InteractiveFly: GeneBrief

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Gene name - Paf1

Synonyms - antimeros (atms: accepted Flybase term)

Cytological map position- 82D5-82D5

Function - chromatin component

Keywords - Paf1 complex, histone methylation, modification of chromatin structure, transcription

Symbol: Paf1

FlyBase ID: FBgn0010750

Genetic map position - 3R:601,323..603,213 [+]

Classification - Paf1 domain

Cellular location - nuclear



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Bahrampour, S. and Thor, S. (2016). Ctr9, a key component of the Paf1 complex affects proliferation and terminal differentiation in the developing Drosophila nervous system. G3 (Bethesda). PubMed ID: 27520958
Summary:
The Paf1 protein complex (Paf1C) promotes H3K4 and H3K36 trimethylation, H2BK123 ubiquitination, RNA Pol II transcriptional termination, and also RNA-mediated gene silencing. Paf1C contains five canonical protein components including Paf1 and Ctr9, that are critical for overall complex integrity, as well as , Leo1 and Cdc73/Hyrax. This study provide the first detailed phenotypic study of Ctr9 function in Drosophila. Ctr9 mutants die at late embryogenesis or early larval life, but can be partly rescued by nervous system re-expression of Ctr9. A number of phenotypes are observed in Ctr9 mutants, including increased neuroblast numbers, increased nervous system proliferation, as well as down-regulation of many neuropeptide genes. Analysis of cell cycle and regulatory gene expression reveals up-regulation of the E2f1 cell cycle factor, as well as changes in Antennapedia and Grainy head expression. Reduction of H3K4me3 modification was found in the embryonic nervous system. Genome-wide transcriptome analysis points to additional downstream genes that may underlie these Ctr9 phenotypes, revealing gene expression changes in Notch pathway target genes, cell cycle genes and neuropeptide genes. In addition, significant effects were found on the gene expression of metabolic genes. These findings reveal that Ctr9 is an essential gene that is necessary at multiple stages of nervous system development, and provides a starting point for future studies of the Paf1C in Drosophila.
BIOLOGICAL OVERVIEW

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. Paf1 therefore directs the histone methyltransferase activity and links 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; full text of article).

Proper control of gene expression is necessary for the development, differentiation, and survival of the cell, and transcription regulation is a cornerstone of this process. The formation of mRNA in eukaryotes involves a complex multistep pathway wherein each step provides an opportunity for regulation. Once RNA polymerase II (Pol II) has been recruited to a promoter and initiates transcription, it must efficiently escape from the promoter-proximal region and transcribe through a gene that is covered with nucleosomes. The nascent RNA must also be capped, spliced, polyadenylated, and exported to the cytoplasm before it can serve as a template for protein translation. Recent evidence from many laboratories indicates that there is a dynamic interplay between the protein complexes that carry out mRNA transcription, processing, and export, such that the efficiency of one step can have significant consequences for other steps in the pathway. For this reason, many factors that are required for the production of functional, mature RNA and were initially thought to directly stimulate Pol II transcription elongation have since been shown to elicit their primary effects on cotranscriptional processing or RNA export. Thus, a major goal towards understanding the mechanisms of transcription regulation requires the identification of both the direct and indirect activities of the numerous factors implicated in RNA production (Adelman, 2006).

The yeast Paf1 complex is one example of a factor that has been linked to a number of transcription-related activities. Yeast Paf1 is a complex of at least five polypeptides (Paf1, Rtf1, Cdc73, Leo1, and Ctr9) that has been implicated in processes as divergent as transcription initiation and elongation, modification of histone tails, phosphorylation of the Pol II C-terminal domain (CTD), RNA processing, and export. Although yeast Paf1 was originally thought to be an alternate mediator based upon its direct interactions with Pol II, it has since been found to be recruited throughout the body of active genes and to associate with the elongation-competent form of Pol II (Krogan, 2002; Mueller, 2004; Pokholok, 2002; Simic, 2003). Additional roles for the Paf1 complex have been suggested by the association of Paf1 with several RNA processing and export factors, such as Ccr4, the major yeast deadenylase, and Hpr1, a component of the THO complex that is involved in the export of mRNAs (Adelman, 2006).

Components of the Paf1 complex are nonessential in yeast, but mutations in Paf1 subunits confer sensitivity to 6-azauracil and generate Spt phenotypes, which are generally thought to signify defects in transcription elongation (Costa, 2000; Squazzo, 2002). In vitro transcription assays with naked DNA templates suggested that Paf1 and Cdc73 might directly stimulate transcription elongation (Rondon, 2004); however, it is not clear what effects Paf1 has on elongation rates in vivo. In Saccharomyces cerevisiae, deletion of Paf1 or Cdc73 did not alter the distribution of Pol II on an active gene but dramatically decreased the chromatin immunoprecipitation (ChIP) signal observed for serine 2-phosphorylated (Ser2-P) Pol II. Consistent with a Ser2 phosphorylation defect, recruitment of 3' cleavage and processing factors was impaired in the paf1Delta strain and poly(A) tail length was modestly shortened (Adelman, 2006).

A link between the Paf1 complex and the chromatin architecture within transcribed regions has been suggested by genetic interactions between Paf1 components and Chd1, subunits of the yeast FACT complex, and histone assembly factors in the Hir/Hpc pathway (Formosa, 2002: Simic, 2003; Squazzo, 2002). The packaging of template DNA into nucleosomes is known to represent a formidable obstacle to Pol II elongation in vitro, an obstacle that is overcome in vivo by a number of proteins that facilitate Pol II elongation by modifying chromatin structure and/or stability. Examples of factors that have been implicated in transcription through nucleosomes are chromatin remodeling enzymes, such as Chd1 and Swi/Snf, and histone-binding proteins like Spt6 and FACT. The ensemble of these complexes appear to help disassemble nucleosomes to promote efficient Pol II transcription through bound DNA and then to reassemble nucleosomes after the passage of Pol II. Both Spt6 and FACT have recently been shown to help maintain the proper balance between assembly and disassembly of nucleosomes during active transcription by Pol II (Kaplan, 2003), with the loss of these factors leading to a net failure to reassemble nucleosomes in the wake of transcription (Adelman, 2006).

The yeast Paf1 complex is required for ubiquitination of histone H2B at lysine 123 in the promoter-proximal region of activated genes (Ng, 2003; Sun, 2002; Wood, 2003). This ubiquitination event is a prerequisite for the methylation of histone H3 (at lysine residues 4 and 79) that accompanies active transcription in yeast; thus, the latter processes are defective in cells lacking functional Paf1 (Ng, 2003; Sun, 2002; Wood, 2003). A model has been 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). In addition, the Paf1 complex has been reported to be critical for the recruitment of the yeast SET2 histone methyltransferase complex to actively transcribed genes, leading to methylation of histone H3 at residue lysine 36 (Krogan, 2003; Xiao, 2005; Adelman, 2006 and references therein)

Although the yeast Paf1 complex has been studied extensively, a number of important questions remain unanswered. Key questions concern the nature of the interactions between the subunits of the Paf1 complex and their associations with Pol II, as well as the importance of Pol II binding in Paf1 function. A pivotal issue concerns the fact that deletion of Rtf1 or Cdc73 has been reported to reduce the association of all Paf1 components with the Pol II and chromatin yet lead to much weaker phenotypes than does deletion of the other Paf1 components. These results have led some to propose that the critical role of Paf1 occurs when the complex is not chromatin associated; however, the other potential activities of Paf1 have yet to be clearly identified. Furthermore, the subunit composition of the Paf1 complex in human cells appears to differ from that in yeast, since the human Rtf1 protein does not appear to stably associate with the other members of the Paf1 complex (Adelman, 2006).

To address these issues and to investigate the activity of Paf1-associated proteins in Drosophila, the Drosophila homologs of the yeast Paf1, Rtf1, and Cdc73 proteins were identified and characterized. In vivo analyses of the Drosophila Paf1 complex uncover both important similarities to and differences from the reported functions of Paf1 in yeast and provide insight into the connections among histone methylation, nucleosome stability, and transcription activation in a metazoan organism. Strikingly, the Drosophila Paf1 homolog is a previously annotated gene that encodes an essential protein, suggesting that the role of Paf1 has evolved and become more critical in metazoans. Rtf1 is not stably associated with the Drosophila Paf1 and Cdc73 proteins in vivo and shows only a weak interaction with Pol II. Moreover, when Paf1-depleted cells are assayed by tandem RNA interference (RNAi)-ChIP, no changes were observed in the level of Ser2-P Pol II on the Hsp70 gene, in contrast to results obtained with yeast. Interestingly, it appears that major effects of Paf1 depletion are the loss of H3-K4 trimethylation near the Hsp70 promoter and a significant decrease in the recruitment of Spt6 and FACT to the body of the Hsp70 gene, suggesting that Drosophila Paf1 may coordinate the activities of elongating Pol II with factors that maintain the proper chromatin architecture during transcription (Adelman, 2006).

This study shows that 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. 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).

The role of Parafibromin/Hyrax as a nuclear Gli/Ci-interacting protein in Hedgehog target gene control

The Hedgehog (Hh) pathway, an evolutionarily conserved key regulator of embryonic patterning and tissue homeostasis, controls its target genes by managing the processing and activities of the Gli/Ci transcription factors. Little is known about the nuclear co-factors the Gli/Ci proteins recruit, and how they mechanistically control Hh target genes. This study provides evidence for the involvement of Parafibromin/Hyx as a positive component in Hh signaling. hyx RNAi impaired Hh pathway activity in Drosophila cell culture. Consistent with an evolutionarily conserved function in Hh signaling, RNAi-mediated knockdown of Parafibromin in mammalian cell culture experiments diminished the transcriptional activity of Gli1 and Gli2. In vivo, in Drosophila, genetic impairment of hyx decreased the expression of the high-threshold Hh target gene knot/collier. Conversely, hyx overexpression ameliorated the inhibitory activity of Ptc and Ci(75) misexpression during Drosophila wing development. It was subsequently found that Parafibromin can form a complex with all three Glis, and evidence is provided that Parafibromin/Hyx directly binds Region 1, the Su(fu) interaction domain, in the N-terminus of all Glis and Ci. Taken together, these results suggest a target gene-selective involvement of the PAF1 complex in Hh signaling via the Parafibromin/Hyx-mediated recruitment to Gli/Ci (Mosimann, 2009).

knot encodes a Col/Olf-1/EBF (COE) family helix-loop-helix-containing transcription factor controlling specification of the intervein region between L3 and L4. Compared to dpp, ptc, and en, the Hh-dependent transcriptional regulation of kn is less well analyzed. kn can be induced in the normally Ci-devoid P-compartment by Ci155 misexpression. Additionally, cells with amorphic PKA alleles in the wing pouch upregulate kn cell-autonomously when not situated close to the D/V compartment boundary, while ptc loss of function clones induce kn expression irrespective of their location in the anterior wing pouch. Unfortunately, these findings do not allow the drawing of an unambiguous picture of kn control by Ci at the A/P boundary, as no study involved complete ci loss of function. However, in the developing wing pouch, the expression of kn is clearly induced by highest Hh output. The selective effect of Hyx impairment on kn expression suggests that Hyx is a context-dependent co-factor of Ci required for selective target genes (Mosimann, 2009).

One potential caveat is that the hypomorphic allele hyxEY6895, which was used in most of the experiments, does not reduce Hyx levels sufficiently to detectably affect expression of lower threshold targets such as ptc or dpp. Arguing against this is that ptc expression was also not affected in a stronger hyx loss of function situation using the hyx2 allele (Mosimann, 2009).

Interestingly, Hyx is not the first reported seemingly target gene-restricted Ci co-factor. The Mediator complex subunits Skuld (Skd) and Kohtalo (Kto) are involved in the control of cell affinity-regulating genes by Ci155, yet not ptc and dpp transcription (Janody, 2003). It remains to be seen if Skd or Kto are also involved in kn control and if they directly interact with Ci. In contrast, the histone acetyl-transferase CBP is required for ptc expression and has been suggested to be an obligate Ci155 partner, but in-depth genetic analysis is hampered by its broad involvement as general transcriptional co-factor (Akimaru, 1997). Together with these results, the current findings strongly suggest that during development, Ci155 assembles differential sets of co-factors dependent on the respective target gene context (Mosimann, 2009).

When Hyx overexpression was analyzed in genetic systems sensitized for Hh signaling, it was found that Hyx partially counter-acts the strong effect caused by ptc misexpression on the developing wing. Anticipating a nuclear function together with Ci, an effect on phenotypes mediated by direct Ci overexpression was subsequently assayed. It was found that Hyx severely attenuates the effects of Ci75 overproduction, but has no effect on overexpressed CiPKA or overexpressed wild-type Ci, which only shows transactivating activity upon Hh input (Mosimann, 2009).

This finding is interpreted as an indication that overexpressed Hyx dominant-negatively interferes with the repressive activity of overexpressed Ci75. Surplus Ci75 may act primarily by occupying the promoters of Hh target genes, and overexpressed Hyx interferes with this property. In contrast, in a wild-type situation the negative activity of endogenous Ci75 may be mediated by the binding of repressive co-factors. This binding is not effectively competed off by additional Hyx, explaining the lack of a detectable effect in a wild-type background (Mosimann, 2009).

Region 1 of Ci/Gli has never revealed any autonomous transactivation potential when tethered to DNA, in contrast to C-terminal Gli fragments. Parafibromin/Hyx binding to Region 1 would not necessarily stimulate transcription on its own, as DNA-tethered Hyx shows no detectable transactivation effect, suggesting that it is not sufficient for triggering RNAPII-mediated transcription. Instead, in agreement with these results, the recruitment of Hyx to Hh target genes by binding to Region 1 probably helps to ensure efficient reoccurring transcription. This function might be particularly important for certain genes induced at high Hh levels and might involve particular chromatin modifications dependent on the PAF1 complex (Mosimann, 2009).

Region 1 is also the minimal interaction site for Su(fu). While competitive Su(fu) binding is an intriguing possibility, the idea of consecutive binding is favored since Parafibromin/Hyx appears to be principally required for high signal output -- conditions under which, due to Fu action, Su(fu) binding is believed not to occur. Su(fu) plays a critical negative regulatory role in the Hh pathway, especially in mammals. How this factor functions is unclear, but it may regulate Gli processing, act as a co-repressor, and/or regulate Gli/Ci localization. The finding that positive and negative regulators bind to Region 1 may explain why its deletion in Ci only had a minor effect (Mosimann, 2009).

In Wnt/Wg signaling, Parafibromin/Hyx seems to participate in a sequence of co-factor exchanges that occurs on β-catenin/Armadillo. This potentially reflects the need for priming chromatin remodeling steps before PAF1 complex function. Interestingly, β-catenin/Arm has overlapping binding sites for its co-activators such as CBP, Brg-1/Brahma (Brm), and Parafibromin/Hyx. This contrasts with Gli/Ci, on which Parafibromin/Hyx occupies a different binding site than CBP. Gli/Ci therefore could organize multiple recruitment steps for auxiliary components via separate domains rather than solely by sequential binding (Mosimann, 2009).

Considering the impact of Hyx impairment on the analyzed Hh target genes in vivo, combined with the overexpression data and RNAi results, it is predicted that Parafibromin/Hyx is a factor involved in maximal Gli/Ci target gene induction. Parafibromin/Hyx, as part of the PAF1 complex, could implement efficient RNAPII control at Hh target genes when sustainable transcriptional induction is needed. On other targets, such as ptc, this process might be redundant with other ways to guide RNAPII. One possibility could be recruitment of the PAF1 complex by a module other than Parafibromin/Hyx, or potentially even via another transcription factor that binds in the vicinity of the Gli/Ci binding site (Mosimann, 2009).

Drosophila stem cells share a common requirement for the histone H2B ubiquitin protease scrawny: Scrawny interacts with PAF1 and likely opposes BRE1 action on H2B thus participating in a conserved pathway of chromatin regulation linking H2B ubiquitylation with H3K4me3 methylation

Stem cells within diverse tissues share the need for a chromatin configuration that promotes self-renewal, yet few chromatin proteins are known to regulate multiple types of stem cells. A Drosophila gene, scrawny (scny), encoding a ubiquitin-specific protease, is required in germline, epithelial, and intestinal stem cells. Like its yeast relative UBP10, Scrawny deubiquitylates histone H2B and functions in gene silencing. Consistent with previous studies of this conserved pathway of chromatin regulation, scny mutant cells have elevated levels of ubiquitinylated H2B and trimethylated H3K4. These findings suggest that inhibiting H2B ubiquitylation through scny represents a common mechanism within stem cells that is used to repress the premature expression of key differentiation genes, including Notch target genes (Buszczak, 2009).

Stem cells are maintained in an undifferentiated state by signals they receive within the niche and are subsequently guided toward particular fates upon niche exit. Within ES cells and during differentiation, cell state changes are controlled at the level of chromatin by alterations involving higher order nucleosome packaging and histone tail modifications. Polycomb group (PcG) and Trithorax group (trxG) genes influence key histone methylation events at the promoters of target genes, including H3K27 and H3K4 modifications associated with gene repression and activation, respectively, but few other genes with a specific role in stem cells are known (Buszczak, 2009).

Histone H2A and H2B mono-ubiquitylation play fundamental roles in chromatin regulation, and H2A ubiquitylation has been linked to PcG-mediated gene repression and stem cell maintenance. The mammalian Polycomb repressive complex 1 (PRC1) component RING1B is a H2A ubiquitin ligase that is required to block the elongation of poised RNA polymerase II on bivalent genes in ES cells. Mutations in the PRC1 component, BMI-1, the ortholog of Psc in the mammalian PRC1, complexes with RING1B, and causes multiple types of adult stem cells to be prematurely lost. The role of H2B ubiquitylation in stem cells is unclear, however. In yeast, ubiquitylation of Histone H2B by the RAD6 and BRE1 ligases controls H3K4 methylation (H3K4me3), a process that requires the polymerase accessory factor PAF1. Conversely, H2B deubiquitylation by the ubiquitin-specific protease (USP) family member UBP10 is required for silencing telomeres, rDNA and other loci. The Drosophila homolog of BRE1, dBRE1, also is needed for H3K4 methylation, suggesting that this pathway is conserved. Furthermore, the Drosophila ubiquitin-specific protease USP7 is part of a complex that selectively deubiquitylates H2B and genetically interacts with PcG mutations. Mutations in another USP family member, Nonstop, increase H2B ubiquitylation and cause axon targeting defects in the eye (Buszczak, 2009).

In order to gain further insight into the role of H2B ubiquitylation in stem cells, a novel Drosophila gene, scrawny (scny) (CG5505), was identified, whose encoded USP family protein shares homology with human USP36 and among yeast USPs closely matches UBP10 within the core protease domain. Strains bearing scny insertions, except for a viable GFP protein trap (CA06690), were female sterile or lethal, and proved to be allelic. Transposon excision or expression of a scny-RB cDNA reverts the phenotype of tested alleles. An anti-SCNY antibody raised against a domain common to all SCNY isoforms recognizes wild type and SCNY-GFP on a Western blot. SCNY protein levels in homozygous third instar larvae are greatly reduced in lethal mutants, and SCNY expression is also lower in stem cell-enriched ovarian tissue from adults homozygous for the sterile d06513 allele. Consistent with a role in gene silencing, several scny mutations act as dominant suppressors of position effect variegation (Buszczak, 2009).

Further studies strongly suggested that SCNY functions in vivo as an H2B-ubiquitin protease. Recombinant full-length SCNY protein, but not a version bearing a point mutation in the protease domain, efficiently deubiquitylates histone H2B in vitro. scnyf01742 homozygous tissue contains levels of Ub-H2B that are elevated at least twofold compared to wild type. As expected if Ub-H2B is required for H3K4 methylation, clones of homozygous scnye00340 mutant cells stain more strongly for H3K4me3 than heterozygous cells. Consistent with a direct rather than an indirect action on Ub-H2B levels, anti-SCNY antibodies co-immunoprecipitate H2B from Drosophila embryonic nuclear extracts. Moreover, epitope-tagged SCNY co-immunoprecipitates Drosophila PAF1, but not Cyclin T (or several other tested chromatin proteins) when co-expressed in S2 tissue culture cells. Together, these data support the view that SCNY participates in a conserved pathway of chromatin regulation linking H2B ubiquitylation with H3K4me3 methylation. Because the effects of scny mutation on Ub-H2B and H3K4me3 are opposite to those of dBre1 mutation, SCNY likely opposes dBRE1 action on H2B, just as UBP10 opposes BRE1 action on H2B in yeast (Buszczak, 2009).

Drosophila male and female gonads contain well characterized germline stem cells (GSCs) that allow the effects of genes on stem cell maintenance to be quantitatively analyzed. High levels of scny expression were observed in female and male GSCs using SCNY-GFP and identical staining was observed using anti-SCNY immunofluoresence. SCNY protein resides in cell nuclei and is enriched in nucleoli. In sterile or semi-fertile scny mutant adults, the numbers of germline stem cells surrounding the testis hub and within germaria were clearly reduced. The half-lives of female GSCs bearing clones of three different scny alleles were all sharply reduced. Later follicular development was also abnormal suggesting that scny continues to function after the stem cell stage. However, previous studies indicate that accelerated GSC loss is a specific phenotype, and hence that scny has a preferential requirement in GSCs (Buszczak, 2009).

A known mechanism of increased GSC loss is the premature activation of differentiation genes. Staining germaria with an antibody specific for multiple sites of histone H3 acetylation (H3-Ac) suggested that scny mutation affects the global chromatin organization of GSCs. Wild type GSCs contain lower levels of H3-Ac than slightly older germ cells within cysts. Presumptive GSCs located in the GSC niche in scny mutants frequently stained more strongly, suggesting that they have begun to upregulate general transcription. Some scny GSC-like cells also expressed bag-of-marbles (bam), a key cystoblast differentiation gene, and GSC-like cells in scnyd06513; bamΔ86 mutant females persist in the germarium . However, it could not be completely ruled out that the observed increases in H3-Ac levels and bam expression were a result rather than a cause of the premature differentiation and loss of scny GSCs (Buszczak, 2009).

To determine if scny is also required in a very different type of stem cell, the epithelial follicle stem cell (FSC), the persistence of individual scny mutant FSCs was quantitatively. The half-life of FSCs mutant for scnyl(3)02331 was reduced more than 10-fold, while the scnyf01742 mutation also caused a sharp decline. However, mutant follicle cells continued to develop normally at later stages. Thus, scny is preferentially required to maintain FSCs as well as GSCs (Buszczak, 2009).

The largest population of Drosophila stem cells are the hundreds of multipotent intestinal stem cells (ISCs) that maintain the adult posterior midgut. ISCs signal to their daughters via Delta-Notch signaling to specify enterocyte vs. enteroendocrine cell fate, but the pathway must remain inactive in the ISCs themselves to avoid differentiation. Most ISCs (those about to produce enterocytes) express high levels of the Notch ligand Delta, allowing them to be specifically distinguished from other diploid gut cells. This study found that SCNY-GFP is expressed in ISCs suggesting that SCNY plays a role in these stem cells as well. While 7-day old normal adult midguts contain a high density of ISCs, as revealed by Delta staining, it was found that corresponding tissue from 7-day-old scnyf01742 or scnyf01742/scnyl(3)02331 escaper adults possess very few Delta-positive cells. ISCs are present in near normal numbers at eclosion, but are rapidly lost in the mutant adults, indicating that scny is required for ISC maintenance (Buszczak, 2009).

It is suspected that inappropriate Notch pathway activation was responsible for the premature ISC loss in scny mutants. dBre1 mutations strongly reduce Notch signaling, suggesting that Notch target genes are particularly dependent on H2B mono-ubiquitylation and H3K4 methylation. Consequently, scny mutations, which have the opposite effects on Ub-H2B and H3K4me3 levels, might upregulate Notch target genes, stimulating ISCs to differentiate prematurely. This idea was tested by supplementing the food of newly eclosed scnyf01742/scnyl(3)02331 adults with 8 mM DAPT, a gamma-secretase inhibitor that blocks Notch signaling and phenocopies Notch mutation when fed to wild type animals. scnyf01742/scnyl(3)02331 DAPT-treated adults remained healthy and the guts of 7-day old animals still contained many ISCs, although not as many as wild type. Tumors like those produced in wild type animals fed DAPT were not observed. Thus, in these animals endogenous stem cell loss can be slowed by drug treatment (Buszczak, 2009).

These experiments provide strong evidence that a pathway involving the ubiquitin protease Scrawny and the ubiquitin ligase dBRE1 controls the levels of Ub-H2B, and H3K4me3 at multiple target sites in the Drosophila genome. Although, other ubiquitin proteases also act on Ub-H2B in Drosophila, the direct interaction between SCNY and H2B, and the strong effects of scny mutations argue that it plays an essential, direct role in silencing genomic regions critical for cellular differentiation, including Notch target genes. SCNY interacts with the RNA polymerase accessory factor complex component, PAF1. Upregulation of H2B ubiquitinylation and H3 methylation in yeast is mediated by the PAF1 complex and is associated with elongating RNA Pol II. Drosophila PAF1 is required for normal levels of H3K4me3 at the hsp70 gene, and another PAF1 complex member, RTF1, is needed for H3K4 methylation and Notch target gene expression. Indeed, the pathway connecting Ub-H2B, H3K4me3 and gene silencing appears to be conserved in organisms as distant as Arabidopsis. A human protein closely related to SCNY, USP36, is overexpressed in ovarian cancer cells, and the results suggest it may act as an oncogene by suppressing differentiation (Buszczak, 2009).

Above all, these experiments indicate that SCNY-mediated H2B deubiquitylation is required to maintain multiple Drosophila stem cells, including progenitors of germline, epithelial and endodermal lineages. In ES cells and presumably in adult stem cells, many differentiation genes contain promoter-bound, arrested RNA Pol II and are associated with Polycomb group proteins. It is envisioned that in the niche environment SCNY activity overrides that of dBRE1, keeping levels of Ub-H2B (and hence H3K4me3) low at key differentiation genes. Upon exit from the niche, the balance of signals shifts to favor H2B ubiquitylation, H3K4 trimethylation, and target gene activation. Thus, the control of H2B ubiquitylation, like H2A ubiquitylation, plays a fundamental interactive role in maintaining the chromatin environment of the stem cell state (Buszczak, 2009).


REFERENCES

Search PubMed for articles about Drosophila Paf1

Adelman, K., Wei, W., Ardehali, M. B., Werner, J., Zhu, B. Reinberg, D. and Lis, J. T. (2006). Drosophila Paf1 modulates chromatin structure at actively transcribed genes. Mol. Cell. Biol. 26(1): 250-60. PubMed ID: 16354696

Akimaru, H., et al. (1997). Drosophila CBP is a co-activator of cubitus interruptus in hedgehog signalling. Nature 386: 735-738. PubMed ID: 9109493

Buszczak, M., Paterno, S. and Spradling, A. C. (2009). Drosophila stem cells share a common requirement for the histone H2B ubiquitin protease scrawny. Science 323: 248-251. PubMed ID: 19039105

Costa, P. J., and Arndt, K. M. (2000). Synthetic lethal interactions suggest a role for the Saccharomyces cerevisiae Rtf1 protein in transcription elongation. Genetics 156: 535-547. PubMed ID: 11014804

Formosa, T., S. Ruone, M. D. Adams, A. E. Olsen, P. Eriksson, Y. Yu, A. R. Rhoades, P. D. Kaufman, and D. J. Stillman. 2002. Defects in SPT16 or POB3 (yFACT) in Saccharomyces cerevisiae cause dependence on the Hir/Hpc pathway: polymerase passage may degrade chromatin structure. Genetics 162: 1557-1571. PubMed ID: 12524332

Janody, F., Martirosyan, Z., Benlali, A. and Treisman, J. E. (2003). Two subunits of the Drosophila mediator complex act together to control cell affinity. Development 130: 3691-3701. PubMed ID: 12835386

Kaplan, C. D., Holland, M. J. and Winston, F. (2005). Interaction between transcription elongation factors and mRNA 3'-end formation at the Saccharomyces cerevisiae GAL10-GAL7 locus. J. Biol. Chem. 280: 913-922. PubMed ID: 15531585

Krogan, N. J., et al. (2002). RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach. Mol. Cell. Biol. 22: 6979-6992. PubMed ID: 12242279

Mosimann, C., Hausmann, G. and Basler, K. (2009). The role of Parafibromin/Hyrax as a nuclear Gli/Ci-interacting protein in Hedgehog target gene control. Mech. Dev. 126(5-6): 394-405. PubMed ID: 19368795

Mueller, C. L., et al. (2004). The Paf1 complex has functions independent of actively transcribing RNA polymerase II. Mol. Cell 14: 447-456. PubMed ID: 15149594

Ng, H. H., Dole, S. and Struhl, K. (2003). The Rtf1 component of the Paf1 transcriptional elongation complex is required for ubiquitination of histone H2B. J. Biol. Chem. 278: 33625-33628. PubMed ID: 12876293

Pokholok, D. K., Hannett, N. M. and Young, R. A. (2002). Exchange of RNA polymerase II initiation and elongation factors during gene expression in vivo. Mol. Cell 9: 799-809. PubMed ID: 11983171

Porter, S. E., Penheiter, K. L. and Jaehning, J. A. (2005). Separation of the Saccharomyces cerevisiae Paf1 complex from RNA polymerase II results in changes in its subnuclear localization. Eukaryot. Cell 4: 209-220. PubMed ID: 15643076

Rondon, A. G., et al. (2004). Molecular evidence indicating that the yeast PAF complex is required for transcription elongation. EMBO Rep. 5: 47-53. PubMed ID: 14710186

Rozenblatt-Rosen, O., et al. (2005). The parafibromin tumor suppressor protein is part of a human Paf1 complex. Mol. Cell. Biol. 25: 612-620. PubMed ID: 15632063

Shi, X., et al. (1996). Paf1p, an RNA polymerase II-associated factor in Saccharomyces cerevisiae, may have both positive and negative roles in transcription. Mol. Cell. Biol. 16: 669-676. PubMed ID: 8552095

Simic, R., D. L. Lindstrom, H. G. Tran, K. L. Roinick, P. J. Costa, A. D. Johnson, G. A. Hartzog, and K. M. Arndt. 2003. Chromatin remodeling protein Chd1 interacts with transcription elongation factors and localizes to transcribed genes. EMBO J. 22:1846-1856. PubMed ID: 12682017

Smith, S. T., et al. (2004). Modulation of heat shock gene expression by the TAC1 chromatin-modifying complex. Nat. Cell Biol. 6: 162-167. PubMed ID: 14730313

Squazzo, S. L., et al. (2002). The PAF1 complex physically and functionally associates with transcription elongation factors in vivo. EMBO J. 21: 1764-177. PubMed ID: 11927560

Sun, Z. W., and Allis, C. D. (2002). Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast. Nature 418: 104-108. PubMed ID: 12077605

Wood, A., Schneider, J., Dover, J., Johnston, M. and Shilatifard, A. (2003). The Paf1 complex is essential for histone monoubiquitination by the Rad6-Bre1 complex, which signals for histone methylation by COMPASS and Dot1p J. Biol. Chem. 278: 34739-34742. PubMed ID: 12876294

Xiao, T., et al. (2005). Histone H2B ubiquitylation is associated with elongating RNA polymerase II. Mol. Cell. Biol. 25: 637-651. PubMed ID: 15632065

Yart, A., et al. (2005). The HRPT2 tumor suppressor gene product parafibromin associates with human PAF1 and RNA polymerase II. Mol. Cell. Biol. 25: 5052-5060. PubMed ID: 15923622


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date revised: 28 November 2007

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