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

Gene name - Retinoblastoma-family protein

Synonyms - RBF

Cytological map position - 1C-1D

Function - transcription factor

Keywords - cell cycle, tumor suppressor

Symbol - Rbf

FlyBase ID: FBgn0015799

Genetic map position -

Classification - Retinoblastoma family

Cellular location - presumably nuclear



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

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).


PROTEIN STRUCTURE

Amino Acids - 797

Structural Domains

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).


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

date revised: 26 April 2001 

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