Rett syndrome mutations abolish the interaction of MeCP2 with the NCoR/SMRT co-repressor

Abstract

Rett syndrome (RTT) is a severe neurological disorder that is caused by mutations in the MECP2 gene. Many missense mutations causing RTT are clustered in the DNA-binding domain of MeCP2, suggesting that association with chromatin is critical for its function. We identified a second mutational cluster in a previously uncharacterized region of MeCP2. We found that RTT mutations in this region abolished the interaction between MeCP2 and the NCoR/SMRT co-repressor complexes. Mice bearing a common missense RTT mutation in this domain exhibited severe RTT-like phenotypes. Our data are compatible with the hypothesis that brain dysfunction in RTT is caused by a loss of the MeCP2 'bridge' between the NCoR/SMRT co-repressors and chromatin.

Figure 1

Missense RTT mutations in the MECP2 gene occur predominantly in two discrete clusters. Missense mutations present in RTT patients, but absent in their parents, map to the MBD and the C terminus of the TRD. The asterisk indicates the domain that we analyzed. The map below shows neutral variants that are present in a population sampled by exome sequencing.

Figure 2

MeCP2 interacts with the NCoR/SMRT corepressor complex via a domain that coincides with a previously unknown cluster of RTT mutations. (a) Immunoprecipitation (IP) of EGFP, NCoR1, SMRT, TBLR1, HDAC3 and SIN3A from extracts of Mecp2-EGFP (MG) knock-in mouse brain using an antibody to EGFP. Immunoprecipitations from an extract from wild-type (M) brain (lacking fused EGFP) and H2B-EGFP (HG) brain are controls. i, inputs. (b) Mapping of the NID by expression of FLAG-tagged fragments of MeCP2 in HeLa cells. Immunoprecipitations with antibody to FLAG were probed on western blots. Arrows compare immunoprecipitation efficiency of the 143–309 and 143–308 fragments. (c) Map summarizing the results shown in b. Vertical lines denote missense mutations and the striped bar corresponds to the minimal NID. (d) Missense mutations that cause RTT abolish the interaction between MeCP2 and NCoR/SMRT. Full-length FLAG-tagged MeCP2 containing the point mutations shown was expressed in HeLa cells, purified with antibody to FLAG and probed on western blots. (e) A biotin-tagged peptide comprising residues 285–319 of MeCP2 bound (B) to an in vitro–translated FLAG-tagged N-terminal domain of NCoR1 (197–598) and SMRT (187–586) and to full-length TBL1 and TBLR1, but failed to bind the C-terminal domain of NCoR1 (2,000–2,337). Interactions were all greatly reduced or abolished by the R306C mutation. WT, wild type. All blots in this figure are representative of at least two biological replicate experiments involving recovery based on protein affinity from independent mouse brains, HeLa cell transfections or in vitro translation reactions. Uncropped blots are presented in Supplementary Figure 8.

Figure 3

Mice with an Mecp2^R306C knock-in allele lose the MeCP2-NCoR/SMRT interaction and exhibit a severe neurological phenotype. (a) Representative western blot of whole brain wild-type MeCP2 and MeCP2^R306C from 5-week-old mice. Histone H3 is a control for equal loading. The histogram shows the ratio of MeCP2^R306C to wild-type protein (1.05) averaged over three biological replicates based on separate protein preparations from independent pairs of mouse brains (s.e.m. = 0.17). The ratios from individual pairs of brains are also plotted. (b) Extracts from brain nuclei of 5-week-old wild-type and Mecp2^R306C mice were immunoprecipitated with antibody to MeCP2 and the resulting western blots probed with antibodies to the NCoR/SMRT components NCoR1, TBLR1 and HDAC3. The weak interaction with the SIN3A co-repressor was not affected by this mutation. Blots are representative of two biological replicate experiments involving separate immunopurifications from independent mouse brain samples. Uncropped blots are presented in Supplementary Figure 8. (c) Mice expressing knocked-in Mecp2^R306C showed a hindlimb clasping phenotype at 6 weeks of age. (d) Phenotypic scoring (see Online Methods) was performed blind to genotype on littermates from two independent cohorts of mice at postnatal ages of 6 (n = 11) and 9 (n = 12) weeks. Phenotypic scores are mean ± s.e.m. (e) Distance traveled in an open field during a 30-min interval was measured and the data were analyzed for wild-type (n = 9) and Mecp2^R306C (n = 10) 15–18-week-old littermates. Plotted data points correspond to individual mice. (f) Latency to fall from an accelerating rotarod was measured and the data analyzed for wild-type (n = 9) and Mecp2^R306C (n = 10) 15–18-week-old littermates. Plotted data points correspond to individual mice. (g) Brain weight (left) and whole body weight (right) were determined for wild-type (n = 11) and Mecp2^R306C (n = 11) 15–18-week-old mice. Plotted data points correspond to individual mice. The P values shown in e–g were obtained by two-sided t test.

Figure 4

Mutations in either the MBD or NID interfere with localization and transcriptional repression by MeCP2. (a) Mouse ES cells carrying knocked-in wild-type Mecp2, Mecp2^T158M or Mecp2^R306C expressed as a fusion with EGFP differentiated to >85% neurons as measured by NeuN staining. DAPI-stained spots correspond to heterochromatin rich in 5-methylcytosine. Scale bar represents 10 μm. (b) Immunoprecipitation with antibody to EGFP from neuronal nuclear extracts of the three genotypes. MeCP2-bound endogenous proteins were assayed on western blots. i, input. Uncropped blots are presented in Supplementary Figure 8. (c) Wild-type MeCP2-EGFP recruited the shared NCoR/SMRT subunit TBL1-mCherry to densely methylated heterochromatic foci in mouse fibroblasts (top row), but the MeCP2^R306C NID mutation prevented recruitment (middle row). Data is from duplicate experiments showing efficient TBL1 recruitment in 55% of cells expressing wild-type MeCP2-EGFP (n = 219) compared with 0% recruitment by both MeCP2^R306C-EGFP (n = 106) and EGFP alone (n = 100). Scale bar represents 10 μm. (d) N-terminal and C-terminal halves of MeCP2 were fused to GAL4 DNA-binding domains and co-transfected with a luciferase reporter gene containing GAL4-binding sites. The effect of RTT missense mutations on luciferase expression was monitored (dark gray bars). TSA, trichostatin A. Error bars represent s.e.m. on the basis of 4–9 replicate experiments. **P < 0.01 and ***P < 0.001, compared with repression by the wild-type C-terminal half of MeCP2 (two-sided t test).