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Genetic and epigenetic determinants of reactivation of Mecp2 and the inactive X chromosome in neural stem cells (MeD-seq)

ABSTRACT: Rett Syndrome is a neurodevelopmental disorder in girls that is caused by heterozygous inactivation of the chromatin remodeler gene MECP2. Rett Syndrome may therefore be treated by reactivation of the wild type copy of MECP2 from the inactive X chromosome. Most studies that model Mecp2 reactivation have used mouse fibroblasts rather than neural cells, which would be critical for phenotypic reversal, and rely on fluorescent reporters that lack adequate sensitivity. Here, we present a mouse model system for monitoring Mecp2 reactivation that is more sensitive and versatile than any bioluminescent and fluorescent system currently available. The model consists of neural stem cells derived from female mice with a dual reporter system where MECP2 is fused to NanoLuciferase and TdTomato on the inactive X chromosome. We show by bioluminescence and fluorescence that Mecp2 is synergistically reactivated by 5-Aza treatment and Xist knockdown. As expected, other genes on the inactive X chromosome are also reactivated, the majority of which overlaps with genes reactivated early during reprogramming of mouse embryonic fibroblasts to iPSCs. Genetic and epigenetic features such as CpG density, SINE elements, distance to escapees and CTCF binding are consistent indicators of reactivation, whereas different higher order chromatin areas are either particularly prone or resistant to reactivation. Our MeCP2 reactivation monitoring system thereby suggests that genetic and epigenetic features on the inactive X chromosome affect reactivation of its genes, irrespective of cell type or procedure of reactivation.

ORGANISM(S): Mus musculus

PROVIDER: GSE166146 | GEO | 2022/01/24

REPOSITORIES: GEO

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Genetic and epigenetic determinants of reactivation of Mecp2 and the inactive X chromosome in neural stem cells (RNA-seq)

Project description:Rett Syndrome is a neurodevelopmental disorder in girls that is caused by heterozygous inactivation of the chromatin remodeler gene MECP2. Rett Syndrome may therefore be treated by reactivation of the wild type copy of MECP2 from the inactive X chromosome. Most studies that model Mecp2 reactivation have used mouse fibroblasts rather than neural cells, which would be critical for phenotypic reversal, and rely on fluorescent reporters that lack adequate sensitivity. Here, we present a mouse model system for monitoring Mecp2 reactivation that is more sensitive and versatile than any bioluminescent and fluorescent system currently available. The model consists of neural stem cells derived from female mice with a dual reporter system where MECP2 is fused to NanoLuciferase and TdTomato on the inactive X chromosome. We show by bioluminescence and fluorescence that Mecp2 is synergistically reactivated by 5-Aza treatment and Xist knockdown. As expected, other genes on the inactive X chromosome are also reactivated, the majority of which overlaps with genes reactivated early during reprogramming of mouse embryonic fibroblasts to iPSCs. Genetic and epigenetic features such as CpG density, SINE elements, distance to escapees and CTCF binding are consistent indicators of reactivation, whereas different higher order chromatin areas are either particularly prone or resistant to reactivation. Our MeCP2 reactivation monitoring system thereby suggests that genetic and epigenetic features on the inactive X chromosome affect reactivation of its genes, irrespective of cell type or procedure of reactivation.

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A screen for reactivation of MeCP2 on the inactive X-chromosome identifies BMP/TGF-b superfamily as a regulator of maintenance of XIST expression

Project description:X-chromosome inactivation (XCI) is an epigenetic phenomenon that renders one of the two X-chromosomes in female cells transcriptionally silent, ensuring that X-linked gene dosage matches that in males, who have only one copy of the X chromosome. When a mutation of an X-linked gene is heterozygous, as it is in most girls with Rett syndrome, a neurodevelopmental disorder caused by a mutation MeCP2 gene, the presence of the mutated allele on the active X chromosome entails transcriptional inactivation of the wild type allele on the inactive X, resulting in complete loss of gene function. Reactivation of the silenced wild-type copy of MeCP2 therefore presents a potential therapeutic strategy for Rett syndrome. To identify genes that silence MeCP2 on the Xi that could prove useful therapeutic targets, we carried out a screen for genes whose downregulation reactivated a MeCP2 reporter on the Xi. The 30 genes we have identified comprise seven functional groups revealing a genetic circuitry required for maintenance of X-chromosome inactivation in differentiated cells and a large number of targets suitable for pharmacologic intervention.

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2011-11-01 | E-GEOD-8720 | biostudies-arrayexpress

Transcriptional regulation in pluripotent stem cells by Methyl CpG binding protein 2 (MeCP2)

Project description:Rett syndrome (RTT) is one of the most prevalent female mental disorders. De novo mutations in methyl CpG binding protein 2 (MeCP2) are a major cause of RTT. MeCP2 regulates gene expression as a transcription regulator as well as through long-range chromatin interaction. Because MeCP2 is present on the X chromosome, RTT is manifested in a X-linked dominant manner. Investigation using murine MeCP2 null models and post-mortem human brain tissues has contributed to understanding the molecular and physiological function of MeCP2. In addition, RTT models using human induced pluripotent stem cells derived from RTT patients (RTT-iPSCs) provide novel resources to elucidate the regulatory mechanism of MeCP2. Previously, we obtained clones of female RTT-iPSCs that express either wild type or mutant MECP2 due to the inactivation of one X chromosome. Reactivation of the X chromosome also allowed us to have RTT-iPSCs that express both wild type and mutant MECP2. Using these unique pluripotent stem cells, we investigated the regulation of gene expression by MeCP2 in pluripotent stem cells by transcriptome analysis. We found that MeCP2 regulates genes encoding mitochondrial membrane proteins. In addition, loss of function in MeCP2 results in de-repression of genes on the inactive X chromosome. Furthermore, we showed that each mutation in MECP2 affects a partly different set of genes. These studies suggest that fundamental cellular physiology is affected by mutations in MECP2 from very early fetal development and that a therapeutic approach targeting to unique forms of mutant MeCP2 is needed. RNA samples from normal ESCs/iPSCs, RTT-iPSCs and MeCP2 KD iPSCs were obtained. Gene expression of those cells were analyzed.

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Xi-reactivation: A mixed modality approach to MECP2 restoration for Rett Syndrome and other X-linked disorders

Project description:The X-chromosome harbors hundreds of disease genes whose associated diseases predominantly affect males. However, a subset — including neurodevelopmental disorders, Rett, Fragile X, and CDKL5 Syndromes — also affects females. These disorders lack disease-specific treatment. Because female cells carry two X-chromosomes, an emerging treatment strategy has been to reawaken the healthy allele on the inactive X (Xi). Here we focus on MECP2 restoration for Rett Syndrome and combinatorially target factors in the interactome of Xist, the noncoding RNA responsible for X-inactivation. We identify a mixed modality approach combining an Xist antisense oligonucleotide and a small molecule inhibitor of DNA methylation, which together achieve 30,000-fold MECP2 upregulation from the Xi in cultured cells. Combining a brain-specific genetic Xist ablation with short-term Aza treatment models the synergy in vivo without evident toxicity. The Xi is selectively reactivated. These experiments provide proof of concept for a mixed modality approach for treating X-linked disorders in females.

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Project description:Rett syndrome is a human intellectual disability disorder that is associated with mutations in the X-linked MECP2 gene. Theepigenetic reader MeCP2 binds to methylated cytosines on the DNA and regulates chromatin organization. We have shownpreviously that MECP2 Rett syndrome missense mutations are impaired in chromatin binding and heterochromatinreorganization. Here, we performed a proteomics analysis of post-translational modifications of MeCP2 isolated from adult mousebrain. We show that MeCP2 carries various post-translational modifications, among them phosphorylation on S80 and S421, whichlead to minor changes in either heterochromatin binding kinetics or clustering. We found that MeCP2 is (di)methylated on severalarginines and that this modification alters heterochromatin organization. Interestingly, we identified the Rett syndrome mutationsite R106 as a dimethylation site. In addition, co-expression of protein arginine methyltransferases 1 and 6 lead to a decrease ofheterochromatin clustering. Altogether, we identified and validated novel modifications of MeCP2 in the brain and show that thesecan modulate its ability to bind as well as reorganize heterochromatin, which may play a role in the pathology of Rett syndrome.

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