Characterization of yeast Rtf1 and the Paf1 complex

Regulated transcription initiation requires, in addition to RNA polymerase II and the general transcription factors, accessory factors termed mediators or adapters. Affinity chromatography was used to identify a collection of factors that associate with Saccharomyces cerevisiae RNA polymerase II. A gene encoding one of these factors, PAF1 (for RNA polymerase-associated factor 1), was identifed and characterized. PAF1 encodes a novel, highly charged protein of 445 amino acids. Disruption of PAF1 in S. cerevisiae leads to pleiotropic phenotypic traits, including slow growth, temperature sensitivity, and abnormal cell morphology. Consistent with a possible role in transcription, Paf1p is localized to the nucleus. By comparing the abundances of many yeast transcripts in isogenic wild-type and paf1 mutant strains, genes were identified whose expression is affected by PAF1. In particular, disruption of PAF1 decreases the induction of the galactose-regulated genes three- to fivefold. In contrast, the transcript level of MAK16, an essential gene involved in cell cycle regulation, is greatly increased in the paf1 mutant strain. Paf1p may therefore be required for both positive and negative regulation of subsets of yeast genes. Like Paf1p, the GAL11 gene product is found associated with RNA polymerase II and is required for regulated expression of many yeast genes including those controlled by galactose. A gal11 paf1 double mutant has a much more severe growth defect than either of the single mutants, indicating that these two proteins may function in parallel pathways to communicate signals from regulatory factors to RNA polymerase II (Shi, 1996).

The products of the yeast CDC73 and PAF1 genes were originally identified as RNA polymerase II-associated proteins. Paf1p is a nuclear protein important for cell growth and transcriptional regulation of a subset of yeast genes. The product of CDC73 is a nuclear protein that interacts directly with purified RNA polymerase II in vitro. Deletion of CDC73 confers a temperature-sensitive phenotype. Combination of the cdc73 mutation with the more severe paf1 mutation does not result in an enhanced phenotype, indicating that the two proteins may function in the same cellular processes. To determine the relationship between Cdc73p and Paf1p and the recently described holoenzyme form of RNA polymerase II, yeast strains were created containing glutathione S-transferase (GST)-tagged forms of CDC73, PAF1, and TFG2 functionally replacing the chromosomal copies of the genes. Isolation of GST-tagged Cdc73p and Paf1p complexes has revealed a unique form of RNA polymerase II that contains both Cdc73p and Paf1p but lacks the Srbps found in the holoenzyme. The Cdc73p-Paf1p-RNA polymerase II-containing complex also includes Gal11p, and the general initiation factors TFIIB and TFIIF, but lacks TBP, TFIIH, and transcription elongation factor TFIIS as well as the Srbps. The Srbp-containing holoenzyme does not include either Paf1p or Cdc73p, demonstrating that these two forms of RNA polymerase II are distinct. In confirmation of the hypothesis that the two forms coexist in yeast cells, it was found that a TFIIF-containing complex isolated via the GST-tagged Tfg2p construct contains both (1) the Srbps and (2) Cdc73p and Paf1p. The Srbps and Cdc73p-Paf1p therefore appear to define two complexes with partially redundant, essential functions in the yeast cell. Using the technique of differential display, several genes have been identified whose transcripts require Cdc73p and/or Paf1p for normal levels of expression. This analysis suggests that there are multiple RNA polymerase II-containing complexes involved in the expression of different classes of protein-coding genes (Shi, 1997; full text of article).

Strong evidence indicates that transcription elongation by RNA polymerase II (pol II) is a highly regulated process. Genetic results are presented that indicate a role for the Saccharomyces cerevisiae Rtf1 protein in transcription elongation. A screen for synthetic lethal mutations was carried out with an rtf1 deletion mutation to identify factors that interact with Rtf1 or regulate the same process as Rtf1. The screen uncovered mutations in SRB5, CTK1, FCP1, and POB3. These genes encode an Srb/mediator component, a CTD kinase, a CTD phosphatase, and a protein involved in the regulation of transcription by chromatin structure, respectively. All of these gene products have been directly or indirectly implicated in transcription elongation, indicating that Rtf1 may also regulate this process. In support of this view, RTF1 was shown to functionally interact with genes that encode known elongation factors, including SPT4, SPT5, SPT16, and PPR2. A deletion of RTF1 causes sensitivity to 6-azauracil and mycophenolic acid, phenotypes correlated with a transcription elongation defect. Collectively, these results suggest that Rtf1 may function as a novel transcription elongation factor in yeast (Costa, 2000; full text of article).

In yeast cells, the Rtf1 and Paf1 components of the Paf1 transcriptional elongation complex are important for recruitment of Set1, the histone H3-lysine 4 (H3-Lys4) methylase, to a highly localized domain at the 5' portion of active mRNA coding regions. Rtf1 is essential for global methylation of H3-Lys4 and H3-Lys79, but not H3-Lys36. This role of Rtf1 resembles that of Rad6, which mediates ubiquitination of histone H2B at lysine 123. Indeed, Rtf1 is required for H2B ubiquitination, suggesting that its effects on H3-Lys4 and H3-Lys79 methylation are an indirect consequence of its effect on H2B ubiquitination. Rtf1 is important for telomeric silencing, with loss of H3-Lys4 and H3-Lys79 methylation synergistically reducing Sir2 association with telomeric DNA. Dot1, the H3-Lys79 methylase, associates with transcriptionally active genes, but unlike the association of Set1 and Set2 (the H3-Lys36 methylase; see Drosophila Set2), this association is largely independent of Rtf1. It is suggested that Rtf1 affects genome-wide ubiquitination of H2B by a mechanism that is distinct from its function as a transcriptional elongation factor (Ng, 2003).

The yeast Paf1 complex (Paf1C), composed of Paf1, Ctr9, Cdc73, Rtf1, and Leo1, associates with RNA polymerase II (Pol II) at promoters and in the actively transcribed portions of mRNA genes. Loss of Paf1 results in severe phenotypes and significantly reduced levels of the other Paf1C components. In contrast, loss of Rtf1 causes relatively subtle phenotypic changes and no reduction in the other Paf1C factors but disrupts the association of these factors with Pol II and chromatin. To elucidate the fate of the Paf1C when dissociated from Pol II, the localization of the Paf1C components in paf1 and rtf1 mutant yeast strains was examined. Although the Paf1C factors remain nuclear in paf1 and rtf1 strains, loss of Paf1 or Rtf1 results in a change in the subnuclear distribution of the remaining factors. In wild-type cells, Paf1C components are present in the nucleoplasm but not the nucleolus. In contrast, in both paf1 and rtf1 strains, the remaining factors are found in the nucleolus as well as the nucleoplasm. Loss of Paf1 affects nucleolar function; it is observed that expression of MAK21 and RRP12, important for rRNA processing, is reduced concomitant with an increase in rRNA precursors in a paf1 strain. However, these changes are not the result of relocalization of the Paf1C because loss of Rtf1 does not cause similar changes in rRNA processing. Instead, it is speculated that the change in localization may reflect a link between the Paf1C and newly synthesized mRNAs as they exit the nucleus (Porter, 2005; full text of article).

The Paf1 complex has functions independent of actively transcribing RNA polymerase II

The yeast Paf1 complex, minimally composed of Paf1, Ctr9, Cdc73, Rtf1, and Leo1, was originally isolated in association with RNA polymerase II (Pol II). Paf1 complex components are abundant and colocalize with Pol II on chromatin at promoters and in the coding regions of actively transcribed genes. Loss of Paf1 results in severe phenotypes and reduced amounts of other Paf1 factors, with little effect on abundance or chromatin distribution of Pol II, proteins important for transcriptional elongation (Spt5, Spt16), or RNA processing (Sub2). Loss of Paf1 factors causes a reduction of Pol II Ser2 phosphorylation and shortened poly(A) tails, suggesting that the complex facilitates linkage of transcriptional and posttranscriptional events. Surprisingly, loss of Rtf1 or Cdc73, with little phenotypic consequence, results in loss of Paf1 factors from chromatin and a significant reduction in Paf1/Pol II association. Therefore, the major functions of Paf1 can be independent of actively transcribing Pol II (Mueller, 2004).

Yeast Rtf1 interacts with Chd1

Transcription in eukaryotes is influenced by the chromatin state of the template, and chromatin remodeling factors have well-documented roles in regulating transcription initiation by RNA polymerase (pol) II. Chromatin also influences transcription elongation; however, little is known about the role of chromatin remodeling factors in this process. Evidence is presented that the Saccharomyces cerevisiae chromatin remodeling factor Chd1 [Drosophila homolog; Chromodomain-helicase-DNA-binding protein)] functions during transcription elongation: (1) Chd1 was identified in a two-hybrid screen for proteins that interact with Rtf1, a member of the Paf1 complex that associates with RNA pol II and regulates transcription elongation; (2) co-immunoprecipitation studies show that that Chd1 also interacts with components of two essential elongation factors, Spt4-Spt5 and Spt16-Pob3; (3) it was demonstrated that deletion of CHD1 suppresses a cold-sensitive spt5 mutation that is also suppressed by defects in the Paf1 complex and RNA pol II. Finally, Chd1, Rtf1 and Spt5 associate with actively transcribed regions of chromatin. Collectively, these findings suggest an important role for Chd1 and chromatin remodeling in the control of transcription elongation (Simic, 2003; full text of article).

Histone methylation downstream of Rtf1 in yeast

The DNA of eukaryotes is wrapped around nucleosomes and packaged into chromatin. Covalent modifications of the histone proteins that comprise the nucleosome alter chromatin structure and have major effects on gene expression. Methylation of lysine 4 of histone H3 by COMPASS is required for silencing of genes located near chromosome telomeres and within the rDNA. To learn about the mechanism of histone methylation, the genome of the yeast Saccharomyces cerevisiae was surveyed for genes necessary for this process. By analyzing approximately 4800 mutant strains, each deleted for a different non-essential gene, it was discovered that the ubiquitin-conjugating enzyme Rad6 is required for methylation of lysine 4 of histone H3. Ubiquitination of histone H2B on lysine 123 is the signal for the methylation of histone H3, which leads to silencing of genes located near telomeres (Dover, 2002; full text of article).

In eukaryotes, the DNA of the genome is packaged with histone proteins to form nucleosomal filaments, which are, in turn, folded into a series of less well understood chromatin structures. Post-translational modifications of histone tail domains modulate chromatin structure and gene expression. Of these, histone ubiquitination is poorly understood. The ubiquitin-conjugating enzyme Rad6 (Ubc2) mediates methylation of histone H3 at lysine 4 (Lys 4) through ubiquitination of H2B at Lys 123 in yeast (Saccharomyces cerevisiae). Moreover, H3 (Lys 4) methylation is abolished in the H2B-K123R mutant, whereas H3-K4R retains H2B (Lys 123) ubiquitination. These data indicate a unidirectional regulatory pathway in which ubiquitination of H2B (Lys 123) is a prerequisite for H3 (Lys 4) methylation. An H2B-K123R mutation perturbs silencing at the telomere, providing functional links between Rad6-mediated H2B (Lys 123) ubiquitination, Set1-mediated H3 (Lys 4) methylation, and transcriptional silencing. Thus, these data reveal a pathway leading to gene regulation through concerted histone modifications on distinct histone tails. This is referred as 'trans-tail' regulation of histone modification, a stated prediction of the histone code hypothesis (Sun, 2002).

Methylation of histone proteins is one of their many modifications that affect chromatin structure and regulate gene expression. Methylation of histone H3 on lysines 4 and 79, catalyzed by the Set1-containing complex COMPASS and Dot1p, respectively, is required for silencing of expression of genes located near chromosome telomeres in yeast. The Paf1 protein complex, which is associated with the elongating RNA polymerase II, is required for methylation of lysines 4 and 79 of histone H3 and for silencing of expression of a telomere-associated gene. The Paf1 complex is required for recruitment of the COMPASS methyltransferase to RNA polymerase II, and the subunits of these complexes interact physically and genetically. Collectively, these results suggest that the Paf1 complex is required for histone H3 methylation, therefore linking transcriptional elongation to chromatin methylation (Krogan, 2003a).

Set2 methylates Lys36 of histone H3. Yeast Set2 copurifies with RNA polymerase II (RNAPII). Chromatin immunoprecipitation analyses demonstrated that Set2 and histone H3 Lys36 methylation are associated with the coding regions of several genes that were tested and correlate with active transcription. Both depend, as well, on the Paf1 elongation factor complex. The C terminus of Set2, which contains a WW domain, is also required for effective Lys36 methylation. Deletion of CTK1, encoding an RNAPII CTD kinase, prevents Lys36 methylation and Set2 recruitment, suggesting that methylation may be triggered by contact of the WW domain or C terminus of Set2 with Ser2-phosphorylated CTD. A set2 deletion results in slight sensitivity to 6-azauracil and much less beta-galactosidase produced by a reporter plasmid, resulting from a defect in transcription. In synthetic genetic array (SGA) analysis, synthetic growth defects were obtained when a set2 deletion was combined with deletions of all five components of the Paf1 complex, the chromodomain elongation factor Chd1, the putative elongation factor Soh1, the Bre1 or Lge1 components of the histone H2B ubiquitination complex, or the histone H2A variant Htz1. SET2 also interacts genetically with components of the Set1 and Set3 complexes, suggesting that Set1, Set2, and Set3 similarly affect transcription by RNAPII (Krogan, 2003b; full text of article).

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 is presented 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, it is shown that the Paf1 complex is 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 (Wood, 2003; full text of article).

Spt6 is a conserved transcription factor that associates with RNA polymerase II (pol II) during elongation. Spt6 is essential for viability in Saccharomyces cerevisiae and regulates chromatin structure during pol II transcription. Evidence is presented that mutations that impair Spt6, a second elongation factor, Spt4, and pol II can affect 3'-end formation at GAL10. Additional analysis suggests that Spt6 is required for cotranscriptional association of the factor Ctr9, a member of the Paf1 complex, with GAL10 and GAL7, and that Ctr9 association with chromatin 3' of GAL10 is regulated by the GAL10 polyadenylation signal. Overall, these results provide new evidence for a connection between the transcription elongation factor Spt6 and 3'-end formation in vivo (Kaplan, 2005; full text of article).

Characterization of the mammalian Paf1 complex

Parafibromin, the product of the HRPT2 (hyperparathyroidism-jaw tumor syndrome 2) tumor suppressor gene, is the human homologue of yeast Cdc73, part of the yeast RNA polymerase II/Paf1 complex known to be important for histone modification and connections to posttranscriptional events. By purifying cellular parafibromin and characterizing its associated proteins, a human counterpart to the yeast Paf1 complex was identified including homologs of Leo1, Paf1, and Ctr9. Like the yeast complex, the parafibromin complex associates with the nonphosphorylated and Ser2 and Ser5 phosphorylated forms of the RNA polymerase II large subunit. Immunofluorescence experiments show that parafibromin is a nuclear protein. In addition, cotransfection data suggest that parafibromin can interact with a histone methyltransferase complex that methylates histone H3 on lysine 4. Some mutant forms of parafibromin lack association with hPaf1 complex members and with the histone methyltransferase complex, suggesting that disruption of these complexes may correlate with the oncogenic process (Rozenblatt-Rosen, 2005; full text of article).

Inactivation of the HRPT2 tumor suppressor gene is associated with the pathogenesis of the hereditary hyperparathyroidism-jaw tumor syndrome and malignancy in sporadic parathyroid tumors. The cellular function of the HPRT2 gene product, parafibromin, has not been defined yet. This study shows that parafibromin physically interacts with human orthologs of yeast Paf1 complex components, including PAF1, LEO1, and CTR9, that are involved in transcription elongation and 3' end processing. It also associates with modified forms of the large subunit of RNA polymerase II, in particular those phosphorylated on serine 5 or 2 within the carboxy-terminal domain, that are important for the coordinate recruitment of transcription elongation and RNA processing machineries during the transcription cycle. These interactions depend on a C-terminal domain of parafibromin, which is deleted in ~80% of clinically relevant mutations. Finally, RNAi-induced downregulation of parafibromin promotes entry into S phase, implying a role for parafibromin as an inhibitor of cell cycle progression. Taken together, these findings link the tumor suppressor parafibromin to the transcription elongation and RNA processing pathway as a PAF1 complex- and RNA polymerase II-bound protein. Dysfunction of this pathway may be a general phenomenon in the majority of cases of hereditary parathyroid cancer (Yart, 2005; full text of article)

The yeast PAF (yPAF) complex interacts with RNA polymerase II and coordinates the setting of histone marks associated with active transcription. This study reports the isolation and functional characterization of the human PAF (hPAF) complex. hPAF shares four subunits with yPAF (hCtr9, hPaf1, hLeo1, and hCdc73), but contains a novel higher eukaryotic-specific subunit, hSki8. RNAi against hSki8 or hCtr9 reduces the cellular levels of other hPAF subunits and of mono- and trimethylated H3-Lys 4 and dimethylated H3-Lys 79. The hSki8 subunit is also a component of the human SKI (hSKI) complex. Yeast SKI complex is cytoplasmic and together with Exosome mediates 3'-5' mRNA degradation. However, hSKI complex localizes to both nucleus and cytoplasm. Immunoprecipitation experiments revealed that hPAF and hSKI complexes interact, and ChIP experiments demonstrated that hSKI associates with transcriptionally active genes dependent on the presence of hPAF. Thus, in addition to coordinating events during transcription (initiation, promoter clearance, and elongation), hPAF also coordinates events in RNA quality control (Zhu, 2005).

Rtf1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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