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. We report that 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. We show that the Paf1 complex is required for recruitment of the COMPASS methyltransferase to RNA polymerase II and that the subunits of these complexes interact physically and genetically. Collectively, our results suggest that the Paf1 complex is required for histone H3 methylation, therefore linking transcriptional elongation to chromatin methylation.

Ubiquitination of histone H2B catalyzed by Rad6 is required for methylation of histone H3 by COMPASS. We identified Bre1 as the probable E3 for Rad6's role in transcription. Bre1 contains a C3HC4 (RING) finger and is present with Rad6 in a complex. The RING finger of Bre1 is required for ubiquitination of histone H2B, methylation of lysine 4 and 79 of H3 and for telomeric silencing. Chromatin immunoprecipitation experiments indicated that both Rad6 and Bre1 are recruited to a promoter. Bre1 is essential for this recruitment of Rad6 and is dedicated to the transcriptional pathway of Rad6. These results suggest that Bre1 is the likely E3 enzyme that directs Rad6 to modify chromatin and ultimately to affect gene expression.

. To learn about the mechanism of histone methylation, we surveyed the genome of the yeast Saccharomyces cerevisiae for genes necessary for this process. By analyzing ϳ4800 mutant strains, each deleted for a different non-essential gene, we 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.

Monoubiquitination of histone H2B, catalyzed byRad6-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. We present evidence 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. We also show that Paf1 complex is required for the interaction of Rad6 and COMPASS with RNA polymerase II. Finally, we show 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 ubiquitinprotein ligase at the promoters of active genes.The DNA of eukaryotic organisms assembles around histone proteins in nucleosomes to form highly organized structures known as chromatin (1). Alterations in chromatin structure play a major role in regulating gene expression, and for this reason much attention has been focused recently on the covalent modifications of histone proteins and their outcomes in transcriptional elongation (1-4). Essential to this process are the N-terminal tails of histone proteins. Because they protrude from the globular body of the nucleosome and are available for interactions with other proteins, the tails are the site of many covalent modifications that alter nucleosome structure. A myriad of modifications, such as acetylation, phosphorylation, ubiquitination, and methylation, decorate each histone tail (2, 5) The combinatorial effects of such modifications can produce an array of different responses involved in transcriptional activation and repression (1-6).One histone modification of major consequence is the methylation of histone H3 at lysines 4 and 79, catalyzed by the Set1-containing complex COMPASS 1 and Dot1p, respectively (7)(8)(9)(10)(11)(12)(13)(14). It has been shown that methylation of both lysine residues impacts the expression of genes within the rDNA loci and telomeric regions of DNA in Saccharomyces cerevisiae (7-8, 15, 16). It has been demonstrated that some of the components of the Paf1 complex, a complex that associates with the initiating and elongating RNA polymerase II, is also required for histone H3 methylation on lysines 4 and 79 (17, 18). Accordingly, previous studies have demonstrated a role for the Paf1 complex in transcriptional elongation and initiation (19 -22). A further requirement for the methylation of both lysines 4 and 79 of hi...

The Set1-containing complex COMPASS, which is the yeast homolog of the human MLL complex, is required for mono-, di-, and trimethylation of lysine 4 of histone H3. We have performed a comparative global proteomic screen to better define the role of COMPASS in histone trimethylation. We report that both Cps60 and Cps40 components of COMPASS are required for proper histone H3 trimethylation, but not for proper regulation of telomere-associated gene silencing. Purified COMPASS lacking Cps60 can mono- and dimethylate but is not capable of trimethylating H3(K4). Chromatin immunoprecipitation (ChIP) studies indicate that the loss subunits of COMPASS required for histone trimethylation do not affect the localization of Set1 to chromatin for the genes tested. Collectively, our results suggest a molecular requirement for several components of COMPASS for proper histone H3 trimethylation and regulation of telomere-associated gene expression, indicating multiple roles for different forms of histone methylation by COMPASS.

To date, several classes of enzymes have been shown to affect transcription by catalyzing the modifications of nucleosomes via methylation. Employing our global proteomic screen, GPS, we have determined that the loss of Bur2, a component of the Bur1/Bur2 cyclin-dependent protein kinase, results in a decrease in histone H3(K4) methylation catalyzed by COMPASS. Furthermore, Bur1/Bur2 is required for histone H2B monoubiquitination by Rad6/Bre1. The effect on histone monoubiquitination and methylation is the result of defective Bur1/Bur2-mediated phosphorylation of Rad6 on its serine residue 120 and proper recruitment of the Paf1 complex to chromatin. We have also demonstrated that serine 120 of Rad6 is required for histone H2B monoubiquitination and the regulation of gene expression in vivo. Our results identify in vivo substrates for Bur1/Bur2, thus linking its role to transcriptional elongation and demonstrating a potential activation mechanism for histone H2B monoubiquitination by the Rad6/Bre1 complex.

Low levels of histone covalent modifications are associated with gene silencing at telomeres and other regions in the yeast S. cerevisiae. Although the histone deacetylase Sir2 maintains low acetylation, mechanisms responsible for low H2B ubiquitylation and low H3 methylation are unknown. Here, we show that the ubiquitin protease Ubp10 targets H2B for deubiquitylation, helping to localize Sir2 to the telomere. Ubp10 exhibits reciprocal Sir2-dependent preferential localization proximal to telomeres, where Ubp10 serves to maintain low H2B Lys123 ubiquitylation in this region and, through previously characterized crosstalk, maintains low H3 Lys4 and Lys79 methylation in a slightly broader region. Ubp10 is also localized to the rDNA locus, a second silenced domain, where it similarly maintains low histone methylation. We compare Ubp10 to Ubp8, the SAGA-associated H2B deubiquitylase involved in gene activation, and show that telomeric and gene-silencing functions are specific to Ubp10. Our results suggest that these H2B-deubiquitylating enzymes have distinct genomic functions.

During normal DNA replication, the proliferating cell nuclear antigen (PCNA) enhances the processivity of DNA polymerases at the replication fork. When DNA damage is encountered, PCNA is monoubiquitinated on Lys-164 by the Rad6 -Rad18 complex as the initiating step of translesion synthesis. DNA damage bypass by the translesion synthesis polymerase Rev1 is enhanced by the presence of ubiquitinated PCNA. Here we have carried out a mutational analysis of Rev1, and we have identified the functional domain in the C terminus of Rev1 that mediates interactions with PCNA. We show that a unique motif within this domain binds the ubiquitin moiety of ubiquitinated PCNA. Point mutations within this ubiquitin-binding motif of Rev1 (L821A,P822A,I825A) abolish its functional interaction with ubiquitinated PCNA in vitro and strongly attenuate damageinduced mutagenesis in vivo. Taken together, these studies suggest a specific mechanism by which the interaction between Rev1 and ubiquitinated PCNA is stabilized during the DNA damage response.During normal progression of the cell cycle, replicative DNA polymerases are charged with the task of faithfully replicating host DNA. In eukaryotes, the B-family DNA polymerase ␦ (pol ␦) 3 and DNA polymerase ⑀ synthesize the bulk of newly formed DNA (reviewed in Ref. 1). Both DNA polymerases possess a highly restrictive active site to promote proper Watson-Crick base pairing between the template strand and incoming bases (2). However, the restrictive nature of these enzymes also makes it inherently difficult for them to deal with DNA damage in the template DNA, and in general, the presence of DNA damage blocks the progression of the replication fork. This stalling activates one of several post-replication repair mechanisms that are designed to bypass damage in either an error-free or error-prone manner (reviewed in Refs. 3-5). These pathways are initiated by monoubiquitination of the proliferating cell nuclear antigen (PCNA) on Lys-164 by Rad6 -Rad18.During normal replication, PCNA serves as a processivity factor for the replicative DNA polymerases and coordinates the functions of enzymes on the lagging strand that are involved in the maturation of Okazaki fragments (reviewed in Refs. 6, 7). In order to carry out its DNA-associated functions, PCNA is loaded by the clamp loader replication factor C (RFC) in an ATP-dependent reaction (reviewed in Ref. TLS in yeast consists of two branches, both of which require mono-ubiquitinated PCNA (PCNA Ubi ) for function. Bypass of UV damage, particularly cis-syn pyrimidine dimers, is mediated by pol , the xeroderma pigmentosum variant DNA polymerase (13,14). In mammalian cells, pol specifically interacts with PCNA Ubi at sites of damage (15, 16). Although unmodified PCNA has been shown to serve as a cofactor for damage bypass by pol , ubiquitination of PCNA increases the rate of bypass in vitro (17,18).TLS of most forms of DNA damage involves the participation of three DNA polymerases, pol ␦, pol , and Rev1, and may require additional activation by the Cd...