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• W2004406206 abstract "Histone methylation is a reversible modification regulated by the antagonistic functions of residue-specific histone methyltransferases and demethylases. Although methylation of histone H3 at lysines 4 and 36 is linked to transcription, the roles of histone demethylases in transcription regulation are not understood. Here we show that overexpression of either Jhd1 or Rph1, two JmjC-domain proteins, bypasses the requirement for the positive elongation factor gene BUR1. Biochemical analysis and chromatin immunoprecipitation experiments indicate that Rph1 functions as a specific demethylase for H3 K36me3 and K36me2, directly regulating Lys36 methylation in transcribed regions. Both Jhd1 and Rph1 are required for normal levels of RNA polymerase II cross-linking to genes. Taken together, these findings indicate that a general function of histone demethylases for H3 Lys36 is to promote transcription elongation by antagonizing repressive Lys36 methylation by Set2. Histone methylation is a reversible modification regulated by the antagonistic functions of residue-specific histone methyltransferases and demethylases. Although methylation of histone H3 at lysines 4 and 36 is linked to transcription, the roles of histone demethylases in transcription regulation are not understood. Here we show that overexpression of either Jhd1 or Rph1, two JmjC-domain proteins, bypasses the requirement for the positive elongation factor gene BUR1. Biochemical analysis and chromatin immunoprecipitation experiments indicate that Rph1 functions as a specific demethylase for H3 K36me3 and K36me2, directly regulating Lys36 methylation in transcribed regions. Both Jhd1 and Rph1 are required for normal levels of RNA polymerase II cross-linking to genes. Taken together, these findings indicate that a general function of histone demethylases for H3 Lys36 is to promote transcription elongation by antagonizing repressive Lys36 methylation by Set2. Posttranslational modifications of histones, including phosphorylation, acetylation, ubiquitylation, and methylation regulate gene expression by affecting chromatin architecture (1Nelson C.J. Santos-Rosa H. Kouzarides T. Cell. 2006; 126: 905-916Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 2Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6679) Google Scholar, 3Dyson M.H. Rose S. Mahadevan L.C. Front. Biosci. 2001; 6 (–D865): D853Crossref PubMed Google Scholar, 4Berger S.L. Curr. Opin. Genet. Dev. 2002; 12: 142-148Crossref PubMed Scopus (991) Google Scholar, 5Zhang Y. Reinberg D. Genes Dev. 2001; 15: 2343-2360Crossref PubMed Scopus (1245) Google Scholar). Histone methylation has been implicated in diverse biological processes including X-chromosome inactivation, heterochromatin formation, and gene silencing (6Kouzarides T. Curr. Opin. Genet. Dev. 2002; 12: 198-209Crossref PubMed Scopus (748) Google Scholar, 7Lachner M. Jenuwein T. Curr. Opin. Cell Biol. 2002; 14: 286-298Crossref PubMed Scopus (702) Google Scholar, 8Margueron R. Trojer P. Reinberg D. Curr. Opin. Genet. Dev. 2005; 15: 163-176Crossref PubMed Scopus (609) Google Scholar, 9Martin C. Zhang Y. Nat. Rev. Mol. Cell Biol. 2005; 6: 838-849Crossref PubMed Scopus (1615) Google Scholar). Methylations occur on both lysine (Lys) and arginine (Arg) residues. Five lysine residues (Lys4, Lys9, Lys27, Lys36, and Lys79) of histone H3 and Lys20 of histone H4 are methylated by specific histone methyltransferases (5Zhang Y. Reinberg D. Genes Dev. 2001; 15: 2343-2360Crossref PubMed Scopus (1245) Google Scholar, 8Margueron R. Trojer P. Reinberg D. Curr. Opin. Genet. Dev. 2005; 15: 163-176Crossref PubMed Scopus (609) Google Scholar). These marks can be binding sites for effector proteins that possess domains that recognize and bind to methylated lysines, such as chromodomains, tudor domains, WD40-repeats, and PHD fingers (9Martin C. Zhang Y. Nat. Rev. Mol. Cell Biol. 2005; 6: 838-849Crossref PubMed Scopus (1615) Google Scholar, 10Wysocka J. Swigut T. Xiao H. Milne T.A. Kwon S.Y. Landry J. Kauer M. Tackett A.J. Chait B.T. Badenhorst P. Wu C. Allis C.D. Nature. 2006; 442: 86-90Crossref PubMed Scopus (891) Google Scholar, 11Li H. Ilin S. Wang W. Duncan E.M. Wysocka J. Allis C.D. Patel D.J. Nature. 2006; 442: 91-95Crossref PubMed Scopus (186) Google Scholar, 12Pray-Grant M.G. Daniel J.A. Schieltz D. Yates J.R. II I Grant P.A. Nature. 2005; 433: 434-438Crossref PubMed Scopus (409) Google Scholar). Furthermore, methylation states (mono-, di-, and tri-) within the same residue can produce different biological and transcriptional consequences. Specific histone methylations have been correlated with either activation or repression of transcription (4Berger S.L. Curr. Opin. Genet. Dev. 2002; 12: 142-148Crossref PubMed Scopus (991) Google Scholar, 6Kouzarides T. Curr. Opin. Genet. Dev. 2002; 12: 198-209Crossref PubMed Scopus (748) Google Scholar, 9Martin C. Zhang Y. Nat. Rev. Mol. Cell Biol. 2005; 6: 838-849Crossref PubMed Scopus (1615) Google Scholar). Histone methylations at Lys4, Lys36, and Lys79 are generally associated with active transcription. In contrast, transcriptionally inactive regions are methylated at H3 Lys9 and Lys27 as well as H4 Lys20. In yeast, H3 Lys4, Lys36, and Lys79 are methylated by Set1, Set2, and Dot1 methyltransferases, respectively (13Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (857) Google Scholar, 14Strahl 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 (433) Google Scholar, 15van Leeuwen F. Gafken P.R. Gottschling D.E. Cell. 2002; 109: 745-756Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar). Methylations at Lys4 and Lys36 are closely linked to C-terminal domain (CTD) phosphorylation of RNA Pol II 2The abbreviations used are: Pol II, polymerase II; CTD, C-terminal domain; HA, hemagglutinin; 5-FOA, 5-fluoroorotic acid; 6-AU, 6-azauracil; MPA, mycophenolic acid; PHD, plant homeodomain; ChIP, chromatin immunoprecipitation. subunit Rpb1 (13Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (857) Google Scholar, 16Li B. Howe L. Anderson S. Yates J.R. II I Workman J.L. J. Biol. Chem. 2003; 278: 8897-8903Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 17Li J. Moazed D. Gygi S.P. J. Biol. Chem. 2002; 277: 49383-49388Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 18Krogan 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. Mol. Cell. Biol. 2003; 23: 4207-4218Crossref PubMed Scopus (519) Google Scholar). Kin28, a catalytic subunit of TFIIH, phosphorylates CTD serine 5, and this modification recruits the Set1 COMPASS complex to 5′ ends of genes (13Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (857) Google Scholar). During elongation, Ctk1 phosphorylates CTD serine 2 (in addition to serine 5), which targets Set2 methyltransferase and H3 Lys36 methylation to the body of genes (16Li B. Howe L. Anderson S. Yates J.R. II I Workman J.L. J. Biol. Chem. 2003; 278: 8897-8903Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 18Krogan 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. Mol. Cell. Biol. 2003; 23: 4207-4218Crossref PubMed Scopus (519) Google Scholar, 19Xiao 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 (326) Google Scholar, 20Kizer K.O. Phatnani H.P. Shibata Y. Hall H. Greenleaf A.L. Strahl B.D. Mol. Cell. Biol. 2005; 25: 3305-3316Crossref PubMed Scopus (367) Google Scholar, 21Schaft D. Roguev A. Kotovic K.M. Shevchenko A. Sarov M. Shevchenko A. Neugebauer K.M. Stewart A.F. Nucleic Acids Res. 2003; 31: 2475-2482Crossref PubMed Scopus (128) Google Scholar). Generally, H3 Lys4 trimethylation is enriched at the 5′ end of genes, while Lys36 trimethylation peaks at the 3′ end of genes (18Krogan 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. Mol. Cell. Biol. 2003; 23: 4207-4218Crossref PubMed Scopus (519) Google Scholar, 22Pokholok D.K. Harbison C.T. Levine S. Cole M. Hannett N.M. Lee T.I. Bell G.W. Walker K. Rolfe P.A. Herbolsheimer E. Zeitlinger J. Lewitter F. Gifford D.K. Young R.A. Cell. 2005; 122: 517-527Abstract Full Text Full Text PDF PubMed Scopus (1105) Google Scholar). Although histone methylation was originally thought to be a stable mark, recent studies show that methylation can be reversed by histone demethylases. Mammalian lysine-specific demethylase 1 (LSD1) specifically reverses mono- and dimethylation of H3 Lys4 and functions as a transcriptional repressor (23Shi Y. Lan F. Matson C. Mulligan P. Whetstine J.R. Cole P.A. Casero R.A. Shi Y. Cell. 2004; 119: 941-953Abstract Full Text Full Text PDF PubMed Scopus (3214) Google Scholar, 24Shi Y.J. Matson C. Lan F. Iwase S. Baba T. Shi Y. Mol. Cell. 2005; 19: 857-864Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar). The chemical mechanism of Lsd1 precludes demethylation of trimethyl lysines. Recently, a family of histone demethylases characterized by the presence of a JmjC domain was identified. Unlike LSD1, JmjC demethylases are found from bacteria to humans and can theoretically reverse all three lysine methylation states by a Fe(II)- and α-ketoglutarate-dependent mechanism (25Tsukada Y. Fang J. Erdjument-Bromage H. Warren M.E. Borchers C.H. Tempst P. Zhang Y. Nature. 2006; 439: 811-816Crossref PubMed Scopus (1638) Google Scholar). Mammalian JHDM1 and JHDM2A have been shown to antagonize mono- and dimethylation of H3 Lys36 and H3 Lys9, respectively, and the JMJD2/JHDM3 family preferentially reverses di- and trimethylation of both H3 Lys36 and Lys9 (25Tsukada Y. Fang J. Erdjument-Bromage H. Warren M.E. Borchers C.H. Tempst P. Zhang Y. Nature. 2006; 439: 811-816Crossref PubMed Scopus (1638) Google Scholar, 26Yamane K. Toumazou C. Tsukada Y. Erdjument-Bromage H. Tempst P. Wong J. Zhang Y. Cell. 2006; 125: 483-495Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 27Whetstine J.R. Nottke A. Lan F. Huarte M. Smolikov S. Chen Z. Spooner E. Li E. Zhang G. Colaiacovo M. Shi Y. Cell. 2006; 125: 467-481Abstract Full Text Full Text PDF PubMed Scopus (818) Google Scholar, 28Klose R.J. Yamane K. Bae Y. Zhang D. Erdjument-Bromage H. Tempst P. Wong J. Zhang Y. Nature. 2006; 442: 312-316Crossref PubMed Scopus (527) Google Scholar, 29Fodor B.D. Kubicek S. Yonezawa M. O'Sullivan R.J. Sengupta R. Perez-Burgos L. Opravil S. Mechtler K. Schotta G. Jenuwein T. Genes Dev. 2006; 20: 1557-1562Crossref PubMed Scopus (312) Google Scholar). The function of JHDM1 in transcription has not been studied, but JHDM2A-dependent demethylation of H3 Lys9 positively affects transcription (26Yamane K. Toumazou C. Tsukada Y. Erdjument-Bromage H. Tempst P. Wong J. Zhang Y. Cell. 2006; 125: 483-495Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar). In contrast, JMJM2A/JHDM3A, a trimethyl-specific demethylase for Lys9 and Lys36, negatively regulates ASCL2 transcription (28Klose R.J. Yamane K. Bae Y. Zhang D. Erdjument-Bromage H. Tempst P. Wong J. Zhang Y. Nature. 2006; 442: 312-316Crossref PubMed Scopus (527) Google Scholar). The downstream functions of H3 Lys36 methylation in yeast have been partially elucidated. This mark acts as a docking site for the chromodomain of Eaf3, a component of the Rpd3C(S) histone deacetylase complex (30Carrozza M.J. Li B. Florens L. Suganuma T. Swanson S.K. Lee K.K. Shia W.J. Anderson S. Yates J. Washburn M.P. Workman J.L. Cell. 2005; 123: 581-592Abstract Full Text Full Text PDF PubMed Scopus (980) Google Scholar, 31Keogh M.C. Kurdistani S.K. Morris S.A. Ahn S.H. Podolny V. Collins S.R. Schuldiner M. Chin K. Punna T. Thompson N.J. Boone C. Emili A. Weissman J.S. Hughes T.R. Strahl B.D. Grunstein M. Greenblatt J.F. Buratowski S. Krogan N.J. Cell. 2005; 123: 593-605Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar, 32Joshi A.A. Struhl K. Mol. Cell. 2005; 20: 971-978Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). Histone deacetylation by Rpd3C(S) inhibits transcription initiation by RNA Pol II at cryptic start sites within open reading frames (30Carrozza M.J. Li B. Florens L. Suganuma T. Swanson S.K. Lee K.K. Shia W.J. Anderson S. Yates J. Washburn M.P. Workman J.L. Cell. 2005; 123: 581-592Abstract Full Text Full Text PDF PubMed Scopus (980) Google Scholar). The Set2/Rpd3C(S) pathway also inhibits elongation by RNA Pol II, and this inhibition is counteracted by the positive elongation factor Bur1 (31Keogh M.C. Kurdistani S.K. Morris S.A. Ahn S.H. Podolny V. Collins S.R. Schuldiner M. Chin K. Punna T. Thompson N.J. Boone C. Emili A. Weissman J.S. Hughes T.R. Strahl B.D. Grunstein M. Greenblatt J.F. Buratowski S. Krogan N.J. Cell. 2005; 123: 593-605Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar). Although BUR1 is a nearly essential gene (33Prelich G. Winston F. Genetics. 1993; 135: 665-676Crossref PubMed Google Scholar, 34Yao S. Neiman A. Prelich G. Mol. Cell. Biol. 2000; 20: 7080-7087Crossref PubMed Scopus (58) Google Scholar, 35Keogh M.C. Podolny V. Buratowski S. Mol. Cell. Biol. 2003; 23: 7005-7018Crossref PubMed Scopus (126) Google Scholar), the severe growth defect of bur1Δ can be suppressed by deletions of either SET2 or genes encoding components of Rpd3C(S) (31Keogh M.C. Kurdistani S.K. Morris S.A. Ahn S.H. Podolny V. Collins S.R. Schuldiner M. Chin K. Punna T. Thompson N.J. Boone C. Emili A. Weissman J.S. Hughes T.R. Strahl B.D. Grunstein M. Greenblatt J.F. Buratowski S. Krogan N.J. Cell. 2005; 123: 593-605Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar, 36Chu Y. Sutton A. Sternglanz R. Prelich G. Mol. Cell. Biol. 2006; 26: 3029-3038Crossref PubMed Scopus (61) Google Scholar). To further understand connections between H3 Lys36 methylation and transcription, we isolated high copy suppressors of bur1Δ. We reasoned that such suppressors may have a positive role in transcription and/or antagonize the Set2-Rpd3C(S) pathway. Two isolated suppressors were JHD1 and RPH1, both of which have a JmjC domain motif for histone demethylases. Jhd1 has previously been shown to specifically demethylate H3 Lys36 (25Tsukada Y. Fang J. Erdjument-Bromage H. Warren M.E. Borchers C.H. Tempst P. Zhang Y. Nature. 2006; 439: 811-816Crossref PubMed Scopus (1638) Google Scholar). Here we show that Rph1 can reverse both tri- and dimethylation at Lys36. We present evidence that the Jhd1 and Rph1 Lys36 demethylases promote transcription by RNA Pol II through repressive chromatin generated by Set2 and Rpd3C(S). Antibodies—The following histone antibodies were used: anti-H3K36Me1 (Abcam 9048), anti-H3K36Me3 (Abcam 9050), anti-H3 (Abcam 1791), and anti-H3K36Me2 (Upstate Biotechnology 07-369). Anti-Rpb3 was from Neoclone. Protein A- and protein G-Sepharose-4 Fastflow were from Amersham Biosciences, and IgG-agarose was from Sigma. Yeast Strains and Plasmids—Yeast strains used in this study are listed in supplemental Table S1 and plasmids in supplemental Table S2. To generate pRS424 plasmids containing JHD1, RPH1, YJR119c/JHD2, GIS1, or ECM5, the entire ORF and 1kb of upstream region was amplified by PCR using oligonucleotide primers that create terminal NotI/SmaI (for RPH1, YJR119c/JHD2, and ECM5) or NotI/BamHI (for JHD1 and GIS1) sites. To construct pRS424-JHD1-3XHA, the JHD1 fragment digested with NotI and BamHI was cloned with a 500-bp BamHI/XhoI fragment containing 3× HA and SSN6 terminator from pBSSK(+)-3XHA/SSN6 terminator into the NotI/XhoI sites of pRS424. For cloning of RPH1, YJR119c/JHD2, ECM5 and GIS1, PCR fragments were gel-purified, digested with the appropriate enzymes for the new terminal sites, and cloned into the corresponding sites of pRS424-JHD1-3XHA. The downstream primers removed the stop codon and produced an in frame fusion to express triple-HA epitope tagged proteins. JHD1 and RPH1 point mutants were constructed by PCR using Pfu polymerase (Stratagene) and confirmed by sequencing. The sequences of oligonucleotides used in this study are listed in supplemental Table S3. Phenotype Analyses—To isolate high copy suppressors of bur1Δ, the BUR1 shuffle strain YSB787 was transformed with pRS424 plasmids (2 micron, TRP1) containing different genes, and the resulting transformants were patched on synthetic complete media lacking uracil (as a positive growth control) or SC media containing 5-FOA (to select against the BUR1/URA3 plasmid). The plates were incubated for 2–6 days as indicated. Spotting analyses were performed as previously described (35Keogh M.C. Podolny V. Buratowski S. Mol. Cell. Biol. 2003; 23: 7005-7018Crossref PubMed Scopus (126) Google Scholar). Chromatin Pull-down Assay—Chromatin pull-down assays were carried out as described by Howe et al. (37Howe L. Kusch T. Muster N. Chaterji R. Yates J.R. II I Workman J.L. Mol. Cell Biol. 2002; 22: 5047-5053Crossref PubMed Scopus (50) Google Scholar). Whole cell extracts were made from wild type or set2Δ cells and nucleosomes were isolated via an Hhf2-TAP fusion protein by precipitation with IgG-agarose. A non-tagged strain served as a negative control. The bead-bound nucleosomes were incubated with whole cell extracts from cells containing either Jhd1-HA or Rph1-HA tagged proteins. After overnight incubation at 4 °C, the complexes were washed four times with lysis buffer (10 mm Tris-Cl (pH 8.0), 150 mm NaCl, 0.1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml pepstatin A) and resolved by SDS-PAGE followed by immunoblotting analysis with the indicated antibodies. Chromatin Immunoprecipitations—Chromatin immunoprecipitations were carried out as previously described with minor modifications (35Keogh M.C. Podolny V. Buratowski S. Mol. Cell. Biol. 2003; 23: 7005-7018Crossref PubMed Scopus (126) Google Scholar, 38Ahn S.H. Kim M. Buratowski S. Mol. Cell. 2004; 13: 67-76Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). For histone H3 and H3K36Me IPs, 1 μl of anti-H3 or 0.5 μl of anti-H3 K36Me3 were bound to protein A-agarose beads and used to precipitate chromatin. For K36Me3 antibody, binding was done overnight in FA lysis buffer (50 mm HEPES-KOH (pH 7.5), 1 mm EDTA, 1% Triton X-100, 0.01% deoxycholate, and 1 mm phenylmethylsulfonyl fluoride) containing 1 m NaCl. The precipitates were washed with the same buffer, once with FA lysis buffer containing 1.5 m NaCl, once with buffer containing 10 mm Tris-HCl (pH 8.0), 0.25 m LiCl, 1 mm EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and once with TE (10 mm Tris-HCl (pH 8.0), 1 mm EDTA). Elution and decross-linking was performed as previously described (35Keogh M.C. Podolny V. Buratowski S. Mol. Cell. Biol. 2003; 23: 7005-7018Crossref PubMed Scopus (126) Google Scholar, 38Ahn S.H. Kim M. Buratowski S. Mol. Cell. 2004; 13: 67-76Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). The sequences of oligonucleotides for PCR amplification are in supplemental Table S3. In Vitro Demethylase Assay—Recombinant Rph1 proteins were expressed as hexahistidine fusion proteins in Escherichia coli and purified using nickel-agarose. 4–6 μg of purified wild-type or mutated Rph1 proteins were incubated with 10 μgof calf thymus type II-A histones (Sigma) in reaction buffer (50 mm Tris-Cl (pH 7.5), 50 μm Fe(NH4)2(SO4)2, 1 mm α-ketoglutarate, and 2 mm ascorbate) for 2 h at 37 °C. The reaction was stopped by adding SDS-PAGE sample buffer and boiling. The reaction mixtures were subject to SDS-PAGE (15%) followed by Western blot analysis with the indicated antibodies. Overexpression of JmjC-domain Proteins Jhd1 or Rph1 Suppresses bur1Δ—Yeast cells grow poorly or not at all if they lack the Bur1/Bur2 kinase complex, but this requirement can be bypassed by mutation of H3 Lys36 to alanine, deletion of the SET2 methyltransferase gene, or by deletions of genes encoding components of the Rpd3C(S) HDAC complex (31Keogh M.C. Kurdistani S.K. Morris S.A. Ahn S.H. Podolny V. Collins S.R. Schuldiner M. Chin K. Punna T. Thompson N.J. Boone C. Emili A. Weissman J.S. Hughes T.R. Strahl B.D. Grunstein M. Greenblatt J.F. Buratowski S. Krogan N.J. Cell. 2005; 123: 593-605Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar, 36Chu Y. Sutton A. Sternglanz R. Prelich G. Mol. Cell. Biol. 2006; 26: 3029-3038Crossref PubMed Scopus (61) Google Scholar). These deletion mutants also exhibit increased resistance to 6-azauracil (6-AU) and mycophenolic acid (MPA), two chemicals that inhibit RNA Pol II transcription elongation by reducing nucleotide pools (16Li B. Howe L. Anderson S. Yates J.R. II I Workman J.L. J. Biol. Chem. 2003; 278: 8897-8903Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 20Kizer K.O. Phatnani H.P. Shibata Y. Hall H. Greenleaf A.L. Strahl B.D. Mol. Cell. Biol. 2005; 25: 3305-3316Crossref PubMed Scopus (367) Google Scholar, 31Keogh M.C. Kurdistani S.K. Morris S.A. Ahn S.H. Podolny V. Collins S.R. Schuldiner M. Chin K. Punna T. Thompson N.J. Boone C. Emili A. Weissman J.S. Hughes T.R. Strahl B.D. Grunstein M. Greenblatt J.F. Buratowski S. Krogan N.J. Cell. 2005; 123: 593-605Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar). These results indicate that Set2-dependent Lys36 methylation is a repressive mark that negatively regulates transcription. We therefore predicted that histone demethylases that can reverse Lys36 methylation should have a positive role in transcription. Furthermore, overexpression of histone demethylases for Lys36 might also suppress the growth defect of bur1Δ. To explore this possibility, we tested whether overexpression of JmjC domain containing proteins could suppress the poor growth phenotype of a bur1Δ strain. Budding yeast has five JmjC proteins: Jhd1, Rph1, Gis1, Ecm5, and Yjr119c/Jhd2 (supplemental Fig. S1). Among these, two genes were considered likely candidates for bur1Δ suppressors. Jhd1 is most similar to mammalian JHDM1A, which antagonizes Lys36 mono- and dimethylation. Jhd1 can remove methyl groups from Set2 methylated H3 Lys36 in vitro, although no in vivo activity has been assigned (25Tsukada Y. Fang J. Erdjument-Bromage H. Warren M.E. Borchers C.H. Tempst P. Zhang Y. Nature. 2006; 439: 811-816Crossref PubMed Scopus (1638) Google Scholar). Recently, the PHD (plant homeodomain) finger of Ecm5 was reported to bind to trimethylated Lys36 in vitro (39Shi X. Kachirskaia I. Walter K.L. Kuo J.H. Lake A. Davrazou F. Chan S.M. Martin D.G. Fingerman I.M. Briggs S.D. Howe L. Utz P.J. Kutateladze T.G. Lugovskoy A.A. Bedford M.T. Gozani O. J. Biol. Chem. 2006; 282: 2450-2455Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Since the Eaf3 subunit of Rpd3C(S) also binds methylated Lys36, Ecm5 overexpression might also suppress bur1Δ by competing with Eaf3 for binding to nucleosomes. High copy plasmids expressing Jhd1, Rph1, Gis1, Ecm5, or Yjr119/Jhd2 were introduced into a BUR1-shuffling strain, and transformants were grown on synthetic complete (SC) media or media containing 5-FOA to test the ability to grow in the absence of BUR1. As predicted from its in vitro activity, Jhd1 overexpression allowed bur1Δ cells to grow (Fig. 1A), suggesting that Jhd1 acts as a H3 Lys36 demethylase in vivo. Surprisingly, high copy RPH1, but not GIS1, ECM5 or YJR119c/JHD2, also bypassed the requirement for BUR1 (Fig. 1A and data not shown). Rph1 was originally identified as a transcriptional repressor for the PHR1 gene (40Jang Y.K. Wang L. Sancar G.B. Mol. Cell. Biol. 1999; 19: 7630-7638Crossref PubMed Scopus (58) Google Scholar). Interestingly, this protein has sequence similarity to mammalian JHDM3A/JMJD2A, a trimethyl-specific histone demethylase for H3 Lys9 and H3 Lys36 (27Whetstine J.R. Nottke A. Lan F. Huarte M. Smolikov S. Chen Z. Spooner E. Li E. Zhang G. Colaiacovo M. Shi Y. Cell. 2006; 125: 467-481Abstract Full Text Full Text PDF PubMed Scopus (818) Google Scholar, 28Klose R.J. Yamane K. Bae Y. Zhang D. Erdjument-Bromage H. Tempst P. Wong J. Zhang Y. Nature. 2006; 442: 312-316Crossref PubMed Scopus (527) Google Scholar, 41Klose R.J. Kallin E.M. Zhang Y. Nat. Rev. Genet. 2006; 7: 715-727Crossref PubMed Scopus (970) Google Scholar). These findings suggest that both Jhd1 and Rph1 may bypass the requirement of BUR1 by removing the repressive Lys36 methylation. To test whether the histone demethylase activities of Jhd1 and Rph1 were required to bypass the requirement for BUR1, point mutants in a key catalytic histidine (41Klose R.J. Kallin E.M. Zhang Y. Nat. Rev. Genet. 2006; 7: 715-727Crossref PubMed Scopus (970) Google Scholar) were constructed. The Jhd1 H305A mutation abrogates demethylase activity in vitro (25Tsukada Y. Fang J. Erdjument-Bromage H. Warren M.E. Borchers C.H. Tempst P. Zhang Y. Nature. 2006; 439: 811-816Crossref PubMed Scopus (1638) Google Scholar), and this allele was unable to suppress bur1Δ (Fig. 1B). Similarly, overexpression of the H235A mutant Rph1 protein also failed to bypass the requirement for BUR1 (Fig. 1B). The mutant proteins were expressed at levels comparable with wild type (Fig. 1C), arguing that the loss of suppression is due to the loss of catalytic activity rather than defects in protein folding or stability. Neither protein suppressed as strongly as deletion of SET2 (Fig. 1B), suggesting that some Lys36 methylation persists even when the demethylases are overexpressed (see below). Rph1 Reverses Trimethylation of H3 Lys36 in Vivo and in Vitro—To test whether overexpression of Jhd1 or Rph1 results in reduction of Lys36 methylation in vivo, immunoblot analysis with antibodies against different methylated forms of H3 Lys36 was carried out on chromatin fractions. Unfortunately, mono- and dimethylated Lys36 were not detectable under these conditions. However, trimethylation of H3 Lys36 was readily detected, and this signal was completely absent in set2Δ cells (Fig. 2A). Triple HA-tagged Jhd1 or Rph1 was overexpressed from the GAL10 promoter and equal amounts of chromatin fractions from the indicated strains were analyzed. Levels of Rpb3, histone H3, and dimethylated Lys4 were unaffected by overexpression of Jhd1 or Rph1. In contrast, Lys36 trimethylation levels were greatly reduced upon overexpression of Rph1 (Fig. 2B). Interestingly, overexpression of Jhd1 at best caused only a small decrease in Lys36 trimethylation. To determine whether Rph1 directly acts as a Lys36 histone demethylase, recombinant Rph1 protein expressed in E. coli (shown in Fig. 2E) was tested for in vitro demethylase activity on purified bulk histones. Rph1 catalyzed demethylation of both the di- and trimethylated H3 Lys36, while mono-methylated Lys36 was unaffected (Fig. 2, C and D). This observation is consistent with previous reports that mammalian JHDM3A/JMJD2A is also capable of removing di- and trimethylation of H3 Lys36 in vitro (27Whetstine J.R. Nottke A. Lan F. Huarte M. Smolikov S. Chen Z. Spooner E. Li E. Zhang G. Colaiacovo M. Shi Y. Cell. 2006; 125: 467-481Abstract Full Text Full Text PDF PubMed Scopus (818) Google Scholar, 28Klose R.J. Yamane K. Bae Y. Zhang D. Erdjument-Bromage H. Tempst P. Wong J. Zhang Y. Nature. 2006; 442: 312-316Crossref PubMed Scopus (527) Google Scholar). An Rph1 protein mutated in a key residue for Fe(II) binding (H235A) was also tested for histone demethylase activity. This mutant protein had no effect on methylated Lys36 levels (Fig. 2, C and D). Jhd1 and Rph1 Associate with Nucleosomes Independently of H3 Lys36 Methylation—In most cases it is unclear how histone demethylases are recruited to their substrates. Some may be recruited to specific genes by interacting with sequence-specific DNA-binding proteins. Alternatively, they could be recruited directly or indirectly by other histone modifications such as acetylation, phosphorylation, ubiquitylation, or methylations at different residues. It was recently shown that JMJD2A/JHDM3A binds to methylated H3 Lys4 through its double Tudor domain (42Huang Y. Fang J. Bedford M.T. Zhang Y. Xu R.M. Science. 2006; 312: 748-751Crossref PubMed Scopus (370) Google Scholar). Jhd1 contains a PHD finger and Rph1 has two zinc fingers (supplemental Fig. S1). The Rph1 zinc domains have been implicated in sequence-specific binding (40Jang Y.K. Wang L. Sancar G.B. Mol. Cell. Biol. 1999; 19: 7630-7638Crossref PubMed Scopus (58) Google Scholar). To monitor the association of Jhd1 and Rph1 with histones, a chromatin binding assay was performed. Nucleosomes were isolated via a Hhf2-TAP protein from SET2 or set2Δ strains and incubated with whole cell extracts from cells expressing Jhd1-HA or Rph1-HA. Both Jhd1 and Rph1 co-precipitated with nucleosomes regardless of whether Lys36 methylation by Set2 was present (Fig. 3). We performed chromatin immunoprecipitation to map the positions of Jhd1 and Rph1 along transcribed and transcriptionally inactive regions. However, no enrichment of Jhd1 or Rph1 was observed with either TAP-tagged or triple HA-tagged proteins (data not shown). Therefore, the association of these two JmjC proteins with histones might be equivalent throughout the genome or else too weak or transient to detect by ChIP. Since these two demethylases might be working together, we tested whether Jhd1 and Rph1 are associated. However, precipitations of TAP-tagged Rph1 did not co-precipitate Jhd1. The converse experiment also gave negative results (data not shown). Rph1 Regulates H3 Lys36 Methylation at Actively Transcribed Regions—Set2-depend" @default.
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