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• W2153383004 abstract "MeCP2 is a transcription factor that recognizes and binds symmetrically methylated CpG dinucleotides to repress transcription. MeCP2 can associate with the Sin3a/histone deacetylase corepressor complex and mediate repression in a histone deacetylase-dependent manner. In extracts from rodent tissues, cultured cells, and Xenopus laevis oocytes, we find that only a small amount of mammalian MeCP2 interacts with Sin3a and that this interaction is not stable. Purification of rat brain MeCP2 (53 kDa) indicates no associated proteins despite an apparent molecular mass by size exclusion chromatography of 400–500 kDa. Biophysical analysis demonstrated that the large apparent size was not because of homo-multimerization, as MeCP2 consistently behaves as a monomeric protein that has an elongated shape. Our findings indicate the MeCP2 is not an obligate component of the Sin3a corepressor complex and may therefore engage a more diverse range of cofactors for repressive function. MeCP2 is a transcription factor that recognizes and binds symmetrically methylated CpG dinucleotides to repress transcription. MeCP2 can associate with the Sin3a/histone deacetylase corepressor complex and mediate repression in a histone deacetylase-dependent manner. In extracts from rodent tissues, cultured cells, and Xenopus laevis oocytes, we find that only a small amount of mammalian MeCP2 interacts with Sin3a and that this interaction is not stable. Purification of rat brain MeCP2 (53 kDa) indicates no associated proteins despite an apparent molecular mass by size exclusion chromatography of 400–500 kDa. Biophysical analysis demonstrated that the large apparent size was not because of homo-multimerization, as MeCP2 consistently behaves as a monomeric protein that has an elongated shape. Our findings indicate the MeCP2 is not an obligate component of the Sin3a corepressor complex and may therefore engage a more diverse range of cofactors for repressive function. Cytosine methylation is an important epigenetic mark on vertebrate genomes (1Li E. Bestor T.H. Jaenisch R. Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3159) Google Scholar). In mammals, methylation occurs mostly in the context of the CpG dinucleotide and can account for about 70–80% of genomic CpGs (2Bird A. Genes Dev. 2002; 16: 6-21Crossref PubMed Scopus (5254) Google Scholar). CpG methylation acts as an additional means of controlling genome function, beyond DNA base pair sequence. Because DNA methylation is copied faithfully to the newly replicating DNA strands during cell division, these marks can be maintained across development and act as a form of epigenetic memory. There are two general mechanisms by which CpG methylation is believed to function. First, modification of cytosines in the recognition sequence of DNA-binding proteins can inhibit their binding to cognate sequences and thus deny access to regulatory regions. Second, proteins have been identified that specifically bind the methyl-CpG dinucleotide via a methyl-CpG binding domain (MBD) 1The abbreviations used are: MBD, methyl-CpG binding domain; HDAC(s), histone deacetylase(s); DTT, dithiothreitol; Ni-NTA, nickel-nitrilotriacetic acid; BSA, bovine serum albumin; EGS, ethylene glycolbis succinimidylsuccinate; ADH, alcohol dehydrogenase; AE, anion exchange; CE, cation exchange; r, recombinant.1The abbreviations used are: MBD, methyl-CpG binding domain; HDAC(s), histone deacetylase(s); DTT, dithiothreitol; Ni-NTA, nickel-nitrilotriacetic acid; BSA, bovine serum albumin; EGS, ethylene glycolbis succinimidylsuccinate; ADH, alcohol dehydrogenase; AE, anion exchange; CE, cation exchange; r, recombinant. (3Hendrich B. Bird A. Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1049) Google Scholar) or, in the case of Kaiso (4Prokhortchouk A. Hendrich B. Jorgensen H. Ruzov A. Wilm M. Georgiev G. Bird A. Prokhortchouk E. Genes Dev. 2001; 15: 1613-1618Crossref PubMed Scopus (371) Google Scholar), by a zinc finger domain. These proteins can interact with methylated CpGs and affect nearby genes by repressing transcription and modulating chromatin structure (5Wade P.A. Oncogene. 2001; 20: 3166-3173Crossref PubMed Scopus (170) Google Scholar). There are five mammalian members of the MBD family: MeCP2 and MBD1–4. With the exception of MBD3, all MBD family members bind methylated CpG dinucleotide specifically. MBD1 (6Ng H.H. Jeppesen P. Bird A. Mol. Cell. Biol. 2000; 20: 1394-1406Crossref PubMed Scopus (215) Google Scholar, 7Cross S.H. Meehan R.R. Nan X. Bird A. Nat. Genet. 1997; 16: 256-259Crossref PubMed Scopus (213) Google Scholar, 8Jorgensen H.F. Ben-Porath I. Bird A.P. Mol. Cell. Biol. 2004; 24: 3387-3395Crossref PubMed Scopus (138) Google Scholar), MBD2 (9Boeke J. Ammerpohl O. Kegel S. Moehren U. Renkawitz R. J. Biol. Chem. 2000; 275: 34963-34967Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 10Ng H.H. Zhang Y. Hendrich B. Johnson C.A. Turner B.M. Erdjument-Bromage H. Tempst P. Reinberg D. Bird A. Nat. Genet. 1999; 23: 58-61Crossref PubMed Scopus (0) Google Scholar, 11Hutchins A.S. Mullen A.C. Lee H.W. Sykes K.J. High F.A. Hendrich B.D. Bird A.P. Reiner S.L. Mol. Cell. 2002; 10: 81-91Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 12Feng Q. Zhang Y. Genes Dev. 2001; 15: 827-832PubMed Google Scholar), MBD3, and MeCP2 (13Nan X. Campoy F.J. Bird A. Cell. 1997; 88: 471-481Abstract Full Text Full Text PDF PubMed Scopus (1014) Google Scholar) are all transcriptional repressors. Elucidation of the relationship between MBD proteins and their partner corepressors is a prerequisite for understanding how DNA methylation represses transcription and modulates chromatin structure. Several methyl-CpG-binding proteins have been shown to associate with histone deacetylases (HDACs) (10Ng H.H. Zhang Y. Hendrich B. Johnson C.A. Turner B.M. Erdjument-Bromage H. Tempst P. Reinberg D. Bird A. Nat. Genet. 1999; 23: 58-61Crossref PubMed Scopus (0) Google Scholar, 14Nan X. Ng H.H. Johnson C.A. Laherty C.D. Turner B.M. Eisenman R.N. Bird A. Nature. 1998; 393: 386-389Crossref PubMed Scopus (2716) Google Scholar, 15Wade P.A. Gegonne A. Jones P.L. Ballestar E. Aubry F. Wolffe A.P. Nat. Genet. 1999; 23: 62-66Crossref PubMed Scopus (703) Google Scholar) or histone methyltransferases (16Fujita N. Watanabe S. Ichimura T. Tsuruzoe S. Shinkai Y. Tachibana M. Chiba T. Nakao M. J. Biol. Chem. 2003; 278: 24132-24138Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 17Fuks F. Hurd P.J. Wolf D. Nan X. Bird A.P. Kouzarides T. J. Biol. Chem. 2003; 278: 4035-4040Abstract Full Text Full Text PDF PubMed Scopus (784) Google Scholar). MBD2 and MBD3 are stable components of the NuRD chromatin remodeling complex (10Ng H.H. Zhang Y. Hendrich B. Johnson C.A. Turner B.M. Erdjument-Bromage H. Tempst P. Reinberg D. Bird A. Nat. Genet. 1999; 23: 58-61Crossref PubMed Scopus (0) Google Scholar, 12Feng Q. Zhang Y. Genes Dev. 2001; 15: 827-832PubMed Google Scholar), and Kaiso can be purified in a complex containing NCoR (18Yoon H.G. Chan D.W. Reynolds A.B. Qin J. Wong J. Mol. Cell. 2003; 12: 723-734Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). In this study, we have asked whether MeCP2 is also part of a stable repression complex. MeCP2 dysfunction is the sole identified determinant of Rett syndrome, the most common inherited form of mental retardation in females (19Amir R.E. Van den Veyver I.B. Wan M. Tran C.Q. Francke U. Zoghbi H.Y. Nat. Genet. 1999; 23: 185-188Crossref PubMed Scopus (3699) Google Scholar). The majority of Rett syndrome point mutations in the MECP2 gene cluster in the MBD and the transcriptional repression domain (20Kriaucionis S. Bird A. Hum. Mol. Genet. 2003; 12: R221-R227Crossref PubMed Scopus (101) Google Scholar), suggesting that methyl-CpG binding and transcriptional repression are important functional determinants of MeCP2 in vivo. MeCP2 has been shown to interact with the Sin3a-HDAC chromatin remodeling complex (14Nan X. Ng H.H. Johnson C.A. Laherty C.D. Turner B.M. Eisenman R.N. Bird A. Nature. 1998; 393: 386-389Crossref PubMed Scopus (2716) Google Scholar). In Xenopus laevis it was reported that MeCP2 partially cofractionated with the Sin3a complex and proposed that xMeCP2 occurs in a stable complex with xSin3a (21Jones P.L. Veenstra G.J. Wade P.A. Vermaak D. Kass S.U. Landsberger N. Strouboulis J. Wolffe A.P. Nat. Genet. 1998; 19: 187-191Crossref PubMed Scopus (2207) Google Scholar, 22Jones P.L. Wade P.A. Wolffe A.P. Methods Mol. Biol. 2001; 181: 297-307PubMed Google Scholar). The link between MeCP2 and Sin3a has been strengthened recently with the identification of MeCP2 target genes. Both MeCP2 and Sin3a bind the promoter region of the brain-derived neurotrophic factor (Bdnf) gene (23Chen W.G. Chang Q. Lin Y. Meissner A. West A.E. Griffith E.C. Jaenisch R. Greenberg M.E. Science. 2003; 302: 885-889Crossref PubMed Scopus (998) Google Scholar, 24Martinowich K. Hattori D. Wu H. Fouse S. He F. Hu Y. Fan G. Sun Y.E. Science. 2003; 302: 890-893Crossref PubMed Scopus (1147) Google Scholar, 25Klose R. Bird A. Science. 2003; 302: 793-795Crossref PubMed Scopus (60) Google Scholar) and modulate its expression. A similar situation occurs at the X. laevis xHairy2a gene, where MeCP2 and Sin3a bind upstream of the promoter region and repress transcription (26Stancheva I. Collins A.L. Van den Veyver I.B. Zoghbi H. Meehan R.R. Mol. Cell. 2003; 12: 425-435Abstract Full Text PDF PubMed Scopus (144) Google Scholar). 2I. Stancheva, personal communication.2I. Stancheva, personal communication. In addition to Sin3a, several other factors have been reported to bind mammalian MeCP2, including DNMT1, CoREST, Suv39H1, and c-SKI (14Nan X. Ng H.H. Johnson C.A. Laherty C.D. Turner B.M. Eisenman R.N. Bird A. Nature. 1998; 393: 386-389Crossref PubMed Scopus (2716) Google Scholar, 27Kimura H. Shiota K. J. Biol. Chem. 2003; 278: 4806-4812Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 28Kokura K. Kaul S.C. Wadhwa R. Nomura T. Khan M.M. Shinagawa T. Yasukawa T. Colmenares C. Ishii S. J. Biol. Chem. 2001; 276: 34115-34121Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 29Lunyak V.V. Burgess R. Prefontaine G.G. Nelson C. Sze S.H. Chenoweth J. Schwartz P. Pevzner P.A. Glass C. Mandel G. Rosenfeld M.G. Science. 2002; 298: 1747-1752Crossref PubMed Scopus (389) Google Scholar), although the contribution of these factors to MeCP2-mediated repression is not known. Native MeCP2 has not been purified previously from mammalian sources, leaving open the possibility that it may exist in a novel multiprotein complex. Here we investigate the association of MeCP2 with the Sin3a complex from both mammalian sources and X. laevis, and we purify native MeCP2 from rat brain. We conclude that MeCP2 does not stably associate with the Sin3a complex. Moreover, we find no evidence that MeCP2 forms a stable association either with itself or with other proteins in nuclear extracts. Hydrodynamic analysis of MeCP2 shows that it behaves as an elongated monomeric molecule. These findings raise the possibility that DNA-bound MeCP2 interacts differently with partner proteins compared with its unbound form. In addition, the results suggest that MeCP2 might interact with a range of cofactors in addition to Sin3a. Cell Culture—NG-108 cells were a gift of Rod Bremner (University of Toronto) and were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% bovine calf serum, nonessential amino acids, sodium pyruvate, and antibiotics (Invitrogen). Chromatography Solutions—Chromatography buffers were filtered through a 0.2-μm filter before application to fast protein liquid chromatography or disposable columns. Anion exchange buffer 20 mm Tris-HCl (pH 7.9), 0.2 mm EDTA, 1 mm DTT, 10% glycerol, supplemented with 100 mm NaCl (AE100), or 1000 mm NaCl (AE1000). Cation exchange buffer was 20 mm Hepes (pH 7.6), 0.2 mm EDTA, 1 mm DTT, 10% glycerol, supplemented with 100 mm NaCl (CE100), or 150 mm NaCl (CE150), or 200 mm NaCl (CE200), or 1000 mm NaCl (CE1000). Ni-Nta affinity buffers (N) were 50 mm NaH2PO4, 300 mm NaCl, 10% glycerol (pH 8.0), supplemented with 20 mm imidazole (N20), 100 mm imidazole (N100), or 250 mm imidazole (N250). Gel filtration buffers were made in 20 mm Hepes-KOH (pH 7.9), 3 mm MgCl2, 10% glycerol, supplemented with 150 mm KCl (GF150) or 500 mm KCl (GF500). Isolation of Rat Brain Nuclei and Nuclear Protein Extraction—Rat brains (∼450 brains obtained from Pel-Freez Biologicals) were ground to a fine powder in liquid nitrogen with a mortar and pestle. The brain powder was diluted 5 volumes to 1 in ice-cold buffer A containing 10 mm Hepes (pH 7.5), 25 mm KCl, 0.15 mm spermine, 0.5 mm spermidine, 1 mm EDTA, 2 m sucrose, 10% glycerol, and complete protease inhibitors (Roche Applied Science) followed by homogenization in a 60-ml Dounce (Braun) on a Potter S (Braun) motorized homogenizer (five strokes at 1100 rpm). The homogenate was layered onto a 10-ml cushion of buffer A and centrifuged in pre-chilled SW28 rotor at 24,000 rpm in an Beckman XL100 ultracentrifuge for 40 min at 3 °C. Recovered nuclei were resuspended in 5 volumes of buffer B containing 10 mm Hepes (pH 7.9), 1.5 mm MgCl2, 10 mm KCl, 0.5 mm DTT, complete protease inhibitors (Roche Applied Science) and incubated on ice 10 min. The nuclei were pelleted at 250 × g and resuspended in 1 volume of buffer C containing 5 mm Hepes (pH 7.9), 26% glycerol, 1.5 mm MgCl2, 0.2 mm EDTA, and complete protease inhibitors (Roche Applied Science) supplemented 400 mm NaCl. The extraction was allowed to proceed for 1 h on ice, and then the nuclei were pelleted at 13,000 rpm for 20 min at 4 °C. The supernatant was taken as the nuclear extract and dialyzed to the indicated salt concentration. Immunoprecipitation—MeCP2 or Gal4 antibodies were incubated with 500 μg of rat brain nuclear extract at 4 °C for 4 h. Protein A-Sepharose beads (Amersham Biosciences) were added to the reaction and incubated for 1 h at 4 °C. Beads were washed four times with 20 mm Hepes (pH 7.9), 0.1 m NaCl, 10% glycerol, 0.2 mm EDTA, 0.01% Triton X-100. Bound proteins were eluted in Laemmli buffer and run on an 8% SDS-polyacrylamide gel. MeCP2 (14Nan X. Ng H.H. Johnson C.A. Laherty C.D. Turner B.M. Eisenman R.N. Bird A. Nature. 1998; 393: 386-389Crossref PubMed Scopus (2716) Google Scholar), Sin3a (Santa Cruz Biotechnology SC994 K20), and topoisomerase I (SC10783 H300) were identified by Western blotting. Large Scale Purification of Native MeCP2—All chromatography and dialyses were performed at 4 °C, and MeCP2 (14Nan X. Ng H.H. Johnson C.A. Laherty C.D. Turner B.M. Eisenman R.N. Bird A. Nature. 1998; 393: 386-389Crossref PubMed Scopus (2716) Google Scholar), Sin3a (Santa Cruz Biotechnology SC994 K20), and HDAC 1/2 (Santa Cruz Biotechnology SC7879 H51/SC7899 H54) containing fractions were identified by Western blotting. Rat brain nuclear extract (26 ml of 400 mm NaCl extraction) was dialyzed against CE150, and the insoluble material was pelleted at 13,000× g for 20 min at 4 °C. Soluble material (140 mg) was loaded onto an 8-ml SP-Sepharose (Amersham Biosciences) column and eluted with a 160-ml linear gradient of CE100 to CE1000 collecting 4-ml fractions. MeCP2-containing fractions were combined (8.8 mg) and dialyzed against AE100. Dialyzed protein was loaded onto a 1-ml MonoQ (Amersham Biosciences) column and eluted with a 40-ml linear gradient of AE100 to AE1000. MeCP2-containing fractions from the flow-through and wash were combined (4 mg) and dialyzed against CE100. The protein was loaded onto a 1-ml heparin (Amersham Biosciences) affinity column and eluted with a 40-ml linear gradient of CE100 to CE1000. MeCP2-containing fractions (0.408 mg) were combined and dialyzed into N20. Ni-NTA resin (0.65 ml; Qiagen) pre-equilibrated with N20 was added to the dialyzed protein and mixed at 4 °C for 1 h. The Ni-NTA was applied to a 10-ml disposable column (Bio-Rad). The column was washed thoroughly with N20 and then eluted in batch with N100 and N250. MeCP2 eluted at the N100 step, and these fractions were pooled (0.148 mg). An aliquot of the N100 elution was applied to a Superose 12 (Amersham Biosciences) column and eluted from the column in GF500 collecting 0.5-ml fractions. Proteins from the gel filtration column were Western-blotted to identify MeCP2-containing fractions, and subsequently trichloroacetic acid-precipitated, subjected to SDS-PAGE, and stained with Sypro-Ruby stain (Bio-Rad). Bands were excised and analyzed by mass spectrometry. Mass Spectrometry—To identify purified proteins, excised bands were in-gel trypsinized, and peptides were eluted from the gel slice. Mass spectrometry analysis was carried out on an Applied Biosystems Voyager DE-STR matrix-assisted laser desorption ionization time-of-flight instrument using α-cyano-4-hydroxycinnamic acid matrix. Spectra were analyzed in MS-Fit and then submitted to Protein Prospector (prospector.ucsf.edu/) for peptide matching. Xenopus Oocyte Extract and xMeCP2 Chromatography—Oocyte extract was prepared from one female as described previously (21Jones P.L. Veenstra G.J. Wade P.A. Vermaak D. Kass S.U. Landsberger N. Strouboulis J. Wolffe A.P. Nat. Genet. 1998; 19: 187-191Crossref PubMed Scopus (2207) Google Scholar). The extract (90 mg) was then loaded onto a 10-ml Bio-Rex 70 column exactly as described previously (21Jones P.L. Veenstra G.J. Wade P.A. Vermaak D. Kass S.U. Landsberger N. Strouboulis J. Wolffe A.P. Nat. Genet. 1998; 19: 187-191Crossref PubMed Scopus (2207) Google Scholar) in Bio-Rex 70 exchange buffer (BE100) containing 100 mm NaCl, 20 mm Hepes (pH 7.5), 10 mm β-glycerophosphate, 1.5 mm MgCl2, 1 mm EGTA, 0.5 mm DTT, 10% glycerol, complete protease inhibitors (Roche Applied Science) and washed with three column volumes of BE100. The column was batch-eluted with Bio-Rex 70 exchange buffer containing 500 mm NaCl (BE500), and fractions were analyzed by Western blotting with three independent xMeCP2 antibodies and an xSin3a antibody (a gift of P. L. Jones). The BE500 elution (250 μl) was separated on a Superose 6 (Amersham Biosciences) gel filtration column in GF150 collecting 0.5-ml fractions. Proteins were trichloroacetic acid-precipitated and run on an 8% SDS-polyacrylamide gel and Western blotted for xMeCP2 and xSin3a. MeCP2 Expression Plasmid—A human MeCP2 cDNA was used to PCR-amplify MeCP2 with primer pairs containing a 5′ NdeI site corresponding to the initiating ATG and 3′ EcoRI site downstream of the endogenous MeCP2 stop codon. The PCR fragment was inserted into the NdeI/EcoRI sites of the bacterial expression plasmid pET30b (Novagen) to create an untagged bacterial MeCP2 expression vector pET30bhMeCP2. Expression and Purification of Full-length Untagged MeCP2 in Bacteria—pET30bhMeCP2 was transformed into BL21 codon plus bacteria (a gift from Robin Allshire). Bacterial cultures (usually 0.5 or 1 liter) were grown in LB at 37 °C until the culture reached an A600 of 0.5 absorbance units. Cultures were induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside for 3 h at 30 °C. Cells were pelleted and lysed in 20 mm Tris-HCl (pH 8.0), 500 mm NaCl, 0.1% Nonidet P-40, complete protease inhibitors (Roche Applied Science) by sonicating on output setting 4–5 at 30% for 3 min with a Branson 250 sonifier. Extracts were centrifuged at 4 °C for 20 min at 20,000 × g, and Ni-NTA beads (Qiagen) pre-equilibrated with lysis buffer were added to the supernatant. Recombinant MeCP2 was allowed to bind the beads mixing for 1 h at 4 °C and then applied to a 10-ml disposable column. The column was washed with 20 column volumes of N20 and batch-eluted with N250. MeCP2-containing fractions were identified by Coomassie Blue staining and dialyzed into CE200. The protein from the Ni-NTA elutions was loaded onto an Sp-Sepharose column and eluted with a linear gradient of CE200 to CE1000, and the MeCP2-containing fractions were combined and directly loaded onto a Sephacryl S-300 26/60 (Amersham Biosciences) column and eluted with GF500. MeCP2-containing fractions were combined and dialyzed against CE200 and then loaded onto a 1-ml MonoS column. Proteins were eluted with a linear gradient of CE200 to CE1000, and MeCP2-containing factions were combined and dialyzed into CE200 and stored at –20 °C. Bandshift Analysis—Increasing concentrations of rMeCP2 (50, 100, 250, 500, and 750 ng) were bound to the CG11 probe (30Meehan R.R. Lewis J.D. McKay S. Kleiner E.L. Bird A.P. Cell. 1989; 58: 499-507Abstract Full Text PDF PubMed Scopus (519) Google Scholar) either methylated or unmethylated. The CG11 probe containing 27 CpGs was generated by digesting out an EcoRI/HindIII (New England Biolabs) fragment from the plasmid pCG11 (30Meehan R.R. Lewis J.D. McKay S. Kleiner E.L. Bird A.P. Cell. 1989; 58: 499-507Abstract Full Text PDF PubMed Scopus (519) Google Scholar). The 135-bp CG11 fragment was purified by gel electrophoresis and agarose gel extraction. The CpGs in the resulting fragments were methylated with SssI methyltransferase (New England Biolabs), and the methylated or unmethylated CG11 probe was end-labeled with [32P]dCTP using Klenow (Roche Applied Science). The binding reactions were assembled in buffer containing 6 mm MgCl2, 3% glycerol, 1 mm DTT, 150 mm KCl, 100 ng/μl poly(dA-dT) (Sigma) for 10 min at room temperature in the absence of probe, and then the probe was added and incubated a further 25 min. The reactions were run on a 1.5% agarose gel and dried onto DE-81 anion exchange paper (Whatman). The bandshifts were exposed on a phosphor screen and analyzed on a Storm 840 PhosphorImager (Amersham Biosciences). Competition with cold methylated and unmethylated probe at ∼100-fold excess was used to demonstrate the specificity of the bandshift. EGS Cross-linking—An EGS (Pierce) stock solution was made fresh to 25 mm in Me2SO. 5 μg of rMeCP2 was incubated in CE200 (without DTT or EDTA) with increasing concentrations of EGS (0.25, 0.5, 1.0, 2.5, 5.0 mm) in a 50-μl reaction volume. BSA (5 μg, monomer) and ADH (5 μg, tetramer) were included as internal controls for cross-linking efficiency. Cross-linking reactions were carried out at room temperature for 30 min. Tris-HCl (pH 8.8) (5 μl of 1.5 m) was added to quench the reaction for 15 min at room temperature. SDS-PAGE loading buffer (4×) was added, and the proteins separated on an 8% SDS-polyacrylamide gel followed by Coomassie Blue staining to visualize the proteins. Gel Filtration and Sucrose Gradient Analysis of rMeCP2—A Superose 12 HR 10/30 gel filtration column was pre-equilibrated with gel filtration standards thyroglobin (669 kDa, RS = 8.5), apoferritin (443 kDa, RS = 6.1), B-amylase (200 kDa, RS = 5.4), ADH (150 kDa), BSA (66 kDa, RS = 3.55), and carbonic anhydrase (29 kDa, RS = 2). Recombinant MeCP2 (125 μg) was loaded onto the column pre-equilibrated with buffer GF500. Fractions (0.5 ml) were collected, and 50 μl of each fraction was separated on an 8% SDS-polyacrylamide gel and Coomassie Blue-stained to verify the identity of protein observed from the A280 trace. Relative absorbance at 280 nm was plotted as the protein eluted from the Superose 12 column, and the radius was calculated by using an equation derived from the plotted standards. For sucrose gradient sedimentation ∼15 μg of rMeCP2, 50 μg of apoferritin (17.7 S), 30 μg of B-amylase (8.9 S), 50 μg of ADH (7.4 S), and 50 μg of BSA (4.3 S) were loaded onto a 13-ml linear 5–20% sucrose gradient made in 0.3 m KCl, 20 mm Hepes (pH 7.9), 2 mm EDTA, 10% glycerol, 10 mm β-mercaptoethanol. The gradient was centrifuged for 19 h at 40,000 rpm in a Beckman SW40 rotor at 4 °C. Fractions (0.5 ml) were taken from the top of the gradient, trichloroacetic acid-precipitated, run on an 8% SDS-polyacrylamide gel, Western-blotted using an anti-MeCP2 antibody or on a 10% SDS-polyacrylamide gel, and Coomassie Blue-stained for the indicated standards. Densitometric analysis utilized Gene Tools Analysis Software package (SynGene), and values were adjusted for background. Intensity was plotted by fraction to determine the relative sedimentation coefficient of rMeCP2. Molecular Weight and Frictional Coefficient Calculations—Calculations to determine molecular weight and frictional coefficient (f/f0) were applied as described (31Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1542) Google Scholar, 32Kolb S.J. Hudmon A. Ginsberg T.R. Waxham M.N. J. Biol. Chem. 1998; 273: 31555-31564Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) using Equations 1 and 2,Mr=6πη20,w·s20,w·RS·N/(1−ρ20,wν)(Eq. 1) f/f0=6πη20,w·RS/6πη20,w·(3νMr/4πN)1/3(Eq. 2) where RS is the Stoke's radius (cm), s20,w is the sedimentation velocity (S × 10–13), η20,w is the viscosity of water at 20 °C (0.01002 g·s–1 cm–1), N = Avogadro's number (6.022 × 1023·mol–1), ρ20,w is the density of water at 20 °C (0.9981 g·cm3), ν is the partial specific volume (used 0.725 cm3/g). Biochemical Analysis of MeCP2 in Nuclear Extracts—Previous studies have demonstrated that Xenopus (21Jones P.L. Veenstra G.J. Wade P.A. Vermaak D. Kass S.U. Landsberger N. Strouboulis J. Wolffe A.P. Nat. Genet. 1998; 19: 187-191Crossref PubMed Scopus (2207) Google Scholar) and mammalian MeCP2 can associate with the Sin3a complex (14Nan X. Ng H.H. Johnson C.A. Laherty C.D. Turner B.M. Eisenman R.N. Bird A. Nature. 1998; 393: 386-389Crossref PubMed Scopus (2716) Google Scholar), but the properties of the mammalian Sin3a/MeCP2 interaction remain to be investigated. We confirmed that in rat brain nuclear extracts Sin3a is coimmunoprecipitated by antibodies against MeCP2 (Fig. 1a), but the amounts are small relative to input. In order to evaluate the relative amount of MeCP2 residing in a stable Sin3a complex, nuclear extracts from either rat brain or NG108 (mouse-rat neural-glial fusion) tissue culture cells were separated by size exclusion chromatography, and the resulting fractions were analyzed by Western blot with antibodies against MeCP2, Sin3a, and HDACs 1 and 2, which are components of the Sin3a complex. To our surprise most of Sin3a-containing fractions were devoid of detectable MeCP2. The majority of Sin3a eluted over the apparent molecular mass range of 500 kDa to 2 mDa, showing significant overlap with HDAC 1/2-containing fractions (Fig. 1b). In contrast, MeCP2 eluted with an apparent molecular mass of 400–500 kDa (Fig. 1b). Biochemical Purification of MeCP2 from Rat Brain—The predicted molecular mass of MeCP2 based on its amino acid sequence is between 52.4 and 53 kDa, depending on species. This is much smaller than the apparent molecular weight observed by size exclusion chromatography, indicating that MeCP2 may exist in a multiprotein complex. To test this possibility, a large scale biochemical purification of rat brain MeCP2 was devised (Fig. 2a). During the purification, MeCP2, Sin3a, and HDAC 1/2 were tracked by Western blotting. Sp-Sepharose and MonoQ columns efficiently separated MeCP2 from the majority of the Sin3a and HDAC 1/2-containing fractions, confirming that the majority of MeCP2 from nuclear extract is absent from the Sin3a complex. After four purification steps, three polypeptides were detected by SDS-PAGE, of which the middle band was identified as MeCP2 by Western blot (Fig. 2c). A final size exclusion chromatography step was applied to fractions eluted from the Ni-NTA column. SDS-PAGE of the resulting fractions (Fig. 2d) demonstrated that the apparent molecular weight of MeCP2 (400–500 kDa) remained constant over the purification. The SDS-PAGE analysis also showed a 90-kDa band whose elution profile overlapped with MeCP2. This protein was identified by mass spectrometry (data not shown) as topoisomerase I. We were able to rule out the possibility that MeCP2 associated with topoisomerase I as MeCP2 antibodies were unable to immunoprecipitate topoisomerase I from rat brain nuclear extract (Fig. 2e). Furthermore, an independent purification starting with 10 mg of rat brain extract yielded MeCP2 with the same Superose 12 size exclusion profile but in the absence of detectable topoisomerase I (data not shown). We conclude that MeCP2 in rat brain extracts, despite its large apparent molecular weight, does not exist in a complex with other proteins. Biochemical Analysis of MeCP2 from X. laevis Oocytes—A previous report (21Jones P.L. Veenstra G.J. Wade P.A. Vermaak D. Kass S.U. Landsberger N. Strouboulis J. Wolffe A.P. Nat. Genet. 1998; 19: 187-191Crossref PubMed Scopus (2207) Google Scholar) suggested that X. laevis MeCP2 cofractionated with X. laevis Sin3a (xSin3a). To re-visit this observation, we made extract from X. laevis oocytes and separated it by Bio-Rex 70 ion exchange chromatography (Fig. 3, a and b), using the procedures described previously (21Jones P.L. Veenstra G.J. Wade P.A. Vermaak D. Kass S.U. Landsberger N. Strouboulis J. Wolffe A.P. Nat. Genet. 1998; 19: 187-191Crossref PubMed Scopus (2207) Google Scholar). Western blots using antibodies directed against xMeCP2 and xSin3a were used to monitor the elution of these proteins (Fig. 3b). Proteins from the Bio-Rex 70 step were separated by size exclusion chromatography, and fractions were analyzed by Western blot (Fig. 3c). The xSin3a protein elutes in a complex with an apparent mass of 500 kDa to 2 mDa, whereas xMeCP2, like mammalian MeCP2, elutes with an apparent molecular mass of 400–500 kDa. In agreement with our observations in mammalian extracts (Fig. 1 and Fig. 2) and with an independent study of Xenopus oocyte extracts (33Ryan J. Llinas A.J. White D.A. Turner B.M. Sommerville J. J. Cell Sci. 1999; 112: 2441-2452PubMed Google Scholar), X. laevis MeCP2 does not coelute with fractions containing the majority of the xSin3a corepressor complex. Purification of Recombinant Untagged Human MeCP2—To examine further the discrepancy between the observed mol" @default.
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• W2153383004 title "MeCP2 Behaves as an Elongated Monomer That Does Not Stably Associate with the Sin3a Chromatin Remodeling Complex" @default.
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