InteractiveFly: GeneBrief

Bre1: Biological Overview | References

Notch signaling controls numerous cell fate decisions during animal development. These typically involve a Notch-mediated switch in transcription of target genes, although the details of this molecular mechanism are poorly understood. dBre1 has been identified as a nuclear component required cell autonomously for the expression of Notch target genes in Drosophila development. dBre1 affects the levels of Su(H) in imaginal disc cells, and it stimulates the Su(H)-mediated transcription of a Notch-specific reporter in transfected Drosophila cells. Strikingly, dBre1 mutant clones show much reduced levels of methylated lysine 4 on histone 3 (H3K4m), a chromatin mark that has been implicated in transcriptional activation. Thus, dBre1 is the functional homolog of yeast Bre1p, an E3 ubiquitin ligase required for the monoubiquitination of histone H2B and, indirectly, for H3K4 methylation. These results indicate that histone modification is critical for the transcription of Notch target genes (Bray, 2005).

It has been suggested that the Paf1 complex (see Drosophila Paf1) functionally activates activation Rad6-Bre1 complex in ubiquitination of histone H2B at promoters (Wood, 2003) Monoubiquitination of histone H2B, catalyzed by Rad6-Bre1, is required for methylation of histone H3 on lysines 4 and 79, catalyzed by the Set1-containing complex COMPASS and Dot1p, respectively. The Paf1 protein complex, which associates with RNA polymerase II, is known to be required for these histone H3 methylation events. During the early elongation stage of transcription, the Paf1 complex is required for association of COMPASS with RNA polymerase II, but the role the Paf1 complex plays at the promoter has not been clear. Evidence that the Paf1 complex is required for monoubiquitination of histone H2B at promoters. Strains deleted for several components of the Paf1 complex are defective in monoubiquitination of histone H2B, which results in the loss of methylation of lysines 4 and 79 of histone H3. Paf1 complex is required for the interaction of Rad6 and COMPASS with RNA polymerase II. Finally, the Paf1 complex is shown to be required for Rad6-Bre1 catalytic activity but not for the recruitment of Rad6-Bre1 to promoters. Thus, in addition to its role during the elongation phase of transcription, the Paf1 complex appears to activate the function but not the placement of the Rad6-Bre1 ubiquitin-protein ligase at the promoters of active genes. A model is presented demonstrating the role of the Paf1 complex in the functional activation of the Rad6-Bre1 complex in ubiquitination of histone H2B at promoters (Wood, 2003; full text of article).

The hallmark of the Bre1 proteins is a C-terminal RING finger domain linked to an extensive N-terminal coiled-coil region. The 39 amino acid C3HC4 RING domain is flanked on both sides by 15 conserved amino acids, suggesting that the fly and mammalian proteins are true orthologs of yeast Bre1p (Hwang, 2003). RING domains are typically found in E3 ubiquitin ligases and frequently mediate the interaction with the E2 ubiquitin-activating enzyme while the other parts of the protein are involved in substrate recognition. The RING domains are therefore critical to catalyze the transfer of ubiquitin from the E2 to the substrate. To confirm the functional importance of the RING domain in dBre1, tests were performed to see whether an N-terminal fragment of dBre1 that lacks the RING domain (δ RING) could rescue dBre1 mutants. No rescue was observed with any of the 4 transgenic lines (from a total of 814 flies scored), confirming that the RING domain is essential for the function of dBre1 as it is for yeast Bre1p (Hwang, 2003; Wood, 2003; Bray, 2005).

To examine the subcellular location of full-length dBre1 and the derivative that lacks the RING domain, both forms of the protein were tagged with GFP at the N terminus. Both GFP-dBre1 and GFP-δRING are predominantly nuclear in embryonic and imaginal disc cells, although a low level of protein is also detectable in the cytoplasm. This nuclear-cytoplasmic distribution is similar to that of a δ RING derivative of human Bre1-B when it is overexpressed in mammalian cells. Thus dBre1 appears to be a nuclear protein, like its mammalian counterpart, and deletion of the RING domain does not alter its subcellular distribution even though it abolishes its ability to rescue the mutants (Bray, 2005).

The lethal allele E132 was fortuitously identified among a collection of mutants that modify the wing notching phenotype caused by Armadillo depletion. Genetic mapping of the lethality associated with E132 placed this at 64E8, and it was found to be allelic to an existing mutation, l(3)01640, caused by the P element insertion P1541. Using plasmid rescue of the P element, the site of insertion was localized to the first intron of the open reading frame CG10542, which encodes a predicted protein of 1044 amino acids. The insertion site is 48 nucleotides upstream of the translation initiation codon. Precise excision of P1541 restores viability, confirming that the P element insertion and, by inference, E132 are lethal alleles of CG10542. In support of this, ubiquitous overexpression of the full-length protein encoded by CG10542 rescues the lethality of E132 or P1541 mutant embryos and sustains development to give essentially normal adult flies (with a few minor defects including slightly reduced bristles). CG10542 encodes a conserved protein with close relatives in mammals, C. elegans, plants, and fungi. The Drosophila protein has been named dBre1, after its relative Bre1p in the yeast S. cerevisiae (Bray, 2005).

The hallmarks of the Bre1 proteins are a C-terminal RING finger domain linked to an extensive N-terminal coiled-coil region. The 39 amino acid C3HC4 RING domain is flanked on both sides by ~15 conserved amino acids, suggesting that the fly and mammalian proteins are true orthologs of yeast Bre1p. RING domains are typically found in E3 ubiquitin ligases and frequently mediate the interaction with the E2 ubiquitin-activating enzyme while the other parts of the protein are involved in substrate recognition. The RING domains are therefore critical to catalyze the transfer of ubiquitin from the E2 to the substrate. To confirm the functional importance of the RING domain in dBre1, tests were performed to see whether an N-terminal fragment of dBre1 that lacks the RING domain (ΔRING) could rescue dBre1 mutants. No rescue was observed with any of the 4 transgenic lines (from a total of 814 flies scored), confirming that the RING domain is essential for the function of dBre1 as it is for yeast Bre1p (Bray, 2005).

To examine the subcellular location of full-length dBre1 and the derivative that lacks the RING domain, both forms of the protein were tagged with GFP at the N terminus. Both GFP-dBre1 and GFP-ΔRING are predominantly nuclear in embryonic and imaginal disc cells, although a low level of protein is also detectable in the cytoplasm. This nuclear-cytoplasmic distribution is similar to that of a ΔRING derivative of human Bre1-B when it is overexpressed in mammalian cells. Thus dBre1 appears to be a nuclear protein, like its mammalian counterpart, and deletion of the RING domain does not alter its subcellular distribution even though it abolishes its ability to rescue the mutants (Bray, 2005).

To investigate the role of dBre1 in the fly, homozygous mutant clones were generated in the imaginal disc precursors of the adult structures. Surprisingly, it was found that the majority of defects were similar to those caused by defects in Notch signaling. Thus, adult flies bearing E132 or P1541 mutant clones show notches in the wing margin and aberrant spacing of wing margin bristles, wing blistering and vein defects, fusions of leg segments, and loss of notal bristles and rough eyes. Most of these phenotypes are characteristic of reduced Notch signaling and are distinct from those produced by loss-of-function of other signaling pathways, such as Wingless, Dpp, or Hedgehog signaling that also operate during imaginal disc development. The phenotypic data suggest therefore that dBre1 has a role in promoting Notch signaling (Bray, 2005).

To confirm this, the expression of Notch target genes was examined in dBre1 mutant clones. Since dBre1 mutant clones are considerably smaller than their matched wild-type twin clones, the Minute technique was used to compensate for the growth defect of the mutant clones. In wing imaginal discs, cut and Enhancer of split [E(spl)] are expressed along the prospective wing margin, and their expression depends directly on Notch signaling. Cut expression is absent in large E132 mutant clones, and is lost (3/11) or reduced (6/11) in most P1541 mutant clones. Likewise, E(spl) expression is lost cell autonomously from all E132 mutant clones in the wing. Conversely, expression of spalt, a target of Dpp signaling in the wing, is not reduced in P1541 and E132 mutant cells, indicating that the effects of dBre1 mutation are relatively specific. Similar results are obtained in the eye, where E(spl) expression is also disrupted in E132 clones. Expression in the neurogenic region at the furrow is lost, and elsewhere it is absent or severely reduced, except in the basal layer of undifferentiated cells where expression is independent of Notch. In addition, a derepression of the neuronal cell marker Elav was observed in eye disc clones. The latter indicates excessive neuronal recruitment due to diminished Notch-mediated lateral inhibition (note, however, that the phenotypes are not identical to those produced by complete absence of Notch, which in the eye results in loss of neuronal markers because Notch is needed to promote neural development by alleviating Su(H)-mediated repression. These results demonstrate that dBre1 functions in multiple developmental contexts and, specifically, that it is required for the subset of Notch functions that involve Su(H)-dependent activation of Notch target genes (Bray, 2005).

To further confirm the importance of dBre1 during Notch signaling, it was asked whether any genetic interactions could be detected between overexpressed dBre1 or ΔRING and mutations in Notch (N) or its ligand Delta (Dl). Indeed, overexpression of either protein in the wing disc results in adult phenotypes. In each of 5 ΔRING-expressing lines, mild if consistent mutant phenotypes were observed in both males and females, namely upward-curved wings (due to stronger expression in the dorsal wing compartment), tiny vein deltas, and a significant decrease in wing size. These defects are more severe after overexpression of ΔRING in dBre1 heterozygotes, indicating that ΔRING acts as a weak dominant-negative. Consistent with this, excess ΔRING significantly enhances the phenotypes of N/+ and Dl/+ heterozygotes, resulting in increased vein thickening and additional vein material and, in the case of N/+, also in more frequent wing notching. These genetic interactions support the link between dBre1 and Notch signaling (Bray, 2005).

Excess full-length dBre1 in wing discs causes vein defects whose strength, however, varies considerably between different dBre1-expressing lines, and between males and females (probably because the ms1096.GAL4 driver produces higher expression levels in males). In most lines (4/6), vein thickening and additional vein material were observe only in males, while female wings appear normal. These vein defects in male wings are suppressed to almost normal in dBre1 heterozygotes, suggesting that they are due to increased levels of functional dBre1 protein. The remaining 2 lines produce similar vein defects also in females. Unexpectedly, these defects are enhanced in N/+ and Dl/+ heterozygotes, suggesting that the overexpressed dBre1 interferes with Notch signaling, rather than enhancing it as might have been expected. This anomalous result could be explained if dBre1 is part of a multiprotein complex, in which case its overexpression might interfere with the function of this complex by titrating one of its components. Nevertheless, the genetic interactions between overexpressed dBre1 and Notch and Delta further underscore the link between dBre1 and Notch signaling (Bray, 2005).

To test whether dBre1 directly influences Notch-dependent transcription, Drosophila S2 cells were transfected with Flag-tagged or untagged dBre1, and the activity of a Notch-specific reporter containing 4 Su(H) binding sites [NRE, a luciferase derivative of Gbe+Su(H)_m8] was measured in the presence or absence of low levels of N^ICD. As a control, a reporter was used with mutant Su(H) binding sites [NME, or Gbe+Su(H)_mut]. These experiments reveal a significant stimulation of the NRE reporter by dBre1, especially in the presence of N^ICD. The degree of stimulation is similar to that observed when the ubiquitin ligase Hdm2 is added to transcription assays of Tat activity. dBre1 also elicits a slight stimulation of NME. The fact that overexpressed dBre1 has stimulatory effects on Notch in the transfection assays but not in imaginal discs presumably reflects differences either in the levels of dBre1 or in the amounts of other limiting factors in the two cell contexts. Nevertheless, the transfection assays reveal an intrinsic potential of dBre1 in stimulating the transcription mediated by Su(H) and its coactivator N^ICD (Bray, 2005).

All these results point to a role of dBre1 in promoting Notch signaling. Since other ubiquitin ligases have been shown to influence the levels of specific protein components of the Notch pathway, whether there were any alterations to Notch, Delta, or Su(H) levels in dBre1 mutant clones was investigated. While there are no detectable changes in Notch or Delta staining in dBre1 mutant cells, the levels of Su(H) staining are enhanced slightly but consistently, and cell autonomously, in mutant clones of both dBre1 alleles, regardless of the location of these clones within the disc. This is also obvious in clones induced early in larval development in a non-Minute background in which the mutant dBre1 clones remain small. As an aside, these clones reveal that individual dBre1 mutant cells are enlarged, reminiscent of the yeast bre1p mutant which also shows a 'large cell'phenotype. This phenotype has not been observed in cells lacking Notch signaling, so this aspect of dBre1 function appears distinct from its role in the Notch pathway, and suggests that there are additional molecular targets. Nevertheless, the elevated levels of Su(H) in the dBre1 mutant clones identify Su(H) as one molecular target of dBre1 and suggest that, in the wild-type, dBre1 may expose Su(H) to ubiquitin-mediated degradation. The effects on Su(H) are consistent with the cell-autonomous action of dBre1 on Notch target gene expression, but the fact that removal of dBre1 has a stabilizing effect on Su(H) appears to contradict its stimulating effect on Notch-dependent transcription. Since Su(H) functions as both a repressor and an activator, this may be explained if loss of dBre1 specifically stabilizes the repressor complex. Alternatively, the effect of dBre1 mutations on Su(H) may reflect an indirect bystander activity of dBre1 (Bray, 2005).

Finally, it was asked whether dBre1 has a similar molecular function as its relative yeast Bre1p. The latter is required for the monoubiquitination of histone H2B, which is a prerequisite for the subsequent methylation of histone H3 at K4 by SET1-containing complexes. H3K4 methylation appears to be a chromatin mark for transcriptionally active genes, and yeast bre1p mutants show defects in the transcription of inducible genes that have been ascribed to the lack of H2B ubiquitination and H3K4 methylation at the promoters of these genes. Since there are no in vitro assays for H2B ubiquitination and no antibodies that detect this modified form of H2B, effects of dBre1 mutations on the linked H3K4 methylation were investigated. Wing discs bearing dBre1 mutant clones were stained with an antibody specific for trimethylated H3K4 (H3K4m). This revealed a significant reduction of H3K4m in P1541 mutant clones. More strikingly, in clones of the stronger E132 allele, H3K4m is barely detectable. In contrast, staining of these clones with an antibody against H3K9m does not show any changes in the mutant territory, indicating that the effect in dBre1 mutant clones on the methylation of H3K4 is relatively specific. It is noted that, in wild-type wing discs, there is slight modulation of trimethylated H3K4, with higher levels at the dorsoventral boundary where Notch is activated. However, Notch mutant cells retain robust H3K4m staining, although occasionally show slightly lowered levels compared to adjacent wild-type cells. Thus, the reduced H3K4m staining in dBre1 mutant cells is primarily due to an activity loss of dBre1 rather than due to loss of Notch signaling. Based on its effects on tri-methylated H3K4, it is concluded that dBre1 is indeed the functional homolog of yeast Bre1p. Furthermore, it appears that the activity of dBre1 is essential for the bulk of trimethylated H3K4 in imaginal disc cells (Bray, 2005).

In yeast, H2B ubiquitination and H3K4 methylation are associated with sites of active transcription, but the only identified natural target gene is GAL1. In Drosophila, the target genes of dBre1 evidently include genes regulated by Notch, given the requirement of dBre1 for their transcription. It is therefore conceivable that Su(H) may have a role in targeting dBre1 to their promoters (although it was not possible to detect direct binding or coimmunoprecipitation between dBre1 and Su(H). It is puzzling that dBre1 has a slight destabilizing effect on Su(H), despite being an activating component of Notch signaling. It is believed that this could be a bystander effect of dBre1: evidence suggests that the Bre1p-mediated monoubiquitination of H2B leads to a transient recruitment of proteasome subunits to chromatin, and that the subsequent methylation of H3K4 depends on the activity of these proteasome subunits. Their transient presence at specific target genes may have a destabilizing effect on nearby DNA binding proteins, and the mildly increased levels of Su(H) in dBre1 mutant cells could therefore reflect a failure of proteasome recruitment due to loss of H2B monoubiquitination (Bray, 2005).

Perhaps the most interesting implication of the results is that the dBre1-mediated monoubiquitination of H2B and methylation of H3K4 may be critical steps in the transcription of Notch target genes. Indeed, it appears that the Notch target genes belong to a group of genes whose transcription is particularly susceptible to the much reduced levels of H3K4m in dBre1 mutant cells. Based on the dBre1 mutant phenotypes, there are likely to be other genes in this group, including for example genes controlling cell survival and cell size. Nevertheless, it would appear that the transcription of Notch target genes is particularly reliant on the activity of dBre1. Other examples are emerging where the transcriptional activity of a subset of signal responsive genes is particularly sensitive to the function of a particular chromatin modifying and/or remodelling factor. This sensitivity presumably reflects the molecular mechanisms used by signaling pathways to activate transcription at their responsive enhancers. Understanding why Notch-induced transcription is particularly susceptible to loss of dBre1 function will require knowledge of these underlying molecular mechanisms (Bray, 2005).

Search PubMed for articles about Drosophila Bre1

Bray, S., Musisi, H. and Bienz, M. (2005). Bre1 is required for Notch signaling and histone modification. Dev. Cell 8(2): 279-86. Medline abstract: 15691768

Hwang, W. W., et al. (2003). A conserved RING finger protein required for histone H2B monoubiquitination and cell size control. Mol. Cell 11: 261-266. Medline abstract: 12535538

Wood, A., et al. (2003). Bre1, an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter. Mol. Cell 11: 267-274. Medline abstract: 12535539

date revised: 10 December 2007

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