Retinoblastoma-family protein
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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