MeCP2 Dependent Heterochromatin Reorganization during Neural Differentiation of a Novel Mecp2-Deficient Embryonic Stem Cell Reporter Line

PLOS ONE, Dec 2019

The X-linked Mecp2 is a known interpreter of epigenetic information and mutated in Rett syndrome, a complex neurological disease. MeCP2 recruits HDAC complexes to chromatin thereby modulating gene expression and, importantly regulates higher order heterochromatin structure. To address the effects of MeCP2 deficiency on heterochromatin organization during neural differentiation, we developed a versatile model for stem cell in vitro differentiation. Therefore, we modified murine Mecp2 deficient (Mecp2−/y) embryonic stem cells to generate cells exhibiting green fluorescent protein expression upon neural differentiation. Subsequently, we quantitatively analyzed heterochromatin organization during neural differentiation in wild type and in Mecp2 deficient cells. We found that MeCP2 protein levels increase significantly during neural differentiation and accumulate at constitutive heterochromatin. Statistical analysis of Mecp2 wild type neurons revealed a significant clustering of heterochromatin per nuclei with progressing differentiation. In contrast we found Mecp2 deficient neurons and astroglia cells to be significantly impaired in heterochromatin reorganization. Our results (i) introduce a new and manageable cellular model to study the molecular effects of Mecp2 deficiency, and (ii) support the view of MeCP2 as a central protein in heterochromatin architecture in maturating cells, possibly involved in stabilizing their differentiated state.

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MeCP2 Dependent Heterochromatin Reorganization during Neural Differentiation of a Novel Mecp2-Deficient Embryonic Stem Cell Reporter Line

et al. (2012) MeCP2 Dependent Heterochromatin Reorganization during Neural Differentiation of a Novel Mecp2-Deficient Embryonic Stem Cell Reporter Line. PLoS ONE 7(10): e47848. doi:10.1371/journal.pone.0047848 MeCP2 Dependent Heterochromatin Reorganization during Neural Differentiation of a Novel Mecp2 -Deficient Embryonic Stem Cell Reporter Line Bianca Bertulat 0 Maria Luigia De Bonis 0 Floriana Della Ragione 0 Anne Lehmkuhl 0 Manuela Milden 0 Christian Storm 0 K. Laurence Jost 0 Simona Scala 0 Brian Hendrich 0 Maurizio D'Esposito 0 M. Cristina Cardoso 0 Pierre-Antoine Defossez, Universite Paris-Diderot, France 0 1 Department of Biology, Technische Universita t Darmstadt , Darmstadt, Germany , 2 Institute of Genetics and Biophysics ''A Buzzati Traverso'' , Naples , Italy , 3 Istituto di Ricovero e Cura a Carattere Scientifico Neuromed , Pozzilli , Italy , 4 Wellcome Trust - Medical Research Council Stem Cell Institute and Department of Biochemistry, University of Cambridge , Cambridge , United Kingdom The X-linked Mecp2 is a known interpreter of epigenetic information and mutated in Rett syndrome, a complex neurological disease. MeCP2 recruits HDAC complexes to chromatin thereby modulating gene expression and, importantly regulates higher order heterochromatin structure. To address the effects of MeCP2 deficiency on heterochromatin organization during neural differentiation, we developed a versatile model for stem cell in vitro differentiation. Therefore, we modified murine Mecp2 deficient (Mecp22/y) embryonic stem cells to generate cells exhibiting green fluorescent protein expression upon neural differentiation. Subsequently, we quantitatively analyzed heterochromatin organization during neural differentiation in wild type and in Mecp2 deficient cells. We found that MeCP2 protein levels increase significantly during neural differentiation and accumulate at constitutive heterochromatin. Statistical analysis of Mecp2 wild type neurons revealed a significant clustering of heterochromatin per nuclei with progressing differentiation. In contrast we found Mecp2 deficient neurons and astroglia cells to be significantly impaired in heterochromatin reorganization. Our results (i) introduce a new and manageable cellular model to study the molecular effects of Mecp2 deficiency, and (ii) support the view of MeCP2 as a central protein in heterochromatin architecture in maturating cells, possibly involved in stabilizing their differentiated state. - Funding: This work was supported in part by grants of the European E-RARE EuroRett network (BMBF grant 01GM0811) and the Deutsche Forschungsgemeinschaft (grant CA198/7-1) to M.C. Cardoso. M. DEsposito was supported by the UE Initial Training Network Project nu238242 DISCHROM and by the EPIGENOMICS FLAGSHIP PROJECT EPIGEN, MIUR-CNR. S. Scala was supported by a Neuromed fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Heterochromatin is defined as chromatin that stays densely packed during interphase. Cytologically, heterochromatin can be further subdivided into constitutive and facultative heterochromatin [1,2], with facultative heterochromatin usually differing between cell types [36]. Hence, heterochromatin is amongst the key features of cellular differentiation and transdifferentiation [7,8]. Heterochromatin is commonly associated with transcriptional silencing and characteristic epigenetic marks on the level of histone [9] and nucleotide modifications [10]. Together both kinds of marks act on the degree of compaction and accessibility of DNA, thereby creating sub-nuclear compartments of less dense euchromatin and more compacted heterochromatin. In mouse cells the majority of constitutive heterochromatin is constituted by AT rich tandem repeats (major satellites) adjacent to the centric region of the chromosomes [11]. It is well known that pericentric heterochromatin domains of different chromosomes are organized in so called chromocenters [12,13] during interphase which reorganize during cellular differentiation [5,6,14]. One characteristic epigenetic mark of chromocenters is the covalent methylation at the carbon 5 position of cytosine (5 mC) in CpG dinucleotides. This epigenetic mark is read and interpreted by the methyl cytosine binding protein 2 (MeCP2) [1518]. The Mecp2 gene is encoded on the X chromosome [19] and its product was initially identified as a selective 5-methyl cytosine binding protein [2022]. Meanwhile it is one of the best-studied members of the methyl cytosine binding protein (MDB) family [2327] and was found to be mutated in the neurological disorder Rett syndrome (RTT, OMIM #321750) occurring with a frequency of 1 in 10,000 female birth [2831]. Children affected by RTT show an apparently normal development up to 618 month (...truncated)


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Bianca Bertulat, Maria Luigia De Bonis, Floriana Della Ragione, Anne Lehmkuhl, Manuela Milden, Christian Storm, K. Laurence Jost, Simona Scala, Brian Hendrich, Maurizio D’Esposito, M. Cristina Cardoso. MeCP2 Dependent Heterochromatin Reorganization during Neural Differentiation of a Novel Mecp2-Deficient Embryonic Stem Cell Reporter Line, PLOS ONE, 2012, 10, DOI: 10.1371/journal.pone.0047848