Genetic and epigenetic determinants of reactivation of Mecp2 and the inactive X chromosome in neural stem cells.

H Mira-Bontenbal,B Tan,S Goossens,J Gribnau,C Dupont,J.B Boers,A Bedalov,C Gontan,W.F.J Ijcken,M.E Van Royen,P French,R.G Boers

doi:10.1016/j.stemcr.2022.01.008

H Mira-Bontenbal, B Tan + Show 10 more

Open Access

https://doi.org/10.1016/j.stemcr.2022.01.008

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Abstract

SummaryRett syndrome may be treated by reactivating the silent copy of Mecp2 from the inactive X chromosome in female cells. 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 based on a dual bioluminescent and fluorescent reporter to assess the level of reactivation of Mecp2 and the inactive X chromosome by treating neural stem cells with 5-azacytidine and Xist knockdown. We show that reactivation of Mecp2 and other X-linked genes correlates with CpG density, with distance from escapees, and, very strongly, with the presence of short interspersed nuclear elements. In addition, X-linked genes reactivated in neural stem cells overlap substantially with early reactivating genes by induced pluripotent stem cell reprogramming of fibroblasts or neuronal progenitors, indicating that X chromosome reactivation follows similar paths regardless of the technique or cell type used.

Highlights

Rett syndrome (RTT) is the second most prevalent cause of intellectual disability in girls after Down syndrome, affecting 1 in 10,000 live female births (Weaving et al, 2005)
We show that 5-Aza treatment in combination with Xist knockdown in neural stem cells (NSCs) leads to X chromosome reactivation (XCR) with a striking resemblance to induced pluripotent stem cell-reprogramming-specific XCR (Janiszewski et al, 2019; Bauer et al, 2021), suggesting a general pattern in the capability of X-linked genes to reactivate independent of the mechanism or cell type
Immunofluorescence (IF) for NLuc and Tomato fluorescence analysis in a Mecp2+/LucTom fully cast female brain shows that methyl-CpG-binding protein 2 (MECP2)-NLuc and Tomato are expressed in 45% of the cells, as expected from random X chromosome inactivation (XCI) (Figure S1B)

Summary

Introduction

Rett syndrome (RTT) is the second most prevalent cause of intellectual disability in girls after Down syndrome, affecting 1 in 10,000 live female births (Weaving et al, 2005). It is caused by heterozygous mutations in the methyl-CpG-binding protein 2 (MECP2), whose gene is X linked and subject to random X chromosome inactivation (XCI) during early embryogenesis. RTT-affected girls are mosaic in terms of MECP2 expression: half of their cells will express the wild-type (WT) copy of MECP2, while the other half will express the mutant MECP2 allele. One way of achieving this is by reactivation of the endogenous WT copy of MECP2 on the Xi in RTT cells

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