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

Bertulat, Bianca; Bonis, Maria Luigia De; Ragione, Floriana Della; Lehmkuhl, Anne; Milden, Manuela; Storm, Christian; Jost, K. Laurence; Scala, Simona; Hendrich, Brian; D’Esposito, Maurizio; Cardoso, M. Cristina

doi:10.1371/journal.pone.0047848

Cited by 35 publications

(53 citation statements)

References 76 publications

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“…Apparently, the difference in results obtained on cells in native tissues of Mecp2 - /y and Mecp2 + /y mice and on cultured cells derived from these mice [36] is analogous to the observations on Suv3-9/Suv4-20 h double knockout cells and might be tentatively explained by compensatory mechanisms operating in vivo but not in vitro .…”

Section: Resultssupporting

confidence: 58%

DNA methylation reader MECP2: cell type- and differentiation stage-specific protein distribution

Song

Feodorova

Guy

et al. 2014

Epigenetics & Chromatin

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BackgroundMethyl-CpG binding protein 2 (MECP2) is a protein that specifically binds methylated DNA, thus regulating transcription and chromatin organization. Mutations in the gene have been identified as the principal cause of Rett syndrome, a severe neurological disorder. Although the role of MECP2 has been extensively studied in nervous tissues, still very little is known about its function and cell type specific distribution in other tissues.ResultsUsing immunostaining on tissue cryosections, we characterized the distribution of MECP2 in 60 cell types of 16 mouse neuronal and non-neuronal tissues. We show that MECP2 is expressed at a very high level in all retinal neurons except rod photoreceptors. The onset of its expression during retina development coincides with massive synapse formation. In contrast to astroglia, retinal microglial cells lack MECP2, similar to microglia in the brain, cerebellum, and spinal cord. MECP2 is also present in almost all non-neural cell types, with the exception of intestinal epithelial cells, erythropoietic cells, and hair matrix keratinocytes. Our study demonstrates the role of MECP2 as a marker of the differentiated state in all studied cells other than oocytes and spermatogenic cells. MECP2-deficient male (Mecp2-/y ) mice show no apparent defects in the morphology and development of the retina. The nuclear architecture of retinal neurons is also unaffected as the degree of chromocenter fusion and the distribution of major histone modifications do not differ between Mecp2-/y and Mecp2 wt mice. Surprisingly, the absence of MECP2 is not compensated by other methyl-CpG binding proteins. On the contrary, their mRNA levels were downregulated in Mecp2-/y mice.ConclusionsMECP2 is almost universally expressed in all studied cell types with few exceptions, including microglia. MECP2 deficiency does not change the nuclear architecture and epigenetic landscape of retinal cells despite the missing compensatory expression of other methyl-CpG binding proteins. Furthermore, retinal development and morphology are also preserved in Mecp2-null mice. Our study reveals the significance of MECP2 function in cell differentiation and sets the basis for future investigations in this direction.

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Section: Resultssupporting

confidence: 58%

DNA methylation reader MECP2: cell type- and differentiation stage-specific protein distribution

Song

Feodorova

Guy

et al. 2014

Epigenetics & Chromatin

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“…Another structural attribute of Alu elements is their GC-richness and high CpG content (~1/3 of all genomic CpG sites are in Alu, 14 with about 75% of Alu CpG methylated, 26 accounting for ~25% of the total DNA methylation in the human genome 21 ). These methylated DNA sites are clear candidates for the binding of MeCP2, 27 which is associated with heterochromatin formation.…”

Section: Discussionmentioning

confidence: 99%

Retrotransposon Alu is enriched in the epichromatin of HL-60 cells

Olins

Ishaque

Chotewutmontri

et al. 2014

Nucleus

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“…In mouse, pericentric heterochromatin clusters are located distant from the periphery inside the nucleus. These so-called chromocenters consist of highly condensed, repetitive DNA, are mostly transcriptionally silent and have been described in mouse [ 18 , 19 ], Drosophila [ 10 ] and plants [ 20 – 22 ].…”

Section: Introductionmentioning

confidence: 99%

Gene repositioning within the cell nucleus is not random and is determined by its genomic neighborhood

Jost

Bertulat

Rapp

et al. 2015

Epigenetics & Chromatin

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BackgroundHeterochromatin has been reported to be a major silencing compartment during development and differentiation. Prominent heterochromatin compartments are located at the nuclear periphery and inside the nucleus (e.g., pericentric heterochromatin). Whether the position of a gene in relation to some or all heterochromatin compartments matters remains a matter of debate, which we have addressed in this study. Answering this question demanded solving the technical challenges of 3D measurements and the large-scale morphological changes accompanying cellular differentiation.ResultsHere, we investigated the proximity effects of the nuclear periphery and pericentric heterochromatin on gene expression and additionally considered the effect of neighboring genomic features on a gene’s nuclear position. Using a well-established myogenic in vitro differentiation system and a differentiation-independent heterochromatin remodeling system dependent on ectopic MeCP2 expression, we first identified genes with statistically significant expression changes by transcriptional profiling. We identified nuclear gene positions by 3D fluorescence in situ hybridization followed by 3D distance measurements toward constitutive and facultative heterochromatin domains. Single-cell-based normalization enabled us to acquire morphologically unbiased data and we finally correlated changes in gene positioning to changes in transcriptional profiles. We found no significant correlation of gene silencing and proximity to constitutive heterochromatin and a rather unexpected inverse correlation of gene activity and position relative to facultative heterochromatin at the nuclear periphery.ConclusionIn summary, our data question the hypothesis of heterochromatin as a general silencing compartment. Nonetheless, compared to a simulated random distribution, we found that genes are not randomly located within the nucleus. An analysis of neighboring genomic context revealed that gene location within the nucleus is rather dependent on CpG islands, GC content, gene density, and short and long interspersed nuclear elements, collectively known as RIDGE (regions of increased gene expression) properties. Although genes do not move away/to the heterochromatin upon up-/down-regulation, genomic regions with RIDGE properties are generally excluded from peripheral heterochromatin. Hence, we suggest that individual gene activity does not influence gene positioning, but rather chromosomal context matters for sub-nuclear location.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0025-5) contains supplementary material, which is available to authorized users.

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

Cited by 35 publications

References 76 publications

DNA methylation reader MECP2: cell type- and differentiation stage-specific protein distribution

DNA methylation reader MECP2: cell type- and differentiation stage-specific protein distribution

Retrotransposon Alu is enriched in the epichromatin of HL-60 cells

Gene repositioning within the cell nucleus is not random and is determined by its genomic neighborhood

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