The MeCP1 complex represses transcription through preferential binding, remodeling, and deacetylating methylated nucleosomes

Abstract

Histone deacetylation plays an important role in methylated DNA silencing. Recent studies indicated that the methyl-CpG-binding protein, MBD2, is a component of the MeCP1 histone deacetylase complex. Interestingly, MBD2 is able to recruit the nucleosome remodeling and histone deacetylase, NuRD, to methylated DNA in vitro. To understand the relationship between the MeCP1 complex and the NuRD complex, we purified the MeCP1 complex to homogeneity and found that it contains 10 major polypeptides including MBD2 and all of the known NuRD components. Functional analysis of the purified MeCP1 complex revealed that it preferentially binds, remodels, and deacetylates methylated nucleosomes. Thus, our study defines the MeCP1 complex, and provides biochemical evidence linking nucleosome remodeling and histone deacetylation to methylated gene silencing.

Figure 1

MBD2 and NuRD copurify with a methyl-CpG-binding activity as a 1MD protein complex. (A) Western blot analysis of fractions derived from a gel filtration Superose-6 column. The elution profiles of the protein size markers and the proteins analyzed are indicated. (B,C) Gel mobility shift assays using the same fractions as in panel A. The probes used in panels B and C are methylated and nonmethylated, respectively. Methylation-dependent shift and free probes are indicated.

Figure 2

The MeCP1 complex contains MBD2 and NuRD. (A) Schematic representation of the steps used to purify the MeCP1 complex. The range of salt concentrations where the MeCP1 complex eluted out of the columns is indicated. (B) Western blot analysis of the fractions derived from DEAE-5PW column. (C) Gel mobility shift assay of the fractions derived from the last purification step. Methylation-dependent shift and free probes are indicated. (D) A silver-stained gel showing that a group of 10 polypeptides copurify with the methyl-CpG-binding activity. The identities of these polypeptides are confirmed by Western blot analysis, shown in panel E.

Figure 3

MBD2 and NuRD exist in the same protein complex. (A) A silver-stained gel showing that the same 10 polypeptides are immunoprecipitated by both MTA2 and MBD2 antibodies. MBD2 has characteristically faint staining with silver (Figs. 2D,3A), but stains better with Coomassie (Fig. 3C). The identities of the proteins and size markers are indicated. (B) Western blot analysis of the same samples used for silver staining in panel A. Antibodies used for immunoprecipitation and Western blotting are indicated on the top and left of the panel, respectively. (C) Coomassie staining of the MeCP1 complex. (D) Mi2 enhances MBD2-mediated transcription repression. The amount of reporter and effector plasmids used in each transfection is indicated. Transfection efficiencies were normalized using β-galactosidase assays. The data shown represent the average of two independent experiments. Variations between experiments are depicted by the error bars.

Figure 4

MeCP1 is able to preferentially bind, remodel, deacetylate and repress transcription from methylated nucleosomal templates. (A) Gel mobility shift assay comparing the binding of MeCP1 to methylated and nonmethylated DNA and nucleosomes. The four probes used are indicated on top of the panel. (B) Mononucleosome disruption assay comparing the efficiency of MeCP1 to disrupt methylated and nonmethylated nucleosomes. End-labeled mononucleosomes with a concentration of 10 nM were incubated with increasing concentrations of MeCP1, followed by DNase I digestion. The presence or absence of ATP in the reactions is indicated. Open circles indicate enhanced DNase I digestion; filled circles indicate reduced DNase I digestion. (C) Histone deacetylase assays comparing the efficiency of MeCP1 to deacetylate methylated or nonmethylated nucleosomal histones. Approximately 300 ng of ³H-labeled acetylated core histones was assembled into mononucleosomes with methylated or nonmethylated DNA (final concentration of 80 nM) and were used in a 30 μL reaction with or without the presence of 5 μL MeCP1 (final concentration of 20 nM) and 2 mM of ATP. The bar graph represents an average of two independent experiments. Variations between the two experiments are depicted by the error bars. (D) Expression of an Mi2 mutant, crippled in ATP-binding, partially relieved methylation-dependent transcriptional repression. Shown are relative luciferase activities of different transfections. M− and M + indicate that the reporter plasmid CG11–pGL12 is mock- or HhaI-methylated, respectively. The data shown represent the average of two independent experiments. Variations between experiments are depicted by the error bars. The insert is a Western blot probed with antibody against Flag.

Figure 5

Model depicting the relationship between the NuRD and the MeCP1 complexes and how each might be recruited to distinct gene promoters by either methylation-specific or sequence-specific DNA-binding proteins.