Abstract

X chromosome inactivation (XCI) silences most genes on one X chromosome in female mammals, but some genes escape XCI. To identify escape genes in vivo and to explore molecular mechanisms that regulate this process we analyzed the allele-specific expression and chromatin structure of X-linked genes in mouse tissues and cells with skewed XCI and distinguishable alleles based on single nucleotide polymorphisms. Using a binomial model to assess allelic expression, we demonstrate a continuum between complete silencing and expression from the inactive X (Xi). The validity of the RNA-seq approach was verified using RT-PCR with species-specific primers or Sanger sequencing. Both common escape genes and genes with significant differences in XCI status between tissues were identified. Such genes may be candidates for tissue-specific sex differences. Overall, few genes (3–7%) escape XCI in any of the mouse tissues examined, suggesting stringent silencing and escape controls. In contrast, an in vitro system represented by the embryonic-kidney-derived Patski cell line showed a higher density of escape genes (21%), representing both kidney-specific escape genes and cell-line specific escape genes. Allele-specific RNA polymerase II occupancy and DNase I hypersensitivity at the promoter of genes on the Xi correlated well with levels of escape, consistent with an open chromatin structure at escape genes. Allele-specific CTCF binding on the Xi clustered at escape genes and was denser in brain compared to the Patski cell line, possibly contributing to a more compartmentalized structure of the Xi and fewer escape genes in brain compared to the cell line where larger domains of escape were observed.

Highlights

  • Dosage compensation in mammals is achieved by upregulation of the X chromosome in both sexes and random inactivation of one of the two X chromosomes in females [1]

  • X inactivation is a female-specific phenomenon that occurs during early development and results in the silencing of one X chromosome in female mammals

  • In the resulting F1 XistΔ/+ female progeny the maternal BL6 X chromosome fails to inactivate, leading to completely skewed X chromosome inactivation (XCI) in all tissues [26]. This was further verified based on allelic expression of a gene known to be subject to XCI (Ubqln2), as determined by Sanger sequencing of RT-PCR products (S1A Fig)

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Summary

Introduction

Dosage compensation in mammals is achieved by upregulation of the X chromosome in both sexes and random inactivation of one of the two X chromosomes in females [1]. The future inactive X (Xi) is coated by the long non-coding RNA Xist (X inactive specific transcript), a process essential for the onset of silencing [2]. Inactive chromatin marks such as tri-methylation of lysine 27 on histone H3 (H3K27me3) are put in place along with DNA methylation at CpG islands, macroH2A modification, and late replication, which represent late and possibly secondary events that lock in silencing of most genes in somatic cells. Despite efficient silencing some genes escape X chromosome inactivation (XCI) and remain bi-allelically expressed in females [3,4]. The presence of a single X chromosome (45,X) results in Turner syndrome characterized by poor viability in utero, infertility, short stature, and an array of other abnormalities [8,9]

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