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Activity-dependent phosphorylation of MeCP2 threonine 308 regulates interaction with NCoR

Abstract

Rett syndrome (RTT) is an X-linked human neurodevelopmental disorder with features of autism and severe neurological dysfunction in females. RTT is caused by mutations in methyl-CpG-binding protein 2 (MeCP2), a nuclear protein that, in neurons, regulates transcription, is expressed at high levels similar to that of histones, and binds to methylated cytosines broadly across the genome1,2,3,4,5. By phosphotryptic mapping, we identify three sites (S86, S274 and T308) of activity-dependent MeCP2 phosphorylation. Phosphorylation of these sites is differentially induced by neuronal activity, brain-derived neurotrophic factor, or agents that elevate the intracellular level of 3′,5′-cyclic AMP (cAMP), indicating that MeCP2 may function as an epigenetic regulator of gene expression that integrates diverse signals from the environment. Here we show that the phosphorylation of T308 blocks the interaction of the repressor domain of MeCP2 with the nuclear receptor co-repressor (NCoR) complex and suppresses the ability of MeCP2 to repress transcription. In knock-in mice bearing the common human RTT missense mutation R306C, neuronal activity fails to induce MeCP2 T308 phosphorylation, suggesting that the loss of T308 phosphorylation might contribute to RTT. Consistent with this possibility, the mutation of MeCP2 T308A in mice leads to a decrease in the induction of a subset of activity-regulated genes and to RTT-like symptoms. These findings indicate that the activity-dependent phosphorylation of MeCP2 at T308 regulates the interaction of MeCP2 with the NCoR complex, and that RTT in humans may be due, in part, to the loss of activity-dependent MeCP2 T308 phosphorylation and a disruption of the phosphorylation-regulated interaction of MeCP2 with the NCoR complex.

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Figure 1: Phosphotryptic mapping of MeCP2 identifies activity-dependent phosphorylation sites.
Figure 2: Phosphorylation of T308 regulates MeCP2’s interaction with the NCoR complex.
Figure 3: MeCP2 T308A knock-in mice have altered activity-dependent gene expression.
Figure 4: Phosphorylation of MeCP2 T308 is a key mechanism underlying RTT.

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Acknowledgements

This work was supported by NIH grant RO1NS048276 and the Rett Syndrome Research Trust to M.E.G. D.H.E was supported by NIH grant K08MH90306, the Dupont–Warren Fellowship in the Department of Psychiatry at Harvard Medical School, and the Nancy Lurie Marks Fellowship in Autism at Harvard Medical School. H.W.G. was supported by Damon Runyon Cancer Research Foundation Grant DRG-2048-10. The Mouse Gene Manipulation Facility of the Boston Children’s Hospital Intellectual and Developmental Disabilities Research Center (IDDRC), funded by NIH grant P30-HD 18655, assisted in generation of the knock-in mice. We thank members of the Greenberg laboratory, particularly C. Mandel-Brehm and E. Griffith, and also G. Mandel and R. S. Greenberg for helpful discussions.

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D.H.E. and M.E.G. conceived and designed the experiments and wrote the manuscript. D.H.E. performed or directed all the experiments in the manuscript. D.H.E., N.D.R. and N.R.K. generated and characterized the MeCP2 T308A knock-in and R306C knock-in mice and characterized activity-dependent phosphorylation of MeCP2. H.W.G. performed the ChIP analysis, and H.W.G. and S.C. performed experiments investigating activity-dependent phosphorylation of MeCP2 that informed this study. D.H.E., L.S.H. and N.D.R. developed the phospho-site-specific antibodies, and A.J.N. assisted in early work with these antibodies during summer rotations. M.J.L., R.E. and A.P.B. discovered that the RTT missense mutation R306C disrupted both the interaction with NCoR and MeCP2’s ability to provide transcription repression using the luciferase reporter assay. All authors reviewed the manuscript.

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Correspondence to Michael E. Greenberg.

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Ebert, D., Gabel, H., Robinson, N. et al. Activity-dependent phosphorylation of MeCP2 threonine 308 regulates interaction with NCoR. Nature 499, 341–345 (2013). https://doi.org/10.1038/nature12348

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