Proper transcription by RNA polymerase II is dependent on the modification state of the chromatin template. The Paf1 complex is associated with RNA polymerase II during transcription elongation and is required for several histone modifications that mark active genes. To uncover additional factors that regulate chromatin or transcription, we performed a genetic screen for mutations that cause lethality in the absence of the Paf1 complex component Rtf1. Our results have led to the discovery of a previously unstudied gene, RKR1. Strains lacking RKR1 exhibit phenotypes associated with defects in transcription and chromatin function. These phenotypes include inositol auxotrophy, impaired telomeric silencing, and synthetic lethality with mutations in SPT10, a gene that encodes a putative histone acetyltransferase. In addition, deletion of RKR1 causes severe genetic interactions with mutations that prevent histone H2B lysine 123 ubiquitylation or histone H3 lysine 4 methylation. RKR1 encodes a conserved nuclear protein with a functionally important RING domain at its carboxy terminus. In vitro experiments indicate that Rkr1 possesses ubiquitin-protein ligase activity. Taken together, our results identify a new participant in a protein ubiquitylation pathway within the nucleus that acts to modulate chromatin function and transcription.Progression of the RNA polymerase II (Pol II) transcription cycle involves the coordinated functions of a large number of regulatory proteins. During transcription elongation, proteins that associate with Pol II assist it in overcoming obstacles to transcription, including DNA damage and condensed chromatin structure. Transcription elongation factors use a variety of mechanisms to facilitate movement of Pol II through a nucleosomal template and coordinate transcription with RNA processing. The Paf1 complex is a Pol II-associated factor that alters the state of the chromatin template during transcription elongation. Biochemical purification of the Saccharomyces cerevisiae Paf1 complex showed that it minimally contains five proteins: Paf1, Ctr9, Rtf1, Cdc73, and Leo1 (37,44,76). Chromatin immunoprecipitation experiments demonstrated that this complex is associated with the open reading frames (ORFs) of actively transcribed genes (37,59,72). Strains lacking members of the Paf1 complex are sensitive to the base analogs 6-azauracil and mycophenolic acid and exhibit altered RNA levels for a large number of genes (10,44,57,76). These results, combined with genetic and physical interactions with the elongation factors Spt4-Spt5 and Spt16-Pob3 (yFACT complex) (76), suggest that the Paf1 complex is important for transcription elongation. Members of the Paf1 complex are also required for proper 3Ј end formation of both polyadenylated and nonpolyadenylated transcripts (57, 68).It is now well established that the arrangements of posttranslational modifications on nucleosomal histones, along with interactions between histones and nonhistone proteins, coordinately affect chromatin structure. Histones can...

c Across diverse eukaryotes, the Paf1 complex (Paf1C) plays critical roles in RNA polymerase II transcription elongation and regulation of histone modifications. Beyond these roles, the human and Saccharomyces cerevisiae Paf1 complexes also interact with RNA 3=-end processing components to affect transcript 3=-end formation. Specifically, the Saccharomyces cerevisiae Paf1C functions with the RNA binding proteins Nrd1 and Nab3 to regulate the termination of at least two small nucleolar RNAs (snoRNAs). To determine how Paf1C-dependent functions regulate snoRNA formation, we used high-density tiling arrays to analyze transcripts in paf1⌬ cells and uncover new snoRNA targets of Paf1. Detailed examination of Paf1-regulated snoRNA genes revealed locus-specific requirements for Paf1-dependent posttranslational histone modifications. We also discovered roles for the transcriptional regulators Bur1-Bur2, Rad6, and Set2 in snoRNA 3=-end formation. Surprisingly, at some snoRNAs, this function of Rad6 appears to be primarily independent of its role in histone H2B monoubiquitylation. Cumulatively, our work reveals a broad requirement for the Paf1C in snoRNA 3=-end formation in S. cerevisiae, implicates the participation of transcriptional proteins and histone modifications in this process, and suggests that the Paf1C contributes to the fine tuning of nuanced levels of regulation that exist at individual loci.

The conserved Paf1 complex (Paf1C) carries out multiple functions during transcription by RNA polymerase (pol) II, and these functions are required for the proper expression of numerous genes in yeast and metazoans. In the elongation stage of the transcription cycle, the Paf1C associates with RNA pol II, interacts with other transcription elongation factors, and facilitates modifications to the chromatin template. At the end of elongation, the Paf1C plays an important role in the termination of RNA pol II transcripts and the recruitment of proteins required for proper RNA 3′ end formation. Significantly, defects in the Paf1C are associated with several human diseases. In this paper, we summarize current knowledge on the roles of the Paf1C in RNA pol II transcription.

The conserved multifunctional Paf1 complex is important for the proper transcription of numerous genes, and yet the exact mechanisms by which it controls gene expression remain unclear. While previous studies indicate that the Paf1 complex is a positive regulator of transcription, the repression of many genes also requires the Paf1 complex. In this study we used ARG1 as a model gene to study transcriptional repression by the Paf1 complex in Saccharomyces cerevisiae. We found that several members of the Paf1 complex contribute to ARG1 repression and that the complex localizes to the ARG1 promoter and coding region in repressing conditions, which is consistent with a direct repressive function. Furthermore, Paf1 complex-dependent histone modifications are enriched at the ARG1 locus in repressing conditions, and histone H3 lysine 4 methylation contributes to ARG1 repression. Consistent with previous reports, histone H2B monoubiquitylation, the mark upstream of histone H3 lysine 4 methylation, is also important for ARG1 repression. To begin to identify the mechanistic basis for Paf1 complex-mediated repression of ARG1, we focused on the Rtf1 subunit of the complex. Through an analysis of RTF1 mutations that abrogate known Rtf1 activities, we found that Rtf1 mediates ARG1 repression primarily by facilitating histone modifications. Other members of the Paf1 complex, such as Paf1, appear to repress ARG1 through additional mechanisms. Together, our results suggest that Rtf1-dependent histone H2B ubiquitylation and H3 K4 methylation repress ARG1 expression and that histone modifications normally associated with active transcription can occur at repressed loci and contribute to transcriptional repression.The organization of eukaryotic DNA into chromatin presents a significant obstacle to transcription by RNA polymerase II (Pol II). To allow proper gene expression, a multitude of accessory factors associate with RNA Pol II to facilitate the transcription of a chromatin template. A conserved, multifunctional protein complex that enables proper RNA Pol II transcription is the Paf1 complex. In Saccharomyces cerevisiae, the Paf1 complex consists of Paf1, Ctr9, Cdc73, Rtf1, and Leo1 (36,45,64,66). Many physical and genetic interactions and phenotypes implicate the Paf1 complex in regulating the elongation stage of transcription. Specifically, strains lacking Paf1 complex members exhibit phenotypes associated with transcription elongation defects, such as sensitivity to 6-azauracil and mycophenolic acid (12, 66). During transcription elongation, the Paf1 complex associates with RNA Pol II on open reading frames (ORFs) (36, 54), where it orchestrates modifications to the chromatin template (11,35,49,50,78) and influences the phosphorylation state of the RNA Pol II carboxy-terminal domain (CTD) (46, 51). In addition, the Paf1 complex genetically and physically interacts with elongation factors such as the Spt4-Spt5 (yDSIF) and Spt16-Pob3 (yFACT) complexes, suggesting coordinated functions of these elongation factors during transc...

The conserved Paf1 complex negatively regulates the expression of numerous genes, yet the mechanisms by which it represses gene expression are not well understood. In this study, we use the ARG1 gene as a model to investigate the repressive functions of the Paf1 complex in Saccharomyces cerevisiae. Our results indicate that Paf1 mediates repression of the ARG1 gene independently of the gene-specific repressor, ArgR/Mcm1. Rather, by promoting histone H2B lysine 123 ubiquitylation, Paf1 represses the ARG1 gene by negatively affecting Gcn4 occupancy at the promoter. Consistent with this observation, Gcn5 and its acetylation sites on histone H3 are required for full ARG1 derepression in paf1⌬ cells, and the repressive effect of Paf1 is largely maintained when the ARG1 promoter directs transcription of a heterologous coding region. Derepression of the ARG1 gene in paf1⌬ cells is accompanied by small changes in nucleosome occupancy, although these changes are subtle in comparison to those that accompany gene activation through amino acid starvation. Additionally, conditions that stimulate ARG1 transcription, including PAF1 deletion, lead to increased antisense transcription across the ARG1 promoter. This promoter-associated antisense transcription positively correlates with ARG1 sense transcription. Finally, our results indicate that Paf1 represses other genes through mechanisms similar to those used at the ARG1 gene. E ukaryotic organisms employ several mechanisms to repress gene expression. In some of the best studied cases, transcriptional repressors bind DNA and recruit corepressors that inhibit the basal transcriptional machinery, interfere with activator binding, or recruit histone-modifying proteins (reviewed in reference 55). In addition, nucleosomes within promoters and coding regions can inhibit RNA polymerase II (Pol II) recruitment and impede transcription elongation (reviewed in reference 3). More recently, the synthesis of noncoding RNAs (ncRNAs) has been implicated in transcriptional repression. For example, transcription across the Saccharomyces cerevisiae SER3 promoter inhibits SER3 expression by establishing a chromatin environment that obstructs activator binding (27,42,43).A conserved, globally acting protein complex that has roles in gene repression and activation is the Paf1 complex (Paf1C), which consists of Paf1, Ctr9, Cdc73, Rtf1, and Leo1 in budding yeast (37,46,65,68). Paf1C associates with RNA Pol II on open reading frames (ORFs) (37,44,58,75), regulates the phosphorylation state of the RNA Pol II carboxy-terminal domain (CTD) (47, 54), and facilitates transcription elongation of chromatin templates in vitro (11,35) and in vivo (71). Paf1C subunits are also required for the proper establishment of several histone modifications that mark active genes (12, 36, 52, 53, 78) and inhibition of H3 and H4 acetylation on the coding regions of active genes (12). In yeast, Paf1 and Rtf1 are required for monoubiquitylation of histone H2B lysine (K) 123 (40,78,79) and the subsequent methylation of histo...

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One eukaryotic protein complex involved in regulating RNA polymerase II transcription is the Paf1 complex (Paf1C), which has well‐characterized roles in promoting histone modifications and transcription elongation. Beyond these roles, the human and yeast Paf1 complexes also interact with RNA 3’‐end processing components to affect RNA formation. Specifically, the S. cerevisiae Paf1C is known to promote the proper 3’‐end formation of at least two small nucleolar RNAs (snoRNAs). To determine how Paf1C‐dependent functions regulate formation of these non‐coding RNAs, we used high‐density tiling arrays to analyze transcripts in cells lacking PAF1 and uncover new snoRNA targets of Paf1. Increased levels of RNA polymerase II seen downstream of snoRNA genes in the absence of PAF1 suggest that these snoRNA 3’‐end defects reflect impaired transcription termination. Examination of Paf1‐regulated snoRNA genes revealed locus‐specific requirements for post‐translational histone modifications and for other transcriptional regulators. Cumulatively, our work reveals a broad requirement for Paf1C in snoRNA 3’‐end formation and suggests that Paf1C contributes to the fine‐tuning of transcriptional regulation at individual loci.

The Paf1 complex associates with RNA pol II and couples histone modification to transcription elongation. The Rtf1 subunit of the complex is essential for H2B K123 ubiquitylation and H3 K4 and K79 methylation. We have identified a conserved histone modification domain (HMD) in Rtf1 that is essential for these modifications. Specific amino acid substitutions in the HMD cause differential effects on H2B ubiquitylation and H3 K4 methylation, indicating that the functions of Rtf1 in directing these marks are genetically separable. Interestingly, expression of the isolated HMD in yeast cells is sufficient to promote histone modification. Microarray experiments have revealed broad effects of the Paf1 complex on transcription patterns, including many genes where the Paf1 complex functions as a repressor. Northern analyses demonstrated that Paf1 and Ctr9, and to a lesser extent Rtf1, are required for repression of the model gene ARG1. Analysis of ARG1 expression in single and double mutant strains indicated that Paf1 directs repression through a pathway that operates in parallel to known ARG1 regulators. The Paf1 complex localizes to ARG1 in both repressing and activating conditions, suggesting that it directly regulates ARG1 transcription. Replacement of the ARG1 coding sequence with that of a heterologous gene revealed that the Paf1 complex functions through the promoter or early transcribed region of ARG1. Our results suggest that the Paf1 complex inhibits transcription of ARG1 and potentially other genes by establishing a repressive chromatin structure at the promoters or 5' ends of these genes. Funding source: NIH.