dRtf1 affects transcription of heat shock genes: Rtf1 in S. cerevisiae is required for COMPASS recruitment to elongating RNA Pol II (Krogan, 2003b; Ng, 2003), implicating it in RNA Pol II transcription. Accordingly, the effect of dRtf1 reduction on transcription was tested in flies. Because RNAi knockdown of dRtf1 in cultured S2 cells reduces Hsp70 expression by ~25%, the Hsp70 heat shock genes were used as reporters for the effect of dRtf1 knockdown in whole animals. In actin-Gal4 driven dRtf1 RNAi third instar larvae dRtf1 levels at the 87A and 87C loci (Hsp70 puff sites) was reduced and Hsp70 transcripts were reduced at various time points throughout a 60-min heat shock. This observation indicates that dRtf1 is required for proper regulation of heat shock gene expression in whole flies and suggests that histone methylation is required for full heat shock gene expression (Tenney, 2006).
A previous report showed that dRtf1 is recruited to heat shock loci in polytene chromosomes upon heat shock (Adelman, 2006). To test whether dRtf1 recruitment is associated with H3K4 methylation at heat shock loci, polytene chromosomes from heat shocked larvae were immunostained with an antibody to trimethylated H3K4. Trimethylated H3K4 accumulates at heat shock puff sites (87A, 87C, and 93D) in response to heat shock. Note that extensive H3K4 methylation remains throughout the rest of the genome, consistent with the observation that a significant fraction of dRtf1 remains at non-heat shock loci during heat shock (Tenney, 2006).
The Paf1 complex in yeast has been reported to influence a multitude of steps in gene expression through interactions with RNA polymerase II (Pol II) and chromatin-modifying complexes; however, it is unclear which of these many activities are primary functions of Paf1 and are conserved in metazoans. The Drosophila homologs of three subunits of the yeast Paf1 complex have been identified and characterized and striking differences were found between the yeast and Drosophila complexes. Although Drosophila Paf1, Rtf1, and Cdc73 (Hyrax) colocalize broadly with actively transcribing, phosphorylated Pol II, and all are recruited to activated heat shock genes with similar kinetics; Rtf1 does not appear to be a stable part of the Drosophila Paf1 complex. RNA interference (RNAi)-mediated depletion of Paf1 or Rtf1 leads to defects in induction of Hsp70 RNA, but tandem RNAi-chromatin immunoprecipitation assays show that loss of neither Paf1 nor Rtf1 alters the density or distribution of phosphorylated Pol II on the active Hsp70 gene. However, depletion of Paf1 reduces trimethylation of histone H3 at lysine 4 in the Hsp70 promoter region and significantly decreases the recruitment of chromatin-associated factors Spt6 and FACT, suggesting that Paf1 may manifest its effects on transcription through modulating chromatin structure (Adelman, 2006; full text of article).
Characterization of the Drosophila homologs of yeast Paf1 subunits has revealed several features in common and several critical differences between the yeast and Drosophila Paf1 complexes. The most striking similarities between the yeast and Drosophila Paf1 complexes are their association with elongating RNA Pol II and their roles in gene activation, while the nature of the Pol II association and the composition of the Paf1 complex reflect marked differences between the species (Adelman, 2006).
The global view provided by Drosophila polytene chromosomes shows that the chromosome-associated Paf1 and Rtf1 proteins colocalize with active Pol II. This result supports the idea that these proteins participate in most, if not all, Pol II transcription. Remarkably, Paf1 and Rtf1 do appear to be separable from actively elongating Pol II under conditions of heat shock. Although Paf1 and Rtf1 are recruited actively to heat shock loci upon heat stress, these factors also remain associated with a number of additional sites on the chromosome, while Pol II is localized almost exclusively at heat shock loci under these conditions. These data suggest that Paf1 and Rtf1 may remain bound to the chromosome at activated genes through interactions with additional proteins (Adelman, 2006).
It has been suggested that, in yeast, while the Paf1 complex is entirely nuclear in its localization (Shi, 1997), it has cellular functions that are independent of elongating Pol II (Mueller, 2004; Porter, 2005). The nucleolar association of Paf1 and Rtf1 observed on Drosophila polytene chromosomes could possibly represent such a function. At the nucleolar organizer, Paf1 shows broad labeling while the Rtf1 signal is restricted to the nucleolar periphery in a manner that is largely nonoverlapping. Interestingly, although the yeast Paf1 complex does not show strong nucleolar association normally (Porter, 2005), in an Rtf1 mutant, the Paf1 complex shows a strong association that is postulated to be a manifestation of its normal role in nuclear processing or export (Adelman, 2006).
By using ChIP experiments, this study obtained a higher-resolution view of the localization of Paf1, Rtf1, and Cdc73 at the Hsp70 gene. The lack of a ChIP signal at Hsp70 under uninduced conditions demonstrates that the presence of engaged Ser-5-P Pol II or the associated elongation factors such as Spt5 and TFIIS is not sufficient to recruit Paf1, Rtf1, or Cdc73. Upon heat induction, recruitment of all three proteins was observe primarily within the coding regions of active Drosophila genes, rather than regions upstream of the promoter, or downstream of the site for cleavage and polyadenylation. The reduction in the Paf1 signal downstream of the polyadenylation site, which accompanies a decrease in the Pol II signal, likely signifies that Paf1 dissociates from chromatin within this region, consistent with recent results obtained with yeast. However, it is noted that the absence of a significant Paf1 signal obtained with a given primer pair may simply indicate that the interactions of Paf1 with a particular region are transient (Adelman, 2006).
The Paf1 complex in S. cerevisiae has been reported to be required for full Ser-2 phosphorylation of the Pol II CTD. This role of Paf1 in CTD phosphorylation regulation also appears consistent with the fact that rtf1Delta mutants show synthetic lethality with CTD kinase and phosphatase mutants in CTK1 and FCP1 (Costa, 2000). The lack of a Ser-2-P Pol II signal detected in yeast Paf1 mutants resulted in reduced recruitment of cleavage and polyadenylation factors, causing a defect in the polyadenylation of nascent transcripts (Mueller, 2004). However, although depletion of Drosophila Paf1 or Rtf1 has a marked effect on induced Hsp70 RNA levels, no change was seen in the levels of Ser2-P Pol II on the Hsp70 gene in Paf1 or Rtf1 RNAi-treated cells, indicating a difference between the functions of Paf1 in yeast and a metazoan system (Adelman, 2006).
Another fundamental difference that observed between Drosophila and yeast Paf1 complexes is the relationship of the Paf1 and Rtf1 subunits in providing anchorage of the complex to Pol II. In yeast, Mueller (2004) has shown that the association of Paf1 with Pol II and active chromatin depends on the presence of Rtf1. In contrast, this study found that the recruitment of Paf1 to activated Drosophila Hsp70 is independent of Rtf1, while Rtf1 recruitment is dependent on Paf1. These results may reflect the evolution of a more important role for the Paf1 protein in metazoans in providing affinity of the complex for Pol II, while Rtf1 became a more loosely bound component of the complex (Adelman, 2006).
The role was investigated of Drosophila Paf1 in the modification of histones within actively transcribed regions. Whereas yeast Paf1 has been implicated in regulating the bulk levels of methylation of histone H3 at lysine residues 4 and 79 (Ng, 2003; Sun, 2002, Wood, 2003), an effect was observed of Paf1 depletion on the trimethylation of H3-K4, but not on di- or trimethylation of H3-K79. Similarly, it was observed that trimethylation of H3-K4 occurred within the promoter-proximal region of Hsp70 and Hsp26 upon heat shock and could be seen to increase from 2.5 to 10 min after heat induction, but no significant levels of H3-K79 dimethylation were observed within the active Hsp70 gene. The latter result differs from results from other systems which link H3-K79 dimethylation with active transcription. However, it is consistent with recent data suggesting that both Grappa, the Drosophila H3-K79 methyltransferase, and the signal corresponding to H3-K79 dimethylation are localized to both active and intergenic regions of Drosophila polytene chromosomes. An alternative possibility is that the apparent differences between yeast and Drosophila result from the experimental systems used; RNAi treatments in Drosophila decrease, but do not completely abolish, their target, and thus the small amount of remaining protein may be sufficient to carry out certain functions. Conversely, the deletion mutants used to investigate yeast Paf1 entirely remove an important protein for many generations of cell growth, raising the possibility that some observed effects are indirect or secondary in nature (Adelman, 2006).
It is interesting that although H3-K4 trimethylation depends upon Paf1 and the recruitment of Paf1 is temporally similar to H3-K4 methylation, the distribution of Paf1 appears to be spatially distinct from the promoter region where the strongest trimethylated H3-K4 signals are observed. Thus, the results suggest that the effects of Paf1 mutants on the modification of promoter-proximal nucleosomes (including the ubiquitination of H2B-K123) may occur through indirect mechanisms. These data are consistent with reports on yeast that indicate that the distribution of Paf1 subunits does not strictly correlate with the patterns of ubiquitinated H2B or methylated histone H3 (Ng, 2003). The localization of H3-K4 trimethylation reported in this study is in agreement with the recently described distribution of Trithorax, a Drosophila H3-K4 methyltransferase (Smith, 2004). Furthermore, recent studies employing a Drosophila Trithorax mutant fly line suggest that a multiprotein complex that contains Trithorax plays a role in Hsp70 gene activation. However, whether the role of Trithorax in Hsp70 activation is direct or indirect remains to be established. It is noted that no effect of Paf1 depletion is observed on the rates of Pol II recruitment, or distribution over the gene, suggesting that H3-K4 trimethylation may serve as a mark of transcription activation rather than a prerequisite for gene activation (Adelman, 2006).
These studies have provided new insights into the increased importance of the Paf1 complex in a metazoan system. It is significant that Paf1 is recruited in a manner that is spatially and temporally identical to that of chromatin-associated factors Spt6 and FACT (Smith, 2004). In agreement with the strong colocalization of Paf1 with these nucleosome-associated factors, it was shown that depletion of Paf1 significantly reduces the recruitment of both Spt6 and the FACT subunit SSRP1. A relationship among Paf1, Rtf1, and FACT is consistent with findings that an rtf1Delta mutation shows synthetic lethality with POB3, a subunit of the yeast FACT complex (Costa, 2000). Moreover, the FACT complex has been shown to interact with the Paf1 complex and the chromodomain-containing Chd1 protein at actively transcribed genes (Simic, 2003). In vitro, FACT has been shown to function optimally to facilitate transcription through nucleosomes when it is present at approximately one molecule of FACT per two nucleosomes; the effectiveness of FACT in promoting elongation is decreased dramatically below this threshold. If these results reflect the situation in vivo, the greater than 50% decrease in FACT levels at the active Hsp70 gene in Paf1-depleted cells would result in a rather pronounced effect on transcription through nucleosomes (Adelman, 2006).
Furthermore, recent evidence obtained with yeast has shown that mutations of Spt6 or the FACT subunit Spt16 lead to aberrant chromatin architecture in the wake of elongating Pol II, presumably due to defects in reassembly of nucleosome structure. The failure to efficiently repackage transcribed DNA results in transcription initiation from cryptic sites and a reduction in levels of properly initiated and processed RNA. If a primary role of Drosophila Paf1 is to help stably recruit factors like Spt6 and FACT, then loss of Paf1 activity could also lead to the accumulation of nonfunctional or improperly processed RNA species. In support of this idea, a paper that was published during the preparation of this report states that mutations in yeast Spt6 alter the recruitment of Paf1 subunit Ctr9 and lead to defects in 3'-end processing of nascent RNA (Kaplan, 2005). It is thus tempting to speculate that the vast array of transcription elongation and RNA processing and export defects reported in yeast Paf1 mutant strains could result from perturbation of the nucleosome structure along actively transcribed genes. Moreover, it may be these chromatin and processing defects that account for the decrease in the amount of Hsp70 mRNA that accumulates in response to heat shock in Paf1- or Rtf1-depleted cells (Adelman, 2006).
Finally, the Paf1 gene in yeast is nonessential while the Paf1 gene in Drosophila is essential. This may reflect the more varied and demanding requirements of the transcription machinery in higher eukaryotes, where chromatin frequently plays a greater and more stringent role in regulation. This, in turn, may place a greater demand on the Paf1 complex, which appears to function at the interface between transcription and chromatin, perhaps serving as a platform that stimulates the association of a number of nucleosome-modifying complexes with actively elongating Pol II (Adelman, 2006).
In summary, the gene for Paf1 is a required Drosophila gene that colocalizes with actively elongating Pol II when chromatin associated and plays a critical role in the activation of stress-induced genes. Furthermore, recent data reveal that mutations in parafibromin, the human homolog of the Paf1 complex subunit Cdc73, are associated with an elevated risk of parathyroid carcinomas; thus, the Paf1 complex may be a key regulator of cellular control in metazoans (Rozenblatt-Rosen, 2005; Yart, 2005). The connection between Paf1 and trimethylation of histone H3 at lysine 4 near the promoters of active genes is particularly interesting because a human homolog of Trithorax, the histone methyltransferase implicated in this activity, is ALL-1/MLL-1, which is associated with a number of acute leukemias. Future work to define the way in which Paf1 directs the histone methyltransferase activity of this key enzyme should provide insight into the interaction between active transcription and modifications of chromatin structure. The data support a model in which the Drosophila Paf1 complex plays a key role in coordinating histone modifications and changes in nucleosome structure with transcription activation and Pol II elongation, thereby serving as a critical link between gene expression and chromatin structure (Adelman, 2006).
Rapid induction of the Drosophila melanogaster heat shock gene hsp70 is achieved through the binding of heat shock factor (HSF) to heat shock elements (HSEs) located upstream of the transcription start site. The subsequent recruitment of several other factors, including Spt5, Spt6 and FACT, is believed to facilitate Pol II elongation through nucleosomes downstream of the start site. This study reports a novel mechanism of heat shock gene regulation that involves modifications of nucleosomes by the TAC1 histone modification complex (consisting of Trithorax, Sbf1 and the histone acetyltransferase CBP). After heat stress, TAC1 is recruited to several heat shock gene loci, where its components are required for high levels of gene expression. Recruitment of TAC1 to the 5'-coding region of hsp70 seems to involve the elongating Pol II complex. TAC1 has both histone H3 Lys 4-specific (H3-K4) methyltransferase (HMTase) activity and histone acetyltransferase activity through Trithorax (Trx) and CREB-binding protein (CBP), respectively. Consistently, TAC1 is required for methylation and acetylation of nucleosomal histones in the 5'-coding region of hsp70 after induction, suggesting an unexpected role for TAC1 during transcriptional elongation (Smith, 2004).
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).
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 NICD. 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 NICD. 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 NICD (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).
Paf1 complex regulation of histone methylatiom: Many of the subunits of COMPASS are required for the proper mono-, di-, and/or trimethylation of H3K4. In addition to COMPASS, the E2 conjugating enzyme Rad6 and its E3 ligase Bre1 are required for proper H3K4 methylation via the regulation of H2B monoubiquitination. Also, it has been shown that deletion of components of the Paf1 complex and the Bur1/Bur2 kinase can greatly reduce histone H2B monoubiquitination and, thereby, H3K4 methylation. However, deletion of RTF1, which is required for the activation of Rad6, seems to be required for mono-, di-, and trimethylation mediated by COMPASS. This observation mirrors that of effects observed when either RAD6 or BRE1 is deleted. Although loss of H2B monoubiquitination is not fully required for H3K4 monomethylation by COMPASS, this observation can be explained by the fact that Rtf1 is not only required for the regulation of H2B monoubiquitination but also plays a role in the recruitment of COMPASS to the transcribing RNA Pol II (Wood, 2003). In addition to regulation of H3K4 methylation, different components of the Paf1 complex have varying effects on other types of H3 tail methylation (Tenney, 2006).
Because Paf1 is required for histone H3K4 and K79 methylation, the effect of Paf1 loss on histone methylation stability was examined in vivo. A tetracycline-regulated PAF1 gene strain grown under normal conditions, turning off the expression of Paf1 in the presence of tetracycline, was used. Cell extracts were prepared at different time points, and histone H3 modification stability was tested in the absence of Paf1. After approximately 4 hours in the presence of tetracycline, Paf1 levels were reduced by >95%. However, even 12 hours after Paf1 loss, histone H3K4 and K79 methylation levels appear to be unaffected. This observation substantiates a report by Ng (2003), who found that H3K4 methylation seems to be a stable mark once established on the Gal gene. It was verified that histone H3K4 methylation seems to be stable, further supporting a role for the stability of this type of histone modification (Tenney, 2006).
dRtf1 is required for Histone H3K4 trimethylation on polytene chromosomes: Because Rtf1 loss in yeast results in the abrogation of H3K4 methylation, whether dRtf1 in Drosophila functions in the same pathway was tested. Extracts prepared from both actin-Gal4 driven dRtf1 progeny (Rtf1 RNAi on) and control progeny (Rtf1 RNAi off) were tested for methylated H3K4 levels. dRtf1 RNAi knockdown resulted in a significant reduction in total cellular trimethylated H3K4. To test whether this reduction occurs throughout the genome, fixed polytene chromosomes squashes were prepared from actin-Gal4 driven dRtf1 RNAi and control larvae and were immunostained with an antibody specific for trimethylated H3K4. In WT polytene chromosomes, trimethylated H3K4 is widely distributed throughout the euchromatic chromosome arms and is highly enriched at developmental puffs, sites of active transcription. In contrast, the polytene chromosomes from dRtf1 knockdown larvae consistently show reduced staining with the same antibody. Together, these data suggest that the loss of Rtf1 results in the loss of H3K4 methylation in Drosophila (Tenney, 2006).
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