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Small Region of Rtf1 Protein Can Substitute for Complete Paf1 Complex in Facilitating Global Histone H2B Ubiquitylation in Yeast

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Small Region of Rtf1 Protein Can Substitute for Complete Paf1 Complex in Facilitating Global Histone H2B Ubiquitylation in Yeast Small region of Rtf1 protein can substitute for complete Paf1 complex in facilitating global histone H2B ubiquitylation in yeast Anthony S. Piro1, Manasi K. Mayekar1, Marcie H. Warner, Christopher P. Davis, and Karen M. Arndt2 Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260 Edited* by Robert G. Roeder, The Rockefeller University, New York, NY, and approved May 14, 2012 (received for review October 14, 2011) Histone modifications regulate transcription by RNA polymerase II acetyltransferase and deacetylase complexes, thereby governing and maintain a balance between active and repressed chromatin histone acetylation patterns on genes (17–20). states. The conserved Paf1 complex (Paf1C) promotes specific From its position at the top of a histone modification cascade histone modifications during transcription elongation, but the that determines the methylation and acetylation state of active mechanisms by which it facilitates these marks are undefined. We chromatin, H2B monoubiquitylation and the factors that estab- previously identified a 90-amino acid region within the Rtf1 lish this mark are key regulators of gene expression. In addition to subunit of Paf1C that is necessary for Paf1C-dependent histone Rad6 and Bre1, the conserved Paf1 complex (Paf1C) is required modifications in Saccharomyces cerevisiae. Here we show that this for H2B K123 ubiquitylation. Paf1C—which in budding yeast histone modification domain (HMD), when expressed as the only consists of the subunits Paf1, Ctr9, Cdc73, Rtf1, and Leo1— source of Rtf1, can promote H3 K4 and K79 methylation and H2B impacts RNA synthesis at multiple stages (21). Paf1C associates K123 ubiquitylation in yeast. The HMD can restore histone mod- with RNA pol II from the 5′ end of a gene to the poly(A) site (22, ifications in rtf1Δ cells whether or not it is directed to DNA by 23); interacts functionally and physically with the transcription a fusion to a DNA binding domain. The HMD can facilitate histone elongation factors Spt4–Spt5/DSIF, Spt16–Pob3/FACT, and modifications independently of other Paf1C subunits and does not TFIIS (8, 24–27); regulates the phosphorylation state of RNA bypass the requirement for Rad6–Bre1. The isolated HMD localizes pol II (28, 29); and is required for proper 3′ end formation of to chromatin, and this interaction requires residues important for certain transcripts (30–32). Important to this study, deletion of histone modification. When expressed outside the context of full- RTF1 from yeast cells causes dramatic reductions in H2B K123 length Rtf1, the HMD associates with and causes Paf1C-dependent ubiquitylation and H3 K4 and K79 methylation (33–36), and this histone modifications to appear at transcriptionally inactive loci, role in histone modification, like other Paf1C functions, is con- suggesting that its function has become deregulated. Finally, the served in higher eukaryotes (37–40). Rtf1 HMDs from other species can function in yeast. Our findings The mechanisms by which Paf1C promotes histone mod- suggest a direct and conserved role for Paf1C in coupling histone ifications have yet to be elucidated. Deletion of RTF1, CTR9,or modifications to transcription elongation. PAF1 reduces the occupancies of Rad6 and Set1/COMPASS on coding regions (33, 35, 41), and purified Paf1C interacts with transcription-coupled histone modifications | nucleosome Bre1 in vitro (42, 43), suggesting that Paf1C serves as a platform for recruiting histone-modifying enzymes to RNA pol II during n eukaryotes, transcription occurs within the context of a re- elongation. Whether Paf1C has a more direct role in promoting Istrictive, yet dynamic, chromatin environment. The posttrans- fi fi histone modi cations is not known. We previously reported that lational modi cation of histones represents a major mechanism deletions and amino acid substitutions within a small region of by which cells control the structure of chromatin. Some mod- the S. cerevisiae Rtf1 protein dramatically reduce global levels of fi i cations of histones include acetylation, methylation, and ubiq- H3 K4 and K79 dimethylation and trimethylation and H2B K123 fi uitylation. These modi cations can alter the structural properties ubiquitylation, leading us to define residues 62–152 as the histone of nucleosomes and serve as specific effectors for the recruitment modification domain (HMD) of Rtf1 (32, 44). Here we show that of proteins that further modify the chromatin template and reg- the Rtf1 HMD interacts with chromatin and is sufficient to pro- ulate transcription (1). mote H2B K123 ubiquitylation and H3 K4 and K79 methylation. Monoubiquitylation of histone H2B on lysine (K) 123 in Our findings suggest that Paf1C plays an active role in promoting Saccharomyces cerevisiae is a conserved modification that is conserved, transcription-coupled histone modifications. enriched on active genes but plays roles in both transcriptional repression and activation (2–4). Consistent with a repressive Results role, H2B monoubiquitylation stabilizes nucleosomes at yeast Rtf1 HMD Is Sufficient to Promote Paf1C-Dependent Histone Modi- promoters (5), inhibits the association of the RNA polymerase fications. To investigate the role of Paf1C in histone modification, fi (pol) II kinase Ctk1 with genes in yeast (6), and interferes with we asked whether the S. cerevisiae Rtf1 HMD is suf cient to fi the recruitment of the elongation factor TFIIS to genes in hu- promote histone modi cations in the absence of all other parts man cells (7). In other studies, H2B monoubiquitylation has of the Rtf1 protein. Because the HMD is genetically separable been shown to stimulate transcription of chromatin templates (8), promote nucleosome reassembly during transcription elon- BIOCHEMISTRY Author contributions: A.S.P., M.K.M., M.H.W., C.P.D., and K.M.A. designed research; A.S.P., gation (9), and inhibit chromatin compaction (10). H2B mono- M.K.M., M.H.W., and C.P.D. performed research; A.S.P., M.K.M., M.H.W., C.P.D., and K.M.A. ubiquitylation is also a prerequisite for other histone modifications analyzed data; and A.S.P., M.K.M., M.H.W., and K.M.A. wrote the paper. that mark active genes. Ubiquitylation of H2B K123 by the Rad6– The authors declare no conflict of interest. Bre1 ubiquitin conjugase–ligase proteins in yeast (11–13) is re- *This Direct Submission article had a prearranged editor. quired for dimethylation and trimethylation of H3 K4 and K79 1A.S.P. and M.K.M. contributed equally to this work. by the Set1/COMPASS and Dot1 methyltransferases, respectively 2To whom correspondence should be addressed. E-mail: [email protected]. – (14 16). Histone H3 K4 dimethylation and trimethylation This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. subsequently stimulate the recruitment and activity of histone 1073/pnas.1116994109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1116994109 PNAS | July 3, 2012 | vol. 109 | no. 27 | 10837–10842 Downloaded by guest on September 25, 2021 from regions of Rtf1 required for association with actively A Myc signal at GAL7 UAS B H3 K4 Me3 at GAL7 UAS transcribing RNA pol II and other members of Paf1C (44), we 35 3.5 decided to direct the HMD to chromatin to test its activity. 30 3 Therefore, we constructed a 2-micron–based plasmid that 25 2.5 expresses a fusion of an 89-amino acid HMD (Rtf1 residues 63– 20 2 152) to the Gal4 DNA binding domain (GBD) and a c-Myc 15 1.5 10 1 epitope tag under the control of the ADH1 promoter. We GAL7/TEL-VIR transformed this plasmid into an rtf1Δ gal4Δ strain and analyzed 5 H3 K4 Me3/ 0.5 recruitment of the fusion protein and restoration of Rtf1-de- 0 0 fi pendent histone modi cations at the GAL7 promoter under GBD GBD repressing conditions by chromatin immunoprecipitation (ChIP). GBD-Rtf1 GBD-Myc GBD-Rtf1 Myc-HMD GBD- GBD-Myc Using an antibody to the Myc tag, we detected enrichment of the GBD- Myc-HMD GBD–Myc–HMD protein at the GAL7 upstream activation se- quence (UAS) (Fig. 1A). The increased level of occupancy by the C GAL7 UAS D PYK1 GBD–Myc–HMD protein relative to the similarly expressed H3 K79 Me2 at H3 K4 Me3 at 3 GBD–Myc control protein (Fig. S1A) indicates a potential role 8 2.5 7 for the HMD in facilitating chromatin association, a point dis- 6 2 cussed below. Interestingly, ChIP analysis revealed a modest but 5 reproducible recovery of H3 K4 trimethylation and H3 K79 1.5 4 dimethylation at the GAL7 UAS in strains expressing the GBD– 1 3 H3 K79 Me2/ H3 K4 Me3/ 2 – 0.5 Myc HMD (Fig. 1 B and C). Total H3 levels at the GAL7 UAS 1 were similar in all strains (Fig. S1B). As judged by a comparison 0 0 with a telomere-proximal region, RNA pol II levels across the GBD GAL7 UAS were very low in all strains (Fig. S1C). These results GBD GBD-Rtf1 fi GBD-Rtf1 GBD-Myc GBD-Myc Myc-HMD indicate that the Rtf1 HMD is suf cienttodirecthistonemod- GBD- Myc-HMD GBD- ifications when recruited to DNA and that it may do so in- dependently of active transcription. E F To test whether the activity of the GBD–Myc–HMD required directed recruitment to DNA, we analyzed histone modification H3 K79 Me2 at PYK1 4 levels at the 5′ end of an active gene, PYK1, which lacks known GBD-Myc-HMDGBD-MycGBD-Myc-Rtf1 – – 3.5 Gal4 binding sites (45). Surprisingly, the GBD Myc HMD re- 3 H3 K4 Me3 stored H3 K4 trimethylation and H3 K79 dimethylation at the 2.5 PYK1 5′ region in an rtf1Δ strain (Fig. 1 D and E). Expression of 2 H3 K4 Me2 this plasmid-encoded HMD did not influence H3 or RNA pol II 1.5 1 H3 K79 Me2 levels at PYK1 compared with cells transformed with control H3 K79 Me2/ 0.5 vectors (Fig.
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