Dataset Information

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

ABSTRACT:

Background

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. 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.

Results

To 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.

Conclusion

X-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.

SUBMITTER: Kawashima S

PROVIDER: S-EPMC8244138 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundIn mammals, sex chromosomes pose an inherent imbalance of gene expression between sexes. In each female somatic cell, random inactivation of one of the X-chromosomes restores this balance. While most genes from the inactivated X-chromosome are silenced, 15-25% are known to escape X-inactivation (termed escapees). The expression levels of these genes are attributed to sex-dependent phenotypic variability.ResultsWe used single-cell RNA-Seq to detect escapees in somatic cells. As only one X-chromosome is inactivated in each cell, the origin of expression from the active or inactive chromosome can be determined from the variation of sequenced RNAs. We analyzed primary, healthy fibroblasts (n = 104), and clonal lymphoblasts with sequenced parental genomes (n = 25) by measuring the degree of allelic-specific expression (ASE) from heterozygous sites. We identified 24 and 49 candidate escapees, at varying degree of confidence, from the fibroblast and lymphoblast transcriptomes, respectively. We critically test the validity of escapee annotations by comparing our findings with a large collection of independent studies. We find that most genes (66%) from the unified set were previously reported as escapees. Furthermore, out of the overlooked escapees, 11 are long noncoding RNA (lncRNAs).ConclusionsX-chromosome inactivation and escaping from it are robust, permanent phenomena that are best studies at a single-cell resolution. The cumulative information from individual cells increases the potential of identifying escapees. Moreover, despite the use of a limited number of cells, clonal cells (i.e., same X- chromosomes are coordinately inhibited) with genomic phasing are valuable for detecting escapees at high confidence. Generalizing the method to uncharacterized genomic loci resulted in lncRNAs escapees which account for 20% of the listed candidates. By confirming genes as escapees and propose others as candidates from two different cell types, we contribute to the cumulative knowledge and reliability of human escapees.