Evidence That Phosphorylation of the RNA Polymerase II Carboxyl-terminal Repeats Is Similar in Yeast and Humans
2005; Elsevier BV; Volume: 280; Issue: 36 Linguagem: Inglês
10.1074/jbc.m501546200
ISSN1083-351X
AutoresDaniel P. Morris, Gregory A. Michelotti, Debra A. Schwinn,
Tópico(s)Genomics and Chromatin Dynamics
ResumoUsing an improved chromatin immunoprecipitation assay designed to increase immunoprecipitation efficiency, we investigated changes in RNA polymerase II (Pol II) density and carboxyl-terminal domain (CTD) phosphorylation during transcription of the cyclophilin A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and several androgen-responsive genes in LNCaP cells. As generally observed in higher eukaryotes, promoter proximal pausing of Pol II appeared to occur on the PPIA and GAPDH genes, but apparently not on the androgen-responsive genes PSA and NKX3-1. Unlike some mammalian studies, we found that the CTD of Pol II in promoter regions contains little phosphorylation at Ser-2 of the heptad repeat, suggesting that Ser-2 phosphorylation is not involved in polymerase exit from the promoter region. In contrast, Pol II near the promoter displayed high levels of Ser-5 phosphorylation, which decreased as polymerase transcribed beyond the promoter region of the PPIA and GAPDH genes. However, total Pol II levels appear to decrease as much or more, suggesting that Ser-5 phosphorylation is maintained. In support of this conclusion, a phosphoserine 5-specific antibody quantitatively immunoprecipitates native hyperphosphorylated Pol II, suggesting that all polymerase with phosphoserine 2 also contains phosphoserine 5. Given reports indicating that phosphoserine 5 is present during elongation in yeast, our data suggest that gross changes in CTD phosphorylation patterns during transcription may be more conserved in yeast and humans than recognized previously. Using an improved chromatin immunoprecipitation assay designed to increase immunoprecipitation efficiency, we investigated changes in RNA polymerase II (Pol II) density and carboxyl-terminal domain (CTD) phosphorylation during transcription of the cyclophilin A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and several androgen-responsive genes in LNCaP cells. As generally observed in higher eukaryotes, promoter proximal pausing of Pol II appeared to occur on the PPIA and GAPDH genes, but apparently not on the androgen-responsive genes PSA and NKX3-1. Unlike some mammalian studies, we found that the CTD of Pol II in promoter regions contains little phosphorylation at Ser-2 of the heptad repeat, suggesting that Ser-2 phosphorylation is not involved in polymerase exit from the promoter region. In contrast, Pol II near the promoter displayed high levels of Ser-5 phosphorylation, which decreased as polymerase transcribed beyond the promoter region of the PPIA and GAPDH genes. However, total Pol II levels appear to decrease as much or more, suggesting that Ser-5 phosphorylation is maintained. In support of this conclusion, a phosphoserine 5-specific antibody quantitatively immunoprecipitates native hyperphosphorylated Pol II, suggesting that all polymerase with phosphoserine 2 also contains phosphoserine 5. Given reports indicating that phosphoserine 5 is present during elongation in yeast, our data suggest that gross changes in CTD phosphorylation patterns during transcription may be more conserved in yeast and humans than recognized previously. Phosphorylation of the carboxyl-terminal domain (CTD) 1The abbreviations used are: CTD, carboxyl-terminal domain; CDK, CTD kinase; ChIP, chromatin immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IP, immunoprecipitation; PIC, protease inhibitor cocktail; PMSF, phenylmethylsulfonyl fluoride; Pol II, RNA polymerase II; PSA, prostate-specific antigen; pSer, phosphoserine. of the largest subunit of RNA polymerase II (Pol II) plays an important role in regulating transcription and mRNA processing (1Sims III, R.J. Belotserkovskaya R. Reinberg D. Genes Dev. 2004; 18: 2437-2468Crossref PubMed Scopus (571) Google Scholar, 2Zorio D.A. Bentley D.L. Exp. Cell Res. 2004; 296: 91-97Crossref PubMed Scopus (116) Google Scholar, 3Palancade B. Bensaude O. Eur. J. Biochem. 2003; 270: 3859-3870Crossref PubMed Scopus (202) Google Scholar, 4Orphanides G. Reinberg D. Cell. 2002; 108: 439-451Abstract Full Text Full Text PDF PubMed Scopus (715) Google Scholar). In yeast and humans this domain is composed, respectively, of 26 or 52 seven-amino acid repeats with the consensus sequence YSPTSPS. Phosphorylation is predominantly at Ser-2 or Ser-5 of the repeat but also occurs at Tyr-1 and perhaps Ser-7 in mammals (3Palancade B. Bensaude O. Eur. J. Biochem. 2003; 270: 3859-3870Crossref PubMed Scopus (202) Google Scholar). In both yeast and humans, 19 consecutive repeats are completely conserved except for 1 of 133 amino acids in each species (5Prelich G. Eukaryot. Cell. 2002; 1: 153-162Crossref PubMed Scopus (100) Google Scholar). This level of conservation, at 99.2% identity, rivals the most highly conserved globular proteins such as histones and occurs even though the CTD is sometimes regarded as unstructured (6Noble C.G. Hollingworth D. Martin S.R. Ennis-Adeniran V. Smerdon S.J. Kelly G. Taylor I.A. Ramos A. Nat. Struct. Mol. Biol. 2005; 12: 144-151Crossref PubMed Scopus (84) Google Scholar). Recently, an evolutionary comparison of CTD kinases has suggested that conservation of the CTD was linked to the conservation of the CTD kinases, CDK7 in TFIIH and CDK8 in mediator (7Guo Z. Stiller J.W. BMC Genomics. 2004; 5: 69Crossref PubMed Scopus (69) Google Scholar). A mechanistic role for CTD phosphorylation in transcription and mRNA processing is well established for the addition of the 5′-cap to nascent mRNA. Capping enzymes bind directly to the phospho-CTD and are also allosterically activated by pSer-5 in both yeast (8Cho E.J. Takagi T. Moore C.R. Buratowski S. Genes Dev. 1997; 11: 3319-3326Crossref PubMed Scopus (373) Google Scholar, 9Cho E.J. Rodriguez C.R. Takagi T. Buratowski S. Genes Dev. 1998; 12: 3482-3487Crossref PubMed Scopus (85) Google Scholar) and mammals (10McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (433) Google Scholar, 11Ho C.K. Sriskanda V. McCracken S. Bentley D. Schwer B. Shuman S. J. Biol. Chem. 1998; 273: 9577-9585Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Phosphorylation of the CTD by CDK7, a kinase generally required for transcription (12Holstege F.C. Jennings E.G. Wyrick J.J. Lee T.I. Hengartner C.J. Green M.R. Golub T.R. Lander E.S. Young R.A. Cell. 1998; 95: 717-728Abstract Full Text Full Text PDF PubMed Scopus (1598) Google Scholar), may not be uniquely responsible for enabling the capping process (13Wen Y. Shatkin A.J. Genes Dev. 1999; 13: 1774-1779Crossref PubMed Scopus (165) Google Scholar, 14Liu Y. Kung C. Fishburn J. Ansari A.Z. Shokat K.M. Hahn S. Mol. Cell. Biol. 2004; 24: 1721-1735Crossref PubMed Scopus (145) Google Scholar); however, it is present at the promoter, processively phosphorylates the CTD after initiation (15Lu H. Zawel L. Fisher L. Egly J.M. Reinberg D. Nature. 1992; 358: 641-645Crossref PubMed Scopus (330) Google Scholar, 16Feaver W.J. Svejstrup J.Q. 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Biol. 2003; 23: 1961-1967Crossref PubMed Scopus (69) Google Scholar), and is the only CTD kinase shown genetically to interact with capping enzymes in yeast (18Schroeder S.C. Schwer B. Shuman S. Bentley D. Genes Dev. 2000; 14: 2435-2440Crossref PubMed Scopus (302) Google Scholar, 21Rodriguez C.R. Cho E.J. Keogh M.C. Moore C.L. Greenleaf A.L. Buratowski S. Mol. Cell. Biol. 2000; 20: 104-112Crossref PubMed Scopus (161) Google Scholar). Although Ser-5 phosphorylation clearly plays an important role in 5′-capping, the role of CTD phosphorylation in the transition of polymerase into productive mRNA synthesis is less clear. In yeast, a role for pSer-5 and capping in the entry of Pol II into productive elongation is implied by one study that showed that specific inhibition of CDK7 and CDK8 can reduce transcription (14Liu Y. Kung C. Fishburn J. Ansari A.Z. Shokat K.M. Hahn S. Mol. Cell. Biol. 2004; 24: 1721-1735Crossref PubMed Scopus (145) Google Scholar). In mammals and Drosophila, 5′-capping may be connected to the phenomena of promoter proximal pausing of Pol II because these events occur at similar positions, 20–75 bp from the transcription initiation site (22Lis J. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 347-356Crossref PubMed Scopus (145) Google Scholar). Further, Pol II transcripts at pause locations closer to the transcription initiation site appear to be less capped than transcripts at pause locations slightly downstream (23Rasmussen E.B. Lis J.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7923-7927Crossref PubMed Scopus (284) Google Scholar). Two complexes, DSIF and NELF, have been implicated in promoter proximal pausing. If these complexes are central, pausing may be relieved by phosphorylation of Ser-2 by CDK9 (kinase subunit of P-TEFb) (24Yamaguchi Y. Takagi T. Wada T. Yano K. Furuya A. Sugimoto S. Hasegawa J. Handa H. Cell. 1999; 97: 41-51Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar, 25Yamaguchi Y. Inukai N. Narita T. Wada T. Handa H. Mol. Cell. Biol. 2002; 22: 2918-2927Crossref PubMed Scopus (165) Google Scholar–26Price D.H. Mol. Cell. Biol. 2000; 20: 2629-2634Crossref PubMed Scopus (571) Google Scholar). Capping enzyme itself could also inhibit transcription and lead to pausing (27Myers L.C. Lacomis L. Erdjument-Bromage H. Tempst P. Mol. Cell. 2002; 10: 883-894Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), but confusingly, active and inactive capping enzyme can displace NELF and increase transcription (28Mandal S.S. Chu C. Wada T. Handa H. Shatkin A.J. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7572-7577Crossref PubMed Scopus (135) Google Scholar). Although the processes necessary for exit from the promoter region are not well established, it has been known for some time that Pol II engaged in productive elongation of mRNA is generally hyperphosphorylated on the CTD (29Payne J.M. Laybourn P.J. Dahmus M.E. J. Biol. Chem. 1989; 264: 19621-19629Abstract Full Text PDF PubMed Google Scholar, 30Weeks J.R. Hardin S.E. Shen J. Lee J.M. Greenleaf A.L. Genes Dev. 1993; 7: 2329-2344Crossref PubMed Scopus (163) Google Scholar, 31O'Brien T. Hardin S. Greenleaf A. Lis J.T. Nature. 1994; 370: 75-77Crossref PubMed Scopus (286) Google Scholar). More recently, the elongation-competent hyperphosphorylated state has been shown to include pSer-2 (19Komarnitsky P. Cho E.J. Buratowski S. Genes Dev. 2000; 14: 2452-2460Crossref PubMed Scopus (802) Google Scholar, 20Cheng C. Sharp P.A. Mol. Cell. Biol. 2003; 23: 1961-1967Crossref PubMed Scopus (69) Google Scholar), although pSer-5 is also likely to be present in both yeast (32Jones J.C. Phatnani H.P. Haystead T.A. MacDonald J.A. Alam S.M. Greenleaf A.L. J. Biol. Chem. 2004; 279: 24957-24964Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) and mammals (20Cheng C. Sharp P.A. Mol. Cell. Biol. 2003; 23: 1961-1967Crossref PubMed Scopus (69) Google Scholar, 33Eberhardy S.R. Farnham P.J. J. Biol. Chem. 2001; 276: 48562-48571Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). An increasing body of evidence suggests that CTD phosphorylation is directly linked to numerous events during mRNA synthesis, including splicing (34Kornblihtt A.R. de la Mata M. Fededa J.P. Munoz M.J. Nogues G. RNA (N. Y.). 2004; 10: 1489-1498Crossref PubMed Scopus (378) Google Scholar, 35Goldstrohm A.C. Greenleaf A.L. Garcia-Blanco M.A. Gene (Amst.). 2001; 277: 31-47Crossref PubMed Scopus (144) Google Scholar), 3′-end formation (36Proudfoot N. Curr. Opin. Cell Biol. 2004; 16: 272-278Crossref PubMed Scopus (239) Google Scholar), histone modification (37Li J. Moazed D. Gygi S.P. J. Biol. Chem. 2002; 277: 49383-49388Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 38Li B. Howe L. Anderson S. Yates III, J.R. Workman J.L. J. Biol. Chem. 2003; 278: 8897-8903Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 39Xiao 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), and DNA modification (40Carty S.M. Greenleaf A.L. Mol. Cell. Proteomics. 2002; 1: 598-610Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Putative kinase orthologs, CTK1 in yeast and CDK9 in multicellular eukaryotes, are strongly implicated in the phosphorylation of elongating polymerases. Deletion of CTK1 (41Ahn S.H. Kim M. Buratowski S. Mol. Cell. 2004; 13: 67-76Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar) or inhibition of CDK9 (42Ni Z. Schwartz B.E. Werner J. Suarez J.R. Lis J.T. Mol. Cell. 2004; 13: 55-65Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar) results in decreased pSer-2 but not pSer-5 levels on the CTD of elongating polymerase and causes defects in 3′-end formation. Although evidence from yeast suggests that pSer-2 is introduced only after Pol II has left the promoter region, data in human cells are more limited and controversial with studies showing that pSer-2 may or may not be present near gene promoters (20Cheng C. Sharp P.A. Mol. Cell. Biol. 2003; 23: 1961-1967Crossref PubMed Scopus (69) Google Scholar, 43Mo X. Dynan W.S. Mol. Cell. Biol. 2002; 22: 8088-8099Crossref PubMed Scopus (57) Google Scholar). Using a simple whole cell ChIP procedure with increased immunoprecipitation (IP) efficiency, we have analyzed CTD phosphorylation patterns during transcription in prostate-derived LNCaP cells. On most genes, Pol II near promoters contained high levels of pSer-5 and very little pSer-2, whereas elongating polymerases appeared to be phosphorylated at both Ser-2 and Ser-5. This pattern of phosphorylation is similar to that observed in yeast. Despite this similarity, some mammalian genes display obvious promoter proximal pausing, a behavior yet to be observed in yeast (22Lis J. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 347-356Crossref PubMed Scopus (145) Google Scholar). Although the housekeeping genes cyclophilin A (PPIA) and GAPDH both appear to display promoter proximal pausing, we found that the androgen-inducible genes NKX3-1 and possibly PSA did not. The presence or absence of pausing on these mammalian genes provides additional evidence of distinct transcriptional states as suggested by prior work in Drosophila (30Weeks J.R. Hardin S.E. Shen J. Lee J.M. Greenleaf A.L. Genes Dev. 1993; 7: 2329-2344Crossref PubMed Scopus (163) Google Scholar, 31O'Brien T. Hardin S. Greenleaf A. Lis J.T. Nature. 1994; 370: 75-77Crossref PubMed Scopus (286) Google Scholar). Materials—RPMI 1640 medium and penicillin/streptomycin (15140-122) were from Invitrogen, and heat-inactivated fetal calf serum (SH30071.03) was from Hyclone. PMSF, NaF, sodium orthovanadate (Na3VO4, S-6508), sodium deoxycholate (D-6750), glycogen (G-8751), bovine serum albumin (A-2153), and formaldehyde (F1268) were from Sigma. Protein G-agarose (1 719 416), Complete protease inhibitor tablets (1 836 145), proteinase K (3 115 879), Nonidet P-40 (1 754 599), and anti-mouse IgM-horseradish peroxidase conjugate (605 22) were from Roche Applied Science. Dry milk (170-6404) and polyvinylidene difluoride (162-0177) were from Bio-Rad. Herring sperm DNA (D181B) was from Promega. Goat IgG anti-mouse IgM (M-8644) was from Sigma. 8WG16, H5, and H14 were from BabCO. Pol II N20 (sc-899), A10 (sc-17798), and H224 (sc-9001) were from Santa Cruz Biotechnology. Preparation of Partially Purified Mammalian RNA Pol II from Heart—A liquid nitrogen-frozen heart from a female Sprague-Dawley rat and 10 ml of frozen solubilization buffer (50 mm Tris, pH 7.5, at 25 °C, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, phosphatase inhibitors (1 mm Na3VO4, 10 mm Na4P2O2, and 10 mm NaF), and 1 × Complete protease inhibitor mixture (PIC)) were placed into an industrial Waring blender and ground at high speed for a total of 2 min with liquid nitrogen present. The frozen dust was put in a small beaker until thawing had begun, and an additional 10 ml of room temperature lysis buffer was added followed by incubation in 25 °C water to thaw the entire sample quickly. The sample was spun at 25,000 × g for 5 min at 5 °C, and 10 ml of the supernatant was immediately loaded onto a 100-ml Sephacryl S-500 (Amersham Biosciences) column preequilibrated with solubilization buffer. The column was run rapidly (∼1 h at 5 °C), and each 10-ml fraction was aliquoted, frozen in liquid nitrogen, and stored at –80 °C. Hyperphosphorylated RNA Pol II was almost entirely in elution fraction 8 counting load as fraction 1. Preparation of Antibody Beads—Protein G-agarose was bound to IgG antibodies by incubation of each antibody with a 5% matrix slurry for 1 h at 25 °C with rotation in IP dilution buffer (10 mm Tris, pH 7.5, at 25 °C, 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mm NaCl, protease inhibitors (1 mm PMSF and 1 × Complete PIC), phosphatase inhibitors (1 mm Na3VO4, 10 mm Na4P2O7, and 10 mm NaF)). Buffers with protease inhibitor mixture were assumed to be stable and stored for 36 h at 4 °C (Roche product information); however, in all cases PMSF was added from a 200 mm stock in absolute ethanol within 1 h of solution use. For each 10 μl of matrix the amount of antibody loaded was 10 μg of goat anti-mouse, 5 μg of Pol II N20, 5 μg of Pol II A-10, and 12.5 μl of 8WG16 (raw ascites fluid). After adsorption, beads were pelleted at 500 × g for 1 min and washed with 1 ml of IP dilution buffer. The mouse IgM antibodies, H5 and H14 (12.5 μl of raw ascites fluid) were loaded similarly onto 10 μl of preloaded goat anti-mouse IgM/ protein G-agarose. Beads were stored overnight at 4 °C in IP dilution buffer or for ChIP in ChIP blocking buffer (IP dilution buffer supplemented with 1 mg/ml bovine serum albumin and 1 mg/ml herring sperm DNA). Except for antibody adsorption all IP steps were carried out at 4 °C. Immunoprecipitation and Western Blotting of the Largest Subunit of RNA Polymerase II—Beads (10 μl) with Pol II N20, 8WG16, H14, or H5 were combined with 500 μl of partially purified RNA Pol II to which 0, 0.02, or 0.1% SDS had been added and incubated for 2 h with inversion mixing. Beads were pelleted at 500 × g for 1 min, and all but the beads and 15 μl of the supernatant was moved to a new tube, and both samples were frozen in liquid nitrogen. To limit potential CTD dephosphorylation samples were thawed, combined with 2 × SDS sample buffer with 20 mm dithiothreitol, and immediately heated to 95 °C for 5 min. Samples were run on precast 4–15 or 4 –20% precast gels and transferred in transfer buffer with 15% methanol onto 0.2 μm of polyvinylidene difluoride using a minitrans-Blot unit (Bio-Rad instructions). Western blots were done at room temperature, and buffers were typical (10 mm Tris, pH 8.0, 150 mm NaCl, 0.1% Tween 20 for washes plus 5% dry milk with antibodies) except for the addition of phosphatase inhibitors (1 mm Na3VO4, 10 mm Na4P2O2, and 10 mm NaF). Primary antibodies were at 1:1,000 dilution, whereas secondary anti-IgM horseradish peroxidase conjugate was at 1:10,000 and was visualized with Super Signal West Dura extended duration substrate (Pierce) and Biomax XR film (Kodak). Blocking, primary antibody, and secondary antibody steps were 1 h. Analysis of cross-linked ChIP extracts and IPs was similar except samples were heated for 20 min at 95 °C in SDS sample buffer to reverse cross-links prior to electrophoresis. Beads were sampled by pipetting 20 μl with a clipped pipette tip missing ∼2 mm, following resuspension in wash buffers. LNCaP Cell Growth and Preparation of ChIP Extracts—5 million LNCaP cells were plated in 15-cm culture dishes and grown for 2 days at 37 °C in 10% fetal calf serum in phenol red-free RPMI 1640 under a 5% CO2 atmosphere. The medium was removed quickly, and the cells were fixed with 1% formaldehyde in phosphate-buffered saline with gentle agitation for 10 min at 25 °C, after which cross-linking was stopped by replacing formaldehyde with 125 mm glycine in phosphate-buffered saline for 5 min at 25 °C. After solution removal, plates were placed on ice, and 2 ml of chilled (2 min on ice) lysis buffer (10 mm Tris, pH 7.5, at 25 °C, 0.5% SDS, protease inhibitors (1 mm PMSF and 5 × Complete PIC), phosphatase inhibitors as above) was added. The cells were rapidly scraped from the plate, and the solution (∼2.3 ml) was transferred to a short 5-ml tube in ice water before sonication to an average fragment size of 1100–1300 bp using 12 5-s bursts on a Misonex 3000 with a microtip probe (Farmingdale, NY). After clarification by centrifugation at 10,000 × g for 5 min at 4 °C, supernatants were diluted about 20-fold with 38 ml of IP dilution buffer (10 mm Tris, pH 7.5, at 25 °C, 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mm NaCl, protease inhibitors (1 mm PMSF and 1 × Complete PIC), phosphatase inhibitors as above). The combined solution approximates ChIP buffer (IP dilution buffer with 0.025% SDS). Subaliquoted samples were frozen in 15-ml conical tubes using liquid nitrogen and stored at –70 °C until use. Each ml of extract represents about 400,000 cells. Immunoprecipitation and Preparation of ChIP DNA—Prior to use in ChIP, antibody beads were washed for 2–3 h with 1 ml of blocking buffer plus 0.025% SDS followed by one wash with 10 vol of the same buffer. During the bead washing period LNCaP extracts were thawed in a 25 °C bath until melted and precleared for 1 h by incubation with 15 μl of protein G-agarose (preequilibrated for 1 h with blocking buffer plus 0.025% SDS)/ml of extract. Matrix was removed by centrifugation at 10,000 × g for 5 min. Precleared extract (1.5 ml) was added to tubes containing 10 μl of antibody beads and incubated for 2 h at 4 °C with mixing by slow inversion. Subsequently, for the standard ChIP procedure, beads were washed four times with 1 ml of ChIP buffer each time with 5-min inversions and recovery by centrifugation at 500 × g for 1 min. For the preliminary ChIP procedure, beads were washed one time with ChIP buffer, one time with 20 mm Tris, pH 7.5, at 25 °C, 500 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.025% SDS, 1× PIC, one time with 10 mm Tris, pH 7.5, at 25 °C, 250 mm LiCl, 1% Nonidet P-40, 0.5% deoxycholate, 1 × PIC, and two times with 1 × TE. In each case, after the last wash, beads were resuspended in 100 μl of ChIP buffer to which was added 2.5 μl of 10% SDS and 5 μl of 10 mg/ml proteinase K. Samples were mixed well and incubated for 2 h at 55 °C and then overnight at 65 °C. Samples were spun and the supernatant collected followed by a second extraction with 100 μl of TE that was combined with first supernatant. After phenol/chloroform/IA and then chloroform extraction, 2 μl of 10 μg/μl glycogen was added and the DNA precipitated with sodium acetate/EtOH. Total DNA was prepared similarly without glycogen. ChIPs were resuspended in water, and total DNA was placed into TE with 20 μg/μl RNase. Analysis and Quantitation of ChIP DNA by PCR—Primers were designed using the program Oligo (Molecular Biology Insights) to have a melting temperature of about 70 °C and a high PE number (a proprietary measure of primer pair quality). Primers were obtained from Qiagen in an unpurified salt-free state. PCR was performed with platinum Taq as recommended but with 500 nm primer concentrations using a PTC-225 Tetrad DNA engine equipped with four independently controllable 96-well α sample blocks from M. J. Research (Waltham, MA). Reactions were "hot" started by incubation for 1.5 min at 94 °C followed by 35 cycles of 0.5 min at 94 °C, 0.5 min at 60 °C, and 1 min at 72 °C plus a final 10-min extension at 72 °C. After agarose gel electrophoresis in an Owl gel apparatus (model A6) with 25-well combs suited for use with a multichannel pipette, ethidium bromide-stained PCR products were quantitated with ImageQuant (Molecular Dynamics). The arbitrary pixel intensity units of PCR product bands from dilutions of total extract DNA (usually representing 1/4000, 1/20,000, 1/100,000, and 1/500,000 of the single precipitation) generated a nonlinear curve between input DNA and PCR product DNA. Appropriate amounts of ChIP DNA (1/200–1/2000 of a single precipitation) for each antibody were used to generate PCR products for each primer set so that product intensity values were generally within the range of the response curve. The fraction of DNA precipitated was estimated by interpolation between response curve values bracketing the sample signal. Linearity was not required; however, curves were regarded as invalid if the increase between points was not at least 30%. Interpolation beyond the highest curve concentrations was not done if the highest two concentration points differed by less than 50%, and these estimates were never more than 2.5-fold above that top concentration. For primer sequences see Supplemental Table 1. ChIP has been used to estimate the density of Pol II and the phosphorylation state of the CTD along numerous genes mostly in yeast and Drosophila. In brief, the process involves in vivo formaldehyde cross-linking of the DNA and Pol II (or other proteins), extracting and breaking the DNA into 200–2000-bp fragments, immunoprecipitating the DNA·Pol II complex with antibodies to Pol II, and finally analyzing the amount of DNA precipitated using PCR. The precipitated DNA can also be "quantitated" by comparing the PCR product from immunoprecipitated chromatin with that from total DNA to obtain a relative measure of Pol II density, which depends on both cross-linking and IP efficiencies (44Orlando V. Trends Biochem. Sci. 2000; 25: 99-104Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). Studies on the role of changing CTD phosphorylation have focused on highly expressed genes at least in part because most ChIP protocols currently in use are relatively insensitive and require a large number of cells. Given that PCR is exquisitely sensitive, it seemed possible that the lack of sensitivity observed in ChIP was a consequence of poor IP efficiency. For this reason, we initially investigated ways of improving the IP protocol. Immunoprecipitation of Native RNA Pol II—Four commercially available antibodies, Pol II N20, 8WG16, H14, and H5, are generally used in ChIP to characterize the phosphorylation state of the CTD during transcription. The Pol II N20 antibody is directed against an amino-terminal peptide of the largest subunit of Pol II and provides a measure of total polymerase density. Of the antibodies directed at the CTD, 8WG16 binds the unphosphorylated consensus repeat, YSPTSPS, whereas H14 requires phosphorylation at Ser-5 (32Jones J.C. Phatnani H.P. Haystead T.A. MacDonald J.A. Alam S.M. Greenleaf A.L. J. Biol. Chem. 2004; 279: 24957-24964Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 45Patturajan M. Schulte R.J. Sefton B.M. Berezney R. Vincent M. Bensaude O. Warren S.L. Corden J.L. J. Biol. Chem. 1998; 273: 4689-4694Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 46Cho E.J. Kobor M.S. Kim M. Greenblatt J. Buratowski S. Genes Dev. 2001; 15: 3319-3329Crossref PubMed Scopus (340) Google Scholar, 47Licatalosi D.D. Geiger G. Minet M. Schroeder S. Cilli K. McNeil J.B. Bentley D.L. Mol. Cell. 2002; 9: 1101-1111Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). H5 displays a strong preference for repeats phosphorylated at Ser-2 over Ser-5 (32Jones J.C. Phatnani H.P. Haystead T.A. MacDonald J.A. Alam S.M. Greenleaf A.L. J. Biol. Chem. 2004; 279: 24957-24964Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 45Patturajan M. Schulte R.J. Sefton B.M. Berezney R. Vincent M. Bensaude O. Warren S.L. Corden J.L. J. Biol. Chem. 1998; 273: 4689-4694Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 46Cho E.J. Kobor M.S. Kim M. Greenblatt J. Buratowski S. Genes Dev. 2001; 15: 3319-3329Crossref PubMed Scopus (340) Google Scholar, 47Licatalosi D.D. Geiger G. Minet M. Schroeder S. Cilli K. McNeil J.B. Bentley D.L. Mol. Cell. 2002; 9: 1101-1111Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar) but binds even more tightly to the doubly phosphorylated state (32Jones J.C. Phatnani H.P. Haystead T.A. MacDonald J.A. Alam S.M. Greenleaf A.L. J. Biol. Chem. 2004; 279: 24957-24964Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Although this lack of specificity might have made this antibody less useful, studies in yeast suggest that pSer-5 alone does not enable H5 binding during ChIPs because promoters bound to Pol II rich in pSer-5 yield very little signal with this antibody (19Komarnitsky P. Cho E.J. Buratowski S. Genes Dev. 2000; 14: 2452-2460Crossref PubMed Scopus (802) Google Scholar, 46Cho E.J. Kobor M.S. Kim M. Greenblatt J. Buratowski S. Genes Dev. 2001; 15: 3319-3329Crossref PubMed Scopus (340) Google Scholar). To determine the ability of the commercially available antibodies to precipitate mammalian Pol II under conditions analogous to ChIP, native phosphorylated Pol II was partially purified on a s
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