Methylation status of genes escaping from X-chromosome inactivation in patients with X-chromosome rearrangements

Sayaka Kawashima,Masayo Kagami,Erina Suzuki,Kazuhiko Nakabayashi,Keiko Matsubara,Maki Fukami,Yukihiro Hasegawa,Atsushi Hattori,Rika Kosaki,Machiko Toki

doi:10.1186/s13148-021-01121-6

Abstract

BackgroundX-chromosome inactivation (XCI) is a mechanism in which one of two X chromosomes in females is randomly inactivated in order to compensate for imbalance of gene dosage between sexes. However, about 15% of genes on the inactivated X chromosome (Xi) escape from XCI. The methylation level of the promoter region of the escape gene is lower than that of the inactivated genes. Dxz4 and/or Firre have critical roles for forming the three-dimensional (3D) structure of Xi. In mice, disrupting the 3D structure of Xi by deleting both Dxz4 and Firre genes led to changing of the escape genes list. To estimate the impact for escape genes by X-chromosome rearrangements, including DXZ4 and FIRRE, we examined the methylation status of escape gene promoters in patients with various X-chromosome rearrangements.ResultsTo detect the breakpoints, we first performed array-based comparative genomic hybridization and whole-genome sequencing in four patients with X-chromosome rearrangements. Subsequently, we conducted array-based methylation analysis and reduced representation bisulfite sequencing in the four patients with X-chromosome rearrangements and controls. Of genes reported as escape genes by gene expression analysis using human hybrid cells in a previous study, 32 genes showed hypomethylation of the promoter region in both male controls and female controls. Three patients with X-chromosome rearrangements had no escape genes with abnormal methylation of the promoter region. One of four patients with the most complicated rearrangements exhibited abnormal methylation in three escape genes. Furthermore, in the patient with the deletion of the FIRRE gene and the duplication of DXZ4, most escape genes remained hypomethylated.ConclusionX-chromosome rearrangements are unlikely to affect the methylation status of the promoter regions of escape genes, except for a specific case with highly complex rearrangements, including the deletion of the FIRRE gene and the duplication of DXZ4.

Highlights

X-chromosome inactivation (XCI) is a mechanism in which one of two X chromosomes in females is randomly inactivated in order to compensate for imbalance of gene dosage between sexes
Clinical features Four 46,XX female patients with various X-chromosome rearrangements were included in this study
Patients 2–4 showed no methylation abnormality in the promoter regions of escape genes, and Patient 1 had abnormally hypermethylated promoter regions in only three escape genes on the duplicated region. These results suggest that rearrangements detected in these patients might not include the critical regions for the regulation of the methylation status of almost all escape genes

Summary

Introduction

X-chromosome inactivation (XCI) is a mechanism in which one of two X chromosomes in females is randomly inactivated in order to compensate for imbalance of gene dosage between sexes. Most escape genes are Kawashima et al Clin Epigenet (2021) 13:134 on Xp [2], including pseudoautosomal region 1 (PAR1), which has a homologous sequence at the distal end of Yp. All genes in PAR1 escape from XCI and are expressed from both Xi and activated X chromosome (Xa) [2]. XCI mosaicism caused by random inactivation has made it complicated to study escape genes [7] To overcome these issues, previous studies examined allele-specific expression using somatic cell hybrids [2], compared methylation levels of CpG on the X chromosome using methylation arrays in both sexes [4], and investigated expression of escape genes in somatic cells using single-cell RNA sequencing [8]

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Results

Conclusion