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Originally published In Press as doi:10.1074/jbc.M409070200 on September 1, 2004

J. Biol. Chem., Vol. 279, Issue 47, 49479-49487, November 19, 2004
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Components of the DNA Methylation System of Chromatin Control Are RNA-binding Proteins*

Linda Jeffery and Sara Nakielny{ddagger}

From the Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom

Received for publication, August 9, 2004 , and in revised form, August 31, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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)1 (1–4). 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 (5). Analysis of these embryos showed that DNA methylation is essential for proper imprinting (6, 7), silencing of transposable elements (8), and X chromosome dosage compensation (9, 10). Mice lacking DNMT3a die prematurely, and embryos without DNMT3b do not develop to term (11). Conditional removal of DNMT3a in germ cells revealed that it is essential for imprinting (12). DNMT3L is also necessary for imprinting (13, 14). 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 (3, 15). Methylation is dynamic in a developing mammal: shortly after fertilization, the male genome is actively demethylated by as yet to be identified demethylase(s) (16, 17), 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 (18). 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 (2). 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 (3).

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) (19–21). 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 (22–28). 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 (23, 24, 29–31). MBD1 and methyl-CpG-binding protein-2 (MeCP2) can recruit histone methyltransferases and other proteins that affect chromatin activity (32–34), and MeCP2 can condense unmethylated chromatin in vitro (35). These properties could contribute to histone deacetylase-independent silencing.



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  		FIG. 1.
The MBD protein family. Five proteins containing a MBD have been characterized in mammals. Mouse (MBD1–4) and rat (MeCP2) proteins are shown here. The MBDs share both amino acid and functional homology, whereas the transcription repression domains (TRD) share functional homology. The transcription repression domain recruits histone deacetylase complexes to silence chromatin. The MBD of MBD2 overlaps the transcription repression domain and is indicated by the line above the protein. Data base mining revealed additional mammalian genes with MBD-like domains. It is not known if these proteins participate in DNA methylation-mediated chromatin control (3).

 
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 (36). 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 (37, 38). MBD2 is also needed for intestinal tumorigenesis in mice (39). Lack of MBD3 is lethal for an embryo (37), and MBD4-null mice have increased DNA mutability, reflecting the mismatch repair activity of MBD4 (40–43). Dysfunctional MeCP2 results in neuronal cell defects that cause Rett's syndrome in humans (44, 45) and a similar phenotype in mice (46, 47). A double null of MeCP2 and MBD2 has an additive phenotype, indicating there is no functional overlap between these family members (47).

Genes or intergenic regions of chromatin under the control of MBD proteins have been obscure (2, 3, 48). Recently, two genes controlled by MeCP2 were identified. Meehan and co-workers (49) 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 (50–52). 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 (20, 53). 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 (20, 54, 55).

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 (15). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 (56). 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 {beta}-D-thiogalactopyranoside (pGEX vectors) or 1 mM isopropyl {beta}-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 x 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 2x 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 (2x 0.5 ml/100-mm plate); sonicated; and centrifuged at 16,000 x 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; (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), [{alpha}-32P]UTP, and [{alpha}-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 [{gamma}-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 (19). The DNA was 5'-end-labeled with polynucleotide kinase and [{gamma}-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 0.5x Tris borate/EDTA at 4 °C (polyacrylamide, 2 h at 200 V; and agarose, 90 min at 130 V), dried, and autoradiographed.



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  		FIG. 4.
MBD2 and MeCP2 proteins interact with particular classes of RNA. The indicated radiolabeled RNAs (5 nM) were incubated with purified recombinant MBD proteins or a double-stranded RBD (dsRBD) from staufen for 20 min on ice. The RNA and RNP complexes were analyzed by native 5% PAGE and autoradiography.

 



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  		FIG. 8.
A, 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.

 
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 HAW00010membrane (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 recombinant protein that was competent for binding.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The MBD2 protein contains a long stretch of RG repeats within its N terminus, and MeCP2 has a shorter RG repeat region (Fig. 1). This feature is commonly found in proteins that interact with RNA and in proteins that are components of RNP complexes (57, 58). This prompted us to test whether MBD2, MeCP2, and the other members of the MBD protein family are capable of interacting with RNA. MBD proteins were expressed in and purified from bacteria. Radiolabeled mRNA produced by in vitro transcription was incubated with purified proteins, and the incubations were analyzed by nondenaturing gel electrophoresis (Fig. 2). MBD1, MBD2, and MeCP2 were all able to form RNP complexes under these conditions. We focused on MBD2 and MeCP2, the RG domain-containing MBD family members, because they appeared to bind RNA with high affinity.



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  		FIG. 2.
A subset of the MBD protein family forms RNP complexes. Radiolabeled RNA (5 nM) was incubated with the indicated purified recombinant proteins for 20 min on ice. The RNA and RNP complexes were analyzed by native 5% PAGE and autoradiography.

 
MBD proteins are known to have high affinity for only one nucleic acid in vivo, genomic methyl-CG dsDNA. We therefore wanted to compare the affinity of MBD proteins for methyl-CG dsDNA and RNA. Using a filter binding assay, it is evident that MBD2 and MeCP2 interact with RNA at least as avidly as with methyl-dsDNA (Fig. 3). With these particular reporter nucleic acids, MBD2 had higher affinity for RNA than for methyl-dsDNA.



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  		FIG. 3.
MBD proteins bind RNA with nanomolar affinity. Purified recombinant GST-MBD2 and GST-MeCP2 proteins were incubated with 5 nM radiolabeled mRNA (271 nucleotides) (•) or methyl-dsDNA (40 bp) containing 12 methyl-CGs ({square}) for 30 min at room temperature. The mixture was passed over cellulose acetate/nitrate membrane to retain nucleic acid·protein complexes and to allow unbound nucleic acids to pass through. After washing, the membranes were analyzed on a PhosphorImager. The data points are triplicate assays and are representative of at least three independent experiments.

 
To address the specificity of the RNA·MBD protein interaction, we produced radiolabeled probes representative of each major class of cellular RNA, mRNA, U snRNA, rRNA, tRNA, and siRNA, and investigated whether MBD2 and MeCP2 have any affinity for these RNAs in vitro. The MBD proteins were able to form RNP complexes with mRNA and siRNA, but not with U snRNA, rRNA, or tRNA (Fig. 4). The interactions of MBD2 and MeCP2 with siRNA were as robust as that of the canonical double-stranded RBD of the staufen protein (Fig. 4). We conclude that a subset of the MBD protein family is able to interact with mRNA and double-stranded siRNA molecules in vitro.

Both MBD2 and MeCP2 are basic proteins, and many proteins that are positively charged at physiological pH will bind nucleic acids nonspecifically via interactions with the negatively charged phosphate backbone. However, the RNP complexes formed by MBD proteins depend on specific nucleic acid·protein contacts because they bind only two classes of RNA in vitro, mRNA and double-stranded siRNA. If the interactions were solely via positively charged amino acids and nucleic acid phosphate backbones, MBD proteins would interact with all nucleic acids.

We next investigated whether the MBD is involved in the RNA-binding activity of MBD proteins. Wild-type MeCP2 and a point mutant of MeCP2 that is known to cause defective methyl-dsDNA interactions (59, 60) were FLAG-tagged and expressed in and purified from mammalian cells. These recombinant MeCP2 proteins were analyzed for their ability to form RNP and methyl-dsDNA complexes. As expected, wild-type MeCP2 formed methyl-dsDNA complexes, whereas the point mutant did not (Fig. 5A). However, the methyl-dsDNA binding-defective MeCP2 mutant retained its ability to bind siRNA. The isolated MBD did not bind double-stranded siRNA, whereas, as expected, it bound methyl-dsDNA (Fig. 5B). This shows that the RNA·MBD protein interactions we observed cannot be explained by a low affinity nonspecific interaction with the methyl-dsDNA-binding domain of these proteins. The MBD did not bind RNA under these conditions. These MBD proteins therefore interact with siRNA and methyl-dsDNA using distinct domains.



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  		FIG. 5.
MBD proteins interact with methyl-dsDNA and RNA via distinct domains. Recombinant FLAG-tagged proteins expressed and purified from mammalian cells were incubated with radiolabeled nucleic acids (5 nM) for 20 min on ice. The nucleic acids and nucleic acid·protein complexes were analyzed by native 1% agarose gel electrophoresis and autoradiography. A, FLAG-tagged wild-type MeCP2 (WT) or point mutant MeCP2 (R106W (106), which cannot interact with methyl-dsDNA is still able to form a complex with siRNA. B, the isolated FLAG-tagged MBD (aa 148–221) of MBD2 interacts with methyl-dsDNA as expected, but cannot interact with siRNA.

 
To further characterize the RNA-binding activity of the MBD proteins, we investigated whether the RG domains contribute to RNP complex formation. Using FLAG-MBD2 and FLAG-MBD2 RGdel expressed in and purified from mammalian cells, we analyzed siRNA·protein complex formation. The ability of MBD2 to bind siRNA was abolished when the RG domain was removed (Fig. 6). The MeCP2 RG deletion mutant was similarly compromised in RNP complex formation (data not shown). We conclude that the MBD proteins form RNP and methyl-dsDNA·protein complexes via distinct protein surfaces and that the RG domains are essential for RNP complex formation.



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  		FIG. 6.
The RG domain of MBD2 is essential for siRNA·protein complex formation. FLAG-tagged proteins were expressed in and purified from mammalian cells and incubated with radiolabeled nucleic acids (5 nM) for 20 min on ice. The nucleic acids and nucleic acid·protein complexes were analyzed by 1% agarose gel electrophoresis and autoradiography. TRD, transcription repression domain; RGdel, RG domain deletion.

 
We note that MBD2 and MeCP2 expressed in and purified from mammalian cells (Figs. 5 and 6) need to be used at higher concentrations than their bacterially expressed counterparts. This could be due to post-translational modifications of MBD proteins in the eukaryotic cell expression system or to copurification of the MBD proteins with cellular RNA targets in mammalian cells.

We tested whether the MBD proteins are able to interact with methyl-dsDNA and siRNA simultaneously or in a mutually exclusive way. Methyl-dsDNA·MeCP2 protein complexes were formed and then incubated with unlabeled siRNA. The bound methyl-dsDNA was bumped off the protein by siRNA (Fig. 7). Only the double-stranded siRNA has the ability to compete with methyl-dsDNA for MeCP2 binding. This was also observed for MBD2, when the RNA was radiolabeled and the RNA·MBD protein complex was incubated with unlabeled methyl-dsDNA, and when the incubations were analyzed on gels of a range of polyacrylamide and agarose concentrations (data not shown). We did not detect a trimeric methyl-dsDNA·MBD protein·siRNA complex. Therefore, under these conditions, MBD2 and MeCP2 bind methyl-dsDNA and double-stranded siRNA in a mutually exclusive manner.



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  		FIG. 7.
The RNA and methyl-dsDNA binding properties of MeCP2 are mutually exclusive. FLAG-MeCP2 was expressed in and purified from mammalian cells, and 30 nM was incubated with 5 nM radiolabeled methyl-dsDNA for 20 min on ice. The incubations were continued for an additional 10 min in the absence or presence of a 100, 500, or 1000-fold molar excess (over methyl-dsDNA) of unlabeled 21-mer siRNA, double-stranded or single-stranded (ssRNA). The nucleic acid and nucleic acid·MeCP2 complexes were analyzed by native 4% PAGE and autoradiography.

 
The ability of MBD proteins to form RNP complexes suggests that RNA molecules play some role in the DNA methylation system of chromatin control. We investigated whether any of the mammalian DNMTs (Fig. 8A) are capable of forming RNP complexes. Purified recombinant GST-DNMTs were incubated with a reporter siRNA, and their ability to form stable RNP complexes was analyzed by native gel electrophoresis. DNMT3a and DNMT3b1 interacted with the siRNA probe. At the highest concentrations of protein that we were able to test, DNMT1 domains and DNMT2 did not (Fig. 8B). DNMT3L was also unable to interact with this reporter RNA (data not shown). Importantly, the RNA·DNMT complexes were formed without regard to the pI of the DNMT. The DNMT-C domain has a pI of 9.1, yet it did not bind RNA under these conditions. Conversely, DNMT3a has a pI of 6.3, but readily formed a complex with RNA. We conclude that the RNP complexes formed by DNMTs in vitro are reflective of an ability to interact with RNA in a class- and/or sequence-specific way.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown that several proteins with well defined functions in the DNA methylation system of chromatin control are capable of forming complexes with RNA. Several processes mediated by methylation of chromatin involve noncoding RNA: X chromosome dosage compensation, imprinting, and possibly silencing of repetitive DNA elements. This, together with our identification of a cellular noncoding RNA in MBD2 protein complexes purified from mammalian cells, which will be reported elsewhere,2 indicates that noncoding RNAs are the best candidates for the in vivo RNA components of other MBD protein and DNMT RNP complexes.

The RG region in the MBD proteins is critical for RNP complex formation. We did not characterize the RNA interaction surface any further, and we have not attempted here to identify an RBD in the DNMTs. There are several reasons for this. First, the RNA interaction domain must be defined using a physiologically relevant RNA. The MBD protein RNP complexes that we have purified from cells are allowing us to map the RNA interaction surface using physiological RNA molecules.2 Second, and related to the first reason, it is possible that, in the cell, the MBD protein RNP complexes are formed by a complex of proteins that includes an MBD family member. The complete RNA-binding surface would then be presented by the complex, whose components adjust the specificity and strength of the MBD protein RNA interaction. In the case of U2AF (a heterodimer of 65- and 35-kDa subunits), the 65-kDa subunit binds polypyrimidine tracts, and only when assembled into the 65/35-kDa heterodimer does U2AF specifically recognize 3'-splice sites (61). It is therefore important to define protein·RNA contacts in the context of a physiological RNP complex.

Apart from the RG repeat regions in MBD2 and MeCP2, there is no obvious indication from the primary amino acid sequences of the MBD proteins or the DNMTs that they could be able to interact with RNA. The characteristics of protein·RNA interactions are many and varied (62–65). Lack of homology to any previously defined RBD does not necessarily mean absence of a specific RNA interaction surface. The mRNA export factor TAP shows no indication of an RBD at the primary sequence level. However, the three-dimensional structure reveals a canonical RBD (66). Conversely, sequence homology to a particular class of RBD does not necessarily mean functional homology. This is the case for Y14, a protein that participates in several aspects of mRNA·protein complex processing and localization. The Y14 "RBD" is in fact a protein interaction domain, although an interaction with RNA at some stage of its function in mRNA·protein complex metabolism cannot be discounted (67, 68).

Since recombinant proteins were used in the nucleic acid interaction assays, our results show that RNA binding to these reporter RNAs is an intrinsic property of the MBD proteins and requires no additional protein cofactors. It will be of interest to see if RNA has any impact on the integrity/activity of the MBD protein·histone deacetylase complexes that silence chromatin or if the RG domain mutants are compromised in their silencing function.

Although a number of proteins have been reported to interact with both DNA and RNA in vitro, very few have been shown to have the capacity to interact with both types of nucleic acid in vivo (69). Three proteins clearly have the ability to form specific and functional DNA·protein and RNA·protein complexes. Transcription factor IIIA controls the expression of the 5 S rRNA gene. It also binds to 5 S rRNA, forming a 5 S rRNA·protein storage complex (70). Ku protein binds to telomeric DNA in a protective capacity, but also makes direct contact with telomerase RNA. Bicoid, a homeodomain protein, contacts DNA in its transcription factor capacity and also manifests as a translation control protein in complex with the 3'-untranslated region of mRNA (71).

This leads to a final point of discussion: why would MBD proteins and DNMTs form RNP complexes? There are several possibilities. RNA molecules could provide specificity to DNMTs (which regions of the genome to methylate) or specificity to MBD proteins (which methyl-CG regions to bring histone deacetylase silencing complexes to). RNA could also function in an architectural/nucleation capacity, providing a platform for the assembly of specific chromatin control proteins. In the case of the MBD proteins, our finding that interaction with RNA and methyl-DNA is mutually exclusive suggests that there may be an exchange of RNA for methyl-DNA when the RNP complex comes into contact with chromatin, or that RNA is a negative regulator of MBD proteins, at least with regard to chromatin. It is also conceivable that the RNP complexes formed by MBD proteins or DNMTs function in a different aspect of cell biology that is not directly connected to their well characterized roles in chromatin control.


   FOOTNOTES
 
* This work was supported by Cancer Research UK. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 44-207-269-3459; Fax: 44-207-269-3417; E-mail: Sara.Nakielny{at}cancer.org.uk.

1 The 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. Back

2 S. Kitao and S. Nakielny, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Haruhiko Siomi and Alain Verreault for encouragement and critical reading of the manuscript and the people mentioned under "Experimental Procedures" for plasmids.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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