Gene name - Retinoblastoma-family protein
Synonyms - RBF
Cytological map position - 1C-1D
Function - transcription factor
Symbol - Rbf
FlyBase ID: FBgn0015799
Genetic map position -
Classification - Retinoblastoma family
Cellular location - presumably nuclear
|Recent literature||Payankaulam, S., Yeung, K., McNeill, H., Henry, R.W. and Arnosti, D.N. (2016). Regulation
of cell polarity determinants by the Retinoblastoma tumor suppressor
protein. Sci Rep 6: 22879. PubMed ID: 26971715
In addition to their canonical roles in the cell cycle, RB family proteins regulate numerous developmental pathways, although the mechanisms remain obscure. This study found that Drosophila Rbf1 associates with genes encoding components of the highly conserved apical-basal and planar cell polarity pathways, suggesting a possible regulatory role. It was shown that depletion of Rbf1 in Drosophila tissues is indeed associated with polarity defects in the wing and eye. Key polarity genes aPKC, par6, vang, pk, and fmi are upregulated, and an aPKC mutation suppresses the Rbf1-induced phenotypes. RB control of cell polarity may be an evolutionarily conserved function, with important implications in cancer metastasis.
|Clavier, A., Rincheval-Arnold, A., Baillet, A., Mignotte, B. and Guenal, I. (2016). Two different specific JNK activators are required to trigger apoptosis or compensatory proliferation in response to Rbf1 in Drosophila. Cell Cycle 15: 283-294. PubMed ID: 26825229
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 (Rbf1D253A) have JNK-dependent pro-apoptotic properties. Rbf1D253A 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.
|Dominado, N., La Marca, J.E., Siddall, N.A.,
Heaney, J., Tran, M., Cai, Y., Yu, F., Wang, H., Somers, W.G., Quinn,
L.M. and Hime, G.R. (2016). Rbf
regulates Drosophila spermatogenesis via control of somatic
stem and progenitor cell fate in the larval testis. Stem Cell
Reports 7: 1152-1163. PubMed ID: 27974223
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.
|Greenspan, L. J. and Matunis, E. L. (2018). Retinoblastoma intrinsically regulates niche cell quiescence, identity, and niche number in the adult Drosophila testis. Cell Rep 24(13): 3466-3476.e3468. PubMed ID: 30257208
Homeostasis in adult tissues depends on the precise regulation of stem cells and their surrounding microenvironments, or niches. This study shows that the cell cycle inhibitor and tumor suppressor Retinoblastoma (RB) is a critical regulator of niche cells in the Drosophila testis. The testis contains a single niche, composed of somatic hub cells, that signals to adjacent germline and somatic stem cells. Hub cells are normally quiescent, but knockdown of the RB homolog Rbf in these cells causes them to proliferate and convert to somatic stem cells. Over time, mutant hub cell clusters enlarge and split apart, forming ectopic hubs surrounded by active stem cells. Furthermore, this study shows that Rbf's ability to restrict niche number depends on the transcription factors E2F and Escargot and the adhesion molecule E-cadherin. Together this work reveals how precise modulation of niche cells, not only the stem cells they support, can drive regeneration and disease.
|Zappia, M. P., Rogers, A., Islam, A. and Frolov, M. V. (2019). Rbf activates the myogenic transcriptional program to promote skeletal muscle differentiation. Cell Rep 26(3): 702-719. PubMed ID: 30650361
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.
|Ariss, M. M., Islam, A., Critcher, M., Zappia, M. P. and Frolov, M. V. (2018). Single cell RNA-sequencing identifies a metabolic aspect of apoptosis in Rbf mutant. Nat Commun 9(1): 5024. PubMed ID: 30479347
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 identifgied 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.
|Mouawad, R., Prasad, J., Thorley, D., Himadewi, P., Kadiyala, D., Wilson, N., Kapranov, P. and Arnosti, D. N. (2019). Diversification of retinoblastoma protein function associated with cis and trans adaptations. Mol Biol Evol. PubMed ID: 31418797
Retinoblastoma proteins are eukaryotic transcriptional co-repressors that play central roles in cell cycle control, among other functions. Although most metazoan genomes encode a single retinoblastoma protein, gene duplications have occurred at least twice: in the vertebrate lineage, leading to Rb, p107, and p130, and in Drosophila, an ancestral Rbf1 gene and a derived Rbf2 gene. Structurally, Rbf1 resembles p107 and p130, and mutation of the gene is lethal. Rbf2 is more divergent and mutation does not lead to lethality. However, the retention of Rbf2 over 60 million years in Drosophila points to essential functions, which prior cell-based assays have been unable to elucidate. Using genomic approaches, this study provides new insights on the function of Rbf2. Strikingly, Rbf2 was shown to regulate a set of cell growth related genes and can antagonize Rbf1 on specific genes. These unique properties have important implications for the fly; Rbf2 mutants show reduced egg laying, and lifespan is reduced in females and males. Structural alterations in conserved regions of Rbf2 gene suggest that it was sub- or neofunctionalized to develop specific regulatory specificity and activity. This study defines cis regulatory features of Rbf2 target genes that allow preferential repression by this protein, indicating that it is not a weaker version of Rbf1 as previously thought. The specialization of retinoblastoma function in Drosophila may reflect a parallel evolution found in vertebrates, and raises the possibility that cell growth control is equally important to cell cycle function for this conserved family of transcriptional corepressors.
|Mouawad, R., Himadewi, P., Kadiyala, D. and Arnosti, D. N. (2020). Selective repression of the Drosophila cyclin B promoter by retinoblastoma and E2F proteins. Biochim Biophys Acta Gene Regul Mech 1863(7): 194549. PubMed ID: 32275964
The Cyclin B1 gene encodes a G2/M cyclin that is deregulated in various human cancers, however, the transcriptional regulation of this gene is incompletely understood. The E2F and retinoblastoma family of proteins are involved in this gene's regulation, but there is disagreement on which of the E2F and retinoblastoma proteins interact with the promoter to regulate this gene. This study dissected the promoter region of the Drosophila CycB gene and studied the role of Rbf and E2F factors in its regulation. This gene exhibits remarkable features that distinguish it from G1/S regulated promoters, such as PCNA. The promoter is comprised of modular elements with dedicated repressor and activator functions, including a segment spanning the first intron that interferes with a 5' activator element. A highly active minimal promoter (-464, +100) is repressed by the Rbf1 retinoblastoma protein, but much more potently repressed by the Rbf2 protein, which has been linked in other studies to control of cell growth genes. Unlike many other cell-cycle genes, which are activated by E2F1 and repressed by E2F2, CycB is potently activated by E2F2, and repressed by E2F1. Although the bulk of Rbf binding is associated with a region 5' of the core promoter, E2F and retinoblastoma proteins functionally interact with the basal promoter region, in part through a conserved E2F site at -80 bp. The specific regulatory requirements of this late cell cycle promoter appear to be linked to the unique activities of E2F and retinoblastoma family members acting on a complex cis-regulatory circuit.
|Sheng, Z. and Du, W. (2020). NatB regulates Rb mutant cell death and tumor growth by modulating EGFR/MAPK signaling through the N-end rule pathways. PLoS Genet 16(6): e1008863. PubMed ID: 32559195
Inactivation of the Rb tumor suppressor causes context-dependent increases in cell proliferation or cell death. In a genetic screen for factors that promoted Rb mutant cell death in Drosophila, this study identified Psid, a regulatory subunit of N-terminal acetyltransferase B (NatB). NatB subunits were required for elevated EGFR/MAPK signaling and Rb mutant cell survival. NatB regulates the posttranscriptional levels of the highly conserved pathway components Grb2/Drk, MAPK, and PP2AC but not that of the less conserved Sprouty. Interestingly, NatB increased the levels of positive pathway components Grb2/Drk and MAPK while decreased the levels of negative pathway component PP2AC, which were mediated by the distinct N-end rule branch E3 ubiquitin ligases Ubr4 and Cnot4, respectively. These results suggest a novel mechanism by which NatB and N-end rule pathways modulate EGFR/MAPK signaling by inversely regulating the levels of multiple conserved positive and negative pathway components. As inactivation of Psid blocked EGFR signaling-dependent tumor growth, this study raises the possibility that NatB is potentially a novel therapeutic target for cancers dependent on deregulated EGFR/Ras signaling.
|Parkhitko, A. A., Singh, A., Hsieh, S., Hu, Y., Binari, R., Lord, C. J., Hannenhalli, S., Ryan, C. J. and Perrimon, N. (2021). Cross-species identification of PIP5K1-, splicing- and ubiquitin-related pathways as potential targets for RB1-deficient cells. PLoS Genet 17(2): e1009354. PubMed ID: 33591981
The RB1 tumor suppressor is recurrently mutated in a variety of cancers including retinoblastomas, small cell lung cancers, triple-negative breast cancers, prostate cancers, and osteosarcomas. Finding new synthetic lethal (SL) interactions with RB1 could lead to new approaches to treating cancers with inactivated RB1. This study identified 95 SL partners of RB1 based on a Drosophila screen for genetic modifiers of the eye phenotype caused by defects in the RB1 ortholog, Rbf1. 38 mammalian orthologs of Rbf1 modifiers were evaluated as RB1 SL partners in human cancer cell lines with defective RB1 alleles. It was further shown that for many of the RB1 SL genes validated in human cancer cell lines, low activity of the SL gene in human tumors, when concurrent with low levels of RB1 was associated with improved patient survival. Higher order combinatorial gene interactions were investigated by creating a novel Drosophila cancer model with co-occurring Rbf1, Pten and Ras mutations; targeting RB1 SL genes in this background suppressed the dramatic tumor growth and rescued fly survival whilst having minimal effects on wild-type cells. Finally, it was found that drugs targeting the identified RB1 interacting genes/pathways, such as UNC3230, PYR-41, TAK-243, isoginkgetin, madrasin, and celastrol also elicit SL in human cancer cell lines. In summary, this study identified several high confidence, evolutionarily conserved, novel targets for RB1-deficient cells that may be further adapted for the treatment of human cancer.
|Payankaulam, S., Hickey, S. L. and Arnosti, D. N. (2021). Cell cycle expression of polarity genes features Rb targeting of Vang. Cells Dev 169: 203747. PubMed ID: 34583062
Specification of cellular polarity is vital to normal tissue development and function. Pioneering studies in Drosophila and C. elegans have elucidated the composition and dynamics of protein complexes critical for establishment of cell polarity, which is manifest in processes such as cell migration and asymmetric cell division. Conserved throughout metazoans, planar cell polarity (PCP) genes are implicated in disease, including neural tube closure defects associated with mutations in VANGL1/2. PCP protein regulation is well studied; however, relatively little is known about transcriptional regulation of these genes. Earlier study revealed an unexpected role for the fly Rbf1 retinoblastoma corepressor protein, a regulator of cell cycle genes, in transcriptional regulation of polarity genes. This study analyzes the physiological relevance of the role of E2F/Rbf proteins in the transcription of the key core polarity gene Vang. Targeted mutations to the E2F site within the Vang promoter disrupts binding of E2F/Rbf proteins in vivo, leading to polarity defects in wing hairs. E2F regulation of Vang is supported by the requirement for this motif in a reporter gene. Interestingly, the promoter is repressed by overexpression of E2F1, a transcription factor generally identified as an activator. Consistent with the regulation of this polarity gene by E2F and Rbf factors, expression of Vang and other polarity genes is found to peak in G2/M phase in cells of the embryo and wing imaginal disc, suggesting that cell cycle signals may play a role in regulation of these genes. These findings suggest that the E2F/Rbf complex mechanistically links cell proliferation and polarity (Payankaulam, 2021).
|Sainz de la Maza, D., Hof-Michel, S., Phillimore, L., Bokel, C. and Amoyel, M. (2022). Cell-cycle exit and stem cell differentiation are coupled through regulation of mitochondrial activity in the Drosophila testis. Cell Rep 39(6): 110774. PubMed ID: 35545055
Whereas stem and progenitor cells proliferate to maintain tissue homeostasis, fully differentiated cells exit the cell cycle. How cell identity and cell-cycle state are coordinated during differentiation is still poorly understood. The Drosophila testis niche supports germline stem cells and somatic cyst stem cells (CySCs). CySCs give rise to post-mitotic cyst cells, providing a tractable model to study the links between stem cell identity and proliferation. While cell-cycle progression was shown to be required for CySC self-renewal, the E2f1/Dp transcription factor is dispensable for self-renewal but instead must be silenced by the Drosophila retinoblastoma homolog, Rbf, to permit differentiation. Continued E2f1/Dp activity inhibits the expression of genes important for mitochondrial activity. Furthermore, promoting mitochondrial biogenesis rescues the differentiation of CySCs with ectopic E2f1/Dp activity but not their cell-cycle exit. In sum, E2f1/Dp coordinates cell-cycle progression with stem cell identity by regulating the metabolic state of CySCs.
In mammalian cells, the Retinoblastoma protein (RB) acts as a critical switch, regulating entry into the DNA synthetic phase (S phase) of the cell cycle. RB interacts with and negatively regulates members of a family of factors called E2Fs, which serve to activate transcription of genes required for entry into S phase. Phosphorylation of RB by cyclin/cyclin dependent kinases, the protein dimers that regulate the cell cycle, target RB for destruction, releasing E2F, thereby allowing E2F to carry out its function as a transcriptional activator.
In mammals, RB is represented by a family of three closely related proteins: RB itself, p107 and p130. Drosophila Retinoblastoma-family protein (Rbf) was isolated by virtue of its ability to interact with E2F complexes containing Drosophila E2F and E2F's dimerization partner, DP transcription factor (DP). In Drosophila, ectopic expression of cyclin E activates E2F-dependent transcription but it is thought that cyclin E does not act directly on E2F; rather it targets a negative regulator of E2F activity. The properties of Rbf suggest that it is an intermediary factor, proposed to allow cyclin E induction of E2F activity (Du, 1996a).
During Drosophila development, E2F-dependent transcription is down-regulated when G1 control first appears. 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 stages 12 and 13, and Rbf 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).
The E2F transcription factor and retinoblastoma protein control cell-cycle progression and DNA replication during S phase. Mutations in the Drosophila E2f1 and DP genes affect the origin recognition complex (DmORC) and initiation of replication at the chorion gene replication origin. Mutants of Rbf (a retinoblastoma protein homolog) fail to limit DNA replication. DP, E2f1 and Rbf proteins are located in a complex with ORC, and E2f1 and ORC are bound to the chorion origin of replication in vivo. These results indicate that E2f and Rbf function together at replication origins to limit DNA replication through interactions with ORC (Bosco, 2001).
To explore the possibility that E2f-Rbf is directly involved in controlling ORC activity, a test was performed to see whether a female-sterile mutant of Rbf (Rbf120a) has DNA replication and gene amplification defects in follicle cells of the Drosophila ovary. TheRbf120a mutation is due to a P-element insertion that causes reduced levels of wild-type Rbf protein, and Rbf14 is a null mutant. Ovaries from mutant Rbf120a/Rbf14 and heterozygous Rbf14/+ females were double labelled with 5-bromodeoxyuridine (BrdU) and anti-ORC2. Wild-type Drosophila follicle cells undergo endoreduplication cycles (endo cycles), reaching 16n ploidy by stage 9 or 10A of egg-chamber development. In stage 10B follicle cells, endo cycles have ceased, ORC has been cleared from the nucleus, and ORC is localized to discrete genomic regions undergoing amplification. Amplification is detected by BrdU incorporation at ORC localized foci. By contrast, the Rbf120a/Rbf14 mutant egg-chambers have a mosaic of follicle cells exhibiting striking replication defects: (1) some mutant follicle cells have inappropriate total nuclear ORC2 staining and continued endo cycles instead of amplification; (2) some follicle cells with specific ORC2 localization to replication origins have undergone gene amplification; and (3) some cells perform both amplification and genomic replication. Staining ovaries with anti-Rbf antibodies reveals a uniform absence of Rbf protein, and thus the mosaic phenotype cannot be explained by stochastic differences in Rbf protein levels (Bosco, 2001).
The Rbf120a/Rbf14 follicle cells undergoing gene amplification have large BrdU foci relative to sibling controls. Quantitation has confirmed that Amplification control element ACE3 DNA is amplified ~26-fold in mutant stage 13 egg-chambers, compared with ~16-fold in heterozygous egg-chambers. This phenotype in the Rbf120a/Rbf14 mutant is similar to the overamplification observed in the E2fi2 truncation mutant in which ACE3 is amplified 32-fold. Thus both Rbf and E2f are negative regulators of gene amplification (Bosco, 2001).
There is also a cell-cycle defect in the Rbf120a/Rbf14 mutant follicle cells. Inappropriate genomic replication seen in the mutant follicle cells results from the continuation of S/G endo cycles beyond the developmental stage at which they would normally cease. This predicts the presence of mutant follicle cells with greater DNA content than the wild-type 16n DNA. Fluorescence-activated cell sorting (FACS) analysis was carried out on purified ovarian nuclei, and heterozygous Rbf14/+ ovaries gave follicle cells with 2n, 4n, 8n and 16n DNA content. Rbf120a/Rbf14 ovaries, however, had cells with 32n DNA content, indicating that they had undergone at least one extra S-phase. It is concluded that stage 10B Rbf120a/Rbf14 mutant follicle cells undergo an ectopic S phase, and genomic replication in stage 10B cells is not due to a developmental delay. This result parallels that obtained with mutations in dDP (Bosco, 2001).
DNA replication also persists in later stages of mutant follicle cells. Wild-type stage 13 egg-chambers have no detectable ORC localization and little or no BrdU incorporation. In contrast, Rbf120a/Rbf14 stage 13 egg-chambers continue to undergo amplification and genomic replication that is consistent with persistent nuclear ORC staining. Some stage 13 cells exhibit characteristics of G1 cells, with nuclear ORC but no BrdU staining. This observation also supports the conclusion that Rbf120a/Rbf14 follicle cells continue bona fide G/S endo cycles (Bosco, 2001).
Tests were performed to see whether misregulation of important E2f target genes might account for the replication defects observed in the Rbf mutant follicle cells. Four important E2f target genes, Cyclin E, PCNA, RNR2 and ORC1, as well as ORC2 transcripts, are not normally induced in wild-type stage 10 follicle cells, and their transcripts are not elevated in the truncation E2fi2 mutant follicle cells. However, because overexpression of ORC1 is sufficient for initiating an ectopic endo cycle in stage 10 follicle cells, ORC1 and ORC2 transcripts were analyzed by in situ hybridization in Rbf mutant follicle cells. No significant differences were found in the amount of messenger RNA levels for either gene in Rbf120a/Rbf14 stage 9, 10 or 13 egg-chambers, as compared with Rbf14/+ sibling controls (Bosco, 2001).
Transcription of the reaper gene is highly induced in the follicle cells of wild-type stage 9 and 10 egg-chambers, and thus reaper levels were used as a measure of general transcriptional activity in an experiment in which attempts were made to block transcription of all genes. Egg-chambers were cultured in vitro for up to 6 h with or without alpha-amanitin. Rbf120a/Rbf14 egg-chambers cultured in the presence of alpha-amanitin abolish visible transcript levels of reaper in stage 10B follicle cells, whereas the Rbf120a/Rbf14 controls induce reaper normally. However, alpha-amanitin does not change the pattern of BrdU labelling in Rbf120a/Rbf14 stage 10 or 13 egg-chambers. Thus, the Rbf mutant replication defects persist even when general transcription is inhibited in follicle cells. It is possible that the in situ analysis of transcript levels or the inhibition of transcription by alpha-amanitin fail to uncover an effect of the Rbf mutant. Taken together, however, these data suggest that the gene amplification phenotype seen in Rbf120a/Rbf14 or E2fi2 follicle cells is not due to a misregulation of E2f target gene transcription in stage 10 egg-chambers (Bosco, 2001).
Whether E2f-Rbf complexes execute an S-phase function through a direct interaction with ORC was tested. Immunoprecipitations were carried out on ovary extracts; immunoblots of the pellets show that E2f and Rbf co-immunoprecipitate with Drosophila ORC when either anti-ORC2 or anti-ORC1 antibodies were used. The E2f-Rbf-ORC interaction could also be detected when immunoprecipitation reactions were performed with anti-E2f polyclonal or anti-DP monoclonal antibodies. This complex could be specifically immunoprecipitated from ovary extracts with five different antibodies. It is possible that in extracts the dDP-E2f-Rbf and ORC interaction might be due to dDP-E2f and ORC binding next to each other on DNA fragments. Therefore, immunoprecipitation reactions were carried out in the presence of ethidium bromide or micrococcal nuclease to disrupt protein-DNA interactions or cleave DNA fragments. Treatment of immunoprecipitation reactions with either reagent failed to disrupt the E2f-Rbf-ORC interaction. Furthermore, a mutation in DP predicted to reduce the DNA-binding activity of E2f did not abolish the E2f-Rbf-ORC interaction. It is therefore concluded that E2f and Rbf can co-immunoprecipitate with ORC through interactions that are independent of their respective DNA-binding activities (Bosco, 2001).
What is the functional relevance of this E2f-Rbf-ORC complex? One possible mechanism is that E2f-Rbf helps localize ORC to E2f-binding sites near the chorion replication origin. Another possibility is that ORC localization to the chorion replication origin is independent of its interaction with E2f-Rbf, and instead E2f-Rbf when bound next to an origin regulates replication initiation through its interaction with ORC. ORC binds the critical amplification control element ACE3 in vivoat a specific time in follicle cell development (stages 10A and 10B). Using anti-ORC2 antiserum, ACE3 has been specifically enriched relative to a control locus that does not bind ORC and is not amplified by using chromatin immunoprecipitation (CHIP). Using CHIP it was asked whether E2f also could be shown to localize specifically to ACE3 in vivo. Stabilization of protein-DNA interactions in live tissue is achieved by formaldehyde crosslinking. Subsequent CHIP enriches for specific trans-factors that are bound to genomic loci. The relative amounts of these loci are quantified by polymerase chain reaction (PCR). Sequence analysis reveals that there are several potential E2f-binding sites within 2.5 kilobases (kb) of ACE3. Using anti-E2f antibodies, it has been shown that ACE3 DNA is enriched ~15-fold relative to the rosy locus in stage 10 egg-chambers. Similarly, anti-ORC2 antibodies also enriched ACE3 DNA ~20-fold relative to the rosy locus. Thus, both E2f and ORC localize to ACE3 when amplification is occurring, and E2f binding is limited to sequences immediately adjacent to ACE3. This observation is consistent with E2f-Rbf functioning at replication origins and possibly controlling ORC activity (Bosco, 2001).
Since transactivation and RB-binding activities are known to be located in the C-terminal domain of mammalian E2F, a truncated form of Drosophila E2f predicted to lack the C-terminal transactivation and Rbf-binding domains was characterized. The E2fi2 mutation produces a stable, truncated E2fi2 protein that can still interact with DP. Truncated E2fi2 does not bind Rbf, as it does not co-immunoprecipitate, even when more than 10% of the total Rbf protein is immunoprecipitated. This failure to pellet the truncated E2fi2 protein is not due to low Rbf levels in mutant extracts because comparable amounts of Rbf in wild-type extracts can immunoprecipitate full-length E2f. Failure to detect this interaction is not due to low levels of truncated E2fi2 protein, because the amount of truncated E2fi2 in the supernatant represents 10% of total E2fi2 in the immunoprecipitation reaction and is comparable to full-length E2f that does interact with Rbf (Bosco, 2001).
Previous work has shown that mutant follicle cells producing this truncated E2fi2 protein specifically localize ORC to the amplification regions as in wild type, but that such cells have elevated levels of ACE3 amplification. This elevated level of amplification is probably due to extra rounds of origin initiation events, suggesting that both E2f and Rbf have a negative regulatory function in origin firing during amplification. The DNA-binding domain of the truncated E2fi2 protein might be sufficient to localize ORC, if it could still interact with ORC. Therefore, whether or not the truncated E2fi2 protein complexes with ORC was tested. Immunoprecipitation experiments show that truncated E2fi2 does not interact with ORC. This means that the C-terminal domain of E2f is necessary for its interaction with ORC, and possibly requires Rbf to mediate this interaction. In contrast to the stated hypothesis, however, localization of ORC to the amplification region does not require a physical complex with E2f (Bosco, 2001).
Thus, the Drosophila E2f-Rbf complex functions during S phase, specifically to regulate DNA replication initiation at origins. It is thought that DP-E2f-Rbf are bound near ORC at the amplification origin and regulate initiation by forming a complex with ORC. Although E2f does not direct ORC binding, it restricts its activity through Rbf. Five lines of evidence form the basis for this model: (1) reduced levels of Rbf result in increased gene amplification levels and genomic replication without measurable effects on transcription of E2f target genes; (2) a complex of dDP-E2f-Rbf-ORC is present in ovary extracts; (3) this complex is independent of DNA binding; (4) truncation of the C terminus of E2f eliminates this complex, and (5) in this truncation mutant, ORC is localized but increased amplification occurs. The mechanism by which the dDP-E2f-Rbf complex limits replication initiation at the chorion locus remains to be determined. It is possible that the dDP-E2f-Rbf proteins inhibit the activity of the ORC subunits through a physical interaction. Alternatively, E2f-Rbf might inhibit loading of other replication factors at origins, such as MCM proteins. Finally, Rbf might alter the local chromatin configuration, for example by histone deacetylation, and thereby affect origin firing. Although ORC does not need to be in the E2f-Rbf complex to bind specifically to the chorion replication origin, a mutation in the DNA-binding domain of E2f does result in loss of ORC localization in the follicle cells. This observation needs to be evaluated in the context of the result that ORC is localized in the E2fi2 mutant, in which the truncated E2f protein is able to bind DNA but does not complex with ORC. Thus, DNA binding by E2f seems to be a prerequisite for ORC localization, but ORC localization does not require complex formation with E2f. This may be because when E2f is not bound to the chorion region, E2f2 can bind to sites at ACE3 normally occupied by E2f, and E2f2-Rbf may repel ORC and preclude localization or antagonize ORC binding activity (Bosco, 2001).
The Rbf mutant provides insights into the controls leading to the cessation of the endo cell-cycle during follicle cell development. Both the female-sterile Rbf mutant and the dDP female-sterile mutant show inappropriate continuation of the endo cell cycle beyond stage 10 of egg-chamber development. In contrast, an ectopic S phase does not occur in either of the female-sterile E2f mutants. Like the dDPa1 mutant, the Rbf120a/Rbf14 mutant is expected to have effects on both E2f-Rbf and E2f2-Rbf complexes. Thus, it seems that DP-E2f2-Rbf is needed to exit endo cycles, whereas DP-E2f-Rbf is involved more directly in regulating ORC and gene amplification. Identification of mutations in E2f2 will permit direct analysis of the roles of E2f2 in the endo cell cycle and amplification. Although it has not been shown whether any other specific replication origins may be regulated in this manner, the E2f-Rbf-ORC complex has been found in embryonic extracts, indicating that E2f-Rbf may be a general repressor of replication origins in embryonic tissues. Notably, a region between the DmPolalpha and E2f genes, containing several known E2f-binding sites, has been identified as a replication initiation region. Human RB (and associated HDACs) co-immunolocalize to BrdU foci in early S phase of primary cells, suggesting that RB may have a role in replication initiation. This observation is consistent with the model that suggests that Drosophila E2F1-Rbf localizes to replication origins and regulates ORC activity through a direct protein-protein interaction. It will be of great value to determine whether mammalian E2F-RB complexes can interact with ORC. Such an interaction would allow for a better understanding of how E2F and RB function to regulate DNA replication and cell proliferation during tumor progression (Bosco, 2001).
The Drosophila retinoblastoma family of proteins (RBF1 and RBF2) and their mammalian homologs (pRB, p130, and p107) are best known for their regulation of the G1/S transition via the repression of E2F-dependent transcription. However, RB family members also possess additional functions. This study reports that rbf1 mutant larvae have extensive defects in chromatin condensation during mitosis. A novel interaction is described between RBF1 and CAP-D3, a non-SMC component of the Condensin II complex that links RBF1 to the regulation of chromosome structure. RBF1 physically interacts with dCAP-D3, RBF1 and dCAP-D3 partially colocalize on polytene chromosomes, and RBF1 is required for efficient association of dCAP-D3 with chromatin. dCap-D3 mutants also exhibit chromatin condensation defects, and mutant alleles of dCap-D3 suppress cellular and developmental phenotypes induced by the overexpression of RBF1. Interestingly, this interaction is conserved between flies and humans. The re-expression of pRB into a pRB-deficient human tumor cell line promotes chromatin association of hCAP-D3 in a manner that depends on the LXCXE-binding cleft of pRB. These results uncover an unexpected link between pRB/RBF1 and chromatin condensation, providing a mechanism by which the functional inactivation of RB family members in human tumor cells may contribute to genome instability (Longworth, 2008).
Previous studies have shown that non-SMC proteins are important for the function of Condensin complexes (Hirano, 2005; Nasmyth, 2005). While the SMC proteins contain the ATPase domains that are essential for DNA supercoiling, non-SMC proteins like CAP-D2 and CAP-D3 are thought to help target Condensins to chromatin. Condensin I and Condensin II complexes are known to be selectively recruited to different chromosomal locations (Ono, 2004; Savvidou, 2005), but the mechanisms responsible for specificity have been elusive. The results described in this study show that RB- family members are important for the chromatin association by dCAP-D3, a specific component of the Condensin II complex. One of the most striking features of these data is the specificity of the interactions. RBF1 interacts consistently with dCAP-D3 but not with dCAP-D2. This specificity is illustrated by the polytene staining experiments showing that RBF1 colocalizes with dCAP-D3 but not with dCAP-D2. A similar specificity was observed in the genetic assays, in binding experiments, and in immunostaining experiments and was seen in experiments in both flies and human cells (Longworth, 2008).
While the lack of interaction with dCAP-D2 provides a valuable negative control, it also suggests that there is an important distinction between the two Condensin subunits and, by inference, between the two Condensin complexes. When tested in Xenopus extracts, the depletion of Condensin I resulted in decondensation of sperm chromatin with no chromosome formation while depletion of Condensin II primarily affected the morphology of chromosomes, resulting in kinked chromosomes that lacked the normal degree of rigidity. These differences indicate that Condensin I and Condensin II play different roles in the organization of chromosome structure, but precisely how the functions of these two complexes differ, or how their functions are integrated, is not well understood. One idea, suggested by studies in mammalian cells showing that Condensin II associates with chromatin prior to nuclear envelope breakdown (NEB), whereas Condensin I complexes are excluded from the nucleus and only gain access to chromatin following NEB, is that Condensin II complexes perform the initial compaction of the chromosome in prophase, and that Condensin I helps to complete the process. However, in Drosophila, both Condensin I and II proteins have been found to localize with chromatin prior to NEB and this temporal distinction is less certain. The fact that the two Condensin complexes localize to distinct regions of euchromatin suggests that they determine the architecture of different subdomains. If this model is correct, then this implies that RB family members are important for the structural organization of particular regions of the chromosome. This may explain why the abnormally condensed chromosomes seen in rbf1 mutant animals contain interspersed segments of condensed and hypocondensed chromatin. In a more general sense, this link between RB proteins and chromosome organization suggests that RB family members are poised to control global changes in chromatin architecture, a position that may help them to act as global regulators of proliferation and differentiation (Longworth, 2008).
It is noted that several studies have described roles for Condensins outside of mitosis. The Caenorhabditis elegans SMC4 homolog DPY-27 is required for the regulation of the dosage compensation complex and the transcriptional repression of the autosomal male sex determination gene, her-1. Non-SMC subunits, which have been shown to form complexes independent of the SMC heterodimer, can also effect gene expression. dCAP-G, (a member of both Condensin I and II) has been shown to effect position effect variegation (PEV), suggesting that it may have a role in maintenance of heterochromatin. An interaction between murine CAP-G2 (MTB) and the SCL and E12 transcription factors allows MTB to repress transcription during erythoid cell development. Recently, a kleisin β mutation in mice was shown to be responsible for defects in T-lymphocyte differentiation. It is possible, therefore, that the interaction between RB family members and CAP-D3 protein may be used, in part, to control gene expression, and that the changes in chromosome condensation seen when RBF1 levels are altered may be a consequence of too little/too much chromatin-associated CAP-D3 that was initially recruited for a different purpose, such as transcriptional regulation (Longworth, 2008).
Interestingly, this study found that mutant alleles of dCap-D3 suppress PEV, supporting the idea that this complex can affect chromatin states. dCAP-D3 and RBF1 do not colocalize with bands of constitutive heterochromatin, which stain strongly with DAPI, but are found in the lighter DAPI-stained or non-DAPI-stained regions, raising the possibility that they act together in areas of repressed euchromatin or facultative heterochromatin. Clearly, further studies are needed to identify the regions of dCAP-D3 that are important for this interaction and to identify the chromatin elements that are bound by dCAP-D3/RBF1 (Longworth, 2008).
Several of the current results raise the intriguing possibility that RBF1 may target a subpopulation of dCAP-D3 that acts independently of dSMC proteins. It is striking that the UAS-RBF1 phenotype was not modified by mutant alleles of dsmc2 or dsmc4. Moreover, antibodies to dSMC4 failed to stain several bands on polytene chromosomes that were costained with antibodies to dCAP-D3 and RBF1, and, while a clear decrease was seen in the levels of dCAP-D3 associated with chromatin in the absence of RBF1, no clear reduction was seen in the level of dSMC4. Possibly, dCAP-D3 may exist in several different complexes, some of which contain dSMC proteins and some that do not, and the activity that is targeted by RBF1 may be different from the traditional view of Condensin complexes. Alternatively, since dSMC2 and dSMC4 are components of both Condensin I and Condensin II, one could argue that the lack of interaction with dsmc2 or dsmc4 and the failure to see a clear reduction in dSMC4 levels when RBF1 was removed are simply due to redundancy between the two Condensin complexes. The overlap between dSMC4 and dCAP-D3 staining on polytene chromosomes was surprisingly high and it is possible that the dSMC4 antibody marks some Condensin complexes better than others. Since dSMC4 coimmunoprecipitates with RBF1, since SMC4 binds to Rb, p107, and p130 fusion proteins in vitro, since many of the dCAP-D3/RBF bands on the polytene chromosomes also costain with dSMC4, and mutant alleles of dCap-H2 and dCap-G also modified the UAS-rbf1 phenotype, it seems likely that this activity of RBF1 involves its interaction with multiple components of the Condensin II complex. However, further studies are needed to fully characterize the RBF1/dCAP-D3 complexes and to determine which of the Condensin II proteins are important for this activity (Longworth, 2008).
Analysis of polytene chromosomes shows that RBF1 is needed for chromatin localization of CAP-D3 in interphase cells. In experiments carried out in human cells, the earliest point in the cell cycle when the ability of pRB to promote chromatin association by dCAP-D3 was late anaphase, the point in the cell cycle where pRB becomes dephosphorylated and associates tightly with chromatin. This, together with the observation that recombinant RB polypeptides associate with hCAP-D3 and the finding that the interaction between RB and hCAP-D3 was disrupted by mutations that mimic phosphorylation, leads to a proposal that RBF1/RB proteins recruit CAP-D3 to DNA at the times when they are traditionally thought to act. While the possibility that a subpopulation of RBF1 interacts with dCAP-D3 later in the cell cycle cannot be excluded, the changes in chromatin condensation in rbf1 mutants are most likely a consequence of reduced dCAP-D3 localization earlier in the cell cycle (Longworth, 2008).
The best-known functions of the RB family proteins in both mammalian cells and Drosophila involve interactions with E2F transcription factors. However neuroblast squashes of rbf1/dDP mutants and either dE2F2 or dDP-null larvae show that the hypocondensation caused by the loss of RBF1 does not require dE2F/dDP activity and cannot be generated by simply inactivating dE2F/dDP complexes. In agreement with this, it was found that the striking colocalization of RBF1 and dCAP-D3 is maintained on polytene squashes from dDP-null larvae. This does not preclude the possibility that dCAP-D3 and RBF1 complex may interact in the vicinity of dE2F-regulated promoters, or that the dCAP-D3/RBF1 interaction may be influenced by changes in E2F activity. Clearly though, these results show that RBF1 recruits dCAP-D3 to chromatin in a manner that does not depend on the conventional model of E2F/DP DNA-binding activity. Many different transcription factors have been shown to interact with RB family members, and there is no shortage of possibilities for such an E2F-independent function. Intriguingly, DNMT3b and HDAC1, which have been linked previously to pRB, have also been found to associate with both SMC4 and SMC2 (Geiman, 2004). This raises the possibility that the interaction between Condensin II proteins and RBF1 may involve one or more of the chromatin-associated complexes that have been connected previously to pRB (Longworth, 2008).
The results described in this study provide a very simple explanation for the aneuploidy and anaphase defects observed in Rb-/- and RbΔLXCXE MEFs. Since the inactivation of Condensin II complexes causes global changes in chromatin structure, these findings may also explain the balloon-like, butterfly chromosomes described in TKO MEFs or the changes in chromatin structure previously noted in Rb-/- MEFs (Longworth, 2008 and references therein).
pRB is one of the most well-studied tumor suppressor proteins and is mutated or inactivated in many types of human cancer. The idea that RB family members are required for normal Condensin II function provides a simple route through which the functional inactivation of RB family proteins, either by deletion, phosphorylation, or by viral proteins that bind to the LXCXE-binding cleft, will promote genomic instability. Indeed, there are several independent lines of evidence implicating Condensin activity in tumorigenesis. Mutations in Condensin subunits SMC2 and SMC4 have been identified in cases of pyothorax-associated lymphoma. Strikingly, a recent study of 102 early-onset breast cancer patients showed that 53% of cases had loss of heterozygosity (LOH) occurring in the region that includes the hCAP-D3 locus. This LOH also correlated with higher tumor grade and a more unfavorable prognosis. These observations seem particularly significant when one considers that heterozygosity for dCAP-D3 was not only sufficient to suppress RBF1-induced phenotypes but also caused visible defects in chromatin condensation and segregation. Given the evidence that RB family members promote CAP-D3 association with chromatin and the evidence that LOH at the CAP-D3 locus occurs frequently in breast cancer, it is suggested that Condensin II function is likely to be compromised in many different human tumor cells (Longworth, 2008).
Drosophila RBF combines several of the structural features of mammalian pRB, p107, and p130, suggesting that the Drosophila gene may have evolved from an ancestor common to the three human genes. This homology extends almost throughout the full length of the open reading frame but is most striking in regions homologous to the pocket domains of pRP, p107 and p130, which are essential for binding to viral oncoproteins, to E2F and to other partners. The Drosophila sequence shows slightly higher identity with p107 and p130 than with pRB; however, the organization of the protein resembles pRB more closely than p107 or p130. Most notably, the Drosophila protein lacks the spacer domain that is highly conserved between p107 and p130 and mediates their stable association with cdks. The B-half of the pocket domain of the Drosophila protein resembles pRB rather than p130 and p107, where these sequences differ by a long insertion into otherwise highly conserved sequences. The Drosophila protein contains a cluster of potential cdk phosphorylation sites immediately downstream of the pocket domain in a position similar to the sites that are thought to regulate the activate pRB, p107 and p130 (Du, 1996a).
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date revised: 12 September 2022
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