MeCP2 Binds Cooperatively to Its Substrate and Competes with Histone H1 for Chromatin Binding Sites

Abstract

Sporadic mutations in the hMeCP2 gene, coding for a protein that preferentially binds symmetrically methylated CpGs, result in the severe neurological disorder Rett syndrome (RTT). In the present work, employing a wide range of experimental approaches, we shed new light on the many levels of MeCP2 interaction with DNA and chromatin. We show that strong methylation-independent as well as methylation-dependent binding by MeCP2 is influenced by DNA length. Although MeCP2 is strictly monomeric in solution, its binding to DNA is cooperative, with dimeric binding strongly correlated with methylation density, and strengthened by nearby A/T repeats. Dimeric binding is abolished in the F155S and R294X severe RTT mutants. MeCP2 also binds chromatin in vitro, resulting in compaction-related changes in nucleosome architecture that resemble the classical zigzag motif induced by histone H1 and considered important for 30-nm-fiber formation. In vivo chromatin binding kinetics and in vitro steady-state nucleosome binding of both MeCP2 and H1 provide strong evidence for competition between MeCP2 and H1 for common binding sites. This suggests that chromatin binding by MeCP2 and H1 in vivo should be viewed in the context of competitive multifactorial regulation.

We thank Joseph Wall and Martha Simon of the Brookhaven National Laboratory STEM Facility for carrying out the mass measurements and assisting with the data analysis, David Brown, University of Mississippi Medical Center, Jackson, MS, for the H1⁰-ACA plasmid and the H1⁰-GFP BALB/c 3T3 cell line, Carolyn Schanen, Department of Biological Sciences, University of Delaware, Newark, DE, and Asmita Kumar, Nemours Biomedical Research, Alfred I. duPont Hospital for Children, Wilmington, DE, for BALB/c 3T3 cells expressing MeCP2-GFP, Christina Cardoso, Max Delbruck Center for Molecular Medicine, Germany, for the MeCP2-mRFP overexpression plasmid, Sergei Grigoryev, Pennsylvania State University School of Medicine, Hershey, PA, for the 601-1 and 601-4 plasmids, Tom Misteli, National Cancer Institute, NIH, Bethesda, MD, for CHO cells expressing HP1α-GFP, and Yuri L. Lyubchenko, Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, for AFM facilities and advice.

This work was supported by the Rett Syndrome Research Foundation and NIH GM070897 (C.L.W.), NSF 0701892 (Yuri L. Lyubchenko), and the Nebraska Research Initiative (L.S.S. and Yuri L. Lyubchenko).