasome in a competing, parallel reaction that enables Rbf1 turnover. Alternatively, Rbf1 recruitment of the proteasome may allow this complex to directly mediate repression, in a way opposite to that produced by activation domains(Acharya, 2010).

The C terminus of Rbf1 appears to represent a regulatory nexus for this protein; in addition to the instability/repression activity described in this study, key residues appear to provide a damper to modulate its overall activity, and phosphorylation within this region by cyclin kinases can inactivate the protein. The deep conservation of residues within the Rbf1 IE argues strongly for similar activities in mammalian pocket proteins; indeed, mutations of key residues in p107, the closest homolog to Rbf1, strongly stabilize the levels of this protein. In addition, the spectrum of mutations associated with the human retinoblastoma gene indicates that the C-terminal region correlating to the Rbf1 IE may similarly contain critical functions for the mammalian RB protein. One common class of genetic lesion associated with retinoblastomas are nonsense mutations that cause a truncation of the C terminus of the RB protein, and several cancer-associated missense mutations have similarly been mapped to the region corresponding to the Rbf1 IE (Acharya, 2010).

Previous studies have shown that the RB C terminus interacts with the E3 ligase Skp2 and the anaphase promoting complex (APC/C) to regulate turnover of the p27 cyclin kinase inhibitor. This pathway has been suggested to represent a transcription-independent mechanism by which RB controls the cell cycle, and indeed RB was shown not to be subject to APC/C degradation. The current results indicate that a clean separation of transcription and proteolytic control in the context of RB proteins may be oversimplified; this study saw evidence for a separate route of proteolytic regulation that modulates transcriptional regulatory potential and protein stability of Rbf1, and possibly related mammalian pocket proteins. Interestingly, the regulation of this pathway may involve the evolutionarily conserved COP9 signalosome. Previous biochemical studies indicated that the COP9 signalosome regulatory complex is physically associated with Rbf proteins and limits turnover of these repressors. From the results of the current study, it is postulated that COP9 antagonizes the function of the Rbf1 IE, perhaps by blocking the access of ubiquitin-modifying E3 ligases that would otherwise potentiate Rbf1 activity and turnover. Alternatively, inhibition of E3 ligases may involve the enzymatic activity of COP9, whereby this complex downregulates E3 ligases by deneddylation of their cullin subunits. How the instability of pocket proteins potentiates their activities, and how these processes relate to developmental control of retinoblastoma family proteins and cancer, will be an area of active investigation (Acharya, 2010).

The pro-apoptotic activity of Drosophila Rbf1 involves dE2F2-dependent downregulation of diap1 and buffy mRNA

The retinoblastoma gene, rb, ensures at least its tumor suppressor function by inhibiting cell proliferation. Its role in apoptosis is more complex and less described than its role in cell cycle regulation. Rbf1, the Drosophila homolog of Rb, has been found to be pro-apoptotic in proliferative tissue. However, the way it induces apoptosis at the molecular level is still unknown. To decipher this mechanism, rbf1 expression was induced in wing proliferative tissue. It was found that Rbf1-induced apoptosis depends on dE2F2/dDP heterodimer, whereas dE2F1 transcriptional activity is not required. Furthermore, Rbf1 and dE2F2 downregulate two major anti-apoptotic genes in Drosophila: buffy, an anti-apoptotic member of Bcl-2 family and diap1, a gene encoding a caspase inhibitor. On the one hand, Rbf1/dE2F2 repress buffy at the transcriptional level, which contributes to cell death. On the other hand, Rbf1 and dE2F2 upregulate how expression. How is a RNA binding protein involved in diap1 mRNA degradation. By this way, Rbf1 downregulates diap1 at a post-transcriptional level. Moreover, the dREAM complex has a part in these transcriptional regulations. Taken together, these data show that Rbf1, in cooperation with dE2F2 and some members of the dREAM complex, can downregulate the anti-apoptotic genes buffy and diap1, and thus promote cell death in a proliferative tissue (Clavier, 2014).

Protein Interactions

Rbf associates with Drosophila E2F and DP in vivo and is a stoichiometric component of E2F DNA-binding complexes. Rbf coimmunoprecipates from Drosophila cell extracts with both E2F and DP specific antibodies. The association of the E2F activity is released upon treatment with deoxycholate (Du, 1996a).

Rbf specifically represses E2F-dependent transcription. Cotransfection of cultured cells with Drosophila E2F and DP activates a 50-fold greater transcription rate for an E2F reporter construct. This activation is suppressed completely by the coexpression of Rbf (Du, 1996a).

Rbf is phosphorylated by a cyclin E-associated kinase in vitro. In embryo extracts, cyclin E has been shown to be associated primarily with Drosophila cdc2c. Purified Rbf protein added to immune complexes prepared from embryo extracts with cyclin E or Drosophila cdc2c-specific antibodies is readily phosphorylated with cyclin E and Drosophila cdc2c-associated kinase. These observations are consistent with the idea that Rbf is negatively regulated by cyclin E/cdc2c kinase through phosphorylation (Du, 1996a).

D-type cyclin complexes are thought to be the major Rb kinase in mammalian cells. Therefore it was of interest to compare Rb kinase activity in wild-type and Cyclin-dependent kinase 4/6 mutant embryos. Unfortunately, the antibodies used failed to precipitate Rb kinase activity even from wild-type embryo extracts. Moreover, in contrast to vertebrate Rb, Drosophila RBF does not change its apparent electrophoretic mobility upon phosphorylation. As also observed with mammalian Rb, Drosophila RBF could not be focused on two-dimensional gels. Thus, using da-GAL4 mouse Rb was expressed in Drosophila embryos from a UAS transgene (UAS-mRb). Immunoblotting with an antibody against Rb clearly reveals two forms with different electrophoretic mobility. Phosphatase treatment converts the lower into the higher mobility form. Conversely, co-expression of UAS-Cdk4 and UAS-Cyclin D results in a relative increase in the lower mobility form. These observations suggest that Cyclin D-Cdk4 might in principle function as Rb kinase. However, no decrease in the abundance of the low mobility form could be detected in extracts from embryos lacking both maternal and zygotic Cdk4 function. It appears therefore that kinases other than Cyclin D-Cdk4 can phosphorylate mRb. In fact, experiments involving expression of either UAS-Cyclin E or UAS-dacapo suggest that Cyclin E-Cdk2 is a major Rb kinase in Drosophila embryos. UAS-Cyclin E results in a strong enrichment of the lower mobility form. In contrast, UAS-dacapo results in a severe reduction of the lower mobility form (Meyer, 2000).

Cyclin E-Cdk2 is essential for S phase entry. To identify genes interacting with cyclin E, a genetic screen was carried out using a hypomorphic mutation of Drosophila cyclin E (DmcycEJP), which gives rise to adults with a rough eye phenotype. Among the dominant suppressors of DmcycEJP, brahma (brm) and moira (mor) were identified. These genes encode conserved core components of the Drosophila Brm complex that is highly related to the SWI-SNF ATP-dependent chromatin remodeling complex. Mutations in genes encoding other Brm complex components, including snr1 (BAP45), osa and deficiencies that remove BAP60 and BAP111 can also suppress the DmcycEJP eye phenotype. Brm complex mutants suppress the DmcycEJP phenotype by increasing S phases without affecting DmcycE protein levels. DmcycE physically interacts with Brm and Snr1 in vivo. These data suggest that the Brm complex inhibits S phase entry by acting downstream of DmcycE protein accumulation. The Brm complex also physically interacts weakly with Drosophila retinoblastoma (Rbf1), but no genetic interactions were detected, suggesting that the Brm complex and Rbf1 act largely independently to mediate G1 arrest (Brumby, 2002).

Several recent studies have provided strong connections between metazoan SWI- SNF complexes and regulation of the cell cycle. In yeast, the SWI- SNF complex is not essential for viability, and whole genome analyses of swi/snf mutants have shown roles in activation and repression of transcription. A screen for modifiers of E2F1/DP function in Drosophila identified new alleles of brm and mor as enhancers of the rough eye phenotype associated with ectopic expression of E2F1 and DP in the developing Drosophila eye imaginal disc. In support of this, mammalian homologs of Brm and Mor (hBrm/Brg1 and BAF55, respectively) have been reported to be present in cyclin E complexes and to be phosphorylated by cyclin E- Cdk2. Significantly, human homologs of Brm (hBrm and Brg1) inhibit entry into S phase and achieve this at least in part by cooperation with the tumor suppressor Rb. Furthermore, Rb can bind to Brg1 and hBrm, and the ability of Rb to induce G1 arrest has been shown to depend upon hBrm and Brg1 (Brumby, 2002 and references therein).

The genetic interactions with DmcycE or E2F1/DP and Brm complex genes initially were thought to be due to effects on DmcycE transcription or E2F/DP-dependent transcription, given the role of the Brm complex in transcriptional regulation. Surprisingly, the results of this study suggest that the Brm complex functions downstream of DmcycE transcription and protein accumulation. (1) No significant effect on DmcycE protein levels in DmcycEJP eye discs was observed when the dosage of brm or mor was halved. (2) The rough eye phenotype due to overexpression of DmcycE from the GMR driver is enhanced by halving the dosage of brm and mor, indicating that Brm and Mor act to inhibit S phase entry downstream of DmcycE transcription. (3) DmcycE forms a complex with Brm and Snr1. Taken together, these data provide strong evidence that the Brm complex does not inhibit the G1 to S phase transition by acting to down-regulate DmcycE transcription (Brumby, 2002).

It is also likely that the Brm complex does not act to down-regulate E2F1/DP-dependent gene transcription, since no effect was observed for at least two E2F1/DP targets in brm mutants. Thus, mutations in Brm complex genes suppress the DmcycEJP mutant phenotypes by allowing progression into S phase without increasing either DmcycE protein levels or the expression of E2F1/DP-dependent genes. This suggests that one function of the Drosophila Brm complex is to restrict entry into S phase by inhibiting DmcycE-Cdk2 activity or by acting downstream of DmcycE-Cdk2 function. A function for Brm downstream of DmcycE-Cdk2 is consistent with reports that mammalian cyclin E can bind to and phosphorylate components of the Brm complex and thereby inactivate it. Thus the Brm complex may be acting as a curb to S phase entry that needs to be overcome by phosphorylation and inactivation by cyclin E-Cdk2 (Brumby, 2002).

Consistent with studies in cultured mammalian cells, the Rbf1 protein was found to be present in complexes with Brm or Snr1 in larval and embryonic extracts. However, in embryos, only a small portion of total cellular Rbf1 is present in Snr1 immunoprecipitates, in contrast to a significant fraction of the cellular DmcycE, suggesting that most Brm complexes do not contain Rbf1. The observation that Drosophila Rbf1 and Brm form a complex in vivo is consistent with studies in mammalian cells showing that hBrm and/or Brg1 can bind to and cooperate with Rb in transcriptional repression, and that hBrm and Brg1 are required for Rb-induced G1 arrest. However, in Drosophila, no clear evidence was obtained for cooperation of brm or mor with rbf1 in S phase entry. It is possible that the phenotypes being examining were not sensitive enough for S phase effects to be observed. However, the lack of a strong effect of Brm complex mutants on the rbf1 mutant S phase phenotype, when strong genetic interactions were observed with Brm complex genes and DmcycE, suggests that Rbf1 and Brm primarily function independently in negatively regulating S phase entry. Therefore, the suppression of the S phase defect of DmcycEJP by Brm complex mutants may not involve rbf1. Independent roles for Brm and Rb are also likely in mammalian cells since Rb knockout mice have a different mutant phenotype from that of Brg1 or Brm knockouts (Brumby, 2002).

In mammalian cells, Rb can form a complex containing both Brg1 and Hdac1, which is required to repress DmcycE transcription and may also have a role at replication origins. However, reducing the dose of the Drosophila Hdac gene, rpd3, did not suppress the DmcycEJP rough eye phenotype. It is possible that no interaction was observed for rpd3 and DmcycE, because there are a least three other Hdacs in flies that may perform overlapping functions with rpd3. However, mutations in sin3A, which encodes a Hdac-interacting protein, enhance the DmcycEJP rough eye phenotype, suggesting that Sin3a functions in opposition to Brm in regulating DmcycE or S phase entry. Further studies using specific mutations in other Drosophila Hdacs, and Hdac-interacting proteins are required to analyze further their role in the G1 to S phase transition (Brumby, 2002).

How does the Brm complex mediate negative regulation of the G1 to S phase transition? The results suggest that the Brm complex is playing a role independent of DmcycE transcription and E2F/DP-dependent transcription in negatively regulating the G1 to S phase transition. One way in which this may occur is by transcriptional regulation of other critical G1/S phase genes. For example, there is evidence that in Drosophila, the Brm complex is important in negatively regulating Armadillo-dTCF target genes in the Wingless signaling pathway. Although as yet there have been no studies showing directly that G1/S phase-inducing genes are targets of the Wingless signaling pathway in Drosophila, this is possible based on studies in mammalian cells. Furthermore, the Wingless pathway clearly has a role in cell proliferation in some Drosophila tissues. Whether this is the mechanism by which the Brm complex mediates negative regulation of cell cycle entry requires further investigation (Brumby, 2002).

Another way in which the Brm complex may function is by restricting or regulating access to chromosomal origins of replication. Several studies have shown that ATP-dependent chromatin remodeling is important for modulating the initiation of chromosomal DNA replication. The data are consistent with the view that the Brm complex may play a role in this process, possibly functioning to restrict entry into S phase by acting directly to remodel nucleosomes at replication origins. In this scenario, DmcycE-Cdk2 may then act to phosphorylate and inactivate the Brm complex, allowing assembly or function of the pre-replication complex and replication origin firing. Indeed, cyclin E-Cdk2 has been shown to be recruited by the Cdc6 pre-replication complex protein to replication origins at the G1 to S phase transition (Brumby, 2002).

Intriguingly, recent studies have shown that the E2F/DP complex also acts directly at replication origins. In the amplification of the chorion gene clusters during the ovarian follicle cell endoreplicative cycles, it has been shown that E2F1/DP is important in localizing the origin of replication complex specifically to the chorion gene origins and activating replication, and that Rbf1 is important in limiting DNA replication. This mechanism is not limited to these specialized cycles, since transcription-independent roles for E2F1 in inducing S phase have also been documented in the eye imaginal disc. Taken together, these studies suggest that the E2F1/DP-Rbf1 complex plays a non-transcriptional role in S phase by acting directly at DNA replication origins. In mammalian cells, a similar non-transcriptional role for Rb in DNA replication inhibition has been demonstrated, possibly through its functional association with the pre-replication complex protein Mcm7 and its localization to replication foci (Brumby, 2002).

Given the data for a role for Rb-E2F/DP directly at replication origins and the evidence that chromatin remodeling is important in replication initiation, it is possible that Brm and Rbf1 may both have a role at replication origins to prevent premature origin firing in G1. However, the failure to detect a genetic interaction between brm complex genes and rbf1 suggests that they also have other important roles, independent of each other, in the G1 to S phase transition (Brumby, 2002).

In summary, these results have shown that mutations in genes encoding components of the Brm chromatin remodeling complex can dominantly suppress a DmcycE hypomorphic allele by increasing the number of S phase cells without affecting cyclin E protein levels. Consistent with this view, DmcycE physically interacts with Brm and Snr1. Although a complex was also observed between the Brm complex and Rbf1, no genetic interactions have been detected between Brm complex genes and rbf1, suggesting that Rbf1 and Brm function largely independently in negatively regulating the G1 to S phase transition. Taken together, these data suggest that the Brm complex negatively regulates entry into S phase, possibly in partial collaboration with Rbf1, and that this negative regulation can be abrogated by the action of cyclin E at the G1 to S phase transition (Brumby, 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).

The Drosophila EST database contains two entries (LD02737 and LD15806) that showed similarities to the N-terminal portions of RBF and human p107. Sequencing of these cDNAs and the corresponding genomic region confirmed the presence of an RBF-related gene (DDBJ/EMBL/GenBank accession No. AF197059 and AF195899). The original retinoblastoma family member is referred to as rbf1, and the later isolate as rbf2. rbf2 is an intronless gene coding for a 782 amino acid protein with extensive homology to RBF1 and mammalian pocket proteins. RBF2 contains sequences corresponding to the A and the B halves of the pocket-domain, and has potential CDK phosphorylation sites on both sides of the pocket region. RBF2 also contains an N-terminal sequence (the N-box) that is highly conserved in human p107, human p130 and both Drosophila pocket proteins, but is absent in pRB. In addition, and unlike RBF1, RBF2 contains a short spacer sequence between the two halves of the pocket domain that is homologous to sequences in the p107 and p130 spacer regions. However, RBF2 lacks sequences homologous to the high affinity cdk-binding sites found in the N-terminal and the spacer regions of p107 and p130. Thus, the RBF2 protein shares more sequence identity with RBF1 than with any other pocket protein, and like RBF1, appears more closely related to p107 and p130 than to pRB (Stevaux, 2002).

An N-terminal portion of RBF2 was expressed in bacteria and used to generate monoclonal antibodies that specifically recognize RBF2 and do not cross-react with RBF1. On SDS-PAGE, RBF2 migrates slightly faster than RBF1 and separates as a single band of ~85 kDa. The expression of RBF1 and RBF2 was examined by Western blot analysis using extracts prepared at various stages of Drosophila development. The levels of RBF1 are relatively constant; the highest level of RBF1 is seen in 0- to 4-h-old embryos, and this drops slightly during the later stages of embryogenesis. In contrast, RBF2 levels vary considerably during development. RBF2 levels increase 4-fold in the first 8 h of development, and drop by one to two orders of magnitude at later stages of embryogenesis. Comparatively low levels of RBF2 were detected in whole larval extracts, and on long exposures of the Western blots, in adult females, but not in males. Higher levels of RBF2 were found in dissected larval imaginal discs and in tissue culture cells. These patterns suggest that RBF2 may be most highly expressed in rapidly cycling cells, a fact that has previously been for p107. The RBF2 present in female extracts is provided exclusively by the ovary, raising the possibility that RBF2 may function during oogenesis (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).

Little imaginal discs, a Drosophila homolog of human Rb binding protein 2

The proteins encoded by two groups of conserved genes, the Polycomb and trithorax groups, have been proposed to maintain, at the level of chromatin structure, the expression pattern of homeotic genes during Drosophila development. To identify new members of the trithorax group, a collection of deficiencies were screened for intergenic noncomplementation with a mutation in ash1, a trithorax group gene. Five of the noncomplementing deletions uncover genes previously classified as members of the Polycomb group. This evidence suggests that there are actually three groups of genes that maintain the expression pattern of homeotic genes during Drosophila development. The products of the third group appear to be required to maintain chromatin in both transcriptionally inactive and active states. Six of the noncomplementing deficiencies uncover previously unidentified trithorax group genes. One of these deficiencies removes 25D2-3 to 26B2-5. Within this region, there are two allelic, lethal, P-insertion mutations that identify one of these new trithorax group genes. The gene has been called little imaginal discs based on the phenotype of mutant larvae. The protein encoded by the little imaginal discs gene is the Drosophila homolog of human retinoblastoma binding protein 2 (Gilde, 2000).

The 133 deficiencies examined collectively uncover ~70% of the genome. Of these, only 6 exhibit intergenic noncomplementation with mutations in all 3 of the trithorax group genes tested and do not uncover previously identified trithorax group genes. Either there must be only a small number (i.e., closer to 10 than to 100) of genes in the entire genome in which mutations fail to complement mutations in the trithorax group genes tested or only deficiencies that uncover 2 or more such genes the were detected in this assay. Four of the deficiencies fail to complement mutations in all 3 trithorax group genes but do not suppress the Polycomb mutant phenotype. Perhaps these deficiencies uncover genes whose products act downstream of the homeotic selector genes, for example, as cofactors necessary for the activity or stability of homeotic selector gene products (Gilde, 2000).

Two of these six deficiencies suppressed the Polycomb mutant phenotype and did not uncover a known trithorax group gene. One of these six deficiencies, Df(2L)cl-h3 (25D2-3;26B2-5), uncovers two different trithorax group genes. The distal gene is within 25D4;25E1. It may be identical to E(var)2-25E, which was recovered in a screen for enhancers of position-effect variegation. Several of the mutations recovered in that screen have proven to be allelic to trithorax group genes. The proximal gene is within 25F4-4;26B2-5. Three lines of evidence have been presented, indicating that the allelic mutations l(2)10424 (now known as lid1) and l(2)k06801 (now known as lid2) represent P-element insertion mutations within this proximal gene, which has been named little imaginal discs. (1) Both alleles are lethal in combination with deficiencies that remove 25F4-4;26B2-5. (2) lid2 enhances the phenotype of ash1, brahma, and trithorax mutations and suppresses the phenotype of a Polycomb deletion. (3) Precise revertants of lid1 are homozygous viable and fail to enhance the phenotype of ash1, brahma, or trithorax mutations and fail to suppress the phenotype of a Polycomb deficiency (Gilde, 2000).

Despite the fact that lid mutations satisfy the criteria used for mutations in trithorax group genes, no homeotic transformations were observed in homozygous or trans-heterozygous mutant embryos or larvae. Instead, a small disc phenotype was observed. Certain allelic combinations of ash1 mutations also cause a small disc phenotype. The few lid mutants that survived the pupal stage expressed bristle phenotypes, similar to mutations in the trithorax group gene ash2. Therefore, lid mutations do cause phenotypes similar to those caused by mutations in other trithorax group genes. The failure to detect a high frequency of homeotic transformations in the two lid mutants is interpreted as a consequence of the nature of the mutations caused by the P-element insertions in these alleles (Gilde, 2000).

The predicted lid gene product is extremely similar to the human retinoblastoma binding protein 2 gene product (RBP-2). RBP-2 was discovered in a screen for proteins that interact with the pocket domain of the retinoblastoma protein (pRB). The full-length sequence of RBP-2 was later determined and found to contain nuclear localization motifs as well as sequence motifs characteristic of transcriptional regulators. RBP-2 has been shown to physically interact with mammalian TATA-binding protein as well as with p107 and Rb (also known as p110). No information is available about the molecular mechanism of LID function. However, given the similarity of LID to RBP-2 and the binding of RBP-2 to pRB there are several intriguing possibilities (Gilde, 2000).

The role of pRB in cell cycle regulation and proliferation is mediated, at least in part, by its interaction with the transcription factor E2F. It interacts physically with E2F to repress transcription and cell cycle progression. Overexpression of RBP-2 in cultured cells was shown to overcome the pRB-mediated suppression of E2F activity. A Drosophila mutant of E2F, E(var)3-95E, was discovered as a dominant enhancer of variegation. E2F is necessary for proliferation and differentiation in the Drosophila eye and interacts genetically with a Drosophila homolog of Rb: RBF. The finding that lid mutations cause defects in imaginal disc cell proliferation may be due to the loss of negative regulation of RBF leading to increased E2F repression of cyclin E (Gilde, 2000).

Histone acetylation has profound effects on transcriptional regulation and both global and local chromatin structure. The Rb protein has recently been found to physically associate with a histone deacetylase, HDAC1, and to repress transcription. The function of LID could be to counteract the repressive activity that histone deacetylation has on chromatin. Two multiprotein complexes from yeast, ADA and SAGA, function as nucleosome acetyltransferases, with GCN5 as the catalytic subunit; GCN5 mutations display synthetic lethality with SWI/SNF mutations. This is especially interesting, since brahma is a Drosophila homolog of yeast SWI2/SNF2, and lid interacts genetically with brahma. Further evidence for an association of trithorax group gene products and pRB is that by both two-hybrid and coimmunoprecipitation studies, Hbrm and Brg1, two human homologs of brahma, are associated with pRB family members. The balance between acetylation and deacetylation is clearly implicated in the function of trithorax group genes. Though the role RBP-2 plays in chromatin regulation is not known, the fact that it could be involved in the inactivation or relocation of a histone deacetylase fits well with how it is thought that trithorax group genes help to maintain an open chromatin conformation (Gilde, 2000).

In addition to the connections of pRB with E2F, cyclin E, and the cell cycle and to the connections of pRB with histone deacetylation and repression of transcription, there is a connection of pRB with the nuclear matrix and nuclear matrix-associated proteins. p110Rb is associated with the nuclear matrix in a cell cycle-dependent manner. Many p110Rb-associated factors have been previously found to be associated with the nuclear matrix, including SV40 large T antigen, adenovirus E1a, human papilloma E7 protein, lamin A, p84, and NRP/B. One model posits that functions within the nucleus occur at specific sites, and this functional compartmentalization of the nucleus is accomplished by localizing the machinery for each task to a specific site. For example, a hypothetical scenario consistent with this model would be that once activated, a homeotic selector gene may be bound by one or more trithorax group protein complexes that maintain the activated state by creating a site on the nuclear matrix for the transcription machinery itself and for proteins involved in acetylation and/or nucleosome remodeling and/or phosphorylation that are necessary for optimal expression. In this context, the change in subnuclear localization of the modifier of mdg-4 gene product may be relevant. Modifier of mdg4, also known as E(var)3-93D, is a trithorax group gene. Loss-of-function mutations enhance the phenotype of ash1;trithorax and brahma;trithorax double mutations and suppress the phenotype of Polycomb mutations. The product of this gene, MOD, is normally associated with the nuclear matrix. However, the subnuclear localization of MOD is dramatically altered in both trithorax group and Polycomb group mutant backgrounds. In trithorax group mutants MOD is primarily cytoplasmic; in Polycomb group mutants MOD is present in the central region of the nucleus rather than the nuclear matrix. Many of the models for the organization of higher order chromatin structures are based on associations with nuclear matrix components. It will be interesting to determine the subnuclear localization of LID and observe whether there are changes in this localization during the cell cycle and/or in trithorax group and Polycomb group mutant backgrounds (Gilde, 2000).

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. Expression of the mouse Cul1 (mCul1) in the larval wing disc of Drosophila 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 delays 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 enhances 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. Thus the G1/S transcription factor E2F is an SCFSlmb target in Drosophila. These results argue that the SCFSlmb ubiquitin ligase directs E2F destruction in S phase (Hériché, 2003).

This study provides evidence indicating a role for an SCFSlmb complex in elimination of E2F during S phase in Drosophila. Mouse Cul1 overexpression has a dominant negative effect on SCF function in the wing imaginal disc resulting in apoptotic cell death leading to a small wing phenotype in adult flies. Thus, SCF activity normally suppresses cell death. The cell death that occurs upon reduction of Cul1 function is suppressed when E2F function is reduced by mutation. This genetic interaction might be due to action of E2F and Cul1 in parallel pathways, one suppressing and one enhancing cell death, or they might act in opposite directions in the same pathway. Because SCF complexes are involved in controlling protein degradation, the hypothesis is favored that Cul1 promotes the destruction of E2F and that the inappropriate persistence of E2F leads to cell death. Consistent with this idea, E2F overproduction can induce apoptosis. Furthermore, cell cycle specific destruction of E2F is suggested by the finding that E2F protein becomes undetectable in the synchronized S phase cells of the morphogenetic furrow of the eye imaginal disc. Double labelling analysis extended this finding to the asynchronously cycling cells of the wing imaginal disc and demonstrated that E2F is rapidly degraded prior to significant DNA replication. In contrast, it was found that E2F is detected in a fraction of S phase cells when SCF function is reduced by mCul1. This shows that normal E2F downregulation is delayed or slowed by reduction in SCF function (Hériché, 2003).

Although the dominant negative action of mCul1 in Drosophila was unanticipated, it offers a fortuitously convenient tool for the analysis of SCF function. The Drosophila Cul1 mutant is less useful because maternal contributions of Cul1 are so large that homozygous mutant animals develop to pupariation. Additionally, unlike the spatially restricted expression of mCul1, the strong Cul1 mutant alleles result in lethality. The stage and generality of the defects make analysis of the Cul1 mutant phenotype particularly difficult. In contrast, the induced expression of mCul1 in the wing disc does not compromise viability and it gives a distinctive graded phenotype well suited for the study of genetic modification. While the basis for the dominant negative effect of mCul1 remains unknown, it is important to recognize that inactivation of the endogenous Cul1 is incomplete so that a reduced level of Cul1 activity persists. One likely explanation for the negative effect is that mCul1 overexpression leads to sequestration of some limiting SCF components into weakly active complexes. This hypothesis is consistent with the fact that mCul1 retains some positive function in flies as revealed by its ability to prolong survival of dCul1 mutant flies (J.-K. Hériché and P.H. O'Farrell, unpublished data) (Hériché, 2003).

The essentially complete suppression of the mCul1 phenotype by reduction of the genetic dose of E2F suggests a remarkable level of specificity, in which, among the many targets of Cul1 action, the destruction of E2F appears to be particularly important. Similarly, limitations of Cul1 function in other organisms also uncovered the disproportionate importance of particular substrates. For example, Cul1 mutations in mice result in the accumulation of the SCF substrate cyclin E but not of p27, another well characterized SCF substrate. Among all of the substrates targeted for degradation by SCF in S. cerevisiae, it is the failure to degrade Sic1 that underlies the G1 arrest in cdc53 mutants. Thus, different substrates of the SCF appear to have particularly high dependence on SCF activity in different biological contexts. The experimental context using mCul1 expression in the wing disc is thought to be particularly effective in exposing the involvement of SCF in E2F destruction (Hériché, 2003).

Since SCF complexes function in conjunction with a variety of F-box proteins that act as specificity factors, mutations in individual F-box proteins ought to affect particular subsets of SCF substrates. In an extensive screen for loci that modify the reduction of function phenotype for Cul1, the gene encoding the F-box protein Slimb as a modifier was identified, but no contributions of other F-box encoding genes to the phenotype were detected. This implicates Slimb in the action of SCF on E2F. Analysis of E2F levels reveals that cell cycle oscillations in E2F levels were absent when Slimb function was severely reduced. Note that the severity of this E2F destruction phenotype in comparison to the mild defect in cell cycle programming of E2F destruction upon mCul1 expression is entirely consistent with the fact that the slimb mutant gives a stronger loss of function than the reduction of function imposed by mCul1 overexpression. The absence of cell cycle oscillation in E2F presence in the slimb mutant suggests that SCFSlmb is responsible for targeting E2F for S phase destruction. If this destruction is the consequence of direct action of SCFSlmb on E2F, the F-box protein would be expected to interact with E2F. This prediction was confirmed by pull-down experiments. Furthermore, it is demonstrated that the interaction between Slimb and E2F is dependent on phosphorylation, as expected for the interaction of F-box proteins with their substrates. The S phase specificity of E2F destruction is regulated by a targeting phosphorylation event, but this level of regulation has yet to be investigated (Hériché, 2003).

Since E2F is a positive regulator of the G1/S transition, it is not clear why S phase destruction of E2F is required. However, it is noted that slimb mutant cells do not replicate DNA normally and the replication defect is correlated with an increase in E2F in individual cells. This observation is consistent with observations indicating that E2F can limit DNA replication both in mammalian cells and in flies. This enigmatic feature of E2F regulation suggests that its continued presence has a negative effect on DNA replication and indicates that there is still much to learn about E2F roles in cell cycle regulation (Hériché, 2003).

SCFSlmb appears to influence other cell cycle events, perhaps by actions on other substrates, or perhaps as a result of events that are secondary to its influence on E2F destruction. For example, a slimb allele shows defects in centrosome duplication control, and slimb null cells undergo apoptosis. Both effects can be explained by a failure to inactivate E2F, since E2F promotes centrosome duplication in mammalian cells and E2F can induce apoptosis. A link between Slimb function and the RB/E2F pathway of cell proliferation control is also suggested by the genetic interaction between the C. elegans slimb ortholog lin-23 and the RBF ortholog lin-35 in which lin-35 function limits the severity of the loss of lin-23 . Since RBF is an important modifier of E2F activity, perhaps this interaction is a reflection of the action of both Lin-23 and Lin-35 on E2F. However, in the Drosophila system there was no modification of the mCul1 overexpression phenotype by mutations in either the RBF or DP gene, two known partners of E2F. This result suggests that these factors are not limiting factors in this situation or that E2F acts independently of RBF and DP. The latter hypothesis is the most likely explanation for the lack of an RBF interaction for the following reason. The E2F/RBF complex has to be disrupted for E2F to drive cells into S phase and E2F degradation occurs after the G1/S phase transition, which only happens after E2F has been released from its association with RBF. According to this view, it would be predicted that the mCul1-induced phenotype should be specifically sensitive to factors impinging on the elimination of E2F activity during S phase, and factors involved in the unknown processes by which persistence of E2F has negative outcome. Consequently, this experimental system may provide an avenue for the genetic dissection of this mysterious facet of E2F function (Hériché, 2003).

Thus, to explore potential roles for Cul1 in cell cycle control in D. melanogaster, mouse Cul1 (mCul1) was overexpressed in the wing imaginal disc. This overexpression has a dominant negative effect leading to a reduction in SCF function. The resulting small wing phenotype was used in a modifier screen to identify mutations in cell cycle genes capable of dominantly modifying the mCul1-induced phenotype. E2F loss-of-function mutations were the strongest modifiers since they completely suppress the phenotype. The reduction in SCF function associated with mCul1 overexpression also correlates with a failure to downregulate E2F normally in S phase. Additionally, mutations in slimb that reduce the function of the F-box protein Slimb enhance the phenotype and lead to persistence of E2F in S phase cells. Finally, this F-box protein interacts with E2F in vitro. These results indicate that an SCFSlmb complex is involved in regulating E2F activity during S phase (Hériché, 2003).

Critical role of active repression by E2F and Rb proteins in endoreplication during Drosophila development

E2F transcription factors can activate or actively repress transcription of their target genes. The role of active repression during normal development has not been analyzed in detail. dE2F1su89 is a novel allele of Drosophila E2f that disrupts E2f's association with RBF Drosophila retinoblastoma protein (Rb) homolog but retains its transcription activation function. Interestingly, the dE2F1su89 mutant, which has E2f activation by dE2F1su89 and active repression by E2f2, is viable and fertile with no gross developmental defects. In contrast, complete removal of active repression in de2f2;dE2F1su89 mutants results in severe developmental defects in macrochaetae and salivary glands, tissues with extensive endocycles, but not in tissues derived from mitotic cycles. The endoreplication defect results from a failure to downregulate the level of cyclin E during the gap phase of the endocycling cells. Importantly, reducing the gene dosage of cyclin E partially suppresses all the phenotypes associated with the endoreplication defect. These observations point to an important role for E2f-Rb complexes in the downregulation of cyclin E during the gap phase of endocycling cells in Drosophila development (Weng, 2003).

A novel allele of E2f1, dE2F1su89, was identified from a genetic screen for suppressors of the Rbf overexpression phenotype. Sequence analysis revealed that dE2F1su89 contains a single base pair mutation in the conserved Rb-binding domain that converts the conserved amino acid leucine at position 786 to glutamine. To test whether this mutation disrupts the interaction between Rbf and E2f, a yeast two-hybrid interaction assay was performed. E2F1su89 is unable to bind to Rbf. To demonstrate further the effect of this mutation with endogenous proteins, a co-immunoprecipitation experiment was carried out. While both E2f and Dp co-immunoprecipitate with Rb from wild-type embryo extracts, no E2f co-immunoprecipitates with Rb from the dE2F1su89 embryo extracts, even though similar levels of E2f protein are present in the two extracts. These results indicate that the endogenous dE2F1su89 and Rb proteins do not form a complex. Interestingly, Dp still co-immunoprecipitates with Rb from the dE2F1su89 embryo extracts, indicating that Dp can still form a complex with Rb in the dE2F1su89 mutant background, probably through the other Drosophila E2F protein, E2f2 (Weng, 2003).

The decreased number of endocycles could be due to a lengthening of the S phase or a lengthening of the gap phase. Lengthening of the S phase would lead to an increased number of cells that are in the S phase, while lengthening of the gap phase would decrease the number of cells that are in S phase at any given time. A decreased number of S phase nuclei was observed in e2f2;dE2F1su89 salivary glands compared with that in wild-type salivary glands. Thus the gap phase of the endocycles in the e2f2;dE2F1su89 mutants is significantly lengthened. e2f2;dE2F1su89 but not wild-type salivary gland cells accumulate high levels of cyclin E in some gap phase cells (cells that are not incorporating BrdU). Since downregulation of cyclin E levels is required for continuous endoreplication, the failure to downregulate cyclin E levels properly in these gap phase cells probably inhibits endoreplication and leads to severe defects in tissues that require extensive endoreplication during development. The observation that decreasing the gene dosage of cyclin E partially suppresses the e2f2;dE2F1su89 phenotypes such as salivary gland endoreplication defects, macrochaetae defects and lethality provides strong support for the idea that the failure to downregulate cyclin E levels in these gap phase cells is a cause for the observed defects in e2f2;dE2F1su89 endocycle tissues (Weng, 2003).

Although previous results established that cyclin E oscillation is critical for continuous endoreplication, it is not clear how cyclin E oscillation in endocycle cells is achieved. No cyclin E oscillation defect is observed in salivary gland cells in the dE2F1su89 mutants, suggesting that active repression by the E2f2-Rb complexes is sufficient to downregulate the level of cyclin E during the gap phase, even in the presence of the unregulated dE2F1su89. In contrast, removal of the dE2F2-Rb complexes in the dE2F1su89 background results in extensive defects in endocycle tissues and defective cyclin E downregulation in the gap phase of endocycling cells. These results argue strongly that the E2f-Rb complexes are required for the normal downregulation of cyclin E in the gap phase of endocycling cells. These results, in conjunction with the observation that E2F activity is required for cyclin E expression and S phase progression of endocycle cells, suggests a model in which E2f activation is required for S phase of the endocycles and active repression by E2f-Rb complexes is required during gap phase. It is interesting to note that even in the complete absence of Rb-E2f active repression, there are still significant levels of endoreplication, suggesting that the oscillation of cyclin E activity, although defective, can still occur to some extent in e2f2;dE2F1su89 mutants. It is possible that additional mechanisms such as protein degradation or binding to inhibitor proteins such as Dacapo can also contribute to the downregulation of cyclin E activity (Weng, 2003).

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).

Retinoblastoma protein regulation by the COP9 signalosome

Similar to their human counterparts, the Drosophila Rbf1 and Rbf2 Retinoblastoma family members control cell cycle and developmentally regulated gene expression. Increasing evidence suggests that Rbf proteins rely on multiprotein complexes to control target gene transcription. The developmentally regulated COP9 signalosome (CSN) physically interacts with Rbf2 during embryogenesis. Furthermore, the CSN4 subunit of the COP9 signalosome co-occupies Rbf target gene promoters with Rbf1 and Rbf2, suggesting an active role for the COP9 signalosome in transcriptional regulation. The targeted knockdown of individual CSN subunits leads to diminished Rbf1 and Rbf2 levels and to altered cell cycle progression. The proteasome-mediated destruction of Rbf1 and Rbf2 is increased in cells and embryos with diminished COP9 activity, suggesting that the COP9 signalosome protects Rbf proteins during embryogenesis. Previous evidence has linked gene activation to protein turnover via the promoter-associated proteasome. These findings suggest that Rbf repression may similarly involve the proteasome and the promoter-associated COP9 signalosome, serving to extend Rbf protein lifespan and enable appropriate programs of retinoblastoma gene control during development (Ullah, 2007).

The role of the COP9 signalosome in gene regulation by Rbf proteins remains imprecisely defined; however, the data suggest that the COP9 signalosome protects Rbf1 and Rbf2 from proteasome-mediated destruction. Rbf protein levels were reduced in csn4 and csn5 mutant embryos, and embryonic levels of both Rbf proteins were restored by inhibiting the proteasome. Similarly, the destruction of Rbf1 and Rbf2 in S2 cells treated with csn4-specific dsRNA was similarly blocked by inhibition of the proteasome. Furthermore, RNAi-mediated reduction of multiple COP9 signalosome subunits lead to reduced Rbf1 and Rbf2 levels, indicating that the entire COP9 signalosome complex is involved in this function. The observed protection of Rbf1 and Rbf2 may extend from two aspects of the COP9 signalosome. (1) Many subunits of the COP9 signalosome share limited sequence homology with components of the 19S proteasome lid complex, and thus the COP9 signalosome may compete with the proteasome for access to Rbf proteins. (2) The COP9 signalosome can deneddylate the cullin subunits of SCF ubiquitin E3 ligase complexes; therefore, altered SCF complex activity in the absence of the COP9 signalosome may be directly responsible for downstream changes in Rbf1 and Rbf2 levels. If so, the decreased levels of Rbf1 and Rbf2 as seen in Drosophila embryos, possibly via a SCF ubiquitin E3 ligase pathway, would be similar to the SCF-mediated destruction of p130, observed in humans. However, SCF deneddylation appears to play both positive and negative roles for SCF activity and subsequent target protein destruction depending on species and cell type examined, and thus, the COP9 signalosome may similarly exhibit bipolar effects on Rbf1 and Rbf2 protection, depending on context. At least in early stages of Drosophila development, the COP9 signalosome plays a protection role in Rbf function (Ullah, 2007).

Previous studies have implicated the COP9 signalosome complex in cell cycle regulatory pathways during development, and individually, the mammalian CSN5 protein, also known as Jab1, has recently been shown to bind E2F1 (Hallstrom, 2006), a protein partner for Rbf1. The newly described linkage between the Drosophila Retinoblastoma protein Rbf2 and COP9 signalosome is consistent with a role for COP9 signalosome in cell cycle progression through its association with Rbf proteins. However, depletion of CSN5 by RNAi resulted in blocked G1/S progression, whereas loss of Rbf1 and Rbf2 function would be expected to facilitate G1/S progression. Thus, in these experiments, it appears likely that impaired function of other cell cycle regulatory proteins such as E2F and cyclin E in the absence of COP9 signalosome activity may play a dominant role in limiting cell cycle progression through G1 phase (Ullah, 2007).

The COP9 signalosome has also been suggested to play an important role in modulating cancer initiation and progression. In this arena, a number of factors that play critical roles in cellular proliferation, including the cyclin/cdk inhibitor p27, cyclin E, c-jun, and the tumor suppressor p53, among others, have been previously linked to the COP9 signalosome. Thus, one mechanism for the tumorigenic control by COP9 is through its targeting of proto-oncoproteins and tumor suppressor proteins that play critical roles in governing cellular proliferation. The data linking the COP9 signalosome to Rbf proteins, homologues of the human Retinoblastoma tumor suppressor protein, strengthens this connection. Interestingly, the CSN4 subunit of the COP9 signalosome co-occupies selected target gene promoters along with Rbf2. The presence of this COP9 complex subunit at Rbf1 and Rbf2 target gene promoters indicates that the complex may play a direct role in transcriptional regulation, or alternatively, the COP9 signalosome may stabilize Rbf proteins against degradation because these proteins regulate gene expression during growth. Interestingly, the presence of proteasome subunits has been documented at actively transcribed genes, and ubiquitylation and proteasome-mediated destruction of transcriptional activators has been linked to increased activator potency. As activator ubiquitylation can serve as a marker for coregulatory protein recruitment, it will be important to determine whether repressor potency and corepressor recruitment are likewise linked to signals that govern their own destruction (Ullah, 2007).

A shared role for RBF1 and dCAP-D3 in the regulation of transcription with consequences for innate immunity

A conserved interaction between RB proteins and the Condensin II protein CAP-D3 is important for ensuring uniform chromatin condensation during mitotic prophase (Longworth, 2008). The Drosophila melanogaster homologs RBF1 and dCAP-D3 co-localize on non-dividing polytene chromatin, suggesting the existence of a shared, non-mitotic role for these two proteins. This study shows that the absence of RBF1 and dCAP-D3 alters the expression of many of the same genes in larvae and adult flies. Strikingly, most of the genes affected by the loss of RBF1 and dCAP-D3 are not classic cell cycle genes but are developmentally regulated genes with tissue-specific functions and these genes tend to be located in gene clusters. The data reveal that RBF1 and dCAP-D3 are needed in fat body cells to activate transcription of clusters of antimicrobial peptide (AMP) genes. AMPs are important for innate immunity, and loss of either dCAP-D3 or RBF1 regulation results in a decrease in the ability to clear bacteria. Interestingly, in the adult fat body, RBF1 and dCAP-D3 bind to regions flanking an AMP gene cluster both prior to and following bacterial infection. These results describe a novel, non-mitotic role for the RBF1 and dCAP-D3 proteins in activation of the Drosophila immune system and suggest dCAP-D3 has an important role at specific subsets of RBF1-dependent genes (Longworth, 2012).

Recent studies have suggested that pRB family members may impact the organization of higher-order chromatin structures, in addition to their local effects on the promoters of individual genes (Longworth, 2010). Mutation of pRB causes defects in pericentric heterochromatin and RBF1 is necessary for uniform chromatin condensation in proliferating tissues of Drosophila larvae (Longworth, 2008). Part of the explanation for these defects is that RBF1 and pRB promote the localization of the Condensin II complex protein, CAP-D3 to DNA both in Drosophila and human cells (Longworth, 2008). Depletion of pRB from human cells strongly reduces the level of CAP-D3 associated with centromeres during mitosis and causes centromere dysfunction (Longworth, 2012).

Condensin complexes are necessary for the stable and uniform condensation of chromatin in early mitosis. They are conserved from bacteria to humans with at least two types of Condensin complexes (Condensin I and II) present in higher eukaryotes. Both Condensin I and II complexes contain heterodimers of SMC4 and SMC2 proteins that form an ATPase which acts to constrain positive supercoils. Each type of Condensin also contains three specific non-SMC proteins that, upon phosphorylation, stabilize the complex and promote ATPase activity. The kleisin CAPH and two HEAT repeat containing subunits, CAP-G and CAP-D2 are components of Condensin I, while the kleisin CAP-H2 and two HEAT repeat containing subunits, CAP-G2 and CAP-D3, are constituents of Condensin II (Longworth, 2012).

Given the well-established functions of Condensins during mitosis, and of RBF1 in G1 regulation, the convergence of these two proteins was unexpected. Nevertheless, mutant alleles in the non-SMC components of Condensin II suppress RBF1-induced phenotypes, and immunostaining experiments revealed that RBF1 displays an extensive co-localization with dCAP-D3 (but not with dCAP-D2) on the polytene chromatin of Drosophila salivary glands (Longworth, 2008). This co-localization occurs in cells that will never divide, suggesting that Condensin II subunits and RBF1 co-operate in an unidentified process in non-mitotic cells. In various model organisms, the mutation of non-SMC Condensin subunits has been associated with changes in gene expression raising the possibility that dCAP-D3 may affect some aspect of transcriptional regulation by RBF1. However, the types of RBF1-regulated genes that might be affected by dCAP-D3, the contexts in which this regulation becomes important, and the consequences of losing this regulation are all unknown (Longworth, 2012).

This study identified sets of genes that are dependent on both rbf1 and dCap-D3. The majority of genes that show altered expression in both rbf1 and dCap-D3 mutants (larvae or adults) are not genes involved in the cell cycle, DNA repair, proliferation, but are genes with cell type-specific functions and many are spaced within 10 kb of one another in 'gene clusters'. To better understand this mode of regulation, the effects were investigated of RBF1 and dCAP-D3 on one of the most highly misregulated clusters which includes genes coding for antimicrobial peptides (AMPs). AMPs are produced in many organs, and one of the major sites of production is in the fat body. Following production in the fat body, AMPs are subsequently dumped into the hemolymph where they act to destroy pathogens. RBF1 and dCAP-D3 are required for the transcriptional activation of many AMPs in the adult fly. Analysis of one such gene cluster shows that RBF1 and dCAP-D3 bind directly to this region and that they bind, in the fat body, to sites flanking the locus. RBF1 and dCAP-D3 are both necessary in the fat body for maximal and sustained induction of AMPs following bacterial infection, and RBF1 and dCAP-D3 deficient flies have an impaired ability to respond efficiently to bacterial infection. These results identify dCAP-D3 as an important transcriptional regulator in the fly. Together, the findings suggest that RBF1 and dCAP-D3 regulate the expression of clusters of genes in post-mitotic cells, and this regulation has important consequences for the health of the organism (Longworth, 2012).

The idea that dCAP-D3 and RBF1 could cooperate to promote tissue development and differentiation is supported by the fact that both proteins are most highly expressed in the late stages of the fly life cycle, and accumulate at high levels in the nuclei of specific cell types in adult tissues. As an illustration of the cell-type specific nature of RBF1/dCAP-D3-regulation this study shows that dCAP-D3 and RBF1 are both required for the constituive expression of a large set of AMP genes in fat body cells. The loss of this regulation compromises pathogen-induction of gene expression and has functional consequences for innate immunity. Interestingly, different sets of RBF1/dCAP-D3-dependent genes were evident in the gene expression profiles of mutant larvae and adults. Given this, and the fact that the gene ontology classification revealed multiple groups of genes, it is suggested that the targets of RBF1/dCAP-D3-regulation do not represent a single transcriptional program, but diverse sets of cell-type specific programs that need to be activated (or repressed) in specific developmental contexts (Longworth, 2012).

The changes in gene expression seen in the mutant flies suggest that RBF1 has a significant impact on the expression of nearly half of the dCAP-D3-dependent genes. This fraction is consistent with previous data showing partial overlap between RBF1 and dCAP-D3 banding patterns on polytene chromatin, and the finding that chromatin-association by dCAP-D3 is reduced, but not eliminated, in rbf1 mutant animals and RBF1-depeleted cells. Although it has been previously shown that RBF1 and dCAP-D3 physically associate with one another (Longworth, 2008), and the current studies illustrate the fact that they each bind to similar sites at a direct target, the molecular events that mediate the co-operation between RBF1 and dCAP-D3 remain unknown (Longworth, 2012).

These results represent the first published ChIP data for the CAP-D3 protein in any organism. Although only a small number of targets were examined, it is interesting to note that the dCAP-D3 binding patterns are different for activated and repressed genes. More specifically, dCAP-D3 binds to an area within the open reading frame of a gene which it represses. However, dCAP-D3 binds to regions which flank a cluster of genes that it activates. Whether or not this difference in binding is true for all dCAP-D3 regulated genes will require a more global analysis (Longworth, 2012).

Human Condensin non-SMC subunits are capable of forming subcomplexes in vitro that are separate from the SMC protein- containing holocomplex, but currently, the extent to which dCAP-D3 relies on the other members of the Condensin II complex remains unclear. It is noted that fat body cells contain polytene chromatin. Condensin II subunits have been shown to play a role in the organization of polytene chromatin in Drosophila nurse cells. Given that RB proteins physically interact with other members of the Condensin II complex (Longworth, 2008), it is possible that RBF1 and the entire Condensin II complex, including dCAP-D3, may be especially important for the regulation of transcription on this type of chromatin template (Longworth, 2012).

A potentially significant insight is that the genes that are deregulated in both rbf1 and dCap-D3 mutants tend to be present in clusters located within 10 kb of one another. This clustering effect seems to be a more general feature of regulation by dCAP-D3, which is enhanced by RBF1, since clustering was far more prevalent in the list of dCAP-D3 target genes than in the list of RBF1 target genes (Longworth, 2012).

These studies focussed on one of the most functionally related families of clustered target genes that were co-dependent on RBF1/dCAP-D3 for activation in the adult fly: the AMP family of genes. AMP loci represent 20% of the gene clusters regulated by RBF1 and dCAP-D3 in adults. ChIP analysis of one such region, a cluster of AMP genes at the diptericin locus, showed this locus to be directly regulated by RBF1 and dCAP-D3 in the fat body and revealed a pattern of RBF1 and dCAP-D3-binding that was very different from the binding sites typically mapped at E2F targets. Unlike the promoter-proximal binding sites typically mapped at E2F-regulated promoters, RBF1 and dCAP-D3 bound to two distant regions, one upstream of the promoter and one downstream of the diptericin B translation termination codon, a pattern that is suggestive of an insulator function. It is hypothesized that RBF1 and dCAP-D3 act to keep the region surrounding AMP loci insulated from chromatin modifiers and accessible to transcription factors needed for basal levels of transcription. The modEncode database shows binding sites for multiple insulator proteins, as well as GATA factor binding sites, at these regions. GATA has been previously implicated in transcriptional regulation of AMPs in the fly, and future studies of dCAP-D3 binding partners in Drosophila fat body tissue may uncover other essential activators. Additionally, the chromatin regulating complex, Cohesin, which exhibits an almost identical structure to Condensin, has been shown to promote looping of chromatin and to bind proteins with insulator functions. Therefore, it remains a possibility that Condensin II, dCAP-D3 may actually possess insulator function, itself. It is proposed that dCAP-D3 may be functioning as an insulator protein, both insulating regions of DNA containing clusters of genes from the spread of histone marks and possibly looping these regions away from the rest of the body of chromatin. This would serve to keep the region in a 'poised state' available for transcription factor binding following exposure to stimuli that would induce activation. In the case of AMP genes, which are made constituitively in specific organs at low levels, dCAP-D3 would bind to regions flanking a cluster, and loop the cluster away from the body of chromatin. Upon systemic infection, these clusters would be more easily accessible to transcription factors like NF-κB. If dCAP-D3 is involved in looping of AMP clusters, then it may also regulate interchromosomal looping which could bring AMP clusters on different chromosomes closer together in 3D space, allowing for a faster and more coordinated activation of all AMPs (Longworth, 2012).

AMP expression is essential for the ability of the fly to recover from bacterial infection. Experiments with bacterial pathogens show that RBF1 and dCAP-D3 are both necessary for induction and maintenance of the AMP gene, drosomycin following infection, but only dCAP-D3 is necessary for the induction of the diptericin AMP gene. Similarly, survival curves indicate, that while dCAP-D3 deficient flies die more quickly in response to both Gram positive and Gram negative bacterial infection, RBF1 deficient flies die faster only in response to Gram positive bacterial infection. The differences seen between RBF1 and dCAP-D3 deficient flies in diptericin induction cannot be attributed to functional compensation by the other Drosophila RB protein family member, RBF2, since results show that loss of RBF2 or both RBF2 and RBF1 do not decrease AMP levels following infection. Since results demonstrate that RBF1 binds most strongly to an AMP cluster prior to infection and regulates basal levels of almost all AMPs tested, it is hypothesized that RBF1 (and possibly RBF2) may be more important for cooperating with dCAP-D3 to regulate basal levels of AMPs. Reports have shown that basal expression levels of various AMPs are regulated in a gene-, sex-, and tissue-specific manner, and it is thought that constitutive AMP expression may help to maintain a proper balance of microbial flora and/or help to prevent the onset of infections. In support of this idea, one study in Drosophila which characterized loss of function mutants for a gene called caspar, showed that caspar mutants increased constitutive transcript levels of diptericin but not transcript levels following infection. This correlated with increased resistance to septic infection with Gram negative bacteria, proving that changes in basal levels of AMPs do have significant effects on the survival of infected flies. Additionally, disruption of Caudal expression, a protein which suppresses NF-κB mediated AMP expression following exposure to commensal bacteria, causes severe defects in the mutualistic interaction between gut and commensal bacteria. It is therefore possible that RBF1 and dCAP-D3 may help to maintain the balance of microbial flora in specific organs of the adult fly and/or be involved in a surveillance-type mechanism to prevent the start of infection. RBF1 deficient flies also exhibit defects in Drosomycin induction following Gram positive bacterial infection. Mutation to Drosophila GNBP-1, an immune recognition protein required to activate the Toll pathway in response to infection with Gram positive bacteria has been show to result in decreased Drosomycin induction and decreased survival rates, without affecting expression of Diptericin. Therefore, it is possible that inefficient levels of Drosomycin, a major downstream effector of the Toll receptor pathway, combined with decreased basal transcription levels of a majority of the other AMPs, would cause RBF1 deficient flies to die faster following infection with Gram positive S. aureus but not Gram negative P. aeruginosa (Longworth, 2012).

Some dCAP-D3 remains localized to DNA in RBF1 deficient flies and it is also possible that other proteins may help to promote the localization of dCAP-D3 to AMP gene clusters following infection. Given that dCAP-D3 regulates many AMPs including some that do not also depend on RBF1 for activation, and given that dCAP-D3 binding to an AMP locus increases with time after infection whereas RBF1 binding is at its highest levels at the start of infection, it may not be too surprising that dCAP-D3 showed a more pronounced biological role in pathogen assays involving two different species of bacteria (Longworth, 2012).

Remarkably, and perhaps unexpectedly, the levels of both RBF1 and dCAP-D3 impact the basal levels of human AMP transcripts, as well. This indicates that the mechanism of RBF1/dCAP-D3 regulation may not be unique to Drosophila. It is striking that many of the human AMP genes (namely, the defensins) are clustered together in a region that spans approximately 1 Mb of DNA. It seems telling that both the clustering of these genes, and a dependence on pRB and CAP-D3, is apparently conserved from flies to humans. The fact that dCAP-D3 and RBF1 dependent activation of Drosomycin was necessary for resistance to Gram positive bacterial infection in flies suggests the same could also be true for the human orthologs in human cells. Human AMPs expressed by epithelial cells, phagocytes and neutrophils are an important component of the human innate immune system. Human AMPs are often downregulated by various microbial pathogenicity mechanisms upon infection. They have also been reported to play roles in the suppression of various diseases and maladies including cancer and Inflammatory Bowel Disease. It is noted that the chronic or acute loss of Rb expression from MEFs resulted in an unexplained decrease in the expression of a large number of genes that are involved in the innate immune system. In humans, the bacterium, Shigella flexneri was recently shown to down regulate the host innate immune response by specifically binding to the LXCXE cleft of pRB, the same site that was previously shown to be necessary for CAP-D3 binding). An improved understanding of how RB and CAP-D3 regulate AMPs in human cells may provide insight into how these proteins are able to regulate clusters of genes, and may also open up new avenues for therapeutic targeting of infection and disease. Further studies of in differentiated human cells may identify additional sets of genes that are regulated by pRB and CAP-D3 (Longworth, 2012).

A dual role for the dREAM/MMB complex in the regulation of differentiation-specific E2F/RB target genes>

E2F and RB proteins regulate the expression of genes involved in cell cycle progression, apoptosis, differentiation, and development. Recent studies indicate that they function as part of an evolutionarily conserved multiprotein complex termed dREAM/DREAM/LINC. This study characterizes the role of the Drosophila complex, dREAM [(Drosophila RBF, dE2F2, and dMyb-interacting proteins), three Myb-interacting proteins (Mip40, Mip120, and Mip130), and p55CAF1 (RbAp46/48)], in the regulation of differentiation-specific E2F target genes in actively proliferating cells. These genes are regulated differently from cell cycle-related E2F targets, they do not depend on E2F activation, and E2F/RB repression is maintained throughout the cell cycle. In proliferating cells, their repression is dependent on dREAM. dREAM was found to play a dual role in their regulation. First, it is required for the stability of the repressive dE2F2/RBF complexes at their promoters during S phase. dREAM was found to be indispensable for both transcriptional repression mechanisms employed at these genes (Lee, 2012).

The identification of native pocket-protein associated complexes in flies, worms and humans called dREAM/MMB/DRM/LINC indicates that such complexes play important roles in RB functions. The mechanisms of action of the complex are not very well understood. The complex has been shown to regulate the expression of G1/S, G2/M, as well as differentiation-specific E2F target genes, and to potentiate RBs tumor suppressive functions. It has been reported to repress as well as activate transcription, regulate site-specific DNA replication, and has been located at a large number of genomic sites. These observations indicate that dREAM/MMB/DRM/LINC plays a vital role in RB functions but also raise the question of how it can support all these diverse E2F/RB activities (Lee, 2012).

This study has explored the means by which the Drosophila complex, dREAM, represses differentiation-specific E2F/RB targets in actively proliferating cells. Differentiation-specific target genes (Group D/E) differ in their regulation from classic, cell cycle regulated targets, and it was found that the function of dREAM is required for some of the unique features of this gene regulation. Specifically, dREAM is not responsible for the target specificity of E2Fs. Instead, it is required for the stability of dE2F2/RBF complexes at Group E gene promoters during S-phase. Our results indicate that it may function, at least in part, by protecting RBF1 from phosphorylation. In addition, the complex is also essential to maintain both mechanisms of repression at these gene (Lee, 2012).

One of the distinctive features of Group E gene regulation is the lack of dependence on E2F activation - the activator dE2F1 does not bind to their promoters or regulate their expression. This poses the question of how dE2F2-specificity is achieved at these promoters. While a comparison of E2F binding sites did not reveal any major differences between sites found at Group E and cell cycle gene promoters, dE2F1 is unable to bind to Group E gene promoters even in the absence of dE2F2. This finding suggests that either dE2F1 is inherently incapable of binding to these promoters or that some factor(s) other the dE2F2 prevents it from binding. It is also possible that dE2F2 is not capable of binding without the assistance of another factor. One candidate for such a factor is the dREAM complex. It could either assist dE2F2 or prevent dE2F1 from binding. This study found that disruption of dREAM did not lead to dE2F1 binding, but disrupted dE2F2 binding, suggesting that neither E2F is capable of binding to these promoters without assistance. However, upon close examination, it was discovered that dREAM is dispensable for dE2F2 binding outside of S-phase, demonstrating that dE2F2 can bind to Group E gene promoters without the assistance of dREAM. These results indicate that dE2F1 is not capable of binding at these promoters. It is possible that there are some subtle differences in the E2F binding sites; alternatively the two E2F proteins may have different affinities for a particular chromatin landscape (Lee, 2012).

In S-phase, dE2F/RBF complexes are replaced by dE2F1 at cell cycle regulated promoters, yet remain bound and functional at Group E gene promoters. Several observations in mammals also indicate that E2F/pocket-protein complexes exist irrespective of cell cycle stage and can function in a CDK-independent manner. It is well established that E2F/RB complexes are disrupted at cell cycle genes at the G1/S transition. Are E2F/RB complexes then protected from CDK phosphorylation in some cases or is the regulation of E2F/pocket-protein interactions even more complex? Structural studies of pRB have suggested that the C-terminus of pRB functions as a molecular sensor that recognizes CDK mediated phosphorylation. When pRB is hypo-phosphorylated, its C-terminal region stabilizes the interaction with E2F1, phosphorylation by CDKs, causes a conformational change, and this intra-molecular interaction is thought to inhibit the interaction between pRB and E2F1. However, it is not clear whether that this is true for other RB family members or that all E2Fs interact in the same manner with pocket proteins. Furthermore, it has been shown that pRB has two distinct E2F binding sites, one being specific for E2F1 and linked to its ability to regulate E2F1-dependent apoptosis. This indicates that pRB interacts with individual E2F proteins in different ways and that regulation of distinct E2F functions are physically separable. Studies in Drosophila also indicate that G1 CDKs are not sufficient to disrupt repressive E2F/RB complexes at cell cycle genes. In the case of differentiation-specific target genes in flies, a simple explanation was found of the stability of E2F/RB complexes in S-phase, namely dREAM. In the absence of a functional dREAM complex, dE2F2 and RBFs exhibit reduced binding in S-phase. The results show that expression of a phospho-mimic RBF mutant results in impaired binding at both cell cycle regulated and differentiation-specific (Group E) gene promoters. Conversely a mutant RBF1 that cannot be phosphorylated is sufficient to bypass the need for a functional dREAM at Group E gene promoters; E2F/RB complexes are stable in S-phase even in the absence of dREAM. These findings indicate that CDK phosphorylation can disrupt dE2F2/RBF repressive complexes at Group E gene promoters and that necessitates their assembly into the dREAM complex. But how does assembly into dREAM ensure stability of E2F/RB complexes? The simplest model is that the phosphorylation sites are blocked, inaccessible to CDKs. Alternatively, dREAM could stabilize either fully or partially phosphorylated RBF protein. Previous studies have shown that Cyc E/cdk2 can phosphorylate RBF1 as part of the dREAM complex in vitro and that phosphorylation does not disrupt the association. However, RBF1 is regulated by both CycD/cdk4 and Cyc2/cdk2 and it is likely that when assembled into dREAM in vivo not all phosphorylation sites are modified. In agreement with this idea, it was found that in cells lacking functional dREAM hyper-phosphorylated RBF1 is increased. Thus, the findings suggest that the complex functions, at least in part, by protecting RBF1 at these promoters from being phosphorylated by CDKs (Lee, 2012).

dREAM, possibly in association with additional factors, may also modify RBF1. For instance, pRB is known to be acetylated during differentiation of monoblastoid cells and keratocytes. Additional post-translational modifications of the protein could either prevent phosphorylation and/or induce a conformational change to promote stability of phosphorylated E2F/RB complexes. It will be interesting to investigate if RBF proteins bound in the dREAM complex have modifications other than phosphorylation (Lee, 2012).

What is the role of the dREAM complex in the regulation of differentiation-specific 424 E2F/RB target genes? The initial identification and characterization of the dREAM/MMB complexes has led to the speculation that dREAM is a repression complex that functions by affecting chromatin structure. The complex is not stably associated with any enzymatic activity, yet this study has demonstrated that Group D/E genes are repressed via two distinct mechanisms in a dREAM-dependent manner. Is the observed dependence on dREAM the result of its ability to affect chromatin structure or is it an indirect consequence of its role in dE2F2/RBF binding? The idea is favored that dREAM plays a direct role in the repression of Group E genes for two reasons. Binding of dE2F2/RBF1 complexes does not require dREAM outside of S-phase. Only about 10% of asynchronously growing SL2 cells are in S-phase, yet disruption of dREAM leads to the same level de-repression as the removal of dE2F2 or RBFs. Moreover, while the expression of phospho-mutant RBF1 restores the binding of dE2F2/RBF1 in S-phase, it does not restore the repression in cells lacking dREAM. A dual role for dREAM in the regulation of these genes is also more consistent with its reported involvement in other RB functions. It would suggest that the complex is capable of potentiating diverse RB activities (Lee, 2012).

S phase-coupled E2f1 destruction ensures homeostasis in proliferating tissues

Precise control of cell cycle regulators is critical for normal development and tissue homeostasis. E2F transcription factors are activated during G1 to drive the G1-S transition and are then inhibited during S phase by a variety of mechanisms. The single Drosophila activator E2F (E2f1) was genetically manipulate to explore the developmental requirement for S phase-coupled E2F down-regulation. Expression of an E2f1 mutant that is not destroyed during S phase drives cell cycle progression and causes apoptosis. Interestingly, this apoptosis is not exclusively the result of inappropriate cell cycle progression, because a stable E2f1 mutant that cannot function as a transcription factor or drive cell cycle progression also triggers apoptosis. This observation suggests that the inappropriate presence of E2f1 protein during S phase can trigger apoptosis by mechanisms that are independent of E2F acting directly at target genes. The ability of S phase-stabilized E2f1 to trigger apoptosis requires an interaction between E2f1 and the Drosophila pRb homolog, Rbf1, and involves induction of the pro-apoptotic gene, hid. Simultaneously blocking E2f1 destruction during S phase and inhibiting the induction of apoptosis results in tissue overgrowth and lethality. It is proposed that inappropriate accumulation of E2f1 protein during S phase triggers the elimination of potentially hyperplastic cells via apoptosis in order to ensure normal development of rapidly proliferating tissues (Davidson, 2012).

Thus stabilizing the single Drosophila activator E2f1 in S phase results in apoptosis is necessary to prevent hypertrophy of wing imaginal discs. It is concluded from these data that hyper-accumulation of E2f1 during S phase represents a form of proliferative stress during development that is sensed by the apoptotic machinery and results in the elimination of cells with excess E2f1 activity to maintain homeostasis during tissue growth (Davidson, 2012).

What might be the function of a Drosophila cell's ability to detect abnormal accumulation of E2f1 protein during S phase and subsequently trigger apoptosis? One possibility is that accumulation of E2f1 during S phase resembles instances of abnormally high E2f1 activity that might occur sporadically during rapid growth of a population of precursor cells such as those in the wing imaginal disc. These events could be caused by stochastic or even genetic changes that affect either E2f1 gene transcription or the ability of the CRL4Cdt2/PCNA pathway to destroy E2f1 after replication factor genes are activated in late G1. The cell's ability to detect E2f1 accumulation in S phase clears these potentially hyperplastic cells from developing tissues via apoptosis, consequently contributing to the balance between cell proliferation and cell death that is necessary for normal tissue growth (Davidson, 2012).

Growing Drosophila imaginal discs possess another mechanism of homeostasis in which a process of compensatory proliferation is activated in order to achieve normal tissue development when as many as 50% of cells are killed by external stimuli like radiation-induced DNA damage. Indeed, in spite of high levels of apoptosis (15% of the cells), 50% of en-Gal4>E2f1Stable progeny survive until adulthood with about 2/3 of these surviving flies containing wings with somewhat mild morphological defects. Blocking apoptosis with baculovirus p35 when E2f1Stable is expressed shifts the cell proliferation/apoptosis balance too strongly in favor of cell proliferation, resulting in massive hypertrophy and 100% lethality (Davidson, 2012).

p35 is a broad caspase inhibitor that blocks effector caspase activity at a step downstream of their proteolytic activation. Therefore, cells expressing p35 can initiate apoptosis, but lack the capacity to complete it and are referred to as 'undead cells.' These undead cells produce signals that stimulate unaffected neighboring cells to proliferate. Thus, the dramatic hypertrophy seen in E2f1Stable/p35 wing discs might be the result of two synergizing growth signals: hyper-active E2f1 and compensatory proliferation from undead cells. The current experiments cannot precisely discern the relative contribution of these two inputs, but E2f1 activity appears to make a larger contribution because E2f1Stable/DBD Mut expression does not cause dramatic overgrowth (Davidson, 2012).

What might explain the 32% of en-Gal4>E2f1Stable discs that displayed a reduced posterior compartment rather than an overgrown one? The DNA damage observed in eye discs experiments provides a possible answer. Perhaps early in development the 'arrest' class of wing discs sustained enough genomic damage to prevent proliferation, resulting in too small a pool of cells that could respond to the hyper-active E2f1 and undead cell signals to support disc overgrowth. Thus, the wide range of phenotypes that were observed in E2f1Stable/p35 wing discs may result from multiple influences that act stochastically within the population (Davidson, 2012).

Because endogenous E2f1 is quantitatively destroyed only in S phase, the relative amount of hyper-accumulation of E2f1Stable is greater during S phase than during any other stage of the cell cycle. Therefore, one possibility is that E2f1Stable-induced phenotypes result from the stability of E2f1 protein in S phase, and not from general over-expression throughout the cell cycle. Failure to detect E2f1Stable induced apoptosis in G1-arrested embryonic cells is consistent with this possibility. However, another difference between these embryonic cells and wing discs cells is that the former are cell cycle arrested and the latter are continuingly proliferating during larval development. Thus, another possibility is that S phase-destruction of E2f1 modulates the levels of E2f1 in proliferating cells, and cells that fail to destroy E2f1 during S phase have an increased chance of activating apoptosis at any point in the cell cycle. In either model, S phase E2f1 destruction is not essential for proliferation per se. In marked contrast, E2f1Stable expression blocks endocycle progression, suggesting that knocking in E2f1Stable to the endogenous locus would be lethal during development, perhaps even dominant lethal (Davidson, 2012).

E2f1Stable induces apoptosis at least in part through expression of the pro-apoptotic gene hid. Surprisingly, these events still occur after expression of an E2f1Stable variant that cannot bind DNA and therefore lacks the ability to stimulate transcription of replication factor genes or cell cycle progression. Instead, E2f1Stable requires the ability to bind Rbf1 to induce hid gene expression and apoptosis. Genetic disruption of Rbf1 is well known to result in increased hid expression. It is therefore proposed that the inappropriate accumulation of E2f1 in S phase disrupts some aspect of Rbf1 function leading to hid expression and apoptosis (Davidson, 2012).

The data do not discern either the function of Rbf1 that is disrupted by E2f1Stable or the mechanism of hid induction. While the mechanism connecting Rbf1/E2f1 function and hid may be indirect, some studies suggest that Rbf1 and/or E2f1 could regulate hid directly. It has been demonstrated that Drosophila wing disc cells undergo apoptosis in response to ionizing radiation independently of p53 and that this response requires E2f1 and is triggered by hid expression. In eye discs, loss of Rbf1 function in the MF results in apoptosis that requires E2f1 transactivation function and is accompanied by hid expression. However, whether these effects represent a direct induction of hid by E2f1 is not clear. E2f1 binding at the hid locus has been observed, but the binding site is located ~1.4 kb upstream of the of the start of hid transcription, which is more distal than in well characterized E2F-regulated promoters. When located this far upstream the hid E2f1 binding site fails to activate gene expression in S2 cell reporter assays. hid is also a target of p53, and so any DNA damage resulting from stabilizing E2f1 during S phase, as was observed in eye discs, may also contribute to the activation of hid expression via p53-mediated DNA damage response pathways (Davidson, 2012).

Another possibility is that E2f1, in combination with Rbf1, plays primarily a repressive role at the hid locus. In this model, the result that E2f1Stable or E2f1Stable/DBD Mut both induce apoptosis would be explained by disruption of Rbf1/E2f1 repressive complexes at the hid locus causing de-repression of hid expression. This model has interesting caveats: what protects the Rbf1/E2f1 complex at the hid locus from being disrupted by Cyclin E/Cdk2, which is active during S phase and inactivates Rbf1-mediated repression of E2f1, or by CRL4Cdt2-mediate destruction of E2f1? Recent data indicate that the dREAM/MMB complex is required for the stability of E2F/Rbf1 repressive complexes in S phase, and acts to protect these complexes from CDK-mediated phosphorylation at non-cell cycle-regulated genes. While there is yet no evidence that dREAM/MMB regulates hid , this work provides precedent for gene specific Rbf1 regulation during S phase (Davidson, 2012).

Finally, while hid might be a critical player in the response to E2f1Stable, there are likely other mechanisms responsible for sensing and modulating the apoptotic response to E2f1 levels. For instance, it has been demonstrated that a micro-RNA, mir-11, which is located within the last intron of the Drosophila E2f1 gene, acts to dampen expression of pro-apoptotic E2f1 target genes following DNA damage. In this way, the normal controls of E2f1 gene expression modulate apoptosis. In addition, transgenic constructs lack the normal E2f1 3' UTR, which serves as a site for suppression of E2f1 expression by pumilio translational repressor complexes. Thus, several modes of E2f1 regulation have been bypassed via transgenic expression of E2f1Stable (Davidson, 2012).

The finding that stabilized Drosophila E2f1 can induce apoptosis independently of transcription and cell cycle progression parallels previous observations made in mammalian cells, albeit with important differences. In mammalian cells, E2F1 can induce apoptosis independently of transcription and cell cycle progression, but apoptosis required E2F1 DNA binding activity, unlike in the current experiments. These studies suggested that DNA binding by E2F1 prevented pro-apoptotic promoters from binding repressor E2F family members (Davidson, 2012).

This comparison of results highlights the way similar phenotypic outcomes in different species can arise from different mechanisms. While mammalian activator E2Fs are also inhibited during S phase, they are not subject to CRL4Cdt2-mediated, S phase-coupled destruction like Drosophila E2f1. Instead, mammalian activator E2Fs are inhibited by direct Cyclin A/Cdk2 phosphorylation, targeted for destruction by SCFSkp2, and functionally antagonized by E2F7 and E2F8. The regulation provided by E2F7 and E2F8 is of particular note, as it is essential for mouse development. These atypical E2Fs homo and hetero-dimerize and act redundantly to repress E2F1 target genes independently of pRb family proteins, thus blocking E2F1 from inducing apoptosis. Moreover, the E2F7 and E2F8 genes are E2F1 targets, consequently creating a negative feedback loop that limits E2F1 activity after the G1/S transition. A similar negative feedback loop among factors that regulate G1/S transcription exists in yeast. The analogous Drosophila negative feedback loop involves E2f1 inducing its own destruction by stimulating Cyclin E transcription, which triggers S phase. Therefore, the evolution of eukaryotes has resulted in the use of different molecular mechanism to achieve negative feedback regulation of G1/S-regulated transcription, and in the case of activator E2Fs this regulation is essential for normal development (Davidson, 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).

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).

Integrated stability and activity control of the Drosophila Rbf1 retinoblastoma protein

Retinoblastoma (RB) family transcriptional corepressors regulate diverse cellular events including cell cycle, senescence, and differentiation. The activity and stability of these proteins are mediated by post-translational modifications, however there is no general understanding of how distinct modifications coordinately impact both of these properties. Previous work has shown that protein turnover and activity are tightly linked through an evolutionarily conserved C-terminal instability element (IE) in the Drosophila RB-related protein Rbf1; surprisingly, mutant proteins with enhanced stability were less, not more active. To better understand how activity and turnover are controlled in this model RB protein, the impact of Cyclin-Cdk kinase regulation on Rbf1 was assessed. An evolutionarily conserved N-terminal threonine residue is required for Cyclin-Cdk response, and showed a dominant impact on turnover and activity, however specific residues in the C terminal IE differentially impacted Rbf1 activity and turnover, indicating an additional level of regulation. Strikingly, specific IE mutations that impaired turnover but not activity induced dramatic developmental phenotypes in the Drosophila eye. Mutation of the highly conserved K774 residue induced hypermorphic phenotypes that mimicked the loss of phosphorylation control; mutation of the corresponding codon of the human RBL2 gene has been reported in lung tumors. These data supports a model in which closely intermingled residues within the conserved IE govern protein turnover, presumably through interactions with E3 ligases, and protein activity, via contacts with E2F transcription partners. Such functional relationships are likely to similarly impact mammalian RB family proteins, with important implications for development and disease (Zhang, 2014).

Mutating RBF can enhance its pro-apoptotic activity and uncovers a new role in tissue homeostasis

The tumor suppressor Retinoblastoma protein (pRb) is inactivated in a wide variety of cancers. While its role during cell cycle is well characterized, little is known about its properties on apoptosis regulation and apoptosis-induced cell responses. pRb shorter forms that can modulate pRB apoptotic properties, resulting from cleavages at caspase specific sites are observed in several cellular contexts. A bioinformatics analysis showed that a putative caspase cleavage site (TELD) is found in the Drosophila homologue of pRb (RBF) at a position similar to the site generating the p76Rb form in mammals. Thus, this study generated a punctual mutant form of RBF in which the aspartate of the TELD site is replaced by an alanine. This mutant form, RBFD253A, conserved the JNK-dependent pro-apoptotic properties of RBF but gained the ability of inducing overgrowth phenotypes in adult wings. This overgrowth is a consequence of an abnormal proliferation in wing imaginal discs, which depends on the JNK pathway activation but not on wingless (wg) ectopic expression. These results show for the first time that the TELD site of RBF could be important to control the function of RBF in tissue homeostasis in vivo (Milet, 2014. PubMed).


Retinoblastoma-family protein: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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