Effects of the Paf1 complex and histone modifications on snoRNA 3'-end formation reveal broad and locus-specific regulation.

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 paf1Δ 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.

Paf1 restricts Gcn4 occupancy and antisense transcription at the ARG1 promoter.

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 paf1Δ 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 paf1Δ 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.

The Paf1 complex represses ARG1 transcription in Saccharomyces cerevisiae by promoting histone modifications.

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 Roles of the Paf1 Complex and Associated Histone Modifications in Regulating Gene Expression.

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.

Identification of Rkr1, a nuclear RING domain protein with functional connections to chromatin modification in Saccharomyces cerevisiae.

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.