Retinoblastoma-family protein

DEVELOPMENTAL BIOLOGY

Embryonic

E2F-dependent transcription is down-regulated when G1 control first appears during Drosophila development. Exit from G1 in the 17th cell cycle after fertilization requires E2F and is accompanied by a transient increase in E2F dependent transcription. Because Rbf is a negative regulator of E2F activity, it is possible that some of these changes in E2F activity might be attributable to changes in Rbf expression. However, Rbf is broadly and uniformily expressed in embryos at stage 12 and 13 and is also abundant in early embryos. Thus, changes in Rbf expression do not appear to play an important role in regulating these early cycles. Because E2F expression is also relatively uniform, it appears that changes in E2F activity are attributable to factors acting upstream of Rbf (Du, 1996a).

Sequential changes at differentiation gene promoters as they become active in a stem cell lineage

Transcriptional silencing of terminal differentiation genes by the Polycomb group (PcG) machinery is emerging as a key feature of precursor cells in stem cell lineages. How, then, is this epigenetic silencing reversed for proper cellular differentiation? This study investigate how the developmental program reverses local PcG action to allow expression of terminal differentiation genes in the Drosophila male germline stem cell (GSC) lineage. It was found that the silenced state, set up in precursor cells, is relieved through developmentally regulated sequential events at promoters once cells commit to spermatocyte differentiation. The programmed events include global downregulation of Polycomb repressive complex 2 (PRC2) components, recruitment of hypophosphorylated RNA polymerase II (Pol II) to promoters, as well as the expression and action of testis-specific homologs of TATA-binding protein-associated factors (tTAFs). In addition, action of the testis-specific meiotic arrest complex (tMAC; Drosophila RB, E2F and Myb), a tissue-specific version of the mammalian MIP/dREAM complex, is required both for recruitment of tTAFs to target differentiation genes and for proper cell type-specific localization of PRC1 components and tTAFs within the spermatocyte nucleolus. Together, the action of the tMAC and tTAF cell type-specific chromatin and transcription machinery leads to loss of Polycomb and release of stalled Pol II from the terminal differentiation gene promoters, allowing robust transcription (Chen, 2011).

The results suggest a stepwise series of developmentally programmed events as terminal differentiation genes convert from a transcriptionally silent state in precursor cells to full expression in differentiating spermatocytes. In precursor cells, differentiation genes are repressed and associated with background levels of hypophosphorylated Pol II and H3K4me3. These genes also display elevated levels of H3K27me3 and Polycomb at the promoter region, suggesting that they are acted upon by the PcG transcriptional silencing machinery. Notably, the differentiation genes studied in precursor cells in this study did not show the hallmark bivalent chromatin domains enriched for both the repressive H3K27me3 mark and the active H3K4me3 mark that have been characterized for a cohort of differentiation genes in mammalian ESCs (Chen, 2011).

The cell fate switch from proliferating spermatogonia to the spermatocyte differentiation program initiates both global and local changes in the transcriptional regulatory landscape, starting a cell type-specific gene expression cascade that eventually leads to robust transcription of the terminal differentiation genes. Globally, soon after the switch from spermatogonia to spermatocytes, core subunits of the PRC2 complex are downregulated, including E(z), the enzyme that generates the H3K27me3 mark. Locally, after male germ cells become spermatocytes, Pol II accumulates at the terminal differentiation gene promoters, although these genes still remain transcriptionally silent, with low H3K4me3 and high Polycomb protein levels near their promoters (Chen, 2011).

The next step awaits the expression of spermatocyte-specific forms of core transcription machinery and chromatin-associated regulators, including homologs of subunits of both the general transcription factor TFIID (tTAFs) and the MIP/dREAM complex (Aly and other testis-specific components of tMAC). The tMAC complex acts either locally or globally, perhaps at the level of chromatin or directly through interaction with tTAFs, to allow recruitment of tTAFs to promoters of target terminal differentiation genes. The action of tTAFs then allows full and robust transcription of the terminal differentiation genes, partly by displacing Polycomb from their promoters (Chen, 2011).

Strikingly, the two major PcG protein complexes appear to be regulated differently by the germ cell developmental program: whereas the PRC2 components E(z) and Su(z)12 are downregulated, the PRC1 components Polycomb, Polyhomeotic and dRing continue to be expressed in spermatocytes. The global downregulation of the epigenetic 'writer' E(z) in spermatocytes might facilitate displacement of the epigenetic 'reader', the PRC1 complex, from the differentiation genes, with the local action of tTAFs at promoters serving to select which genes are relieved of PRC1. In addition, the tTAFs act at a second level to regulate Polycomb by recruiting and accompanying Polycomb and several other PRC1 components to a particular subnucleolar domain in spermatocytes. It is not yet known whether sequestering of PRC1 to the nucleolus by tTAFs plays a role in the activation of terminal differentiation genes, perhaps by lowering the level of PRC1 that is available to exchange back on to differentiation gene promoters. Conversely, recruitment of PRC1 to the nucleolar region might have a separate function, such as in chromatin silencing in the XY body as observed in mammalian spermatocytes (Chen, 2011).

The findings indicate that, upon the switch from spermatogonia to spermatocytes, the terminal differentiation genes go through a poised state, marked by presence of both active Pol II and repressive Polycomb, before the genes are actively transcribed. Stalled Pol II and abortive transcript initiation are emerging as a common feature in stem/progenitor cells. This mechanism may prime genes to rapidly respond to developmental cues or environmental stimuli. Stalled Pol II could represent transcription events that have initiated elongation but then pause and await further signals, as in the regulation of gene expression by the androgen receptor or by heat shock. Alternatively, Pol II might be trapped at a nascent preinitiation complex, without melting open the DNA, as found in some instances of transcriptional repression by Polycomb. Although ChIP analyses did not have the resolution to distinguish whether Pol II was stalled at the promoter or had already initiated a short transcript, the results with antibodies specific for unphosphorylated Pol II suggest that Pol II is trapped in a nascent preinitiation complex. The PRC1 component dRing has been shown to monoubiquitylate histone H2A on Lys119 near or just downstream of the transcription start site. It is proposed that in early spermatocytes, before expression of the tTAFs and tMAC, the local action of PRC1 in causing H2AK119ub at the terminal differentiation gene promoters might block efficient clearing of Pol II from the preinitiation complex and prevent transcription elongation (Chen, 2011).

Removal of PRC1 from the promoter and full expression of the terminal differentiation genes in spermatocytes require the expression and action of tMAC and tTAFs. Cell type-specific homologs of TFIID subunits have been shown to act gene-selectively to control developmentally programmed gene expression. For example, incorporation of one subunit of the mammalian TAF4b variant into TFIID strongly influences transcriptional activation at selected promoters, directing a generally expressed transcriptional activator to turn on tissue-specific gene expression (Chen, 2011).

The local action of the tTAFs to relieve repression by Polycomb at target gene promoters provides a mechanism that is both cell type specific and gene selective, allowing expression of some Polycomb-repressed genes while keeping others silent. Similar developmentally programmed mechanisms may also reverse PcG-mediated epigenetic silencing in other stem cell systems. Indeed, striking parallels between the current findings and recent results from mammalian epidermis suggest that molecular strategies are conserved from flies to mammals. In mouse epidermis, the mammalian E(z) homolog Ezh2 is expressed in stem/precursor cells at the basal layer of the skin. Strikingly, as was observed for E(z) and Su(z)12 in the Drosophila male GSC lineage, the Ezh2 level declines sharply as cells cease DNA replication and the epidermal differentiation program is turned on. Overexpression of Ezh2 in epidermal precursor cells delays the onset of terminal differentiation gene expression, and removal of the Ezh2-generated H3K27me3 mark by the Jmjd3 (Kdm6b) demethylase is required for epidermal differentiation (Chen, 2011 and references therein).

In particular, the results suggest a possible explanation for the conundrum that, although PcG components are bound at many transcriptionally silent differentiation genes in mammalian ESCs, loss of function of PcG components does not cause loss of pluripotency but instead causes defects during early embryonic differentiation. In Drosophila male germ cells, events during the switch from precursor cell proliferation to differentiation are required to recruit Pol II to the promoters of differentiation genes. Without this differentiation-dependent recruitment of Pol II, loss of Polycomb is not sufficient to precociously turn on terminal differentiation genes in precursor cells. Rather, Polycomb that is pre-bound at the differentiation gene promoters might serve to delay the onset of their transcription after the mitosis-to-differentiation switch. Robust transcription must await the expression of cell type- and stage-specific components of the transcription machinery. These might in turn guide gene-selective reversal of Polycomb repression to facilitate appropriate differentiation gene expression in specific cell types (Chen, 2011).

Two different specific JNK activators are required to trigger apoptosis or compensatory proliferation in response to Rbf1 in Drosophila

The Jun Kinase (JNK) signaling pathway responds to diverse stimuli by appropriate and specific cellular responses such as apoptosis, differentiation or proliferation. The mechanisms that mediate this specificity remain largely unknown. The core of this signaling pathway, composed of a JNK protein and a JNK kinase (JNKK), can be activated by various putative JNKK kinases (JNKKK) which are themselves downstream of different adaptor proteins. A proposed hypothesis is that the JNK pathway specific response lies in the combination of a JNKKK and an adaptor protein upstream of the JNKK. Previous studies have showed that the Drosophila homolog of pRb (Rbf1) and a mutant form of Rbf1 (Rbf1^D253A) have JNK-dependent pro-apoptotic properties. Rbf1^D253A is also able to induce a JNK-dependent abnormal proliferation. This study shows that Rbf1-induced apoptosis triggers proliferation which depends on the JNK pathway activation. Taking advantage of these phenotypes, this study investigated the JNK signaling involved in either Rbf1-induced apoptosis or in proliferation in response to Rbf1-induced apoptosis. Two different JNK pathways involving different adaptor proteins and kinases were shown to be involved in Rbf1-apoptosis (i.e. Rac1-dTak1-dMekk1-JNK pathway) and in proliferation in response to Rbf1-induced apoptosis (i.e., dTRAF1-Slipper-JNK pathway). Using a transient induction of rbf1, this study shows that Rbf1-induced apoptosis activates a compensatory proliferation mechanism which also depends on Slipper and dTRAF1. Thus, these 2 proteins seem to be key players of compensatory proliferation in Drosophila (Clavier, 2016).

Rbf regulates Drosophila spermatogenesis via control of somatic stem and progenitor cell fate in the larval testis

The Drosophila testis has-been fundamental to understanding how stem cells interact with their endogenous microenvironment, or niche, to control organ growth in vivo. This study reports the identification of two independent alleles for the-highly conserved tumor suppressor gene, Retinoblastoma-family protein (Rbf), in a screen for testis phenotypes in X chromosome third instar lethal alleles. Rbf mutant alleles exhibit overproliferation of spermatogonial cells, which is phenocopied by the molecularly characterized Rbf11 null allele. Rbf promotes cell-cycle exit and differentiation of the somatic and germline stem cells of the testes. Intriguingly, depletion of Rbf specifically in the germline does not disrupt stem cell differentiation, rather Rbf loss of function in the somatic lineage drives overproliferation and differentiation defects in both lineages. Together, these observations suggest that Rbf in the somatic lineage controls germline stem cell renewal and differentiation non-autonomously via essential roles in the microenvironment of the germline lineage (Dominado, 2016).

Rbf activates the myogenic transcriptional program to promote skeletal muscle differentiation

The importance of the retinoblastoma tumor suppressor protein pRB in cell cycle control is well established. However, less is known about its role in differentiation during animal development. This study investigated the role of Rbf, the Drosophila pRB homolog, in adult skeletal muscles. Depletion of Rbf severely reduced muscle growth and altered myofibrillogenesis but only minimally affected myoblast proliferation. An Rbf-dependent transcriptional program in late muscle development was identified that is distinct from the canonical role of Rbf in cell cycle control. Unexpectedly, Rbf acts as a transcriptional activator of the myogenic and metabolic genes in the growing muscles. The genomic regions bound by Rbf contained the binding sites of several factors that genetically interacted with Rbf by modulating Rbf-dependent phenotype. Thus, these results reveal a distinctive role for Rbf as a direct activator of the myogenic transcriptional program that drives late muscle differentiation (Zappia, 2019).

Effects of Mutation or Deletion

Rbf suppresses the phenotype generated by ectopic expression of dE2F and dDP in the developing Drosophila eye. Coexpression of Drosophila E2F and DP in the fly's retina causes ectopic S-phases in regions of the eye that normally contain only postmitotic cells. The adult eyes of flies carrying two copies of E2F and DP are rough and are characterized by abnormal patterns of photoreceptors and cone cells, and by additional bristles. This phenotype is reversed with expression of Rbf in the eye (Du, 1996a and b). Two copies of ectopically expressed Rbf in the eye result in fused ommatidia that lack bristles almost entirely. The majority of retinas expressing four copies of Rbf contain only three cone cells rather than the normal complement of four cells (Du, 1996b).

Loss-of-function cyclin E mutations enhance an Rbf overexpression phenotype, consistent with the idea that the biological activity of Rbf is negatively regulated by endogenous cyclin E. Whereas eyes carrying one extra copy of Rbf are completely normal, introduction of a null allele of cyclin E into this background results in eyes with fused ommatidia and missing bristles, similar to fly eyes carrying two extra copies of Rbf (Du, 1996a).

While flies carrying either two copies of dap or Rbf transgenes have wild-type eyes, the combination results in extremely rough eyes. Many pigment cells are missing, consistent with a marked deficit of precursor cells; the stripe of DNA synthesizing cells posterior to the morphogenetic furrow is completely blocked. Thus dap and Rbf exhibit significant synergy in arresting cell cycle entry in vivo. This synergy is likely to involve binding of Rbf to E2F and interference of cyclin E/cdk involvement with E2F (de Nooij, 1996).

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

Given that E2f is just one of many potential targets of Rbf, the dramatic suppression of the rbf mutant phenotypes by E2f mutants is very unexpected. (1) Lowering the activity of E2f can suppress the early larval lethality of the rbf mutants as well as the developmental phenotypes observed in the adult eyes and bristles. (2) An allele of E2f with an intact DNA binding domain but with no transactivation domain or Rbf binding domain can suppress the lethality of rbf null mutants, allowing the double mutant flies to develop into viable adults. Furthermore, these suppressed rbf null adults show normal adult structures. Thus the uninhibited E2f in rbf mutants mediates both the lethality as well as the observed eye and bristle phenotypes in adults. These observations provide strong evidence that E2F mediates most, if not all, of the phenotypes of rbf during development (Du, 2000).

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

E2F transcription factors can function both to activate transcription and to repress transcription by recruiting RB family proteins to specific promoters. Although analysis of E2f mutants shows that E2f is required for the coordinated expression of replication functions such as PCNA and Ribonucleoside diphosphate reductase small subunit (RnrS), it is not clear whether the lack of transcription of these set of genes is the cause of the larval lethality. It is formally possible that the lethality of E2f mutant is caused by the failure to repress certain critical E2f target genes. Depending on the function of E2f as a transcription activator or a co-repressor of Rbf, completely different predications are expected regarding the genetic interaction between Rbf and E2f. The observation that lowering the level of Rbf can suppress the larval lethality of E2f mutants and allow the E2f mutants to develop into pharate adults, suggests that during Drosophila development, the function of E2f is mainly to activate transcription and not to recruit Rbf to repress transcription (Du, 2000).

In addition to the first E2f to be identified in Drosophila, a second, termed E2f2, has been identified (Sawado, 1998). Interestingly, E2f2 can bind to E2F binding sites, but the function of E2f2 appears to be distinct from that of E2f. Cotransfection of E2f2 represses the expression from the PCNA gene promoter while cotransfection of E2f activates the expression (Sawado, 1998). Similar findings are also observed in transfection experiments in which E2f strongly activates transcription, while E2f2 fails to activate a reporter with E2F binding sites. These results suggest that E2f2 may function mainly to repress transcription while E2f functions mainly to activate transcription. Taken together, these data suggest a model for the function of Rbf, E2f and E2f2. In this model, E2f functions mainly to activate transcription of the E2f target genes. Rbf negatively regulates the activity of E2f to inhibit the expression of E2f target genes. In addition, Rbf can also repress the expression of E2F targets genes through other targets of Rbf such as E2f2. Thus the expression of E2F target genes will have three different states: activated, when there is free E2f/Dp to activate transcription; repressed, when there is E2f2/Dp/Rbf (and possibly E2f/Dp/Rbf) to repress transcription; and basal, when there is neither activation nor repression. At present, it is not clear whether E2f also has a function to repress transcription during development, nor is it is clear about the function of free E2f2/Dp (Du, 2000).

Many studies have indicated that the major substrates for vertebrate D-type cyclin-Cdk complexes are the Rb family proteins. A Drosophila homolog of Rb, RBF, inhibits cell division when overexpressed in the wing imaginal disc. RBF does not immediately block cell growth, and as a result there is a large increase in cell size. To test interactions between CycD-Cyclin-dependent kinase 4/6) and RBF, all three genes were co-expressed together in wings, salivary glands or eyes. Using the flip-out Gal4 method in the wing, it was found that cells co-expressing CycD-Cdk4 and RBF cycle more rapidly than cells expressing RBF alone, and thus that CycD-Cdk4 attenuates the inhibitory effects of RBF on cell cycle progression. Interestingly, although cell clones co-expressing CycD-Cdk4 and RBF have fewer cells than wild-type controls, they encompassed substantially more area. Flow cytometry and in situ cell size measurements confirm that the increased mass of these clones is due to increased cell size. The large size of cells co-expressing CycD-Cdk4 and RBF, despite a nearly normal division rate, suggests that CycD-Cdk4 promotes extra growth even while RBF slows cell cycle progression. A logical inference is that CycD-Cdk4 promotes growth via targets other than RBF. This effect is less evident at a later time-point (67 h.p.i.), perhaps because cell cycle suppression by RBF eventually throttles even the growth of CycD-Cdk4-expressing clones (Datar, 2000).

RBF-CycD interaction tests were performed in the larval salivary gland, a differentiated tissue in which cell growth is accomplished by cycles of DNA endoreplication. To express UAS-linked target genes, the F4-Gal4 driver was used, that commences its expression late in embryogenesis after cell proliferation in the salivary primordium is complete and stays active throughout the larval stages. Results obtained in the salivary glands are consistent with those described above for the wing. F4-Gal4-driven expression of CycD-Cdk4 results in oversized salivary glands with nuclei containing excessively endoreduplicated polytene chromosomes. Co-expression of cyclin D with the kinase impaired variant Cdk4^D175N or Cdk2 does not induce this size increase, suggesting that the protein kinase activity of cyclin D-Cdk4 complexes is required for the stimulation of salivary gland DNA endoreplication and growth. Although expression of Cdk4^D175N alone has little effect on salivary growth, overexpressed RBF strongly inhibits both growth and DNA endoreplication. Even stronger growth suppression is observed when RBF is co-expressed with Cdk4^D175N. As in wings and eyes, the growth-inhibitory effects of RBF are attenuated by simultaneous co-expression of CycD-Cdk4 (Datar, 2000).

Similar results are obtained when RBF and CycD-Cdk4 are co-expressed in the eye using ey-Gal4, which is expressed throughout the eye primordium beginning very early in eye development. Expression of RBF under ey-Gal4 control causes a dramatic reduction of the adult eye. This loss of eye tissue is suppressed virtually completely when CycD-Cdk4 is co-expressed with RBF, providing further evidence that CycD-Cdk4 can functionally inactivate RBF. Interestingly, suppression of RBF is also observed when CycD is expressed without the Cdk4 kinase subunit or to a lesser extent when CycD is co-expressed with the kinase-impaired variant Cdk4^D175N. This suggests that CycD may suppress RBF function by utilizing an endogenous Cdk, or in a kinase-independent fashion (Datar, 2000).

Although ectopic RBF suppresses growth in developing wings, salivary glands and eyes, growth inhibition by RBF is secondary to its effects on cell cycle progression. The tests described above are consistent with this interpretation, since in all cases both cell cycle progression (DNA replication) and growth are affected coincidentally. However, these tests cannot rule out the possibility that RBF inhibits cellular growth directly. Therefore the late acting eye specific drivers, GMR-Gal4 and sev-Gal4, were used to test whether RBF could suppress growth in non-cycling cells. Expression of RBF alone using these drivers has little effect on eye size, suggesting that RBF cannot suppress the post-mitotic growth that normally occurs in the eye. Moreover, co-expressed RBF does not substantially suppress eye overgrowth caused by GMR-Gal4- or sev-Gal4-driven CycD-Cdk4. Given this, a parsimonious interpretation of all these interaction tests is that whereas CycD-Cdk4 can promote cellular growth in both proliferating and non-cycling cells, RBF can suppress growth only in cells that are undergoing cycles of DNA replication. In this case, growth suppression by ectopic RBF most likely stems from its ability to inhibit cell cycle progression (Datar, 2000).

To further test RBF function, FLP/FRT-mediated mitotic recombination was used to generate cell clones homozygous for a null allele of rbf (rbf¹⁴). Areas of rbf^14/14 cell clones were measured in wing discs, and these were compared with the areas of their rbf^+/+ sister clones ('twin spots'). Cell clones mutant for rbf are slightly smaller than their wild-type twin spots, though the difference is not statistically significant. Thus, loss of RBF does not confer a growth advantage. FACS analysis of rbf^14/14 cells shows a reduced cell size and, surprisingly, an increased G₁ population. Microscopic examination reveals many pyknotic nuclei associated with rbf^14/14 mutant clones, suggesting elevated levels of apoptosis. Reduced cell size and poor viability have also been observed in hyperproliferative cell clones overexpressing E2F/DP, and similar characteristics have been described for mouse embryo fibroblasts lacking pRB, p107 or p130. One explanation for these phenotypes is that they arise when cell division rates outpace rates of cell growth (Neufeld, 1998). This may also be the case for rbf^14/14 cells (Datar, 2000).

Finally, large rbf^14/14 clones were generated in the eye using the Minute technique. Although these clones populate large fractions of the eye they do not exhibit the hypertrophic characteristics noted when CycD-Cdk4 is overexpressed. Instead, rbf^14/14 clones in the adult eye exhibit slight to moderate hypoplasia and mild defects such as missing or duplicated inter-ommatidial bristles and fused ommatidiae. Similar phenotypes have been noted in eyes overexpressing E2F or cyclin E, both of which promote extra cell division in the eye without increasing growth. These results are consistent with the interpretation that RBF functions to inhibit cell cycle progression, rather than to inhibit cellular growth directly. If this is the case, stimulation of cellular growth by CycD-Cdk4 must be, at least in part, independent of RBF (Datar, 2000).

Vertebrate D-type cyclin-cdk complexes inhibit Rb function. The defects observed in Cdk4 mutants, therefore, might reflect increased Rb function. To evaluate this notion, the effects were studied of heterozygosity for mutations in the gene encoding the Drosophila Rb family member (RBF) on the fertility and size of Cdk4 mutant females. For these experiments two putative null alleles, RBF¹¹ and RBF¹⁴, were used. A PCR assay was developed to monitor the presence of the RBF¹¹ allele. Cdk4 mutant females heterozygous for RBF¹¹ (RBF¹¹/+; Cdk4³/Cdk4³) were found to be significantly more fertile than sibling females without RBF¹¹ (+/+; Cdk4³/Cdk4³). In the experiment with RBF¹⁴, the presence of this mutation could not be monitored. Based on the crossing scheme, however, one half of the Cdk4 mutant females were expected to be heterozygous for RBF¹⁴ (RBF¹⁴/+; Cdk4³/Cdk4³), while the other half was expected to have two functional RBF gene copies (+/+; Cdk4³/Cdk4³). As in the RBF¹¹ experiment, an increased fertility was observed in ~50% of the Cdk4 mutant females in the RBF¹⁴ experiment. Therefore, the 50% of females with increased fertility presumably correspond to the RBF¹⁴ heterozygotes. It is concluded that the fertility of Cdk4mutant females is increased by a reduction from two to one in the copy number of functional RBF genes. Similarly, heterozygosity for mutations in RBF was found to increase the weight of Cdk4 mutant females. The antagonistic activities of Cdk4 and RBF could also be demonstrated in experiments involving overexpression using the UAS-GAL4 system (Meyer, 2000).

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

RBF-280 cannot be regulated by Cyclin D- and Cyclin E-dependent kinases. RBF-280 was used to test the role of RBF in developmentally regulated cell proliferation and in Cyclin D/Cdk4-induced cellular growth. Inhibiting the E2F target gene expression in the second mitotic wave of the developing eye, via expression of RBF-280, mainly delays S-phase completion instead of inhibiting S phase entry. These results suggest that cells in the second mitotic wave are driven into S phase through an RB/E2F-independent mechanism. In addition, while RBF-280 completely inhibits cellular growth induced by Cyclin D/Cdk4 in the proliferating wing discs, RBF-280 cannot block cellular growth induced by activated Ras in the wing disc cells or Cyclin D/Cdk4 in the non-dividing eye cells. These observations indicate that the ability of Cyclin D/Cdk4 to induce growth in the proliferating wing discs is distinct from the ability of activated Ras to induce growth or the ability of Cyclin D/Cdk4 to induce growth in the non-dividing eye cells. In addition, these results suggest that RBF may have a role in inhibiting growth (Xin, 2002).

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

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

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

It is interesting to note that delaying the completion of S phase leads to the development of adult eyes with missing bristles and fused ommatidia, similar to the adult eyes developed when the second mitotic wave is inhibited. These observations suggest that not only cell proliferation, but also the rate of cell proliferation, need to be tightly coordinated with differentiation in certain developmental settings. How might delaying the completion of S phase lead to phenotypes similar to inhibition of S phase? It is possible that there is only a very short time window that a specific cell type can be recruited into the ommatidia clusters from the surrounding cells. Delaying the completion of S phase may lead to a lack of cells that can be recruited locally, resulting in the phenotype of missing cone cells, pigment cells and bristles. In addition, it is possible that specific phases of the cell cycle (such as S phase and M phase) are incompatible with the ommatidia recruitment process. Thus, those ommatidia clusters that are surrounded by cells that are still in S phase will not be able to recruit additional cells into the ommatidia. There are some reports that support this idea. For example, the protein serine/threonine kinase Tribbles is required to prevent premature mitosis by inducing specific degradation of String and Twine during Drosophila embryogenesis. Failure to prevent the premature mitosis leads to defects in gastrulation. Similarly, failure to have G1 arrest in roughex mutant eye discs results in defects in cell fate determination, as well as abnormalities in the adult eye. Further studies will be needed to directly test the relationship between cell differentiation and cell cycle phasing (Xin, 2002).

Cyclin D/Cdk4 is able to drive cellular growth in addition to induce cell proliferation. Interestingly, the consequence of Cyclin D/Cdk4 expression is different in different cell types: Cyclin D/Cdk4 primarily induces growth and lead to larger cells in the non-dividing differentiated eye cells (hypertrophy); Cyclin D/Cdk4 induces increased DNA endoreplication and increased cell size (hypertrophy) in the salivary gland cells; Cyclin D/Cdk4 induces growth and division coordinately without affecting cell size in the proliferating wing discs, leading to more rapid cell cycle and more cells (hyperplasia) (Datar, 2000). To test if these biological effects are mediated by RBF, the effect of RBF-280, a form of RBF that cannot be regulated by Cyclin D, was tested on cellular growth and proliferation induced by Cyclin D and Cdk4 (Xin, 2002).

RBF is an important target of Cyclin D/Cdk4 in G1/S regulation. RBF-280 blocks the ability of Cyclin D to induce S phase in G1 arrest eye disc cells and the excessive DNA endoreplication in the salivary gland cells. In addition, RBF-280 also blocks the ability of Cyclin D/Cdk4 to increase the rate of cell proliferation in the proliferating wing discs. These results demonstrate that the ability of Cyclin D/Cdk4 to induce cell proliferation (G1/S transition) is mediated through inactivation of RBF. By contrast, the ability of RBF-280 to block Cyclin D/Cdk4 induced growth varies in different cell types. In the proliferating wing disc cells, Cyclin D/Cdk4 expression leads to more rapid cell cycle and more cells (hyperplasia). RBF-280 completely blocks the effect of Cyclin D/Cdk4. The average size of RBF-280 clones is not significantly different from the average size of Cyclin D+Cdk4+RBF-280 clones (P=0.68). In addition, the number of cells in the clone is also not significantly different (P=0.32). These observations indicate that RBF-280 blocks both cell growth and proliferation induced by Cyclin D/Cdk4 in the proliferating wing discs. The effect of RBF-280 on Cyclin D/Cdk4-induced growth is distinct from the effect of RBF-280 on activated Ras induced growth. Activated Ras induces cellular growth and leads to larger cells without affecting the rate of cell doubling in the wing discs (hypertrophy). The average area of Ras^V12+RBF-280 clones is significantly larger than the area of RBF-280 clones (P<0.0001). The observed increase in clone size is mainly due to increased cell size. Although it is not possible to get enough cells for Facs analysis to determine the cell size directly, because clones with RBF-280 expression are extremely small, cell size estimation using the average cell sizes derived from clone area/cell number has shown that RBF-280+Ras^V12 cells arenoticeably larger than RBF-280 cells, while the size of RBF-280+Cyclin D+Cdk4 cells is similar to the size of RBF-280 cells. These results are consistent with the reported cell size effect of Cyclin D/Cdk4 and activated Ras on RBF-WT. It was shown that the Cyclin D/Cdk4+RBF-WT cells are similar in size as the RBF-WT cells, while the Ras^V12+RBF-WT cells are much larger than the RBF-WT cells. Taken together, this evidence supports the notion that Ras^V12 can stimulate cellular growth in the presence of functional RBF, while Cyclin D/Cdk4 induces growth at least in part through inactivation of RBF in the developing wing discs (Xin, 2002).

Similar to activated Ras, Cyclin D/Cdk4 also induces growth and leads to large eyes as a result of increased cell size in the non-dividing eye cells (hypertrophy). Consistent with the idea that the large eye phenotypes are the consequence of cellular growth induced by Cyclin D/Cdk4, which is mediated through targets distinct from RBF, RBF-280 blocks Cyclin D/Cdk4-induced ectopic S phase in the eye discs but not Cyclin D/Cdk4 induced large eye phenotype. It is likely that Cyclin D/Cdk4 drives growth through distinct targets in the non-proliferating eye cells and in the proliferating wing disc cells. The target in the non-proliferating eye cells can drive growth in the presence of RBF-280, while the target in the proliferating wing disc cells are either RBF itself or a target that can drive growth only when RBF is inactivated. Further studies will be needed to identify the targets that mediate the ability of Cyclin D/Cdk4 to induce growth (Xin, 2002).

In addition to increase cell sizes, the Ras^V12+RBF-280 clones also have more cells (faster cell doubling time) than the RBF-280 clones (P=0.0002). These observations suggest that activated Ras can increase proliferation in addition to growth in the presence of RBF-280. This seems to be contradictory to the observations that Ras^V12 expression alone does not appear to affect the rate of cell doubling significantly. Interestingly, although no statistical difference is observed in cell doubling rate between Ras^V12 and wild-type control (P=0.60), Ras^V12 overexpression clones do have a few more cells than do the wild-type control clones. A plausible explanation is that the observed increase in cell number by Ras^V12 is RBF independent, and the contribution of this difference in cell number may become statistically significant when the total number of cells in each clone is reduced in the presence of RBF-280. It is possible that this observed small increase in cell doubling might be the consequence of increased growth by activated Ras (Xin, 2002).

Although mutations that activate the Hedgehog (Hh) signaling pathway have been linked to several types of cancer, the molecular and cellular basis of Hh's ability to induce tumor formation is not well understood. A mutation in patched (ptc), an inhibitor of Hh signaling, was identified in a genetic screen for regulators of the Retinoblastoma (Rb) pathway in Drosophila. Hh signaling promotes transcription of Cyclin E and Cyclin D, two inhibitors of Rb, and principal regulators of the cell cycle during development in Drosophila. Upregulation of Cyclin E expression, accomplished through binding of Cubitus interruptus (Ci) to the Cyclin E promoter, mediates the ability of Hh to induce DNA replication. Upregulation of Cyclin D expression by Hh mediates the distinct ability of Hh to promote cellular growth. The discovery of a direct connection between Hh signaling and principal cell-cycle regulators provides insight into the mechanism by which deregulated Hh signaling promotes tumor formation (Duman-Scheel, 2002).

During eye development in Drosophila, initiation of neural differentiation, marked by an indentation referred to as the morphogenetic furrow, begins at the posterior end of the disc and passes anteriorly. Cells within the furrow arrest in G1 phase before differentiating. Cells located just posterior to the furrow exit G1 arrest and enter a synchronous S phase referred to as the second mitotic wave. Overexpression of the Drosophila Retinoblastoma-family gene (Rbf), an inhibitor of the S phase promoting transcription factor E2F, produces a 'rough' adult eye phenotype, characterized by loss of bristles and fusion of ommatidia. This phenotype results from delay of S phase progression in cells of the second mitotic wave as a consequence of inhibited E2F target gene expression. Loss of one copy of the ptc gene suppresses this rough eye phenotype and restores E2F target gene expression. The observed genetic interaction between Rbf and ptc suggests that the Hh signaling pathway might regulate the cell cycle during eye development (Duman-Scheel, 2002).

The ability of Hh signaling to induce expression of Cyclin D may explain why increased Hh signaling suppresses phenotypes associated with RBF overexpression. In support of this idea, overexpression of Ci, which induces Cyclin D expression, also induces ectopic expression of PCNA, an E2F target gene, in both the furrow and in G1-arrested cells located in the wing margin. Coexpression with Ci of RBF-280, a constitutively active form of RBF that cannot be regulated by Cyclin D or Cyclin E, blocks the ability of Ci to induce ectopic PCNA expression in the furrow and wing margin. However, although RBF-280 can block the ability of Ci to induce E2F target gene expression, it does not block the ability of Ci to promote S phase in the eye. These results indicate that although Hh signaling induces E2F target gene expression, it must also be capable of inducing S phase independently of E2F (Duman-Scheel, 2002).

Cell cycle-dependent and cell cycle-independent control of transcription by the E2F/RB pathway

To determine which E2F/RB-family members are functionally important at E2F-dependent promoters, RNA interference (RNAi) was used to selectively remove each component of the dE2F/dDP/RBF pathway, and the genome-wide changes in gene expression that occur when each element is missing. The results reveal a remarkable division of labor between family members. Classic E2F targets, encoding functions needed for cell cycle progression, are expressed in cycling cells and are primarily dependent on dE2F1 and RBF1 for regulation. Unexpectedly, there is a second program of E2F/RBF-dependent transcription, in which E2F2/RBF1 or E2F2/RBF2 complexes repress gene expression in actively proliferating cells. These new E2F target genes encode differentiation factors that are transcribed in developmentally regulated and gender-specific patterns and not in a cell cycle-regulated manner. It is proposed that E2F/RBF complexes should not be viewed simply as a cell cycle regulator of transcription. Instead, E2F/RBF-mediated repression is exerted on genes that encode an assortment of cellular functions, and these effects are reversed on sets of functionally related genes in particular developmental contexts. As a result, dE2F/RBF regulation is used to link gene expression with cell cycle progression at some targets while simultaneously providing stable repression at others (Dimova, 2003).

The results challenge the dogma that E2F-regulated genes have cell cycle-regulated patterns of expression. At Group E promoters, E2F2 and RBF proteins provide a repressor activity that is uncoupled from cell cycle progression, and the loss of E2F-mediated repression results in the inappropriate expression of tissue-specific genes and markers of differentiation (Dimova, 2003).

ChIP experiments illustrate two clear-cut differences between the promoters of Group E genes and the more conventional, cell cycle-regulated E2F targets. The first distinction lies in the recruitment of the activator E2F vs. E2F1. E2F2, DP, RBF1, and RBF2 are readily detected at most E2F-dependent genes, and at each of the different groups of E2F targets uncovered in this study, E2F1 is conspicuously and specifically absent from Group E promoters. This specificity does not, at first glance, appear to be due to a simple distinction in the types of E2F binding sites. Computer searches revealed multiple E2F-like binding sites upstream of Group E genes, but each of these variants could also be found in Group A and Group B promoters. It seems likely therefore that the specific recruitment of E2F proteins is influenced by selective interactions with other factors). The absence of dE2F1 at Group E promoters provides a simple mechanism to explain why these promoters escape the cell cycle-regulated burst of dE2F1-mediated activation that occurs during G1/S progression (Dimova, 2003).

Based on these results, a revised view of E2F regulation in Drosophila is presented. It is suggested that dE2F2-repressor complexes occupy the promoters of a diverse variety of genes. Such dE2F2-mediated repression is relieved at particular subsets of genes in response to cues that may come from developmental signals or from cell cycle signals. At cell cycle-regulated, E2F-controlled promoters, the transcriptional activation is mediated by dE2F1, and this switch from repression to activation is likely to involve Cdk-mediated disruption of the repressor complexes. However, dE2F1 fails to target other dE2F2-repressed genes, and the repressor complexes remain stable. Based on the restricted expression patterns of Group E genes, and the failure to detect dE2F1 at Group E genes even when dE2F2 is removed, it is proposed that dE2F2/RBF-mediated repression is relieved at these targets by developmentally regulated signals, and that gene expression is driven by factors other than dE2F1. The notion that not all E2F-regulated genes are expressed at any one time raises the question of whether the set of targets that are induced by activator E2Fs in cycling cells is fixed or variable. Recent studies of mammalian E2F proteins show that the recruitment of activator E2Fs to a promoter involves synergistic interactions with adjacent transcription factors. It is therefore easy to imagine how the expression of genes that have the potential to be induced by activator E2Fs might also be tailored in different cellular situations to favor different subsets of targets (Dimova, 2003).

Extracellular signals responsible for spatially regulated proliferation in the differentiating Drosophila eye

Spatially and temporally choreographed cell cycles accompany the differentiation of the Drosophila retina. The extracellular signals that control these patterns have been identified through mosaic analysis of mutations in signal transduction pathways. All cells arrest in G1 prior to the start of neurogenesis. Arrest depends on Dpp and Hh, acting redundantly. Most cells then go through a synchronous round of cell division before fate specification and terminal cell cycle exit. Cell cycle entry is induced by Notch signaling and opposed in subsets of cells by EGF receptor activity. Unusually, Cyclin E levels are not limiting for retinal cell cycles. Rbf/E2F and the Cyclin E antagonist Dacapo are important, however. All retinal cells, including the postmitotic photoreceptor neurons, continue dividing when rbf and dacapo are mutated simultaneously. These studies identify the specific extracellular signals that pattern the retinal cell cycles and show how differentiation can be uncoupled from cell cycle exit (Firth, 2005).

The EGFR holds R2-R5 cells in G1 phase and promotes G2/M progression of other cells during the second mitotic wave (SMW). Earlier regulation is now found to depend on longer-range signaling by the Hh, DPP, and N signals already known to drive the progression of the morphogenetic furrow. These studies exclude other models that show that Hh, Dpp, or N act indirectly by releasing other, cell cycle-specific signals from differentiating cells, or that patterned cell cycle withdrawal or reentry occur independent of extracellular signals, such as by synchronized growth. Instead, specific signals are necessary or sufficient for each aspect of cell cycle patterning (Firth, 2005).

G1 arrest ahead of the morphogenetic furrow depends on posterior-to-anterior spread of Hh and Dpp. Hh is secreted from differentiating cells, starting at column 0 in the morphogenetic furrow. Dpp is transcribed in ~6 ommatidial columns in the morphogenetic furrow in response to Hh. Cells accumulate in G1 about 16-17 cell diameters anterior to column 0, suggesting an effective range of ~13-17 cells for Hh and Dpp (Firth, 2005).

The contribution of Dpp to this cell cycle arrest is known already, but that of Hh was not suspected. Both Dpp and Hh signaling can promote proliferation in other developmental contexts (Firth, 2005).

S phase entry in the SMW depends on another signal, N. Expression of the N ligand Dl begins at the anterior of the morphogenetic furrow. The first S phase cells are detected 6-8 cell diameters more posteriorly, just behind column 0. The transmembrane protein Dl must act more locally or more slowly than the secreted Hh and Dpp proteins, to explain gaps between S phases (Firth, 2005).

Although N activity has been associated with growth through indirect mechanisms involving the release of other secreted growth factors and also regulates endocycles, this appears to be the first report of a specific role of N in G1/S in diploid Drosophila cells. Notably, deregulated N signaling contributes to at least two human cancers and is oncogenic in mice (Firth, 2005).

At the same time that N promotes S phase entry in the SMW, EGFR activity ensures that R2-R5 cells remain in G1. N is still required in the absence of EGFR, so N activity is a positive signal and is not required only to counteract EGFR activity. Instead, EGFR activity interferes with S phase entry in response to N (Firth, 2005).

Ligands for the EGF receptor are thought to be released from R8 precursor cells, although EGFR-dependent MAPK phosphorylation is detected one ommatidial column before the column where R8 precursor cells can be identified, which is in column 0. This means that EGFR activation begins after Dl expression but before S phase DNA synthesis starts. Later, ligands released from differentiating precluster cells activate EGFR in surrounding cells to permit SMW mitosis around columns 3-5 (Firth, 2005).

Hh and Dpp together promote expression of Dl and of EGFR ligands; in part, this occurs indirectly through Atonal and the onset of differentiation. EGF receptor activity also promotes Dl expression (Firth, 2005).

At least three genetic mechanisms arrest distinct retinal cells in G1. Arrest ahead of the morphogenetic furrow depends on Dpp and Hh. During the SMW, R2-R5 cells are held in G1 by EGFR, which counteracts the SMW-promoting N activity. In addition, R8 cells, which are defined by the proneural gene atonal, remain in G1 independent of EGFR. After the SMW, all cells remain in G1 indefinitely, independent of EGFR. Although cell cycle withdrawal roughly correlates with differentiation, many of the cells that arrest after the SMW are still unspecified (Firth, 2005).

Loss of rbf and dap together overcome all cell cycle blocks, even though cell differentiation continues. This redundancy indicates that Cyclin E/Cdk2 targets other than Rbf are needed for proliferation, consistent with many other studies. Dap may be regulated by EGFR in R2-R5 cells. If rbf regulates the normal SMW, where Cyclin E expression seems not to be limiting, then other E2F targets may be involved. Some cell cycle arrest can also be overridden by forced expression of Cyclin E, E2F/DP, dRef, and ORC1, or by mutation of the Cyclin A antagonist rux (Firth, 2005).

The results show that mechanisms that assure both short- and long-term arrest of retinal cells must operate upstream of (or parallel to) Rbf and Cyclin E activities. They might resemble the barriers to transformation and regeneration that exist in mammals (Firth, 2005).

Synthetic lethality of retinoblastoma mutant cells in the Drosophila eye by mutation of a novel peptidyl prolyl isomerase gene

Mutations that inactivate the retinoblastoma (Rb) pathway are common in human tumors. Such mutations promote tumor growth by deregulating the G1 cell cycle checkpoint. However, uncontrolled cell cycle progression can also produce new liabilities for cell survival. To uncover such liabilities in Rb mutant cells, a clonal screen was performed in the Drosophila eye to identify second-site mutations that eliminate Rbf^- cells, but allow Rbf⁺ cells to survive. This study reports the identification of a mutation in a novel highly conserved peptidyl prolyl isomerase (PPIase) that selectively eliminates Rbf^- cells from the Drosophila eye (Edgar, 2005).

Peptidyl prolyl isomerases belong to an extended protein superfamily whose members all catalyze the cis-trans isomerization of proline imidic bonds in polypeptides. The superfamily includes the cyclophilin-like peptidyl prolyl isomerases (Cyp), the FK-506-binding proteins (immunophilin/FKBP), and the parvulin/Pin proteins. In addition to sequence and structural divergence, differences in substrates and sensitivity to inhibitors distinguish members within these families. Mechanistically, interconversion of x-Pro bond cis-trans conformation can alter protein folding and the conformation of the native state, leading to potential effects on protein function and regulation of serine/threonine phosphorylation events. PPIases have been shown to play diverse functional roles in the cell and some, like Pin1, have been implicated in cellular transformation and human cancer (Edgar, 2005 and references therein).

There is considerable evidence in the literature to support a mechanistic link between the PPIase Pin1 and its regulation of the cell cycle and apoptosis. Pin1 alters the conformation of the p53 family members p53 and p73 and is required for them to induce the DNA damage checkpoint in response to genotoxic stress. Pin1 has also been shown to interact with Cdc25 and Plk1 and to modulate Cyclin D1 expression levels and activity and Rb phosphorylation. In turn, Pin1 itself is a direct target of E2F activity, participating in a positive feedback loop involving cyclin D1/Cdks, E2F, and RB1. Loss of Pin1 in mouse embryonic fibroblasts causes cell cycle defects and decreases the levels of cyclinD1 and phosphorylated RB1. Similarly, Pin1 knockout mice display a range of proliferative defects, many of which are attributed to its effects on Cyclin D1. Although KIAA0073, the human ortholog of CG3511, has not been studied as extensively as Pin1, it is possible that KIAA0073 and other PPIases aside from Pin1 might also interact with components of the cell cycle and checkpoint pathways, as has been suggested from the comparatively mild knockout phenotype observed for murine Pin1 (Edgar, 2005 and references therein).

In summary, a novel conserved gene, CG3511, is described which when mutated or when its transcript levels are reduced in abundance results in the specific loss of Rbf^- cells in the Drosophila eye. Future experiments will elucidate how the PPIase protein family may interact with RB1 to regulate cell survival and/or proliferation. KIAA0073 may represent an efficacious and novel anti-cancer drug target whose inhibition might result in the specific death of RB1 mutant cells. Such a synthetic lethal target would have applications in several RB1 pathway-dependent cancers, such as small cell lung cancers (SCLCs), and may represent a unique opportunity for targeted therapeutics (Edgar, 2005).

Retinoblastoma family 2 is required in vivo for the tissue-specific repression of dE2F2 target genes

In higher eukaryotes, the Retinoblastoma and E2F families of proteins control the transcription of a large number of target genes. The second Drosophila Retinoblastoma family gene (Rbf2) has been mutated, and the in vivo molecular functions of RBF2 and dE2F2, the only E2F partner of RBF2, have been compared. Previous studies failed to uncover a unique role for RBF2 in E2F regulation. This study found that RBF2 functions in concert with dE2F2 in vivo to repress the expression of differentiation markers in ovaries and embryos where RBF2 is highly expressed. The profiles of transcripts that are mis-expressed in ovaries, embryos and S2 cells where RBF2 function has been ablated were examined, and RBF2 and dE2F2 were found to control strikingly different transcriptional programs in each situation. In vivo promoter occupancy studies point to the redistribution of dE2F/RBF complexes to different promoters in different cell types as one mechanism governing the tissue-specific regulation of dE2F/RBF target genes. These results demonstrate that RBF2 has a unique function in repressing E2F-regulated differentiation markers and that dE2F2 and RBF2 are required to regulate different sets of target genes in different tissues (Stevaux, 2005).

In order to understand the molecular basis for the differential effects observed following removal of dE2F2 and RBF2 in ovaries and S2 cells, focus was placed on genes that were upregulated in de2f2 mutant ovaries, but that were unchanged in the equivalent microarrays of S2 cells following the depletion of dE2F or RBF proteins, and the occupancy of the promoters of such targets was compared in ovaries and in S2 cells. Arp53D served as a positive control for this experiment since it was de-repressed in the absence of dE2F2 in both S2 cells and ovaries. As one would expect, the Arp53D promoter is occupied by dE2F2 and RBF2 in both settings (Stevaux, 2005).

Clear-cut results for the four promoters examined. The promoters of two targets, CG10654 and CG14610, are occupied by RBF2 and dE2F2 in ovaries but not in S2 cells. These changes in promoter occupancy provide a very simple explanation for why their expression levels change in ovaries but not in S2 cells following dE2F2/RBF2 removal: these promoters are not occupied by dE2F2/RBF2 repressor complexes in S2 cells, hence removing these proteins in these cells has no effect; but they are occupied by dE2F2/RBF2 complexes in ovaries, hence in this organ, removing these repressor proteins results in increase gene expression. These changes illustrate that dE2F/RBF repressor proteins are physically redistributed to different promoters in different cell types (Stevaux, 2005).

Promoter occupancy analysis of the CG5245 and CG15267 promoters provided an equally clear result, but a different answer. The ChIP experiments show that these promoters are occupied by dE2F2 and RBF2 in ovaries as well as in S2 cells. However, in ovaries the binding by dE2F2 and RBF2 is functionally important since the transcription of these genes is elevated when these proteins are removed. In contrast, removing dE2F2 and RBF2 in S2 cells had no effect on the expression of these genes. Thus, the functional importance of RBF2 and dE2F2 at these promoters varies in different cell types. In S2 cells, dE2F2 and RBF2 can be found at promoters of genes whose expression do not change in the absence of either dE2F2 or RBF2. The results from this study demonstrate that the functional importance of individual dE2F/RBF proteins varies not only between different promoters in a given cell type, but also at the same promoter in different cell types (Stevaux, 2005).

Thus, in vivo promoter-binding studies point to two fundamentally different mechanisms that contribute to the existence of cell-type specific dE2F/RBF transcriptional programs. First dE2F/RBF repressor complexes are physically relocalized to different promoters in different cell types, and second the functional importance of individual RBF and dE2F family members in regulating particular target genes changes in different cell types. Rbf2 and dE2F2 control changing sets of target genes during development. As a further test of the idea that RBF2 controls the expression of different sets of genes in different cellular and developmental contexts, the RBF2 transcriptional program was studied at a different developmental stage. Given that RBF2 is part of the recently described dream embryonic transcriptional repression complex and that RBF2 levels are markedly increased in embryos 4 to 8 hours after egg deposition, the transcriptional profiles of wild type and rbf2 mutant embryos were compared using microarrays at this early embryonic developmental stage. Numerous transcripts were found elevated in early rbf2 mutant embryos, including a number of ovary- and testis-specific differentiation markers. In keeping with earlier observations, the overlap between transcripts elevated in rbf2 mutant embryos, rbf2 mutant ovaries and S2 cells depleted for RBF2 was minimal. Interestingly, S2 cells, which are of embryonic origin, do not display upregulation of the genes elevated in de2f2 and rbf2 mutant embryos. The lack of deregulation in S2 cells may stem from the fact that RBF2 and dE2F2 control different sets of genes at different developmental stages: S2 cells are derived from 20-24 hour-old embryos, whereas in vivo target gene deregulation is observed in 4-8 hour-old embryos. Alternatively, these differences could be due to the fact that the cell lineage in which the dE2F2/RBF2 embryo-specific transcripts are elevated in vivo is different than the lineage from which S2 cells are derived (Stevaux, 2005).

Transcripts identified in the rbf2 embryo array were further analyzed by Northern blot. Transcripts that are repressed in the embryo by RBF2 and dE2F2 (Kek1, CG8607 and CG3509) do not require RBF2 and dE2F2 in ovaries and S2 cells for their proper transcriptional regulation. Conversely, a target gene that requires RBF2/dE2F2 in ovaries (CG4250) no longer requires them in S2 cells and embryos for its repression. These results lead to the conclusion that RBF2 and dE2F2 do not control a single static transcriptional program but, rather, they regulate different sets of genes (Stevaux, 2005).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Capicua regulates proliferation and survival of RB-deficient cells in Drosophila

Mutations in rbf1, the Drosophila homologue of the RB tumour suppressor gene, generate defects in cell cycle control, cell death, and differentiation during development. Previous studies have established that EGFR/Ras activity is an important determinant of proliferation and survival in rbf1 mutant cells. This study reports that Capicua (Cic), an HMG box transcription factor whose activity is regulated by the EGFR/Ras pathway, regulates both proliferation and survival of RB-deficient cells in Drosophila. cic mutations allow rbf1 mutant cells to bypass developmentally controlled cell cycle arrest and apoptotic pressure. The cooperative effect between Cic and RBF1 in promoting G1 arrest is mediated, at least in part, by limiting Cyclin E expression. Surprisingly, evidence was also found to suggest that cic mutant cells have decreased levels of reactive oxygen species (ROS), and that the survival of rbf1 mutant cells is affected by changes in ROS levels. Collectively, these results elucidate the importance of the crosstalk between EGFR/Ras and RBF1 in coordinating cell cycle progression and survival (Krivy, 2013).

Both Dap and Cic are negative regulators of proliferation downstream of the EGFR/Ras pathway. However, EGFR/Ras activity promotes Dap expression while inhibiting Cic expression (Astigarraga, 2007). Accordingly, their expression patterns at the MF show that Cic expression drops where Dap expression is most prominent. Immunostaining with anti-phospho-MAPK, which is a marker for EGFR activity and cells initiating differentiation processes, shows a similar expression pattern to that of Dap. Perhaps, once cells start to differentiate, Dap plays a predominant role over Cic to maintain differentiating cells in the G1 phase. This would also explain the absence of EdU-positive cells in rbf1 cic double mutant clones that express Atonal. It was noticed that Dap expression is slightly increased in rbf1 mutant clones. This is likely in response to the Cyclin E activation. In contrast to the MF, co-expression of Dap and Cic proteins was detected in the anterior region of the eye disc. Perhaps, in this region, EGFR/Ras is activated to a level at which both proteins can coexist. Nevertheless, the results indicate that, at least at the MF, the cellular context in which Dap and Cic act to restrict proliferation is distinct (Krivy, 2013).

In cic homozygous mutant eye discs generated in the rbf1^120a heterozygous background, Cyclin E levels are specifically increased at the MF. In fact, this is the location where dE2F1 proteins are most highly expressed in the eye disc. While this observation suggests a strong cooperation between dE2F1 and Cic, no changes were observed in the dE2F1 protein level nor its activity in cic mutant clones. This result indicates that cic mutations do not affect dE2F1 activity in general. A previous study demonstrated that increases in both Cyclin E and E2F activities are necessary to overcome the cell cycle arrest imposed at the MF. This likely explains why ectopic S-phase cells were not observed in cic mutant cells generated in rbf1^120a heterozygous eye discs despite the elevated level of Cyclin E expression. E2F target genes are consistently expressed at a lower level in cic mutant eye discs generated in the rbf1^120a heterozygous background than rbf1^120a homozygous mutant eye discs. Nevertheless, it cannot be excluded that dE2F1 is required for the effect of cic mutations on Cyclin E expression. It is interesting to note that both Dap and Cic act on Cyclin E/CDK2 and that their mutations cooperate with rbf1 mutations to cause uncontrolled proliferation. Perhaps, such a context in which either E2F1 or Cyclin E/CDK2 activity is elevated represents a sensitised genetic background where regulators of the other protein can be identified. Indeed, haploinsufficiency of rbf1 is shown to be sufficient to dominantly modify the rough eye phenotype induced by p21 overexpression, the mammalian inhibitor of Cyclin E/CDK2 (Krivy, 2013).

The molecular mechanism by which RBF1 and Cic cooperatively regulate Cyclin E expression remains unclear. RT-qPCR did not reveal that cic mutations produce any discernable changes in cyclin E RNA levels, indicating that the effect of cic mutations on Cyclin E expression is post-transcriptional and likely to be indirect. One interesting observation is that heterozygosity of rbf1 seems to have a general effect on the expression level of RBF1 target genes. Transcript levels of mcm2 and rnrS are elevated in the rbf1 heterozygous background compared to the wild type. This raises the possibility that increased expression of RBF1 target genes in general provides a specific context that allows the cic mutation to have an effect on Cyclin E expression. The transcriptional changes that are induced by cic mutations in control and rbf1 mutant backgrounds are currently being investigated to determine if Cic regulates different transcriptional programs depending on the status of RBF1 (Krivy, 2013).

Whether the alteration of Cyclin E levels is the only molecular mechanism by which cic and rbf1 mutations cooperate to promote ectopic S phase is still unclear. Oddly, no discernible change was observed in Cyclin E levels when cic mutant clones were generated in an rbf1^120a homozygous background despite the presence of ectopic S-phase cells. One possible explanation for this is that rbf1 homozygous mutations increase Cyclin E expression to the level higher than what is achieved by cic mutations in rbf1^120a heterozygous backgrounds. This increase is perhaps near, but not over, the threshold that can overcome the G1 arrest at the MF. In this context, cic mutations would provide the additional Cyclin E expression that is required to actually surpass this threshold. However, once cells enter S phase, Cyclin E is rapidly targeted for degradation, making it difficult to detect the increase in Cyclin E level. The lack of an increase in Cyclin E level in the rbf1^120a homozygous background could also indicate that cic mutations can result in additional molecular changes that can cooperate with rbf1 mutations. While it is unclear what these changes might be, it is known that Cic's ability to regulate ROS is not likely to contribute to the cell cycle defect. No changes were observed in the EdU staining pattern when expression levels of SOD2 were altered in an rbf1 mutant background. Presently, transcriptional changes induced by cic mutations are being compared in wild-type and rbf1 mutant backgrounds in order to postulate a molecular mechanism (Krivy, 2013).

An intronic microRNA links Rb/E2F and EGFR signaling

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

Single cell RNA-sequencing identifies a metabolic aspect of apoptosis in Rbf mutant

The function of Retinoblastoma tumor suppressor (pRB) is greatly influenced by the cellular context, therefore the consequences of pRB inactivation are cell type specific. This study employed single cell RNA sequencing (scRNA seq) to profile the impact of an Rbf mutation during Drosophila eye development. First, a catalogue was built of 11,500 wild type eye disc cells containing major known cell types. A transcriptional switch was found occurring in differentiating photoreceptors at the time of axonogenesis. Next, a cell landscape of Rbf mutant was mapped, and a mutant specific cell population was identified that shows intracellular acidification due to increase in glycolytic activity. Genetic experiments demonstrate that such metabolic changes, restricted to this unique Rbf mutant population, sensitize cells to apoptosis and define the pattern of cell death in Rbf mutant eye disc. Thus, these results illustrate how scRNA seq can be applied to dissect mutant phenotypes (Ariss, 2018).

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