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• W2084223479 abstract "The view that autosomal gene expression is controlled exclusively by protein trans-acting factors has been challenged recently by the identification of RNA molecules that regulate chromatin. In the majority of cases where RNA molecules are implicated in DNA control, the molecular mechanisms are unknown, in large part because the RNA·protein complexes are uncharacterized. Here, we identify a novel set of RNA-binding proteins that are well known for their function in chromatin regulation. The RNA-interacting proteins are components of the mammalian DNA methylation system. Genomic methylation controls chromatin in the context of transposon silencing, imprinting, and X chromosome dosage compensation. DNA methyltransferases (DNMTs) catalyze methylation of cytosines in CGs. The methyl-CGs are recognized by methyl-DNA-binding domain (MBD) proteins, which recruit histone deacetylases and chromatin remodeling proteins to effect silencing. We show that a subset of the DNMTs and MBD proteins can form RNA·protein complexes. We characterize the MBD protein RNA-binding activity and show that it is distinct from the methyl-CG-binding domain and mediates a high affinity interaction with RNA. The RNA and methyl-CG binding properties of the MBD proteins are mutually exclusive. We speculate that DNMTs and MBD proteins allow RNA molecules to participate in DNA methylation-mediated chromatin control. The view that autosomal gene expression is controlled exclusively by protein trans-acting factors has been challenged recently by the identification of RNA molecules that regulate chromatin. In the majority of cases where RNA molecules are implicated in DNA control, the molecular mechanisms are unknown, in large part because the RNA·protein complexes are uncharacterized. Here, we identify a novel set of RNA-binding proteins that are well known for their function in chromatin regulation. The RNA-interacting proteins are components of the mammalian DNA methylation system. Genomic methylation controls chromatin in the context of transposon silencing, imprinting, and X chromosome dosage compensation. DNA methyltransferases (DNMTs) catalyze methylation of cytosines in CGs. The methyl-CGs are recognized by methyl-DNA-binding domain (MBD) proteins, which recruit histone deacetylases and chromatin remodeling proteins to effect silencing. We show that a subset of the DNMTs and MBD proteins can form RNA·protein complexes. We characterize the MBD protein RNA-binding activity and show that it is distinct from the methyl-CG-binding domain and mediates a high affinity interaction with RNA. The RNA and methyl-CG binding properties of the MBD proteins are mutually exclusive. We speculate that DNMTs and MBD proteins allow RNA molecules to participate in DNA methylation-mediated chromatin control. The most abundant covalent modification of mammalian DNA is symmetric methylation of cytosines in the context of CGs. DNA methylation is catalyzed after replication by DNA methyltransferases (DNMTs) 1The abbreviations used are: DNMTs, DNA methyltransferases; MBD, methyl-CG-binding domain; MeCP2, methyl-CpG-binding protein-2; RNP, RNA·protein; GST, glutathione S-transferase; FL, full-length; RBD, RNA-binding domain; aa, amino acids; DTT, dithiothreitol; PBS, phosphate-buffered saline; snRNA, small nuclear RNA; siRNA, small interfering RNA; dsDNA, double-stranded DNA. (1Bestor T.H. Hum. Mol. Genet. 2000; 9: 2395-2402Crossref PubMed Scopus (1611) Google Scholar, 2Bird A. Genes Dev. 2002; 16: 6-21Crossref PubMed Scopus (5486) Google Scholar, 3Meehan R.R. Semin. Cell Dev. Biol. 2003; 14: 53-65Crossref PubMed Scopus (86) Google Scholar, 4Hendrich B. Tweedie S. Trends Genet. 2003; 19: 269-277Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). Five DNMTs have been described: DNMT1, DNMT2, DNMT3a, DNMT3b, and DNMT3L. All have a conserved catalytic domain, but no enzyme activity has yet been detected for DNMT2 and DNMT3L. Genetic analyses of DNMTs have amply demonstrated the relevance of CG methylation for genome function. Disruption of DNMT1 in the mouse results in embryonic lethality (5Li E. Bestor T.H. Jaenisch R. Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3246) Google Scholar). Analysis of these embryos showed that DNA methylation is essential for proper imprinting (6Li E. Beard C. Jaenisch R. Nature. 1993; 366: 362-365Crossref PubMed Scopus (1784) Google Scholar, 7Howell C.Y. Bestor T.H. Ding F. Latham K.E. Mertineit C. Trasler J.M. Chaillet J.R. Cell. 2001; 104: 829-838Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar), silencing of transposable elements (8Walsh C.P. Chaillet J.R. Bestor T.H. Nat. Genet. 1998; 20: 116-117Crossref PubMed Scopus (871) Google Scholar), and X chromosome dosage compensation (9Beard C. Li E. Jaenisch R. Genes Dev. 1995; 9: 2325-2334Crossref PubMed Scopus (248) Google Scholar, 10Panning B. Jaenisch R. Genes Dev. 1996; 10: 1991-2002Crossref PubMed Scopus (290) Google Scholar). Mice lacking DNMT3a die prematurely, and embryos without DNMT3b do not develop to term (11Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4574) Google Scholar). Conditional removal of DNMT3a in germ cells revealed that it is essential for imprinting (12Kaneda M. Okano M. Hata K. Sado T. Tsujimoto N. Li E. Sasaki H. Nature. 2004; 429: 900-903Crossref PubMed Scopus (1033) Google Scholar). DNMT3L is also necessary for imprinting (13Hata K. Okano M. Lei H. Li E. Development (Camb.). 2002; 129: 1983-1993PubMed Google Scholar, 14Bourc'his D. Xu G.L. Lin C.S. Bollman B. Bestor T.H. Science. 2001; 294: 2536-2539Crossref PubMed Scopus (1085) Google Scholar). CG methylation is therefore essential for, rather than consequential to, silenced chromatin. Because CG methylation is heritable, it can be properly described as an epigenetic modification. Regions of the genome that are under the control of DNA methylation and the molecular basis of methylation-mediated silencing have been extensively studied. Although a precise map of methyl-CG distribution in any genome is not yet available, some patterns are apparent (3Meehan R.R. Semin. Cell Dev. Biol. 2003; 14: 53-65Crossref PubMed Scopus (86) Google Scholar, 15Jaenisch R. Bird A. Nat. Genet. 2003; 33: 245-254Crossref PubMed Scopus (4739) Google Scholar). Methylation is dynamic in a developing mammal: shortly after fertilization, the male genome is actively demethylated by as yet to be identified demethylase(s) (16Oswald J. Engemann S. Lane N. Mayer W. Olek A. Fundele R. Dean W. Reik W. Walter J. Curr. Biol. 2000; 10: 475-478Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar, 17Mayer W. Niveleau A. Walter J. Fundele R. Haaf T. Nature. 2000; 403: 501-502Crossref PubMed Scopus (1108) Google Scholar), and the female genome is passively demethylated. The embryonic genome then undergoes de novo methylation during implantation and can lose methylation again during differentiation of some tissues (18Reik W. Dean W. Walter J. Science. 2001; 293: 1089-1093Crossref PubMed Scopus (2457) Google Scholar). In the adult, ∼80% of CGs are stably methylated. Coding region, intronic, and extragenic CGs are generally methylated. Promoter region CGs are generally undermethylated, except at imprinted alleles and on the inactive X chromosome (2Bird A. Genes Dev. 2002; 16: 6-21Crossref PubMed Scopus (5486) Google Scholar). How CGs are chosen for, or spared from, DNMT and demethylase attention is not known, although members of the SNF2 helicase family that disrupt DNA/histone contacts are implicated (3Meehan R.R. Semin. Cell Dev. Biol. 2003; 14: 53-65Crossref PubMed Scopus (86) Google Scholar). The mechanisms that translate methyl-CGs into silenced chromatin are several and not fully understood. Some methyl-CGs block DNA·protein interactions, whereas others are attractive. The methyl-CG-binding domain (MBD) family is the best characterized set of proteins that are attracted to methyl-CGs (see Fig. 1) (19Lewis J.D. Meehan R.R. Henzel W.J. Maurer-Fogy I. Jeppesen P. Klein F. Bird A. Cell. 1992; 69: 905-914Abstract Full Text PDF PubMed Scopus (1091) Google Scholar, 20Hendrich B. Bird A. Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1081) Google Scholar, 21Nan X. Meehan R.R. Bird A. Nucleic Acids Res. 1993; 21: 4886-4892Crossref PubMed Scopus (493) Google Scholar). Outside the MBD, these proteins generally share little sequence similarity, but several members of the family appear to use a similar core mechanism to silence chromatin. They recruit histone deacetylases and proteins with homology to ATP-dependent helicases, enzymes that can create a chromatin structure inhospitable to RNA polymerases (22Feng Q. Zhang Y. Genes Dev. 2001; 15: 827-832PubMed Google Scholar, 23Jones 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 (2252) Google Scholar, 24Nan 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 (2804) Google Scholar, 25Ng 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, 26Wade P.A. Gegonne A. Jones P.L. Ballestar E. Aubry F. Wolffe A.P. Nat. Genet. 1999; 23: 62-66Crossref PubMed Scopus (713) Google Scholar, 27Zhang Y. Ng H.H. Erdjument-Bromage H. Tempst P. Bird A. Reinberg D. Genes Dev. 1999; 13: 1924-1935Crossref PubMed Scopus (935) Google Scholar, 28Narlikar G.J. Fan H.Y. Kingston R.E. Cell. 2002; 108: 475-487Abstract Full Text Full Text PDF PubMed Scopus (1256) Google Scholar). A functionally homologous region in the MBD proteins, the transcription repression domain, is essential for nucleating these histone deacetylases at methyl-CGs (see Fig. 1). It has been suggested that MBD proteins can silence chromatin by mechanisms other than histone deacetylase recruitment based on observations that histone deacetylase inhibitors do not totally reverse DNA methylation-mediated or MBD protein-mediated transcription inhibition (23Jones 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 (2252) Google Scholar, 24Nan 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 (2804) Google Scholar, 29Cameron E.E. Bachman K.E. Myohanen S. Herman J.G. Baylin S.B. Nat. Genet. 1999; 21: 103-107Crossref PubMed Scopus (1686) Google Scholar, 30Fujita N. Jaye D.L. Kajita M. Geigerman C. Moreno C.S. Wade P.A. Cell. 2003; 113: 207-219Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, 31Jorgensen H.F. Ben-Porath I. Bird A.P. Mol. Cell. Biol. 2004; 24: 3387-3395Crossref PubMed Scopus (141) Google Scholar). MBD1 and methyl-CpG-binding protein-2 (MeCP2) can recruit histone methyltransferases and other proteins that affect chromatin activity (32Fuks 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 (805) Google Scholar, 33Fujita 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 (230) Google Scholar, 34Fujita N. Watanabe S. Ichimura T. Ohkuma Y. Chiba T. Saya H. Nakao M. Mol. Cell. Biol. 2003; 23: 2834-2843Crossref PubMed Scopus (67) Google Scholar), and MeCP2 can condense unmethylated chromatin in vitro (35Georgel P.T. Horowitz-Scherer R.A. Adkins N. Woodcock C.L. Wade P.A. Hansen J.C. J. Biol. Chem. 2003; 278: 32181-32188Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). These properties could contribute to histone deacetylase-independent silencing. Genetic analyses have shown that each MBD protein has a distinct function. Mice without MBD1 have compromised neurons, with deficits in adult neurogenesis and hippocampal function (36Zhao X. Ueba T. Christie B.R. Barkho B. McConnell M.J. Nakashima K. Lein E.S. Eadie B.D. Willhoite A.R. Muotri A.R. Summers R.G. Chun J. Lee K.F. Gage F.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6777-6782Crossref PubMed Scopus (313) Google Scholar). Mice with defective MBD2 show a behavioral phenotype; mothers do not nurture their off spring well; and the interleukin-4 gene is derepressed in a subset of T cells (37Hendrich B. Guy J. Ramsahoye B. Wilson V.A. Bird A. Genes Dev. 2001; 15: 710-723Crossref PubMed Scopus (398) Google Scholar, 38Hutchins 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). MBD2 is also needed for intestinal tumorigenesis in mice (39Sansom O.J. Berger J. Bishop S.M. Hendrich B. Bird A. Clarke A.R. Nat. Genet. 2003; 34: 145-147Crossref PubMed Scopus (150) Google Scholar). Lack of MBD3 is lethal for an embryo (37Hendrich B. Guy J. Ramsahoye B. Wilson V.A. Bird A. Genes Dev. 2001; 15: 710-723Crossref PubMed Scopus (398) Google Scholar), and MBD4-null mice have increased DNA mutability, reflecting the mismatch repair activity of MBD4 (40Bellacosa A. J. Cell. Physiol. 2001; 187: 137-144Crossref PubMed Scopus (91) Google Scholar, 41Bellacosa A. Cicchillitti L. Schepis F. Riccio A. Yeung A.T. Matsumoto Y. Golemis E.A. Genuardi M. Neri G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3969-3974Crossref PubMed Scopus (222) Google Scholar, 42Hendrich B. Hardeland U. Ng H.H. Jiricny J. Bird A. Nature. 1999; 401: 301-304Crossref PubMed Scopus (528) Google Scholar, 43Millar C.B. Guy J. Sansom O.J. Selfridge J. MacDougall E. Hendrich B. Keightley P.D. Bishop S.M. Clarke A.R. Bird A. Science. 2002; 297: 403-405Crossref PubMed Scopus (261) Google Scholar). Dysfunctional MeCP2 results in neuronal cell defects that cause Rett's syndrome in humans (44Amir 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 (3859) Google Scholar, 45Kriaucionis S. Bird A. Hum. Mol. Genet. 2003; 12: R221-R227Crossref PubMed Scopus (103) Google Scholar) and a similar phenotype in mice (46Chen R.Z. Akbarian S. Tudor M. Jaenisch R. Nat. Genet. 2001; 27: 327-331Crossref PubMed Scopus (1042) Google Scholar, 47Guy J. Hendrich B. Holmes M. Martin J.E. Bird A. Nat. Genet. 2001; 27: 322-326Crossref PubMed Scopus (1229) Google Scholar). A double null of MeCP2 and MBD2 has an additive phenotype, indicating there is no functional overlap between these family members (47Guy J. Hendrich B. Holmes M. Martin J.E. Bird A. Nat. Genet. 2001; 27: 322-326Crossref PubMed Scopus (1229) Google Scholar). Genes or intergenic regions of chromatin under the control of MBD proteins have been obscure (2Bird A. Genes Dev. 2002; 16: 6-21Crossref PubMed Scopus (5486) Google Scholar, 3Meehan R.R. Semin. Cell Dev. Biol. 2003; 14: 53-65Crossref PubMed Scopus (86) Google Scholar, 48Tudor M. Akbarian S. Chen R.Z. Jaenisch R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15536-15541Crossref PubMed Scopus (282) Google Scholar). Recently, two genes controlled by MeCP2 were identified. Meehan and co-workers (49Stancheva 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) reported that the Xenopus Hairy2a gene, the protein product of which is an inhibitor of neuronal cell differentiation, is repressed by MeCP2 in this animal, while in cultured mammalian neurons, MeCP2 represses the brain-derived neurotrophic factor gene, the product of which functions in neuronal cell development and plasticity. MeCP2-mediated repression is overcome by membrane depolarization (50Chen 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 (1022) Google Scholar, 51Martinowich K. Hattori D. Wu H. Fouse S. He F. Hu Y. Fan G. Sun Y.E. Science. 2003; 302: 890-893Crossref PubMed Scopus (1185) Google Scholar, 52Klose R. Bird A. Science. 2003; 302: 793-795Crossref PubMed Scopus (61) Google Scholar). Exactly how control of these two genes translates to neuronal cell function and dysfunction when MeCP2 is mutated is not clear. To explain how specific genes are repressed by any one MBD family member, the observation that each MBD protein tends to be concentrated at sites of constitutive heterochromatin needs to be incorporated (20Hendrich B. Bird A. Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1081) Google Scholar, 53Nan X. Tate P. Li E. Bird A. Mol. Cell. Biol. 1996; 16: 414-421Crossref PubMed Scopus (285) Google Scholar). Interestingly, factors that dictate specificity of methyl-CG·MBD interactions are not known, although there is some evidence that the density of methyl-CGs and DNA sequence context surrounding methyl-CGs may be relevant (20Hendrich B. Bird A. Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1081) Google Scholar, 54Fraga M.F. Ballestar E. Montoya G. Taysavang P. Wade P.A. Esteller M. Nucleic Acids Res. 2003; 31: 1765-1774Crossref PubMed Scopus (189) Google Scholar, 55Yoon 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 (299) Google Scholar). Noncoding RNA molecules function in several epigenetic chromatin controls that also use DNA methylation, e.g. X chromosome inactivation, genomic imprinting and silencing of repetitive DNA elements (15Jaenisch R. Bird A. Nat. Genet. 2003; 33: 245-254Crossref PubMed Scopus (4739) Google Scholar). The molecular mechanisms by which RNA molecules influence any of these processes are not understood. Here, we show that specific MBD proteins and DNMTs are able to form RNA·protein (RNP) complexes. The RNA·MBD protein and methyl-DNA·MBD protein complexes are mutually exclusive. Our observations indicate a new molecular aspect to MBD and DNMT protein function. Plasmids for Protein Expression in Bacteria—Plasmid encoding GST-MBD2(FL)-His was made by Pwo PCR amplification of full-length (FL) mouse MBD2 using a 5′-primer containing an EcoRI site and a 3′-primer containing a SalI site. Following restriction enzyme digestion, the PCR product was inserted into EcoRI/SalI-digested pET-GST-II (56Nakielny S. Shaikh S. Burke B. Dreyfuss G. EMBO J. 1999; 18: 1982-1995Crossref PubMed Scopus (183) Google Scholar). DNAs encoding GST-MBD1(FL)-His, GST-MBD2 (deletion mutants), GST-MeCP2 (full-length and deletion mutants), and His-MBD3(FL) were kindly provided by Dr. Adrian Bird (University of Edinburgh). DNA encoding the GST-fused double-stranded RNA-binding domain (RBD; amino acids (aa) 579–646) of Drosophila staufen was kindly provided by Dr. Daniel St. Johnston (University of Cambridge). The Gateway (Invitrogen)-converted GST expression vector pDEST-GST was kindly provided by Dr. Simon Boulton (Cancer Research UK, London Research Institute). Plasmids encoding GST-DNMT1-N, GST-DNMT1-M, and GST-DNMT1-C were made by Pwo PCR using primers containing attB sites. The PCR template was mouse Dnmt1 cDNA (kindly provided by Dr. En Li, Harvard Medical School). DNMT1-N is aa 1–300; DNMT1-M is aa 300–1000; and DNMT1-C is aa 1000–1620. These were cloned into pDEST-GST via pDONR-221 (Invitrogen). DNA encoding GST-DNMT2 was made by Pwo PCR using primers containing attB sites from a 3T3 cell library and cloned into pDEST-GST via pDONR-221. DNAs encoding GST-DNMT3a and GST-DNMT3b1 (mouse) were provided by Dr. Shoji Tajima (Osaka University). Plasmids for Protein Expression in Mammalian Cells—Mammalian FLAG Gateway expression vector (pcDNA3.1-FLAG-DEST) was made using EcoRI/BamHI-cut and blunt-ended pcDNA3.1-FLAG DNA, which was ligated to a blunt-ended cassette containing attR sites (Invitrogen). DNAs encoding full-length mouse MBD2, the mouse MBD2 MBD (aa 148–221), and rat MeCP2, for cloning in the Gateway system, were made by Pwo PCR using primers containing attB sites. These were cloned into pDONR-221. Deletion of the MBD2 RG domain (aa 48–114) and the MeCP2 (rat) RG domain (aa 160–200) was achieved using a QuikChange mutagenesis Kit (Stratagene). All pDONR-221 clones were then cloned into pcDNA3.1-FLAG-DEST. DNA encoding FLAG-MeCP2 (human) was made by Pwo PCR amplification of IMAGE clone 3956518 using primers containing attB sites and cloned into pDONR-221. A point mutation at amino acid 106 (Arg to Trp) was made using the QuikChange mutagenesis kit. The wild-type and point mutant MeCP2 were cloned into the pcDNA3.1-FLAG-DEST vector via pDONR-221. FLAG-DNMT3L was made by Pwo PCR amplification of IMAGE clone 3138514 using primers containing attB sites and cloned into pcDNA3.1-FLAG-DEST via pDONR-221. Recombinant Proteins Expressed in Bacteria—All proteins were overexpressed in BL21(DE3) cells with the exception of GST-MeCP2(FL), which was expressed in BL21(DE3) Codon Plus cells; GST-MBD2(FL)-His, which was expressed in BL21(DE3) pLysS cells; and GST-DNMT1-M, which was expressed in Tuner(DE3) cells. Protein expression was induced with 0.2 mm isopropyl β-d-thiogalactopyranoside (pGEX vectors) or 1 mm isopropyl β-d-thiogalactopyranoside (pET vectors) for 4 h at 37 °C. All protein purification steps were carried out on ice at 4 °C. Bacterial pellets were resuspended in the buffers indicated below, containing 10 μg/ml each leupeptin, aprotinin, and pepstatin; 0.5 mm phenylmethylsulfonyl fluoride; and 0.1 mm tris(2-carboxyethyl)phosphine hydrochloride for nickel resin purifications or 1 mm dithiothreitol (DTT) for glutathione resin purifications. Thereafter, all buffers contained 1 μg/ml each leupeptin, aprotinin, and pepstatin; 0.2 mm phenylmethylsulfonyl fluoride; and 0.1 mm tris(2-carboxyethyl)phosphine hydrochloride or 1 mm DTT. GST-MBD1(FL)-His was purified on nickel resin, and GST-MBD2(FL)-His and GST-MeCP2(FL) were purified on glutathione resin followed by nickel resin. The MeCP2 protein itself contains an internal stretch of histidines. Single-tag recombinant proteins were purified on glutathione (GST-MBD2 and GST-MeCP2 deletion mutants) or nickel (His-MBD3) resin. Pellets of bacteria expressing GST fusion proteins were resuspended in phosphate-buffered saline (PBS), except for DNMT1, DNMT3a, and DNMT3b1, which were resuspended in PBS, 0.33 m NaCl, and 0.1% Triton X-100; sonicated; and centrifuged at 30,000 × g. The proteins were purified using modified glutathione elution buffer (Amersham Biosciences) according to the manufacturer's instructions, except for DNMT1, DNMT3a, and DNMT3b1, which were eluted in PBS, 0.33 m NaCl, 0.1% Triton X-100, and 20 mm glutathione. Pellets of bacteria expressing His fusion proteins were resuspended in binding buffer (20 mm Tris-HCl (pH 8), 250 mm NaCl, 5 mm imidazole, 0.1% Triton X-100, and 10% glycerol), sonicated, and centrifuged, and the extract was loaded onto nickel resin equilibrated in binding buffer. The resin was washed with 10 column volumes of binding buffer followed by 10 column volumes of binding buffer and 15 mm imidazole. Protein was eluted in 2 column volumes of binding buffer and 500 mm imidazole. For proteins purified over glutathione and nickel resins, an equal volume of 2× binding buffer was added to the glutathione resin elution, and this was purified on nickel resin as described above. All proteins were dialyzed against PBS and 10% glycerol, except for MBD3, which was dialyzed against 50 mm Tris-HCl (pH 8), 0.5 m KCl, and 10% glycerol; quick-frozen in liquid nitrogen; and stored at -80 °C. The protein concentration of each preparation was determined by SDS-PAGE and Coomassie staining by comparison with optical density-determined bovine serum albumin standard titrated alongside. The recombinant proteins that were not susceptible to proteolysis during preparation were >90% pure as judged by SDS-PAGE and Coomassie staining. Proteins that suffered proteolysis in the bacteria or during purification showed additional bands on the gel smaller than that of the full-length protein. We confirmed that these bands were derived from the intact protein by Western blotting with anti-GST or anti-His tag antibody. Recombinant Proteins Expressed in Mammalian Cells—293T cells were transfected with plasmids encoding FLAG-tagged proteins (6 μgof plasmid and 60 μl of Effectene (QIAGEN Inc.) transfection reagent/100-mm plate. All subsequent steps were done on ice at 4 °C, and all buffers contained 2 μg/ml each leupeptin, aprotinin, and pepstatin; 0.2 mm phenylmethylsulfonyl fluoride; and 1 mm DTT. Cells were washed once with PBS; lysed in 10 mm Tris-HCl (pH 7.4), 800 mm NaCl, 2.5 mm MgCl2, and 0.5% Triton X-100 (2× 0.5 ml/100-mm plate); sonicated; and centrifuged at 16,000 × g for 10 min. The supernatant was added to anti-FLAG antibody M2-agarose beads (Sigma; 25-μl bead volume/plate of cells, washed with lysis buffer), and the volume was brought to 0.8 ml with lysis buffer. The slurry was rotated for 1 h, and beads were washed five times with lysis buffer before eluting the FLAG-tagged proteins in 20 mm Tris-HCl (pH 7.4), 100 mm NaCl, 2.5 mm MgCl2, and 0.4 mg/ml 3X FLAG® peptide (Sigma) for 1 h rotating. The eluate was collected by passing the slurry through a Micro Bio-Spin column (Bio-Rad). Proteins were dialyzed into PBS and 10% glycerol, quick-frozen in liquid nitrogen, and stored at -80 °C. Protein concentrations were determined as described for recombinant proteins expressed in bacteria. All FLAG-tagged proteins were >90% pure as judged by SDS-PAGE and Coomassie staining. RNA Probes—The plasmids used for production of 32P-labeled mRNA, U1 small nuclear RNA (snRNA), and tRNA were kindly provided by Dr. Naoyuki Kataoka (Kyoto University) and that for production of 5 S rRNA by Dr. Maarten Fornerod (Netherlands Cancer Institute). The mRNA (mouse immunoglobulin gene, coding exons C3 and C4 of the constant region of the IgM heavy chain) has 271 nucleotides; U1 snRNA (Xenopus) has 164 nucleotides;(tRNAiMet)(human) has 78 nucleotides; and 5 S rRNA (Xenopus) has 121 nucleotides. Plasmid DNA encoding each RNA was linearized with an appropriate restriction enzyme and in vitro transcribed with T7 or SP6 RNA polymerase (Promega), [α-32P]UTP, and [α-32P]GTP according to the manufacturer's instructions. The reactions were treated with DNase for 15 min at 37 °C. mRNA was purified using a G-50 spin column. U1 snRNA, tRNA, and 5 S rRNA were purified on a urea-5% polyacrylamide gel. The small interfering RNA (siRNA) probe is a 21-mer of chemically synthesized annealed sense and antisense nucleotides corresponding to nucleotides 2297–2315 of human NUP153 mRNA. Each strand has a 3′-dTT single-strand overhang. The siRNA probe was 5′-end-labeled with polynucleotide kinase (New England Biolabs Inc.) and [γ-32P]ATP according to the manufacturer's instructions. All radiolabeled RNAs were quantified by absorbance at 260 nm and ethidium bromide staining. DNA Probes—Methyl-dsDNA has 40 bp (GAM12) and contains 12 methyl-CGs (19Lewis J.D. Meehan R.R. Henzel W.J. Maurer-Fogy I. Jeppesen P. Klein F. Bird A. Cell. 1992; 69: 905-914Abstract Full Text PDF PubMed Scopus (1091) Google Scholar). The DNA was 5′-end-labeled with polynucleotide kinase and [γ-32P]ATP according to the manufacturer's instructions. Radiolabeled methyl-dsDNA was quantified by absorbance at 260 nm and ethidium bromide staining. Nucleic Acid·Protein Binding Assay—All reactions were performed in band-shift buffer (40 mm HEPES (pH 7.3), 110 mm KOAc, 6 mm MgOAc, and 250 mm sucrose) plus 1 mm DTT and 0.1% Nonidet P-40. RNA·protein binding assays contained 20 units of RNasin. Incubations with radiolabeled methyl-dsDNA included 0.1 mg/ml tRNA. In Fig. 4, the band-shift buffer included 0.2 mg/ml yeast tRNA and did not contain DTT or Nonidet P-40. In Fig. 8B, the band-shift buffer included 0.1 mg/ml yeast tRNA for the DNMT1 incubations. Reactions were incubated on ice for 20 min and analyzed by native 4 or 5% polyacrylamide (19:1 or 79:1 acrylamide/bisacrylamide) or 1% agarose gel electrophoresis. Gels were run in 0.5× Tris borate/EDTA at 4 °C (polyacrylamide, 2 h at 200 V; and agarose, 90 min at 130 V), dried, and autoradiographed.Fig. 8A, the DNMT protein family. The five DNMT genes have a conserved catalytic domain (green). The cysteine-rich domain (CXXCXXC), the BAH (bromo-adjacent homology) domain, the PWWP (Pro-Trp-Trp-Pro) domain, and the PHD/CXXC (plant homeodomain-like) zinc finger domain are indicated. B, a subset of the DNMT protein family forms RNP complexes. Recombinant GST-DNMT proteins were expressed in and purified from bacteria. DNMT1-N, DNMT1-M, and DNMT1-C are aa 1–300, aa 300–1000, and aa 1000–1620, respectively. Radiolabeled RNA (5 nm) was incubated with the purified proteins for 20 min on ice. The RNA and RNP complexes were analyzed by native 5% PAGE and autoradiography.View Large Image Figure ViewerDownload (PPT) Filter Binding Assay—Recombinant proteins and radiolabeled nucleic acid probes were as described for the nucleic acid·protein binding assay. Filter binding assays were done in filter binding buffer (10 mm Tris-HCl (pH 7.5), 100 mm KCl, and 2.5 mm MgCl2) with 5 nm radiolabeled nucleic acid and 8 units of RNasin for RNA probes. Assays were set up in triplicate, incubated for 30 min room temperature, and then applied to a Millipore HAW00010 membrane (mixture of cellulose acetate and cellulose nitrate) presoaked in filter binding buffer and assembled in a Fisher 96-well dot-blot manifold. After washing with filter binding buffer (∼30 ml/filter), the membrane was air-dried, analyzed by phosphorimaging (Storm 860, Amersham Biosciences), and quantified using ImageQuant software. Typically, maximum binding represents 50–80% input nucleic acid. The assays were not corrected for the fraction of recombin" @default.
• W2084223479 created "2016-06-24" @default.
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• W2084223479 date "2004-11-01" @default.
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• W2084223479 title "Components of the DNA Methylation System of Chromatin Control Are RNA-binding Proteins" @default.
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