Perturbed maintenance of transcriptional repression on the inactive X-chromosome in the mouse brain after Xist deletion

Robin L Adrianse,Joost Gribnau,Uyen Lao,Ruben G Boers,Tonibelle Gatbonton-Schwager,Smitha P Sripathy,Eric J Foss,Kaleb Smith,Joachim B Boers,Antonio Bedalov

doi:10.1186/s13072-018-0219-8

Abstract

BackgroundThe long noncoding RNA Xist is critical for initiation and establishment of X-chromosome inactivation during embryogenesis in mammals, but it is unclear whether its continued expression is required for maintaining X-inactivation in vivo.ResultsBy using an inactive X-chromosome-linked MeCP2-GFP reporter, which allowed us to enumerate reactivation events in the mouse brain even when they occur in very few cells, we found that deletion of Xist in the brain after establishment of X-chromosome inactivation leads to reactivation in 2–5% of neurons and in a smaller fraction of astrocytes. In contrast to global loss of both H3 lysine 27 trimethylation (H3K27m3) and histone H2A lysine 119 monoubiquitylation (H2AK119ub1) we observed upon Xist deletion, alterations in CpG methylation were subtle, and this was mirrored by only minor alterations in X-chromosome-wide gene expression levels, with highly expressed genes more prone to both derepression and demethylation compared to genes with low expression level.ConclusionOur results demonstrate that Xist plays a role in the maintenance of histone repressive marks, DNA methylation and transcriptional repression on the inactive X-chromosome, but that partial loss of X-dosage compensation in the absence of Xist in the brain is well tolerated.

Highlights

The long noncoding RNA Xist is critical for initiation and establishment of X-chromosome inactivation during embryogenesis in mammals, but it is unclear whether its continued expression is required for maintaining X-inactivation in vivo
While MeCP2-EGFP is highly expressed throughout brain of MeCP2-GFP/MeCP2 females, we found no expression in (Xistmut MeCP2)m/ (Xist MeCP2-GFP)p females (Fig. 1b), consistent with uniform silencing of the MeCP2-GFP transgene on the paternal X-chromosome (Xp)
Given complete skewing of X-chromosome inactivation and RT-Quantitative PCR (qPCR) (n = 3 for each group, error bars indicate SD, ***p < 0.001, Student’s t test) absence of MeCP2-GFP expression, we felt that this model would be sufficiently sensitive to detect low levels of the reporter gene reactivation that would be obscured in a MeCP2/MeCP2-GFP animal, in which large fraction of cells expresses MeCP2-GFP as baseline

Summary

Introduction

The long noncoding RNA Xist is critical for initiation and establishment of X-chromosome inactivation during embryogenesis in mammals, but it is unclear whether its continued expression is required for maintaining X-inactivation in vivo. An initial wave of inactivation occurs at the 2–4-cell stage in the embryo, on the paternal X-chromosome (Xp) [4, 5]. X-inactivation initiates with the expression of the long noncoding RNA, Xist (X-inactive-specific transcript), from the chromosome to be inactivated [7, 8], which spreads in cis along the entire chromosome and recruits additional silencing factors, which together establish stable repression of that chromosome for the lifetime of the cell and its progeny (reviewed in [9]). The expression of Xist continues during the maintenance phase, generating a cloud of Xist that surrounds the Xi

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Discussion

Conclusion