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W2027349141 abstract "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. Here, we show that 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), this association is largely independent of Rtf1. We suggest that Rtf1 affects genome-wide ubiquitination of H2B by a mechanism that is distinct from its function as a transcriptional elongation factor. 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. Here, we show that 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), this association is largely independent of Rtf1. We suggest that Rtf1 affects genome-wide ubiquitination of H2B by a mechanism that is distinct from its function as a transcriptional elongation factor. In a variety of eukaryotic organisms, histone H3 is methylated at lysines 4, 36, and 79 (respectively H3-Lys4, H3-Lys36, H3-Lys79). In the budding yeast Saccharomyces cerevisiae, H3-Lys4 is methylated by Set1 (1Briggs S.D. Bryk M. Strahl B.D. Cheung W.L. Davie J.K. Dent S.Y. Winston F. Allis C.D. Genes Dev. 2001; 15: 3286-3295Crossref PubMed Scopus (477) Google Scholar, 2Roguev A. Schaft D. Shevchenko A. Pijnappel W.W. Wilm M. Aasland R. Stewart A.F. EMBO J. 2001; 20: 7137-7148Crossref PubMed Scopus (458) Google Scholar, 3Krogan N. Dover J. Khorrami S. Greenblatt J.F. Schneider J. Johnston M. Shilatifard A. J. Biol. Chem. 2002; 277: 10753-10755Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 4Nagy P.L. Griesenbeck J. Kornberg R.D. Cleary M.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 90-94Crossref PubMed Scopus (265) Google Scholar), H3-Lys36 is methylated by Set2 (5Strahl B.D. Grant P.A. Briggs S.D. Sun Z.W. Bone J.R. Caldwell J.A. Mollah S. Cook R.G. Shabanowitz J. Hunt D.F. Allis C.D. Mol. Cell. Biol. 2002; 22: 1298-1306Crossref PubMed Scopus (431) Google Scholar), and H3-Lys79 is methylated by Dot1 (6van Leeuwen F. Gafken P.R. Gottschling D.E. Cell. 2002; 109: 745-756Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 7Ng H.H. Feng Q. Wang H. Erdjument-Bromage H. Tempst P. Zhang Y. Struhl K. Genes Dev. 2002; 16: 1518-1527Crossref PubMed Scopus (424) Google Scholar, 8Lacoste N. Utley R.T. Hunter J. Poirier G.G. Cote J. J. Biol. Chem. 2002; 277: 30421-30424Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Unexpectedly, genome-wide methylation of H3-Lys4 and H3-Lys79 depends on Rad6, an enzyme that mono-ubiquitinates lysine 123 of histone H2B (9Sun Z.W. Allis C.D. Nature. 2002; 418: 104-108Crossref PubMed Scopus (830) Google Scholar, 10Dover J. Schneider J. Tawiah-Boateng M.A. Wood A. Dean K. Johnston M. Shilatifard A. J. Biol. Chem. 2002; 277: 28368-28371Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 11Ng H.H. Xu R.M. Zhang Y. Struhl K. J. Biol. Chem. 2002; 277: 34655-34657Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 12Briggs S.D. Xiao T. Sun Z.-W. Caldwell J.A. Shabanowitz J. Hunt D.F. Allis C.D. Strahl B.D. Nature. 2002; 418: 498Crossref PubMed Scopus (393) Google Scholar). H3-Lys4 and H3-Lys79 methylation also depends on Bre1, an E3 ubiquitin ligase that is required for substrate selection of Rad6 (13Wood A. Krogan N.J. Dover J. Schneider J. Heidt J. Boateng M.A. Dean K. Golshani A. Zhang Y. Greenblatt J.F. Johnston M. Shilatifard A. Mol. Cell. 2003; 11: 267-274Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 14Hwang W.W. Venkatasubrahmanyam S. Ianculescu A.G. Tong A. Boone C. Madhani H.D. Mol. Cell. 2003; 11: 261-266Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). The relationship between ubiquitination of H2B-Lys123 and methylation of H3 at lysines 4 and 79 is unidirectional, as the loss of H3 methylation does not influence H2B ubiquitination. Set1 and Dot1 are important for heterochromatic silencing (1Briggs S.D. Bryk M. Strahl B.D. Cheung W.L. Davie J.K. Dent S.Y. Winston F. Allis C.D. Genes Dev. 2001; 15: 3286-3295Crossref PubMed Scopus (477) Google Scholar, 3Krogan N. Dover J. Khorrami S. Greenblatt J.F. Schneider J. Johnston M. Shilatifard A. J. Biol. Chem. 2002; 277: 10753-10755Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 6van Leeuwen F. Gafken P.R. Gottschling D.E. Cell. 2002; 109: 745-756Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 7Ng H.H. Feng Q. Wang H. Erdjument-Bromage H. Tempst P. Zhang Y. Struhl K. Genes Dev. 2002; 16: 1518-1527Crossref PubMed Scopus (424) Google Scholar, 15Bryk M. Briggs S.D. Strahl B.D. Curcio M.J. Allis C.D. Winston F. Curr. Biol. 2002; 12: 165-170Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 16Nislow C. Ray E. Pillus L. Mol. Biol. Cell. 1997; 8: 2421-2436Crossref PubMed Scopus (199) Google Scholar), whereas Set2 does not appear to play a role in this process. Dot1-mediated methylation and H3-Lys79 itself are important for association of Sir silencing proteins at telomeres, with the effects being more dramatic at telomere-distal regions than at telomere-proximal regions (6van Leeuwen F. Gafken P.R. Gottschling D.E. Cell. 2002; 109: 745-756Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 7Ng H.H. Feng Q. Wang H. Erdjument-Bromage H. Tempst P. Zhang Y. Struhl K. Genes Dev. 2002; 16: 1518-1527Crossref PubMed Scopus (424) Google Scholar). It is likely, but not yet demonstrated, that Set1-mediated methylation of H3-Lys4 affects Sir protein association. Interestingly, methylation of H3-Lys4 (15Bryk M. Briggs S.D. Strahl B.D. Curcio M.J. Allis C.D. Winston F. Curr. Biol. 2002; 12: 165-170Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 17Bernstein B.E. Humphrey E.L. Erlich R.L. Schneider R. Bouman P. Liu J.S. Kouzarides T. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8695-8700Crossref PubMed Scopus (596) Google Scholar) and H3-Lys79 (18Ng H.H. Ciccone D.N. Morshead K.B. Oettinger M.A. Struhl K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1820-1825Crossref PubMed Scopus (250) Google Scholar) is extremely low at heterochromatic loci, and H3-Lys79 methylation occurs at high levels in bulk chromatin (6van Leeuwen F. Gafken P.R. Gottschling D.E. Cell. 2002; 109: 745-756Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar). These observations suggest that Sir proteins preferentially associate with nucleosomes that are unmethylated at H3-Lys79 (6van Leeuwen F. Gafken P.R. Gottschling D.E. Cell. 2002; 109: 745-756Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 18Ng H.H. Ciccone D.N. Morshead K.B. Oettinger M.A. Struhl K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1820-1825Crossref PubMed Scopus (250) Google Scholar) and that Sir proteins block the ability of Dot1 to methylate H3-Lys79 (18Ng H.H. Ciccone D.N. Morshead K.B. Oettinger M.A. Struhl K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1820-1825Crossref PubMed Scopus (250) Google Scholar). In addition to its role in heterochromatin silencing, Set1 has an important role in transcriptional elongation. Set1 mediates both di- and trimethylation of H3-Lys4, and trimethylation correlates with transcriptional activity (19Santos-Rosa H. Schneider R. Bannister A.J. Sherriff J. Bernstein B.E. Emre N.C. Schreiber S.L. Mellor J. Kouzarides T. Nature. 2002; 419: 407-411Crossref PubMed Scopus (1603) Google Scholar). Set1 is specifically recruited by elongating RNA polymerase II (pol II) 1The abbreviation used is: pol II, polymerase II. to a highly localized domain at the 5′ region of actively transcribed genes, thereby generating a highly localized domain of trimethylated H3-Lys4 (20Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar). Recruitment of Set1 depends on some, but not all, components of the Paf1 complex (20Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar, 21Krogan N.J. Dover J. Wood A. Schneider J. Heidt J. Boateng M.A. Dean K. Ryan O.W. Golshani A. Johnston M. Greenblatt J.F. Shilatifard A. Mol. Cell. 2003; 11: 721-729Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). The Paf1 complex associates with elongating Pol II over the entire mRNA coding region and has a role in transcriptional elongation (22Costa P.J. Arndt K.M. Genetics. 2000; 156: 535-547Crossref PubMed Google Scholar, 23Mueller C.L. Jaehning J.A. Mol. Cell. Biol. 2002; 22: 1971-1980Crossref PubMed Scopus (179) Google Scholar, 24Pokholok D.K. Hannett N.M. Young R.A. Mol. Cell. 2002; 9: 799-809Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 25Krogan N.J. Kim M. Ahn S.H. Zhong G. Kobor M.S. Cagney G. Emili A. Shilatifard A. Buratowski S. Greenblatt J.F. Mol. Cell. Biol. 2002; 22: 6979-6992Crossref PubMed Scopus (409) Google Scholar, 26Squazzo S.L. Costa P.J. Lindstrom D.L. Kumer K.E. Simic R. Jennings J.L. Link A.J. Arndt K.M. Hartzog G.A. EMBO J. 2002; 21: 1764-1774Crossref PubMed Scopus (247) Google Scholar). The Paf1 complex is also required for recruitment of Set2 and elevated levels of H3-Lys36 methylation within mRNA coding regions of transcriptionally active genes (27Krogan N.J. Kim M. Tong A. Golshani A. Cagney G. Canadien V. Richards D.P. Beattie B.K. Emili A. Boone C. Shilatifard A. Buratowski S. Greenblatt J.F. Mol. Cell. Biol. 2003; 23: 4207-4218Crossref PubMed Scopus (516) Google Scholar). Here, we show that the Rtf1 component of the Paf1 complex is required for global dimethylation of H3-Lys4 and H3-Lys79,a result obtained independently (21Krogan N.J. Dover J. Wood A. Schneider J. Heidt J. Boateng M.A. Dean K. Ryan O.W. Golshani A. Johnston M. Greenblatt J.F. Shilatifard A. Mol. Cell. 2003; 11: 721-729Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). Furthermore, we show that Rtf1 is required for ubiquitination of bulk H2B, strongly suggesting that Rtf1 affects H3-Lys4 and H3-Lys79 methylation through its effect on H2B ubiquitination. Rtf1 is required for silencing, and H3-Lys4 and H3-Lys79 act synergistically for stability of Sir2 at the telomeres. Dot1 is also recruited to transcriptionally active mRNA coding regions, but unlike Set1 and Set2, this recruitment is largely independent of Rtf1. We suggest that Rtf1 affects genome-wide ubiquitination of H2B by a mechanism that is distinct from its function as a transcriptional elongation factor. Yeast Strains and Plasmids—Most experiments used strain UCC1111 (28Kelly T.J. Qin S. Gottschling D.E. Parthun M.R. Mol. Cell. Biol. 2000; 20: 7051-7058Crossref PubMed Scopus (114) Google Scholar), and derivatives containing rtf1::KanR, dot1::KanR and set1::HIS5 deletion alleles that were generated by PCR-based gene replacement (29Longtine M.S. McKenzie A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4172) Google Scholar). FLAG-H2B, which contains the FLAG epitope immediately upstream of the ATG initiation codon of H2B, was expressed from the H2B promoter in the context of plasmid FB1251 (11Ng H.H. Xu R.M. Zhang Y. Struhl K. J. Biol. Chem. 2002; 277: 34655-34657Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). For analysis of Dot1 occupancy, we generated a derivative of ZGY005 (30Zhang Z. Shibahara K. Stillman B. Nature. 2000; 408: 221-225Crossref PubMed Scopus (387) Google Scholar) containing a version of Dot1 with 9 copies of the Myc epitope at the C terminus by PCR-based gene replacement (31Cosma M.P. Tanaka T. Nasmyth K. Cell. 1999; 97: 299-311Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar) along with an isogenic strain containing the rtf1::KanR allele. Western Blotting—For analyzing histone ubiquitination, exponentially growing cells were harvested by centrifugation and boiled immediately in sample buffer (50 mm Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.1 m dithiothreitol, 0.001% bromphenol blue, 2 mm EDTA, supplemented with Complete protease inhibitors) for 10 min with occasional vortexing, and the resulting lysates were clarified by centrifugation. For analyzing other histone modifications, cell-free extracts were prepared as described previously (7Ng H.H. Feng Q. Wang H. Erdjument-Bromage H. Tempst P. Zhang Y. Struhl K. Genes Dev. 2002; 16: 1518-1527Crossref PubMed Scopus (424) Google Scholar). The resulting proteins were electrophoretically separated on a 14% denaturing polyacrylamide gel and analyzed by Western blotting using antibodies against the following peptides: FLAG epitope (M2, Eastman Kodak Co.); dimethylated H3-lysine 4; dimethylated H3-lysine 36; di-acetylated H3-lysines 9 and 14; tetra-acetylated H4-lysines 5, 8, 12, and 16 (all obtained from Upstate Biotechnology); dimethylated H3-lysine 79 (7Ng H.H. Feng Q. Wang H. Erdjument-Bromage H. Tempst P. Zhang Y. Struhl K. Genes Dev. 2002; 16: 1518-1527Crossref PubMed Scopus (424) Google Scholar, 32Feng Q. Wang H.H.H.N. Erdjument-Bromage H. Tempst P. Struhl K. Zhang Y. Curr. Biol. 2002; 12: 1052-1058Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar). Transcriptional Analysis—RPL2B, BRE1, and RAD6 mRNA levels were determined with respect to GLT1 mRNA levels by reverse transcription-PCR in real time (11Ng H.H. Xu R.M. Zhang Y. Struhl K. J. Biol. Chem. 2002; 277: 34655-34657Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 33Proft M. Struhl K. Mol. Cell. 2002; 9: 1307-1317Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Chromatin Immunoprecipitation—Formaldehyde-cross-linked chromatin was immunoprecipitated with affinity-purified anti-Sir2 antibody (kindly provided by Danesh Moazed) or with a monoclonal antibody against the myc epitope (9e10, Upstate Biotechnology) and the resulting input and immunoprecipitated samples analyzed by quantitative PCR in real time (18Ng H.H. Ciccone D.N. Morshead K.B. Oettinger M.A. Struhl K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1820-1825Crossref PubMed Scopus (250) Google Scholar). Sir2 and Dot1 occupancy values are presented in arbitrary units that are directly related to the apparent immunoprecipitation efficiency (i. e. the amount of material immunoprecipitated relative to that of the input sample), with a value of 1 being the experimental background. Rtf1 Is Important for Genome-wide Methylation of H3-Lys4 and H3-Lys79, but Not H3-Lys36—Previously, we showed that Set1 recruitment to mRNA coding regions in rtf1 and paf1 deletion strains is ∼15–30% of the wild-type level, even though bulk H3-Lys4 trimethylation is abolished (20Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar). This apparent discrepancy suggested the possibility that the Paf1 complex has a more general effect on methylation of histone H3. Western blot analysis on bulk histones shows that loss of Rtf1 abolishes dimethylated H3-Lys4 and H3-Lys79, whereas it has no significant effect on H3-Lys36 methylation or acetylation of H3 and H4 (Fig. 1). After this work was completed, similar observations were reported for the Rtf1, Paf1, and Ctr9 subunits of the Paf1 complex (21Krogan N.J. Dover J. Wood A. Schneider J. Heidt J. Boateng M.A. Dean K. Ryan O.W. Golshani A. Johnston M. Greenblatt J.F. Shilatifard A. Mol. Cell. 2003; 11: 721-729Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). Interestingly, levels of trimethylated H3-Lys4, dimethylated H3-Lys4, and dimethylated H3-Lys79 are unaffected in strains lacking the Leo1 and Cdc73 components of the Paf1 complex (20Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar, 21Krogan N.J. Dover J. Wood A. Schneider J. Heidt J. Boateng M.A. Dean K. Ryan O.W. Golshani A. Johnston M. Greenblatt J.F. Shilatifard A. Mol. Cell. 2003; 11: 721-729Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). Rtf1 Is Required for H2B Ubiquitination—Ubiquitination of H2B-Lys123 is required for genome-wide methylation of H3-Lys4 and H3-Lys79 (9Sun Z.W. Allis C.D. Nature. 2002; 418: 104-108Crossref PubMed Scopus (830) Google Scholar, 10Dover J. Schneider J. Tawiah-Boateng M.A. Wood A. Dean K. Johnston M. Shilatifard A. J. Biol. Chem. 2002; 277: 28368-28371Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 11Ng H.H. Xu R.M. Zhang Y. Struhl K. J. Biol. Chem. 2002; 277: 34655-34657Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 12Briggs S.D. Xiao T. Sun Z.-W. Caldwell J.A. Shabanowitz J. Hunt D.F. Allis C.D. Strahl B.D. Nature. 2002; 418: 498Crossref PubMed Scopus (393) Google Scholar, 13Wood A. Krogan N.J. Dover J. Schneider J. Heidt J. Boateng M.A. Dean K. Golshani A. Zhang Y. Greenblatt J.F. Johnston M. Shilatifard A. Mol. Cell. 2003; 11: 267-274Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 14Hwang W.W. Venkatasubrahmanyam S. Ianculescu A.G. Tong A. Boone C. Madhani H.D. Mol. Cell. 2003; 11: 261-266Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). This suggested the possibility that Rtf1 might affect ubiquitination of H2B-Lys123, which in turn would account for the failure to observe methylation of H3-Lys4 and H3-Lys79. Western blot analysis of wild-type and rtf1 deletion strains containing FLAG-tagged H2B indicates that the ubiquitinated, slow migrating form of H2B is absent in the rtf1 mutant strain (Fig. 2A). The levels of RAD6 and BRE1 mRNA are not significantly altered in the rtf1 deletion strain (Fig. 2B), excluding the possibility that Rtf1 indirectly affects the expression of the enzymes that mediate H2B ubiquitination. Thus, Rtf1 is required for H2B ubiquitination. Rtf1 Is Important for Telomeric Silencing—Methylation of H3-Lys4, methylation of H3-Lys79, and ubiquitination of H2BLys123 are important for telomeric silencing, because strains individually lacking Dot1 (6van Leeuwen F. Gafken P.R. Gottschling D.E. Cell. 2002; 109: 745-756Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 7Ng H.H. Feng Q. Wang H. Erdjument-Bromage H. Tempst P. Zhang Y. Struhl K. Genes Dev. 2002; 16: 1518-1527Crossref PubMed Scopus (424) Google Scholar), Set1 (1Briggs S.D. Bryk M. Strahl B.D. Cheung W.L. Davie J.K. Dent S.Y. Winston F. Allis C.D. Genes Dev. 2001; 15: 3286-3295Crossref PubMed Scopus (477) Google Scholar, 3Krogan N. Dover J. Khorrami S. Greenblatt J.F. Schneider J. Johnston M. Shilatifard A. J. Biol. Chem. 2002; 277: 10753-10755Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 15Bryk M. Briggs S.D. Strahl B.D. Curcio M.J. Allis C.D. Winston F. Curr. Biol. 2002; 12: 165-170Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 16Nislow C. Ray E. Pillus L. Mol. Biol. Cell. 1997; 8: 2421-2436Crossref PubMed Scopus (199) Google Scholar), Rad6 (9Sun Z.W. Allis C.D. Nature. 2002; 418: 104-108Crossref PubMed Scopus (830) Google Scholar, 34Huang H. Kahana A. Gottschling D.E. Prakash L. Liebman S.W. Mol. Cell. Biol. 1997; 17: 6693-6699Crossref PubMed Scopus (112) Google Scholar), and Bre1 (13Wood A. Krogan N.J. Dover J. Schneider J. Heidt J. Boateng M.A. Dean K. Golshani A. Zhang Y. Greenblatt J.F. Johnston M. Shilatifard A. Mol. Cell. 2003; 11: 267-274Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar) are defective in telomeric silencing. As Rtf1 is essential for H2B ubiquitination, and hence for methylation of H3-Lys4 and H3-Lys79, it should be important for telomeric silencing. Indeed, in a standard telomeric silencing assay in which the URA3 gene is located within the telomeric region (35Gottschling D.E. Aparicio O.M. Billington B.L. Zakian V.A. Cell. 1990; 63: 751-762Abstract Full Text PDF PubMed Scopus (1134) Google Scholar), rtf1 mutant strains show decreased growth in the presence of 5-fluoro-orotic acid (Fig. 3), indicating that loss of Rtf1 impairs telomeric silencing. Similar results were reported after the work described here was completed (21Krogan N.J. Dover J. Wood A. Schneider J. Heidt J. Boateng M.A. Dean K. Ryan O.W. Golshani A. Johnston M. Greenblatt J.F. Shilatifard A. Mol. Cell. 2003; 11: 721-729Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). H3-Lys4 and H3-Lys79 Methylation Synergistically Affect Sir Protein Association at Telomeres—Dot1-mediated methylation and H3-Lys79 itself are important for association of Sir silencing proteins at telomeric regions (6van Leeuwen F. Gafken P.R. Gottschling D.E. Cell. 2002; 109: 745-756Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 7Ng H.H. Feng Q. Wang H. Erdjument-Bromage H. Tempst P. Zhang Y. Struhl K. Genes Dev. 2002; 16: 1518-1527Crossref PubMed Scopus (424) Google Scholar). However, the reduction in Sir protein association in dot1 mutant strains is marginal at the telomere itself and becomes more pronounced at a region 3.5 kb from the telomere (7Ng H.H. Feng Q. Wang H. Erdjument-Bromage H. Tempst P. Zhang Y. Struhl K. Genes Dev. 2002; 16: 1518-1527Crossref PubMed Scopus (424) Google Scholar). This distinction is likely to reflect the fact that Sir proteins are directly recruited to the telomere by Rap1 (and associated proteins), whereas Sir protein association at telomere-distal regions is mediated primarily through interactions with histones (36Grunstein M. Cell. 1998; 93: 325-328Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). As with H3-Lys79, loss of Set1-mediated H3-Lys4 methylation results in a very marginal reduction in Sir2 occupancy at the telomeres. In contrast, Sir2 association at the telomere is reduced 3-fold in the set1, dot1 double mutant strain (Fig. 4), indicating that H3-Lys4 and H3-Lys79 methylation synergistically affects the association of silencing proteins at heterochromatic loci. A similar 3-fold reduction in Sir2 occupancy at the telomere is observed in the rtf1 mutant strain, suggesting that the Paf1 complex and H2B ubiquitination indirectly affect Sir protein association by virtue of their effects on H3 methylation. Several observations suggest that Sir proteins preferentially associate with nucleosomes that are unmethylated at H3-Lys79 (6van Leeuwen F. Gafken P.R. Gottschling D.E. Cell. 2002; 109: 745-756Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 18Ng H.H. Ciccone D.N. Morshead K.B. Oettinger M.A. Struhl K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1820-1825Crossref PubMed Scopus (250) Google Scholar). The synergistic effects of dot1 and set1 mutations suggest that Sir proteins preferentially associate with nucleosomes in which H3 is unmethylated at both Lys4 and Lys79. Our results provide additional evidence for the similar roles of H3-Lys4 and H3-Lys79 in heterochromatic silencing, and they suggest that reciprocal positive feedback loops such as proposed for H3-Lys79 (18Ng H.H. Ciccone D.N. Morshead K.B. Oettinger M.A. Struhl K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1820-1825Crossref PubMed Scopus (250) Google Scholar) might also apply to Set1-mediated methylation of H3-Lys4. By simultaneously affecting the H3-Lys4 and H3-Lys79 feedback loops, Rtf1 and Rad6-mediated ubiquitination of H2B would mutually reinforce the stability of the silenced state and significantly contribute to position-effect variegation. Dot1 Associates with Transcriptionally Active Genes in an Rtf1-independent Manner—In vivo, Set1 and Set2 histone methylases associate with transcriptionally active genes in an Rtf1-dependent manner (20Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar, 21Krogan N.J. Dover J. Wood A. Schneider J. Heidt J. Boateng M.A. Dean K. Ryan O.W. Golshani A. Johnston M. Greenblatt J.F. Shilatifard A. Mol. Cell. 2003; 11: 721-729Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, 27Krogan N.J. Kim M. Tong A. Golshani A. Cagney G. Canadien V. Richards D.P. Beattie B.K. Emili A. Boone C. Shilatifard A. Buratowski S. Greenblatt J.F. Mol. Cell. Biol. 2003; 23: 4207-4218Crossref PubMed Scopus (516) Google Scholar, 37Xiao T. Hall H. Kizer K.O. Shibata Y. Hall M.C. Borchers C.H. Strahl B.D. Genes Dev. 2003; 17: 654-663Crossref PubMed Scopus (325) Google Scholar). As Rtf1 is required for methylation of H3-Lys79, we examined whether Dot1 also associates with active genes in vivo (Fig. 5). Dot1 associates with the RPL2B, PYK1, PGK1, and GAL10 coding regions, but not with the corresponding promoters. In addition, Dot1 association with the GAL10 coding region occurs when the gene is transcriptionally active (galactose medium), but not when it is quiescent (raffinose medium). In all these cases, Dot1 association is quantitatively modest (2–3-fold above the background), but clearly significant. By analogy with Set1 and Set2 histone methylases, the association of Dot1 with transcriptionally active genes strongly suggests that Dot1 interacts with the elongating Pol II machinery. However, Rtf1 is largely dispensable for Dot1 recruitment to active genes, although the level of Dot1 association in an rtf1 deletion strain might be slightly reduced in comparison with the wild-type strain (Fig. 5). Thus, even though Rtf1 is extremely important for global H3-Lys79 methylation, it plays a minor role in recruitment of Dot1 to active coding regions. How Does Rtf1 Affect Global Ubiquitination of H2B and Methylation of H3-Lys4 and H3-Lys79?—It is likely that genome-wide H3-Lys4 and H3-Lys79 methylation is not directly affected by Rtf1, but rather is a consequence of global H2BLys123 ubiquitination. Unlike H3-Lys4 trimethylation, H3-Lys4 dimethylation is relatively constant over the entire genome (except for heterochromatic regions) in a manner that is unrelated to transcriptional activity and to Rtf1-dependent recruitment of Set1 to mRNA coding regions (20Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar). Furthermore, loss of Rad6, and hence H2B ubiquitination, abolishes H3-Lys4 methylation at active mRNA coding regions even though it has no effect on the recruitment of Set1 histone methylase (20Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar). Global H3-Lys79 methylation also occurs at a relatively constant level over euchromatic regions of the genome (18Ng H.H. Ciccone D.N. Morshead K.B. Oettinger M.A. Struhl K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1820-1825Crossref PubMed Scopus (250) Google Scholar), and the physical proximity of H3-Lys79 and H2B-Lys123 within the nucleosome suggests that Dot1 activity in vivo might depend on ubiquitinated H2B (11Ng H.H. Xu R.M. Zhang Y. Struhl K. J. Biol. Chem. 2002; 277: 34655-34657Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Human Dot1L can methylate native, but not recombinant nucleosomes (11Ng H.H. Xu R.M. Zhang Y. Struhl K. J. Biol. Chem. 2002; 277: 34655-34657Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar), and the catalytic domain may interact with ubiquitinated H2B (38Min J. Feng Q. Li Z. Zhang Y. Xu R.M. Cell. 2003; 112: 711-723Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Our results suggest that Rtf1 has distinct global and elongation-related functions. First, although we have not directly assayed the genomic pattern of H2B ubiquitination, the localization of Rtf1 to actively transcribed genes (24Pokholok D.K. Hannett N.M. Young R.A. Mol. Cell. 2002; 9: 799-809Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 25Krogan N.J. Kim M. Ahn S.H. Zhong G. Kobor M.S. Cagney G. Emili A. Shilatifard A. Buratowski S. Greenblatt J.F. Mol. Cell. Biol. 2002; 22: 6979-6992Crossref PubMed Scopus (409) Google Scholar) is very discordant with the relatively constant levels of dimethylated H3-Lys4 and H3-Lys79, both of which depend on H2B ubiquitination. In contrast, the localization pattern of Rtf1 is consistent with and important for targeted recruitment of Set1 and Set2 histone methylases to coding regions of actively transcribed genes and the corresponding high levels of H3-Lys4 trimethylation and H3-Lys36 methylation (20Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar, 21Krogan N.J. Dover J. Wood A. Schneider J. Heidt J. Boateng M.A. Dean K. Ryan O.W. Golshani A. Johnston M. Greenblatt J.F. Shilatifard A. Mol. Cell. 2003; 11: 721-729Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, 27Krogan N.J. Kim M. Tong A. Golshani A. Cagney G. Canadien V. Richards D.P. Beattie B.K. Emili A. Boone C. Shilatifard A. Buratowski S. Greenblatt J.F. Mol. Cell. Biol. 2003; 23: 4207-4218Crossref PubMed Scopus (516) Google Scholar, 37Xiao T. Hall H. Kizer K.O. Shibata Y. Hall M.C. Borchers C.H. Strahl B.D. Genes Dev. 2003; 17: 654-663Crossref PubMed Scopus (325) Google Scholar). Second, Rtf1 is differentially required for recruitment of histone methylases to coding regions and genome-wide methylation. Genome-wide methylation of H3-Lys36 is not affected by Rtf1 (Fig. 1) (21Krogan N.J. Dover J. Wood A. Schneider J. Heidt J. Boateng M.A. Dean K. Ryan O.W. Golshani A. Johnston M. Greenblatt J.F. Shilatifard A. Mol. Cell. 2003; 11: 721-729Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar), even though Rtf1 is critical for Set2 recruitment and elevated H3-Lys36 methylation at transcriptionally active genes (27Krogan N.J. Kim M. Tong A. Golshani A. Cagney G. Canadien V. Richards D.P. Beattie B.K. Emili A. Boone C. Shilatifard A. Buratowski S. Greenblatt J.F. Mol. Cell. Biol. 2003; 23: 4207-4218Crossref PubMed Scopus (516) Google Scholar). Conversely, Rtf1 is essential for genome-wide methylation of H3-Lys79 (Fig. 1) (21Krogan N.J. Dover J. Wood A. Schneider J. Heidt J. Boateng M.A. Dean K. Ryan O.W. Golshani A. Johnston M. Greenblatt J.F. Shilatifard A. Mol. Cell. 2003; 11: 721-729Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar), but it plays a minimal role in recruitment of Dot1 to active genes (Fig. 5). Third, unlike Rtf1 association with active coding regions, the Rad6 and Bre1 enzymes mediating H2B ubiquitination are recruited to promoter regions in an activator-dependent manner (13Wood A. Krogan N.J. Dover J. Schneider J. Heidt J. Boateng M.A. Dean K. Golshani A. Zhang Y. Greenblatt J.F. Johnston M. Shilatifard A. Mol. Cell. 2003; 11: 267-274Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). We cannot exclude the possibility that Rtf1 might mediate global H2B ubiquitination in the context of the elongating Paf1-Pol II complex, but such a model would require additional features. For example, yeast pol II might elongate promiscuously over the entire genome at a low level, thereby permitting genome-wide, Rtf1-dependent ubiquitination of H2B. There is no evidence for such a nonspecific function of elongating Pol II, although this might be difficult to detect by conventional chromatin immunoprecipitation or transcriptional analyses. Alternatively, it is formally possible that H2B ubiquitination may spread from transcribed regions to non-transcribed regions if ubiquitination at a given nucleosome facilitates ubiquitination of a neighboring nucleosome. For these reasons, we suggest that the Paf1 complex, or a subcomplex containing Rtf1, functions independently of its association with elongating pol II to mediate genome-wide ubiquitination of H2B. Such an Rtf1-containing complex might interact with both the Rad6-Bre1 ubiquitinating activity and with histones, thereby acting as a bridge between the enzyme and substrate and facilitating H2B ubiquitination. Although our results are restricted to the Rtf1, it is likely that the Paf1 and Ctr9 components are also important for global H2B ubiquitination, because loss of Paf1 or Ctr9 abolishes H3-Lys4 and H3-Lys79 methylation (20Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar, 21Krogan N.J. Dover J. Wood A. Schneider J. Heidt J. Boateng M.A. Dean K. Ryan O.W. Golshani A. Johnston M. Greenblatt J.F. Shilatifard A. Mol. Cell. 2003; 11: 721-729Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). In contrast, the Leo1 and Cdc73 components of the Paf1 complex do not affect methylation of either Lys4 or Lys79, indicating that they also do not affect H2B ubiquitination. Although the mechanism remains to be resolved, our results demonstrate an unexpected link between specific components of the Paf1 complex and global ubiquitination of H2B. We thank Danesh Moazed for antibodies against Sir2 and Ali Shilatifard for communication of results prior to publication." @default.
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W2027349141 date "2003-09-01" @default.
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W2027349141 title "The Rtf1 Component of the Paf1 Transcriptional Elongation Complex Is Required for Ubiquitination of Histone H2B" @default.
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W2027349141 doi "https://doi.org/10.1074/jbc.c300270200" @default.
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