InteractiveFly: GeneBrief

Retinoblastoma-family protein 2: Biological Overview | References

BIOLOGICAL OVERVIEW

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 have bee contrasted with dE2F2, the only E2F partner of RBF2. Previous studies failed to uncover a unique role for RBF2 in E2F regulation. This study finds 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 were compared of transcripts that are mis-expressed in ovaries, embryos and S2 cells where RBF2 function has been ablated; 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 study the unique functions of Rbf2 advantage was taken of a P-element insertion, P2000, located 5 kb downstream of the Rbf2 ORF, and it was used to generate Rbf2 deletion alleles. Western blot analysis showed that stocks homozygous for two alleles, rbf2^M2 or rbf2^M3, lack the full-length RBF2 protein and express a 30kD amino-terminal fragment of RBF2 (DRBF2). This fragment lacks the entirety of the RBF2 pocket domain and is thus unable to perform any of the functions associated with this signature domain. Hence, animals carrying the rbf2^M2 or rbf2^M3 deletion alleles are deficient for E2F binding and transcriptional repression functions of RBF2 (Stevaux, 2005).

Stocks homozygous for rbf2^M2 and rbf2^M3 are viable and do not display visible developmental abnormalities. Because de2f2 mutant flies exhibit fertility defects, tests were performed to see whether rbf2 mutants also have reduced fertility. It was found that rbf2 mutant females have no fertility defect: they lay an increased number of eggs as compared to wild-type flies and none of the eggs exhibit morphological abnormalities. Furthermore the hatching rate of eggs laid by rbf2 mutant females is comparable to the hatching rate of eggs laid by wild-type flies (Stevaux, 2005).

To test for functional overlap between RBF1 and RBF2 in vivo, the rbf2^M2 mutant was crossed with two rbf1 mutant strains. First a weak loss-of-function allele of rbf1 (rbf1^120a), which harbors a P-element insertion in its promoter region, was used. The rbf1^120a viable allele exhibits normal eye architecture but interacts genetically with mutations in cell cycle regulators such as p21Cip/Kip and de2f1. Flies carrying rbf1^120a and homozygous for rbf2^M2 displayed a rough eye phenotype that was absent in the single rbf2^M2 and rbf1^120a mutant strains indicating that mutation in both genes has a cooperative effect. This rough eye phenotype was rescued by an Rbf2 genomic rescue construct. Next, the genetic interaction between rbf1 and rbf2 was examined using the null allele rbf1^D14. rbf1^D14 larvae are sub-viable, never reach the late pupal stages of development, and display a developmental delay of up to two days at the third instar larval stage. rbf1^D14; rbf2^M2 double mutant larvae exhibit significantly poorer viability than rbf1^D14 larvae at late larval and early pupal stages. Moreover, the double mutants had a greater developmental delay, taking up to nine days to reach the early pupal stages (Stevaux, 2005).

These results show that loss of rbf2 enhances the defects observed in rbf1 mutant animals. Identical genetic interactions were observed with the rbf2^M3 mutant. These results are consistent with previous experiments showing that RBF2 has many of the same properties as RBF1 and with the fact that RBF1 and 2 redundantly repress a number of E2F-regulated genes (Stevaux, 2005).

RBF2 and dE2F2 control minimally overlapping transcriptional programs in vivo. Since rbf2 mutant flies are viable, these animals provided an opportunity to examine the role of RBF2 in transcriptional regulation in vivo. Such experiments have not been possible using null alleles of de2f1 or rbf1 because these mutations cause dramatic developmental defects. Previous studies in tissue culture cells showed that RBF2 acts redundantly with RBF1 at E2F-repressed genes. The fact that RBF2 levels vary greatly during development suggests that its function in vivo may be tissue or stage specific. In adult flies RBF2 protein levels are barely detectable in males but are markedly elevated in ovaries (Stevaux, 2002). To assess the potential role of RBF2 in transcriptional regulation in this tissue, the transcriptional profiles of wild type and rbf2 mutant ovaries were compared (Stevaux, 2005).

Transcripts displaying a statistically significant two-fold increase in rbf2 mutants, as compared to wild type extracts, were compiled and analyzed. The list of genes with increased expression shows that, in ovaries, RBF2 is required for the repression of genes with a wide variety of functions, some of which, remarkably, are male-specific testis differentiation markers. These results reveal a unique role for RBF2. Unlike RBF1, RBF2 does not appear to regulate cell cycle genes. Rather, RBF2 functions to repress the expression of differentiation markers (Stevaux, 2005).

It was asked if the transcripts that are repressed by RBF2 in vivo are also repressed by dE2F2, the only known partner of RBF2. de2f2 mutant flies have reduced viability and fertility. However, the extent and nature of these defects differ for the two reported de2f2 null alleles, suggesting that animals deficient for dE2F2 are highly sensitive to mutations at other loci in the genome. To exclude genetic background-specific and/or allele-specific effects, de2f2 mutant females were used that carry trans-heterozygous combinations of the two alleles. Although these animals are homozygous null for dE2F2, they lay normal eggs and do not exhibit the morphological defects previously described for the homozygous alleles of de2f2. They also display much less pronounced fertility defects -- slightly reduced hatching rate and lower number of eggs laid. Genes that have elevated expression in de2f2 mutant ovaries were also identified. No deregulation of genes with functions in DNA replication were detected, as was observed by Cayirlioglu (2001) who performed an analysis of E2F regulated transcripts in ovarian follicle cells. Two reasons may have precluded detecting these changes: (1) the fold change reported by Cayirlioglu was small, between 1.5 and 2.0, while this study considered only genes that exhibited a fold change of 2.0 or more; (2) the other ovarian cell types may have masked the deregulation of these genes, which occurs in a subset of ovarian cells (Stevaux, 2005).

By comparing the lists of RBF2 and dE2F2 target genes, an unpredicted feature was noted. Although RBF2 physically associates with dE2F2, and the ability of overexpressed RBF2 to repress E2F transcription requires dE2F2 the overlap between the RBF2-dependent genes and the dE2F2-dependent genes was unexpectedly small. Comparisons of the transcripts elevated in de2f2 and rbf2 mutant ovaries show that only 16 transcripts (~8%) were elevated in both mutants. The expression patterns of these differentially regulated genes underline the complexity of dE2F/RBF transcriptional control in vivo with individual target genes requiring either dE2F2 (CG5250), RBF2 (CG17470) or both gene products (CG8316, CG4250, CG10654) to maintain their normal patterns of expression (Stevaux, 2005).

Given the observed functional redundancy between RBF1 and RBF2 at dE2F2-regulated promoters in S2 cells, it is not surprising to find some transcripts upregulated in de2f2 mutant ovaries that remain unchanged in rbf2 mutant ovaries.12 However, the presence of transcripts elevated in rbf2 mutants but unchanged in de2f2 mutants raises a number of possibilities. First, RBF2 could be recruited to particular promoters by factors other than dE2F2. Second, a dE2F1/RBF1 complex might repress these targets in the absence of dE2F2, but be precluded to bind when RBF2 is missing due to the retained presence of dE2F2 at these promoters. Third, these changes in gene expression could be indirect effects of mutating either dE2F2 or Rbf2. To distinguish between these possibilities, chromatin immunoprecipitation (ChIP) assays were performed with ovary chromatin extracts on a number of RBF2- and dE2F2-regulated promoters (Stevaux, 2005).

ChIP analysis revealed that dE2F and RBF proteins bind at the promoters of genes from every category, verifying that these genes are directly regulated by dE2F/RBF. As evidenced by the lack of dE2F1 binding to their promoters, these genes appear to be specifically regulated by dE2F2-mediated repression similar to the genes repressed by dE2F2/RBF1 and dE2F2/RBF2 in S2 cells (Dimova, 2003). Interestingly, dE2F2 and RBF2 were found at these promoters, regardless of whether they are upregulated in de2f2 mutant ovaries, rbf2 mutant ovaries, or both. The fact that dE2F2 and RBF2 are part of a large multi-subunit complex, the dREAM complex (Lewis, 2004; Korenjak, 2004), raises the possibility that RBF2 can be retained within this complex via another subunit and maintain repression in the absence of dE2F2. Studies with de2f2 mutant ovaries rule out this possibility since RBF2 can no longer be detected at any promoter in de2f2 mutant ovaries indicating that RBF2 is recruited to promoters via dE2F2. It is thought unlikely that dE2F1/RBF1 can redundantly repress transcription of these genes, since no binding to their promoters is detected in de2f2 mutant ovaries, even though dE2F1 can bind to some dE2F2-regulated genes in the absence of dE2F2. It is likely that a repression mechanism independent of dE2F1 can function in the absence of dE2F2 to inhibit the expression of these genes (Stevaux, 2005).

These results suggests that the different regulation of these target genes is not determined by the differential binding of dE2F/RBF proteins to their promoters. Rather, the importance of the individual components most likely results from the differential requirement of additional cofactors. RBF2 and dE2F2 control cell-type specific transcriptional programs. Since its discovery, E2F has been viewed as a regulator of cell proliferation. Because the genes regulated by E2F encode core components of the cell cycle machinery, and E2F is a ubiquitous factor, it has been generally assumed that E2F regulates the transcription of these genes in most, if not all cell types. E2F has been studied in a wide variety of tissue culture cells. These tissue culture studies have, for a large part, placed very little emphasis on the types of cells used for experimentation. Although the activity of E2F is understood to vary between cell lines, and at different stages of the cell cycle, many reviews of the E2F literature contain an implicit assumption that E2F and RB like proteins target the same sets of genes in all cell types. This assumption was tested by comparing the ovary RBF2-dependent targets genes with target genes previously identified in S2 cells following RBF2 specific RNA interference. Remarkably, minimal overlap was found between the transcripts that are elevated in RBF2-deficient ovaries and in S2 cells. Only 1 gene (<1%) is commonly elevated in rbf2 mutant ovaries in vivo and in RBF2 depleted S2 cells. This analysis was extended to dE2F2, and using the same methodology the transcripts that have elevated expression in de2f2 mutant ovaries were compared with the genes upregulated in dE2F2-depleted S2 cells. In keeping with the results obtained for RBF2, a small overlap of only 15 genes was found between these two lists. The limited overlap between the genes regulated by dE2F2 and RBF2 in S2 cells and in ovaries indicates that dE2F2 and RBF2 are needed to regulate distinct sets of genes in different cell types (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 were obtained 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. It has been shown that in S2 cells, dE2F2 and RBF2 can be found at promoters of genes whose expression did not change in the absence of either dE2F2 or RBF2. The results from the current 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 examined 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 (Stevaux, 2002), the transcriptional profiles of wild type and rbf2 mutant embryos were compared using microarrays at this early embryonic developmental stage (Stevaux, 2005).

Numerous transcripts were found to be elevated in early rbf2 mutant embryos, including a number of ovary- and testis-specific differentiation markers. In keeping with earlier observations, the overlap was minimal between transcripts elevated in rbf2 mutant embryos, rbf2 mutant ovaries and S2 cells depleted for RBF2. 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 were observed to be deregulation 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. The 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, not only in different cell types, but also at different stages of development (Stevaux, 2005).

Taken together these results demonstrate that RBF2 is required in vivo for the direct transcriptional repression of dE2F2 targets encoding a wide array of biological functions, including genes encoding sex-specific differentiation markers. Remarkably, the dE2F2/RBF2-dependent transcription program appears to be variable, rather than constant (Stevaux, 2005).

Previous work has reported that individual dE2F family members have common as well as specific transcriptional target genes. Among the common targets are genes with functions in cell cycle progression and DNA synthesis. Recent work suggests that the type of regulation of these targets depends on the cellular context. While in S2 cells and in the developing eye disk dE2F1 and dE2F2 play antagonistic roles, activating and repressing their expression respectively, in ovarian follicle cells dE2F1 and dE2F2 cooperate to repress transcription of DNA synthesis genes. This study confirms this idea by showing that dE2F2/RBF2 complexes control the expression of changing sets of genes in different cell types and/or different developmental contexts. Collectively this data indicates that dE2F/RBF proteins provide a diversity of transcriptional regulatory inputs at different target gene promoters in different contexts. Thus, a variety of transcriptional outputs is achieved by a relatively simple set of proteins. This regulation is likely conserved throughout evolution and it is tempting to speculate that the more complex mammalian E2F/RB network will generate even a greater variety of transcriptional responses. Clearly the next challenge will be to identify the cofactors that act in concert with E2F/RB proteins in each context to confer functional importance. Likely candidates include E-Box transcription factors that have recently been shown to associate with E2F3 and allow it to specifically target subsets of promoters (Stevaux, 2005).

The ability of E2F and RB family members to regulate distinct sets of genes in different cell types means that E2F/RB functions should be viewed only in the context of a particular cellular environment and imply that E2F microarray studies based on a single cell type most likely underestimate the full range of E2F target genes. This regulatory plasticity could also explain why the biological responses to perturbations the E2F/RB pathway can be seen to vary so much between different tissues, be that in the context of normal development or in tumorigenesis (Stevaux, 2005).

Widespread regulatory specificities between transcriptional co-repressors and enhancers in Drosophila

Gene expression is controlled by the precise activation and repression of transcription. Repression is mediated by specialized transcription factors (TFs) that recruit co-repressors (CoRs) to silence transcription, even in the presence of activating cues. However, whether CoRs can dominantly silence all enhancers or display distinct specificities is unclear. This work reports that most enhancers in Drosophila can be repressed by only a subset of CoRs, and enhancers classified by CoR sensitivity show distinct chromatin features, function, TF motifs, and binding. Distinct TF motifs render enhancers more resistant or sensitive to specific CoRs, as was demonstrated by motif mutagenesis and addition. These CoR-enhancer compatibilities constitute an additional layer of regulatory specificity that allows differential regulation at close genomic distances and is indicative of distinct mechanisms of transcriptional repression (Jacobs, 2023).

Animal development and homeostasis critically depend on differential gene expression, enabled by the precise regulation of transcriptional activation and repression. Although repression is often associated with heterochromatin, genes can also be silenced in transcriptionally permissive euchromatin by repressive transcription factors (TFs), also termed repressors, that bind to DNA and recruit corepressors (CoRs). As CoRs can suppress transcription even in the presence of activators, this mode of gene silencing is termed active transcriptional repression. Active repression is critical and its failure can cause developmental defects and diseases like cancer; and it is conceptually intriguing as it requires the fast and efficient overriding of activating cues. However, the modes and mechanisms of this process are unclear, and whether a regulatory code coordinates repression and activation, is unknown. Given that transcriptional activation can occur via distinct and mutually incompatible modes, it is intriguing to speculate whether distinct modes of active transcriptional repression exist. Examples of specificities between repressors and activators have indeed been observed. The CoR Retinoblastoma protein (Rb) as part of the DREAM complex for example can repress the TFs E2F, Mip120 and PU.1 but not others like SP-1, and repressors can have different activities in distinct transcriptional contexts. Comprehensive studies allowing to define different modes of active repression and uncover their regulatory rules are however lacking. This study determined the mutual compatibilities between five known CoRs and a genome-wide library of active enhancers by measuring enhancer-activity changes upon CoR tethering in otherwise unperturbed cells, similar to activator-bypass experiments. It was reasoned that testing each enhancer with each CoR in all possible combinations should reveal CoR-enhancer combinations that lead to decreased enhancer activity (enhancers are sensitive) and those that do not (resistant), indicative of compatible and incompatible pairings (Jacobs, 2023).

First, this study tested whether distinct specificities between CoRs and enhancers exist, whereby certain enhancers are sensitive to repression by a given CoR, while other enhancers are resistant. For this, it was necessary to systematically measure the effects that selected CoRs have on the activity of a large number of enhancers. The comprehensive mapping of CoR-enhancer compatibilities, by examining all combinations of CoRs and enhancers, requires highly controllable quantitative high-throughput assays. Therefore the massively parallel enhancer-activity assay STARR-seq was modified to enable the function-based testing of genome-wide enhancer candidate libraries with different CoRs. Briefly, four upstream-activating-sequence (UAS) motifs were introduced immediately downstream of the enhancer library, which leave the enhancer sequence intact yet allows for the direct tethering of selected CoRs via the Gal4 DNA-binding domain. The tethering of CoRs next to active enhancers directly assesses whether CoRs can override existing activating cues, a process akin to active repression (Jacobs, 2023).

Drosophila S2 cells were chosen as a model system, and a panel of five CoRs; CoRest, CtBP, Rbf, Rbf2 and Sin3A was tested. These CoRs represent different protein complexes, repressive pathways, enzymatic functions, and distinct groups with context-specific functions. Testing diverse CoRs casts a wide net and should increase the ability to detect compatible and incompatible CoR-enhancer pairs. For each CoR, two independent UAS-STARR- seq screens were performed where the UAS-STARR-seq library was co-transfect with a vector that expresses the Gal4-CoR (or Gal4-GFP as neutral control), and spike-in-controls were used for normalization. As spike-in controls a distinct STARR-seq library was used containing 18 Drosophila pseudoobscura enhancers, cloned without the UAS motifs, and hence not targeted by the Gal4- -CoRs. In all cases, the two independent replicates correlated well. In order to reliably assess repression (which requires a high baseline activity), it was decided to evaluate enhancer-activity changes for 3094 enhancers that were highly active in Gal4-GFP controls (Jacobs, 2023).

First, which of the 3094 enhancers could be silenced by the highly conserved CoR CtBP was determined, by assessing the enhancer activity changes when tethering Gal4-CtBP versus Gal4-GFP. This revealed that some enhancers, like the enhancers near Orct, Orct2 and Pka-C3, were reproducibly repressed by Gal4-CtBP, whereas others, like the enhancer near CG10516, were unaffected. A differential analysis based on the two replicates per condition using edgeR showed that CtBP significantly repressed 759 enhancers but not the remaining 2335. Thus, CtBP could only repress ~25% of the enhancers, suggesting that CtBP displays preferences or specificities towards some enhancers but not others (Jacobs, 2023).

The enhancer-activity changes upon recruiting Rbf2, a CoR from the retinoblastoma protein family was determined. Interestingly, Rbf2 was also able to repress only a subset of the enhancers (1733, 56%), including the enhancers near Orct and Pka-C3, which were also repressed by CtBP, and CG10516, which was not repressed by CtBP. In contrast, Rbf2 was unable to repress the aforementioned Orct2 enhancer and others, indicating that the specificities of Rbf2 differ from those of CtBP. Indeed, while 502 enhancers were repressed by CtBP and Rbf2, 1231 enhancers were repressed by Rbf2 but not by CtBP and, vice versa, 257 enhancers were repressed by CtBP but not by Rbf2. These enhancer-CoR specificities were validated in luciferase reporter assays, in which the CoRs were recruited upstream of the enhancer and promoter. This assay confirmed that an enhancer near serpent was specifically repressed by Gal4-CtBP but not Gal4-Rbf2, an enhancer near CG2116 was specifically repressed by Gal4-Rbf2 but not Gal4-CtBP, and an intronic enhancer of kay was repressed by both CoRs, each in agreement with the STARR-seq results. Taken together, the CoRs CtBP and Rbf2 are each able to repress a specific subset of enhancers but not others, indicating the existence of distinct CoR-enhancer specificities (Jacobs, 2023).

Screening three additional prominent CoRs; CoRest, Rbf and Sin3A revealed that each of them was able to repress a specific subset of enhancers. CoRest was the strongest repressor, repressing 1452 enhancers and often reducing their activity to background levels. The overall repression profiles of CoRest and Sin3A were similar to that of CtBP and clearly different from Rbf2. Rbf's repression profile was not similar to any of the other tested CoRs. In general, the five tested CoRs were each able to repress a subset of enhancers and displayed distinct specificities (Jacobs, 2023).

Given that enhancers were differentially sensitive to some of the tested CoRs, and resistant to others, whether these differential sensitivities were related to other enhancer properties was examined. For this, the enhancers were clustered into groups of similar repression patterns. A self-organizing tree algorithm (SOTA) was used with the PCC as distance metric to cluster the enhancers into five groups based on their sensitivity to the CoRs. Cluster 1 contains CoRest and CtBP resistant enhancers while enhancers in cluster 2 are resistant to CtBP and Sin3A. Cluster 3 contains enhancers that are resistant to Rbf2, enhancers in cluster 4 are sensitive to all CoRs while enhancers in cluster 5 are overall very sensitive to repression but resistant to Rbf. Hence, the enhancers of S2 cells could be divided into five groups, defined by their differential response to the tested CoRs (Jacobs, 2023).

Enhancers clustered by corepressor sensitivity differ in chromatin marks, transcription factor motif content and binding Next, whether these enhancer clusters differ in additional properties that correlate with their behaviour towards the CoRs was tested. Initial enhancer activity, as measured by UAS- STARR-seq with Gal4-GFP, was similar for all five clusters. Also H3K27ac, a histone modification that marks active enhancers, was similarly enriched at the endogenous enhancer loci of all clusters. It was inferred that the distinct specificities did not stem from differences in initial enhancer strengths. The histone modifications H3K4me1 and H3K4me3 did however show differential and complementary trends: H3K4me1 was the highest enriched at cluster 5, followed by 4 and 3, whereas H3K4me3 was more highly enriched at enhancer clusters 1 and 2. High H3K4me1 levels combined with high H3K27ac levels have been associated with distal regulatory regions or cell type-specific enhancers, suggesting that enhancers with the highest H3K4me1 levels (cluster 5) might be most specific to S2 cells. Analysing chromatin accessibility and H3K27ac levels in other cell types and tissues indeed revealed that enhancers from cluster 5 were highly cell type-specific. Interestingly, these enhancers were also the most strongly repressed by CoRest, CtBP and Rbf2, suggesting that developmental or cell type-specific enhancers might intrinsically be more sensitive to repression by certain CoRs, while more globally active or housekeeping enhancers might be more resistant. As active enhancers are known to function through a variety of different TFs, it was hypothesised that their differential response to the CoRs might be linked to the specific TFs they bind (Jacobs, 2023).

Using published TF ChIP-seq data, it was observed that prominent TFs were bound to enhancers from specific clusters and absent from others. The TFs DREF and M1BP for example bound almost exclusively to enhancers from clusters 1 and 2, with DREF preferring cluster 1 and M1BP cluster 2. Trithorax-like (Trl, also called GAGA factor or GAF) on the other hand was absent from these clusters and instead mainly bound to enhancers from cluster 3 and to a lower extent to cluster 4. The distribution of these three TFs suggests that differential CoR sensitivities might relate to distinct TFs. To identify associations between CoR sensitivities and TFs in a more comprehensive manner, TF-motif enrichment analyses was performed for the enhancers of the five clusters using the 6502 TF motifs from the iRegulon database. Consistent with the ChIP-seq results, motifs for DREF, M1BP and Trl were specifically enriched in clusters 1, 2 and 3, respectively. Additional motifs were also differentially enriched between the clusters: Rsc30 and E-box motifs were enriched in cluster 3, Sim motifs were enriched in cluster 4, while ETS and GATA motifs were enriched in cluster 5. Other prominent TF motifs were enriched over several clusters, including AP-1, Ato and CrebB. Taken together, the different enhancer clusters, defined by their sensitivity towards CoRs, associate with distinct chromatin features and TF motif content (Jacobs, 2023).

To directly test the association between TF motifs and sensitivity towards each of the CoRs,the enrichment of TF motifs within enhancers that were sensitive ) versus those that were resistant to each CoR were evaluated. For CtBP, for example, it was found that AP-1, Trl and GATA motifs were strongly enriched in sensitive enhancers, whereas DREF, Ohler1 and Mip120 motifs were enriched in the resistant enhancers. Motif enrichment profiles across all five CoRs revealed an intricate relationship between enhancer-CoR sensitivity and TF motifs with highly distinctive enrichments. Consistent with the conserved role of Rb proteins as part of the DREAM complex, E2F, DP, and Mip120 motifs were enriched in Rbf sensitive enhancers. Interestingly, these three motifs as well as DREF and M1BP motifs were enriched in enhancers that were resistant to CoRest, CtBP and Sin3A repression. On the other hand, motifs for the developmental TFs GATA, AP1 and TEAD were enriched in enhancers sensitive to CoRest, CtBP and Sin3A and resistant to Rbf and Rbf2. Furthermore, Trl and ETS motifs were specifically enriched in enhancers that were resistant to Rbf2 and Rbf respectively, while sensitive to all other CoRs. These results indicate that, for each CoR, certain TF motifs are specifically and strongly enriched in sensitive and resistant enhancers (Jacobs, 2023).

Given the differential motif enrichments, whether TF motif content is predictive of an enhancer's CoR sensitivity was tested. For this, a Generalized Linear Model (GLM) was trained using TF motif counts as features and enhancer sensitivity to a given CoR as response variable. For each CoR, using 10-fold cross-validation, the models based on motif counts performed well and were able to predict an enhancer's sensitivity and resistance to a given CoR. Overall, these results establish a strong association between CoR sensitivity and TF motif content that is predictive and might correspond to causal relationships (Jacobs, 2023). It was noticed that DREF TF motifs were enriched in enhancers that were resistant to CoRest and CtBP. Indeed, enhancers bound by DREF, such as enhancers near the RYBP and jupiter genes, are specifically resistant to repression by CoRest and CtBP but sensitive to repression by Rbf. Ranking all enhancers based on their sensitivity to repression by CoRest or CtBP confirmed that resistant enhancers were significantly enriched for DREF motifs and bound by DREF according to ChIP-seq. Given the correlation between CoRest and CtBP resistance and presence of DREF, it was considered that DREF might protect enhancers against CoRest and CtBP-mediated repression. To test whether DREF motifs are indeed required for the resistance, 65 DREF-motif containing enhancers, mutated the DREF motifs, were tested, and the enhancers' sensitivity to repression was assessed. The mutated enhancers were significantly more sensitive to repression by CoRest and CtBP, while their sensitivity towards Rbf did not change. It is inferred that DREF motifs are required for resistance to CoRest and CtBP. Several other TF motifs were predicted to confer resistance to repression by specific CoRs. ETS- family motifs, for example, were specifically enriched in enhancers that were resistant to Rbf repression was observed. The ETS motifs were mutated in 157 enhancers and indeed increased sensitivity towards Rbf-mediated repression. Remarkably, the opposite trend was observed for the other CoRs, which were all able to repress the wildtype enhancers containing ETS motifs better than their mutated counterparts, suggesting that ETS TFs are in general sensitive to repression. Similarly, Trl motifs were enriched in enhancers that were specifically resistant to Rbf2 repression and mutating these motifs in 127 enhancers led to a slight but specific increase in sensitivity towards Rbf2, but not towards the other CoRs (Jacobs, 2023).

In general, specific TF motifs are required to protect resistant enhancers against a given CoR. As these motifs and enhancers are however still sensitive to other CoRs, an intricate pattern emerges, which suggests that the interplay between repressors and activators can be highly specific (Jacobs, 2023).

Given that specific TF motifs are required for CoR resistance, it was asked whether these motifs are also sufficient to cause CoR resistance. To test this, two 'resistant' TF motifs (10 bp each) were introduced into CoR-sensitive enhancers at positions deemed unimportant for enhancer activity, and the enhancers' sensitivity to repression was evaluated. As a control for the addition of motifs, two neutral control TF motifs were introduced at the same positions (of the ACE2 and FOX TFs), which were predicted to not impact the sensitivity to repression. Since DREF motifs were required for resistance to CoRest and CtBP-mediated repression, whether they were sufficient to render CoRest and CtBP-sensitive enhancers resistant was tested. Overall, the introduction of two DREF motifs was sufficient to protect the enhancers from CoRest and CtBP-mediated silencing and this protection was specific and due to the DREF motifs, as control motifs had no effect on sensitivity. Similarly, ETS motifs were necessary and sufficient to desensitize enhancers from repression by Rbf, and Trl motifs were necessary and sufficient to specifically generate Rbf2-resistant enhancers (Jacobs, 2023).

In each case, the gain in resistance due to the addition of selected TF motifs was highly CoR specific, as these new motifs had little effect on the repression by other CoRs or could even sensitize the enhancers to other CoRs. The addition of ETS or Trl motifs for example made enhancers more sensitive to all other CoRs except for Rbf or Rbf2 respectively, while DREF motifs increased sensitivity towards Rbf. Hence, introducing specific TF motifs changed the enhancer's sensitivity to repression by a given CoR in a specific and predictable manner. Taken together, it is concluded that specific TFs are necessary and sufficient to confer resistance to repression by a given CoR, while other CoRs are able to repress these TFs, indicating that certain TFs can directly counteract some CoRs and their repressive function or that different modes of transcriptional activation require different modes of repression (Jacobs, 2023).

This work has shown that transcriptional enhancers are differentially susceptible to active repression by prominent CoRs. The regulatory specificities between active enhancers and five CoRs were mappedon a genome-wide scale, and it was discovered that enhancers across the genome are repressed by just a subset of specific CoRs. This additional layer of regulatory specificities enables the differential repression of closely spaced enhancers and genes as they frequently occur in the Drosophila genome. At the fs(1)h/mys locus for example, CoRest could repress the intronic mys enhancer without affecting the neighboring fs(1)h gene, whereas Rbf could do the opposite. Similarly, at the kay/fig locus some CoRs like CtBP could repress both closely spaced enhancers, whereas Rbf2 could specifically repress the enhancer closest to fos intronic gene (fig) but not the others (Jacobs, 2023).

The uncovered specificities between repressors and enhancers not only suggest that repression occurs via distinct mechanisms, but also reveal a previously unappreciated layer of 'resistance' against repression. Enhancers that are sensitive or resistant towards a given CoR display clear differences in their TF motif content, and motif mutagenesis experiments changed enhancer sensitivities in a predictable manner. Specific motif combinations of activating and repressive TFs, together with the regulatory proteins present in each cell type, will largely determine when and where a regulatory region is active or repressed. In addition, the ability of TFs to confer resistance also allows for enhancers and genes to be activated by de- repression via TF recruitment, as has been demonstrated by motif addition. This additional layer of regulation circumvents the requirement to remove the involved repressors and the interplay between TFs and CoRs provides more flexibility to the regulatory system. Importantly, the fact that one mode of activation can be sensitive to one repressor but resistant to another implies that distinct activation and repression mechanisms must converge. Active repression might directly interfere with specific factors or defined steps of transcriptional activation. Certain CoRs might for example inactivate specific TFs but not others (e.g. via posttranslational modifications), or counteract the TFs' downstream activating mechanisms. Alternatively, certain TFs might directly counteract a repressor's function or bypass the rate-limiting step of initiation- or elongation controlled by the CoR. Discerning the distinct mechanisms of activation and repression and how they intersect and coordinate will be of great future interest (Jacobs, 2023).

Retinoblastoma protein regulation by the COP9 signalosome

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

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

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

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

p55, the Drosophila ortholog of RbAp46/RbAp48, is required for the repression of dE2F2/RBF-related genes

Many proteins have been proposed to be involved in retinoblastoma protein (pRB)-mediated repression, but it is largely uncertain which cofactors are essential for pRB to repress endogenous E2F-regulated promoters. Advantage was taken of the stream-lined Drosophila dE2F/RBF pathway, which has only two E2Fs (dE2F1 and dE2F2), and two pRB family members (RBF1 and RBF2). With RNA interference (RNAi), potential corepressors were depleted and the elevated expression of groups of E2F target genes that are known to be directly regulated by RBF1 and RBF2 was sought. Previous studies have implicated histone deacetylase (HDAC) and SWI/SNF chromatin-modifying complexes in pRB-mediated repression. However, the results fail to support the idea that the SWI/SNF proteins are required for RBF-mediated repression and suggest that a requirement for HDAC activities is likely to be limited to a subset of targets. The chromatin assembly factor p55/dCAF-1 is essential for the repression of dE2F2-regulated targets. The removal of p55 deregulates the expression of E2F targets that are normally repressed by dE2F2/RBF1 and dE2F2/RBF2 complexes in a cell cycle-independent manner but has no effect on the expression of E2F targets that are normally coupled with cell proliferation. The results indicate that the mechanisms of RBF regulation at these two types of E2F targets are different and suggest that p55, and perhaps p55's mammalian orthologs RbAp46 and RbAp48, have a conserved function in repression by pRB-related proteins (Taylor-Harding, 2004).

Cell cycle-dependent and cell cycle-independent control of transcription by the Drosophila 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 were examined 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 dE2F/RBF-dependent transcription, in which dE2F2/RBF1 or dE2F2/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 dE2F/RBF complexes should not be viewed simply as a cell cycle regulator of transcription. Instead, dE2F/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).

This study describes the existence of a program of E2F-dependent transcription that is the very antithesis of the conventional view of E2F/RB action. The analysis of RNAi-treated cells and mutant animals reveals a group of E2F-regulated targets, the Group E genes, that are strongly repressed by dE2F2, RBF1, and RBF2 in actively proliferating cells. These dE2F/RBF-regulated genes are expressed in a variety of developmentally regulated, tissue-specific, and/or gender specific patterns (Dimova, 2003).

These results challenge the dogma that E2F-regulated genes have cell cycle-regulated patterns of expression. At Group E promoters, dE2F2 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, dE2F1. Whereas dE2F2, dDP, RBF1, and RBF2 were readily detected at most E2F-dependent genes and at each of the different groups of E2F targets uncovered in this study, dE2F1 was 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 dE2F proteins is influenced by selective interactions with other factors, as has been demonstrated for mammalian E2F proteins. 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).

A second feature of Group E promoters is that dE2F2/RBF1 and dE2F2/RBF2 complexes appear to be stable and persist in S phase, at times when only dE2F1 is bound at cell cycle-regulated promoters. The implication of this result is that the activation of G1 Cdks is not sufficient to disrupt all dE2F2/RBF repressor complexes; hence, there must be an additional level of control that dictates which repressor complexes remain stable and which repressor complexes are disrupted. It is not yet clear whether dE2F2/RBF compexes remain stable despite being phosphorylated or whether they escape Cdk action. Observations that mammalian E2F4/p107 and E2F4/pRB complexes of unknown function exist in S-phase cells suggest that this type of regulation may not be unique to Drosophila. Moreover, human promoters have been identified that are bound by E2F1 and pRB during S phase, and pRB has been found that was poorly phosphorylated at some of these sites. Factors other than Cdks have been found to disrupt E2F/pocket-protein complexes, and future studies are needed to determine how different signals may distinguish between repressor complexes at different promoters (Dimova, 2003).

The discovery of E2F target genes that are regulated by dE2F and RBF proteins in a manner that is so different from the known pattern illustrates the limitations of current models for E2F action. These models are based in large part on the analysis of overexpressed E2F proteins and the detailed analysis of only a few cell cycle-regulated promoters. The mutation of E2F and RB family members in mice, flies, or worms has given a variety of unexpected and unexplained tissue-specific defects. It has been suggested that these differentiation phenotypes are caused by defects in cell cycle exit prior to terminal differentiation and as a consequence of changes in the expression of cell cycle regulators. However, the discovery that dE2F/RBF complexes are needed to repress developmentally regulated genes raises the possibility that many of the changes seen in mutant animals may be due, at least in part, to the inappropriate expression of differentiation factors. For example, it seems likely that the misexpression of genes with known functions in gametogenesis and genes normally expressed in gender-specific patterns, which occurs in de2f2 mutants, contributes to the fertility defects seen in these animals (Dimova, 2003).

Several studies have pointed out that the key to understanding the biological functions of E2F- and RB-related genes lies in understanding how this network operates over its full range of targets. Which E2F proteins are important, at which promoters, and when? And what are the rules that govern how these activities are integrated? By examining the consequences of specifically and systematically removing each dE2F and RBF protein, transcripts were identified that depend on each component of the Drosophila dE2F/RBF network for their normal regulation. The results give the first glimpse of how the functions of these family members are organized. Several general conclusions are summarized below (Dimova, 2003).

(1) It is clear that there is not one program of E2F-dependent transcription. Instead, different groups of functionally related genes are coordinately regulated by subsets of dE2F/RBF family members. The E2F-regulated genes were separated into two fundamentally different categories. The first category consists of genes with functions required for cell cycle progression (DNA replication, DNA repair, chromatin structure, and mitosis) that are highly expressed during cell division. Expression of these targets is mainly dependent on dE2F1 activation. A second category, which includes the Group E genes, contains E2F targets that are not expressed in dividing cells. These genes are strongly repressed by dE2F2, and depend very little if at all on dE2F1 activation. This category includes genes with functions in gametogenesis and markers of differentiation, and these genes have an assortment of dE2F2-restricted expression patterns suggesting that they are normally expressed in distinct developmental programs (Dimova, 2003).

(2) The functional overlap between RBF1 and RBF2 at a given promoter is tightly connected to the relative roles of dE2F1 and dE2F2. Of the 61 genes up-regulated in RBF1-depleted cells, 57 (93%) were decreased in cells lacking dE2F1. Conversely, of the 52 transcripts increased in cells lacking both RBF1 and RBF2, but not in cells lacking RBF1 alone, 37 (71%) were up-regulated in dE2F2-depleted cells. Hence, genes regulated by dE2F1 activation are mostly dependent on RBF1, whereas genes regulated by dE2F2 repression can be equally repressed by RBF1 or RBF2. These results closely parallel the pattern of protein/protein interactions between dE2F/RBF family members (Stevaux, 2002). The observation that RBF2 levels increase in the absence of RBF1 suggests a simple model in which RBF2 can functionally compensate for RBF1 at certain promoters. This situation closely parallels the mammalian pocket-protein network, where p107 is known to compensate for the absence of pRB during myotube formation. Because RBF2 is developmentally regulated (Stevaux, 2002), its normal function may involve the repression of some dE2F2-regulated genes in cell types or tissues where RBF1 is limiting (Dimova, 2003).

(3) E2F proteins clearly do have different target specificities. However, most E2F-regulated promoters bind all family members, and it is impossible to predict from ChIP experiments precisely which dE2F and RBF family members will be rate-limiting for overall levels of gene expression. This distinction between binding and functional significance is not limited to Drosophila cells. Recent studies in yeast and mammals have found discrepancies between promoter binding assays and experiments that test functional significance. Studies in mammalian cells show that particular E2F/RB proteins are important at different sets of promoters. However, ChIP experiments have failed to find any correlation with selective binding to individual E2F or pRB family members. Thus, in most cases the key issue is not 'which E2F or pRB family members bind to a promoter' but 'which, of the many E2F and pRB family members that do bind, are important for function' (Dimova, 2003 and references therein).

Why do the contributions of individual family members vary between promoters that bind the same sets of proteins? A favored explanation highlights the combinatorial nature of transcriptional regulation: The contribution of any one component of the dE2F/RBF network to the transcription of any given promoter is determined by both its recruitment to the promoter and by the availability of other activators and repressors. Thus, promoters that are coordinately regulated by dE2F1/RBF1, for example, share a specific requirement for dE2F1/RBF1, rather than being the only promoters where these proteins bind. In addition, differences in affinity and timing of binding may also be important. Although relative differences in ChIP signals are difficult to interpret, it is noted that the intensity of the dE2F1 and dE2F2 ChIP signals varied between the groups of E2F targets in a manner that paralleled the relative importance of dE2F1 and dE2F2 in the expression patterns. Studies of the mammalian B-Myb promoter have shown that it is bound by activating E2F complexes for only a short window of time at the end of G1, whereas other cell cycle-regulated promoters are occupied by activator E2F throughout S phase. Such differences in occupancy might affect the strength of E2F-mediated repression and/or activation (Dimova, 2003}.

Finally, based on the analysis outlined here, a revised view of E2F regulation in Drosophila is proposed. 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}.

Distinct mechanisms of E2F regulation by Drosophila RBF1 and RBF2

RBF1, a Drosophila pRB family homolog, is required for cell cycle arrest and the regulation of E2F-dependent transcription. RBF2, a second family member, represses E2F transcription and is present at E2F-regulated promoters. Analysis of in vivo protein complexes reveals that RBF1 and RBF2 interact with different subsets of E2F proteins. E2F1, a potent transcriptional activator, is regulated specifically by RBF1. In contrast, RBF2 binds exclusively to E2F2, a form of E2F that functions as a transcriptional repressor. RBF2-mediated repression requires E2F2. Moreover, RBF2 and E2F2 act synergistically to antagonize E2F1-mediated activation, and they co-operate to block S phase progression in transgenic animals. The network of interactions between RBF1 or RBF2 and E2F1 or E2F2 reveals how the activities of these proteins are integrated. These results suggest that there is a remarkable degree of symmetry in the arrangement of E2F and RB family members in mammalian cells and in Drosophila (Stevaux, 2002).

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

An N-terminal portion of RBF2 was expressed in bacteria and used to generate monoclonal antibodies that specifically recognize RBF2 and do not cross-react with RBF1. On SDS-PAGE, RBF2 migrates slightly faster than RBF1 and separates as a single band of ~85 kDa. The expression of RBF1 and RBF2 was examined by Western blot analysis using extracts prepared at various stages of Drosophila development. The levels of RBF1 are relatively constant; the highest level of RBF1 is seen in 0- to 4-h-old embryos, and this drops slightly during the later stages of embryogenesis. In contrast, RBF2 levels vary considerably during development. RBF2 levels increase 4-fold in the first 8 h of development, and drop by one to two orders of magnitude at later stages of embryogenesis. Comparatively low levels of RBF2 were detected in whole larval extracts, and on long exposures of the Western blots, in adult females, but not in males. Higher levels of RBF2 were found in dissected larval imaginal discs and in tissue culture cells. These patterns suggest that RBF2 may be most highly expressed in rapidly cycling cells, a fact that has previously been for p107. The RBF2 present in female extracts is provided exclusively by the ovary, raising the possibility that RBF2 may function during oogenesis (Stevaux, 2002).

Repression of E2F transcription is a hallmark of RB family proteins. To determine whether RBF2 regulates E2F-dependent transcription, its ability to repress E2F-regulated reporter constructs in SL2 cells was assayed. RBF1 and RBF2 expression constructs were generated and transfected into SL2 cells together with a PCNA reporter that had been previously used to measure the activity of dE2F1 and dE2F2. In order to monitor the expression levels from both constructs, RBF1 and RBF2 were HA-tagged on their N-terminal ends. Titration experiments showed that RBF2 represses transcription from the wild-type PCNA promoter but has no effect on the mutant PCNA reporter construct lacking E2F-binding sites. The repression properties of RBF1 and RBF2 were examined. RBF2 represses transcription from the PCNA promoter, as well as the MCM3 and DNA Polalpha promoters, two other E2F-regulated genes. In these reporter assays, RBF1 proved a more effective repressor than RBF2, when expressed at the same level. The reason why RBF1 and RBF2 expression plasmids give different levels of protein expression is not known. This difference may reflect a property of the endogenous proteins, since quantitative blots show that SL2 cells contain ~30 times more RBF1 than RBF2. It is concluded that RBF2 can repress E2F-dependent transcription, but in a less efficient manner than RBF1 (Stevaux, 2002).

To determine whether RBF1 and RBF2 are present at these promoters in vivo under physiological conditions, a chromatin immunoprecipitation (ChIP) assay was used with specific RBF1 or RBF2 antibodies. DNA sequences from the PCNA and the DNA Polalpha promoters are selectively enriched in the RBF1 and RBF2 immunoprecipitations. It is concluded that RBF1 and RBF2 are both able to repress transcription from E2F-regulated promoters, and that the endogenous RBF1 and RBF2 proteins are normally found at these promoters in vivo. The presence of both Drosophila pocket proteins at E2F promoters suggests that RBF1 and RBF2 may have overlapping functions in the regulation of E2F targets genes (Stevaux, 2002).

Since the overexpression of RBF2 is able to repress E2F-dependent transcription, it seemed likely that RBF2 would repress dE2F1-mediated activation in a manner similar to that previously shown for RBF1. To test this possibility, RBF2 and dE2F1 expression constructs were co-transfected together with a PCNA reporter plasmid in SL2 cells. To ensure that dE2F1 was not saturating, small quantities of the dE2F1 expression plasmid were used that resulted in a 4.5-fold activation of the PCNA reporter. Co-transfection of HA-RBF1 completely blocked this dE2F1-induced transcriptional response, and a significant degree of repression was observed when a low amount of HA-RBF1 was transfected (Stevaux, 2002).

Surprisingly, however, HA-RBF2 had no effect on dE2F1-activated transcription. RBF2 also failed to repress the dE2F1 activation of the DNA Pola reporter. In keeping with these transfection results, it was noted that in transgenic animals, contrary to RBF1, RBF2 overexpression fails to suppress phenotypes caused by elevated levels of dE2F1 in various tissues. Since overexpression of RBF2 does not inhibit dE2F1-driven transcription and does not suppress dE2F1-induced phenotypes in vivo, it appears that RBF1 and RBF2 regulate E2F-dependent transcription in a distinct manner (Stevaux, 2002).

To understand the relationship between RBF1, RBF2, and the E2F proteins, the pattern of dE2F-RBF protein interactions that exist in Drosophila SL2 cells was examined. Specific antibodies for dDP, dE2F2, dE2F1 were used to immunoprecipitate protein complexes from SL2 extracts. These immune complexes were analyzed by Western blotting with monoclonal antibodies specific for RBF1 or RBF2. A single 85 kDa band was detected by an anti-RBF2 monoclonal antibody in dDP and dE2F2 immunoprecipitates, but not in the dE2F1 or the control immunoprecipitates. The blot was stripped and re-probed with an anti-RBF1 monoclonal antibody and, as expected, RBF1 was detected in each of the test lanes. In the reciprocal experiment, dE2F1 was detected in RBF1 immune complexes, but not in RBF2 immune complexes, whereas dE2F2 was found in both RBF1 and RBF2 immune complexes. These results indicate that, under physiological conditions, RBF1 forms complexes with dE2F1 or dE2F2. RBF2, however, does not bind dE2F1, the activator Drosophila E2F, but associates exclusively with the repressor Drosophila E2F, dE2F2 (Stevaux, 2002).

This pattern of interactions could explain, in a very simple way, why RBF2 is unable to block dE2F1-mediated activation. This arrangement also predicts that the effects of RBF2 on E2F-regulated transcription are likely to be mediated via dE2F2. To test this hypothesis, the ability of RBF2 to repress transcription in cells depleted for dE2F2 was assessed by RNA-mediated interference (RNAi). Cells were treated with control or dE2F2-specific double-stranded (ds) RNA and subsequently transfected with an E2F reporter construct and RBF1 or RBF2 expression plasmids. Western blots of the dsRNA-treated cells showed that the level of dE2F2 was substantially reduced by the RNAi treatment after 4 days. The ability of RBF2 to repress transcription from the MCM3 promoter is completely inhibited in cells treated with dE2F2 dsRNA. In contrast with RBF2, the ability of RBF1 to repress transcription is unaffected by the depletion of dE2F2, presumably because of the presence of dE2F1, the other E2F partner of RBF1. This experiment indicates that dE2F2 is necessary for the proper repression of E2F target genes by RBF2. To test whether dE2F2 is required for the detection of RBF2 at E2F-regulated promoters, ChIP assays was performed on cells treated with dE2F2 dsRNA. The depletion of dE2F2 from SL2 cells eliminates the binding of dE2F2 and RBF2 to the DNA Pola promoter, while leaving dE2F1 binding unaffected (Stevaux, 2002).

These experiments show that RBF2 can no longer be localized to E2F-regulated promoters or repress transcription in the absence of the DNA-binding activity provided by dE2F2. While the levels of HA-RBF2 expressed in transient transefction assays were unaffected by depletion of dE2F2, it was noticed that levels of endogenous RBF2 protein were slightly reduced when dE2F2 was depleted by RNAi. Interestingly, Western blots of larval extracts prepared from wild-type and de2f2 mutant larvae show that a long-term consequence of removing dE2F2 is that RBF2 becomes barely detectable. The reduction of RBF2 protein is due to post-transcriptional effects, since rbf2 transcripts are readily detectable in total RNA preparations from de2f2 mutant larvae and, indeed, are present at elevated levels in the de2f2 mutants. The most likely explanation is that RBF2 becomes less stable in the absence of its binding partner. At present, the formal possibility that dE2F2 influences the synthesis of RBF2 cannot be excluded. Nevertheless, these observations all support the notion that the function of RBF2 depends on the presence of dE2F2 (Stevaux, 2002).

Previous studies have revealed that dE2F1 and dE2F2 have distinct biochemical and functional properties. Both proteins can be found at endogenous E2F-regulated promoters, but dE2F1 functions primarily as an activator of transcription, whereas dE2F2 is a transcriptional repressor. Studies of de2f1, de2f2 and de2f2;de2f1 double mutants demonstrate that the normal expression patterns of E2F-target genes depends on the integrated activities of both dE2F1 and dE2F2. Thus far, it has been observed that (1) RBF2 interacts specifically with dE2F2, (2) dE2F2 is able to antagonize dE2F1 in a manner dependent upon its interaction with RBF proteins, and (3) RBF2-mediated repression requires dE2F2. These results suggest that transcriptional repression by RBF2 is not mediated via dE2F1. Rather, RBF2 appears to form an RBF2-dE2F2 complex that antagonizes dE2F1 indirectly, by altering the balance between the transcriptional activities of dE2F1 and dE2F2 (Stevaux, 2002).

This idea was tested directly in SL2 cells, where constant levels of transfected dE2F1 were challenged with increasing amounts of transfected dE2F2. The overall level of transcription generated by both dE2Fs at the PCNA promoter were compared with and without co-transfected RBF2. The combined expression of dE2F2 and RBF2 is able to reduce dE2F1-mediated activation of the PCNA reporter far more effectively than dE2F2 alone. Thus, in SL2 cells, RBF2 and dE2F2 cooperate to antagonize the transcriptional activity of dE2F1 (Stevaux, 2002).

The combined expression of dE2F2 and RBF2 generates a small rough eye phenotype. This small eye phenotype is similar to, but weaker than, what is observed with an ey-Gal4/UAS-rbf1 transgenic line. Wing discs overexpressing RBF2 and dE2F2 were examined to determine the physiological basis of the observed phenotype. The co-expression of RBF2 and dE2F2 causes a significant decrease in DNA synthesis. Overexpression of RBF2 and dE2F2 gives a phenotypic range that varies from discs that are shrunken and abnormal to those discs that have relatively normal morphology but display reduced BrdU incorporation. The defects do not appear until sufficient amounts of dE2F2 and RBF2 have accumulated in sensitive tissues (Stevaux, 2002).

To further assess the effects of RBF2-E2F2 expression on the cell cycle, clones of dE2F2-RBF2-overexpressing cells were generated in the wing discs and marked with GFP. The cell cycle profile of these cells was determined by FACS analysis and compared with wild-type cells from the same discs. The overexpression of dE2F2 or RBF2 alone has no effect on cell cycle distribution. However, the co-expression of dE2F2 and RBF2 causes a significant increase in the population of cells with a G₁ DNA content, and a decrease of S phase and G₂ cells. Taken together, these experiments demonstrate that, when overexpressed, dE2F2 and RBF2 act synergistically to antagonize dE2F1-mediated transcriptional activation and to block S phase entry in vivo (Stevaux, 2002).

It is concluded that although RBF1 and RBF2 are both able to repress E2F-dependent transcription, they appear to act in markedly different ways. RBF1 was originally identified by virtue of its ability to physically interact with the transcriptional activation domain of dE2F1. RBF1 is a potent inhibitor of dE2F1-mediated activation and it readily suppresses dE2F1-induced phenotypes in vivo. Unlike RBF1, RBF2 does not associate with dE2F1 in vivo, and it is unable to suppress the effects of overexpressed dE2F1. In vivo, RBF2 associates specifically with dE2F2. The recruitment of RBF2 to E2F-regulated promoters, and its ability to repress transcription, requires dE2F2. In support of the idea that RBF2 acts in a stable complex with dE2F2, these proteins act synergistically when overexpressed in SL2 cells or in transgenic animals, and that RBF2 levels are strongly reduced in de2f2 mutant larvae (Stevaux, 2002).

Of the four proteins, only dE2F1 activates transcription. It is suggested that RBF proteins modulate dE2F1-mediated activation in two distinct ways. (1) dE2F1 can be directly regulated through a physical interaction with RBF1. (2) dE2F1-mediated activation can be antagonized by the presence of RBF2-dE2F2 and RBF1-dE2F2 repressor complexes at the promoter of E2F-regulated genes. Because E2F-regulated promoters often contain multiple E2F-binding sites, it is unclear whether these complexes compete for the same binding element or whether they act antagonistically through adjacent sites. The results described here suggest that there is a hierarchy of effects, with RBF1 being a stronger antagonist of dE2F1 than either of the dE2F2-containing complexes. Nevertheless, the level of dE2F1-dependent transcription is influenced by dE2F2 and RBF2, and changing the levels of RBF2 alters the balance between dE2F1-mediated activation and dE2F2-mediated repression. One of the implications of this arrangement is that a pocket protein does not need to bind directly to an E2F subunit in order to influence its activity (Stevaux, 2002).

Strikingly, a similar arrangement of E2F/pocket proteins exists in mammalian cells. Although mammalian cells contain multiple E2F and pRB family members, recent studies have suggested that the different forms of E2F can be subdivided into two groups depending on whether they appear to be primarily involved in activation or repression. Intriguingly, pRB, like RBF1, interacts with both sets of E2Fs, whereas p107 and p130, like RBF2, interact specifically with the co-repressor E2Fs. Consistent with this, the loss of RBF1 function induces phenotypes that are remarkably similar to the effects of mutating pRB (deregulation of E2F, ectopic S phases, increased apoptosis), and the mutation of dE2F1 gives phenotypes that are very similar to those recently described for the combined mutation of the murine E2F1, E2F2 and E2F3 genes (G₁ arrest and loss of E2F-dependent transcription). Furthermore, the genetic interactions observed between RB and E2f1 or RB and E2f3 alleles in mice are reminiscent of the genetic interactions between rbf and de2f1 in flies. The logical extension of this homology is that the roles of RBF2-E2F2 in Drosophila may be similar to the repressor complexes formed by p107/p130 and E2F4/E2F5 in mammalian cells. Despite the attractions of this analogy, it is noted that the phylogenetic tree of Retinoblastoma-related proteins shows that RBF1 and RBF2 are more closely related to one another than they are to any mammalian protein. This suggests that RBF1 and RBF2 were generated by a gene duplication event, most likely from an ancestral protein that resembled p107 or p130. Consequently, similarities between the arrangement of Drosophila and mammalian RB-E2F complexes are more likely to result from a convergent evolutionary process, rather than the conservation of functional differences between pocket proteins (Stevaux, 2002).

What is the advantage of such an arrangement of complexes, and why might it be selected? The distinction between activator and repressor E2Fs is most important if one considers the effects when the complexes are disrupted. Since dE2F2 appears to be unable to activate transcription, the release of RBF1 or RBF2 from a dE2F2 complex is predicted to de-repress E2F target genes. In contrast, the release of RBF1 from a dE2F1-containing complex would liberate a strong activator of transcription. These types of E2F complexes therefore allow three different types of E2F regulatory transitions: (1) from repression to de-repression (in which RBF2-dE2F2 or RBF1-dE2F2 complexes are disrupted and removed from the promoters); (2) from repression to activation (in which RBF1-dE2F1 complexes are disrupted by phosphorylation liberating dE2F1, a strong activator of transcription); and (3) from repression to de-repression to activation (in which RBF2-dE2F2 or RBF1-dE2F2 repressors are disrupted and replaced by dE2F1) (Stevaux, 2002).

The evolution of multiple E2F-RB complexes may offer several additional advantages. Individual complexes may preferentially regulate different subsets of targets. This specificity might be achieved by different DNA-binding subunits (e.g. E2F1-RBF1 versus E2F2-RBF1) or by different protein-protein interactions with adjacent factors at the promoter. Indeed, several studies have suggested that mammalian E2F/pocket proteins may target specific promoters. A second possibility is suggested by the fact that many chromatin-remodeling activities have been linked to pocket proteins, potentially allowing a wide variety of activities to be recruited to E2F-regulated promoters. Perhaps RBF1 and RBF2 provide a bridge to different types of complexes. A third possibility is that the pocket protein-E2F complexes may be differentially regulated. pRB appears to be uniquely required for DNA damage-induced cell cycle arrest. pRB is specifically required in TGFß and p16-induced cell cycle arrest. In a similar way, RBF2 may be required for cell cycle arrest at a specific stage of development or in particular tissues. The high levels of RBF2 protein in early embryos and in dissected ovaries may reflect specific roles in embryonic cell cycle and oogenesis. Alternatively, RBF1- and RBF2-containing complexes might occupy E2F sites during different phases of the cell cycle. E2F has recently been shown to control the expression of genes encoding mitotic functions whose transcription is induced later than the G₁ to S transition. RBF2, like p107, is expressed at higher levels in actively cycling cells and is a likely E2F target gene. Potentially, this newly synthesized pocket protein may provide a repressor activity during S phase on mitosis-specific promoters or during G₂ at S phase-specific promoters (Stevaux, 2002).

The answer to many of these questions will stem from a careful comparison between rbf1 mutants, rbf2 mutants and rbf1;rbf2 double mutants. This analysis will be needed to separate the specific functions of RBF1 and RBF2 in vivo and to uncover the functions that are redundant between the two Drosophila pocket proteins (Stevaux, 2002).

Search PubMed for articles about Drosophila Rbf2

Cayirlioglu, P., Bonnette, P. C., Dickson, M. R. and Duronio, R. J. (2001). Drosophila E2f2 promotes the conversion from genomic DNA replication to gene amplification in ovarian follicle cells. Development 128: 5085-98. PubMed ID: 11748144

Dimova, D. K., Stevaux, O., Frolov, M. V. and Dyson, N. J. (2003). Cell cycle-dependent and cell cycle-independent control of transcription by the Drosophila E2F/RB pathway. Genes Dev. 17(18): 2308-20. PubMed ID: 12975318

Hallstrom, T. C. and Nevins, J. R. (2006). Jab1 is a specificity factor for E2F1-induced apoptosis. Genes Dev. 20(5): 613-23. PubMed ID: 16481464

Jacobs, J., Pagani, M., Wenzl, C. and Stark, A. (2023). Widespread regulatory specificities between transcriptional co-repressors and enhancers in Drosophila. Science 381(6654): 198-204. PubMed ID: 37440660

Korenjak, M., et al. (2004). Native E2F/RBF complexes contain Myb-interacting proteins and repress transcription of developmentally controlled E2F target genes. Cell 119(2): 181-93. PubMed ID: 15479636

Lewis, P. W., et al. (2004). Identification of a Drosophila Myb-E2F2/RBF transcriptional repressor complex. Genes Dev. 18: 2929-2940. PubMed ID: 15545624

Stevaux, O., et al. (2002). Distinct mechanisms of E2F regulation by Drosophila RBF1 and RBF2. EMBO J. 21: 4927-4937. PubMed ID: 12234932

Stevaux, O., et al. (2005). Retinoblastoma family 2 is required in vivo for the tissue-specific repression of dE2F2 target genes. Cell Cycle 4(9): 1272-80. PubMed ID: 16082225

Taylor-Harding, B., Binne, U. K., Korenjak, M., Brehm, A. and Dyson, N. J. (2004). p55, the Drosophila ortholog of RbAp46/RbAp48, is required for the repression of dE2F2/RBF-related genes. Mol. Cell. Biol. 24: 9124-9136. PubMed ID: 15456884

Ullah, Z., Buckley, M. S., Arnosti, D. N. and Henry, R. W. (2007). Retinoblastoma protein regulation by the COP9 signalosome. Mol. Biol. Cell 18(4): 1179-86. PubMed ID: 17251548

date revised: 10 April 2008

Home page: The Interactive Fly © 2008 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.