MeCP2 is required for global heterochromatic and nucleolar changes during activity-dependent neuronal maturation Malaika K. Singleton, Michael L. Gonzales, Karen N. Leung, Dag H. Yasui, Diane I. Schroeder, Keith Dunaway, Janine M. LaSalle ⁎ Department of Medical Microbiology and Immunology, School of Medicine, Genome Center, and M.I.N.D. Institute, University of California, Davis, CA 95616, USA abstract article info Article history: Received 2 November 2010 Revised 24 January 2011 Accepted 11 March 2011 Available online xxxx Keywords: MeCP2 DNA methylation Maturation Chromatin Nucleoli Neuronal Epigenetic Neurodevelopmental Rett syndrome Mutations in MECP2, encoding methyl CpG binding protein 2, cause the neurodevelopmental disorder Rett syndrome. MeCP2 is an abundant nuclear protein that binds to chromatin and modulates transcription in response to neuronal activity. Prior studies of MeCP2 function have focused on specific gene targets of MeCP2, but a more global role for MeCP2 in neuronal nuclear maturation has remained unexplored. MeCP2 levels increase during postnatal brain development, coinciding with dynamic changes in neuronal chromatin architecture, particularly detectable as changes in size, number, and location of nucleoli and perinucleolar heterochromatic chromocenters. To determine a potential role for MeCP2 in neuronal chromatin maturational changes, we measured nucleoli and chromocenters in developing wild-type and Mecp2-deficient mouse cortical sections, as well as mouse primary cortical neurons and a human neuronal cell line following induced maturation. Mecp2-deficient mouse neurons exhibited significant differences in nucleolar and chromocenter number and size, as more abundant, smaller nucleoli in brain and primary neurons compared to wild-type, consistent with delayed neuronal nuclear maturation in the absence of MeCP2. Primary neurons increased chromocenter size following depolarization in wild-type, but not Mecp2-deficient cultures. Wild-type MECP2e1 over-expression in human SH-SY5Y cells was sufficient to induce significantly larger nucleoli, but not a T158M mutation of the methyl-binding domain. These results suggest that, in addition to the established role of MeCP2 in transcriptional regulation of specific target genes, the global chromatin-binding function of MeCP2 is essential for activity-dependent global chromatin dynamics during postnatal neuronal maturation. © 2011 Elsevier Inc. All rights reserved. Introduction Mutations in the methyl CpG binding protein 2 gene (MECP2) cause Rett Syndrome (RTT), a progressive, neurodevelopmental disorder in females that occurs in 1 in 10000–15000 births (Amir et al., 1999). MECP2 is located on the X-chromosome and a variety of different mutations and duplications are observed in a wider range of neurodevelopmental disorders in addition to RTT (Gonzales and LaSalle, 2010; Zoghbi, 2005). MeCP2 is an abundant nuclear protein expressed in numerous tissues and cell types throughout the body; however, the phenotypic consequences of MECP2 mutation are most apparent in the central nervous system (CNS) during postnatal brain development when expression is elevated (Balmer et al., 2003; Shahbazian et al., 2002; Skene et al., 2010). The neuronal nucleus undergoes dynamic, nonrandom changes in global chromatin during maturation that include changes in the location, size, and number of nuclear structures such as chromocen- ters and nucleoli (Manuelidis, 1984a; Manuelidis, 1985; Martou and De Boni, 2000; Solovei et al., 2004). Heterochromatic chromo- centers consist of transcriptionally silent DNA frequently positioned around nucleoli. Nucleoli are euchromatic structures formed by transcriptionally active ribosomal DNA. Therefore, measurements of nucleolar size can be considered a direct measurement of active rDNA transcription in the neuronal nucleus. Nucleoli have other essential functions in the nucleus including the regulation of mitosis and cellular stress responses (Boisvert et al., 2007). Chromocenters and nucleoli are intimately linked as chromocenters consist of transcrip- tionally silent ribosomal DNA (rDNA) and nucleoli contain chromatin remodeling proteins involved in the formation of heterochromatin domains (Akhmanova et al., 2000; Caperta et al., 2007; Guetg et al., 2010; Santoro et al., 2002). Neuronal nuclei undergo dynamic changes in compaction and histone deacetylation during postnatal neuronal maturation and MeCP2 has been shown to mediate changes to the local chromatin structure (Ishibashi et al., 2008; Nikitina et al., 2007a; Nikitina et al., 2007b; Thatcher and LaSalle, 2006). Brero et al. (2005) investigated Neurobiology of Disease xxx (2011) xxx–xxx Abbreviations: MeCP2, methyl-CpG binding protein 2; MeCP2e1, isoform of MeCP2; MBD, methyl-binding domain; RTT, Rett syndrome; DIV, days in vitro; rDNA, ribosomal DNA. ⁎ Corresponding author at: University of California, Medical Microbiology and Immunology, One Shields Avenue, Davis, CA 95616, USA. Fax: +1 530 752 8692. E-mail address: jmlasalle@ucdavis.edu (J.M. LaSalle). Available online on Direct (www.sciencedirect.com). YNBDI-02383; No. of pages: 11; 4C: 0969-9961/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2011.03.011 Contents lists available at ScienceDirect Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi Please cite this article as: Singleton, M.K., et al., MeCP2 is required for global heterochromatic and nucleolar changes during activity- dependent neuronal maturation, Neurobiol. Dis. (2011), doi:10.1016/j.nbd.2011.03.011