Sex-specific DNA methylation at an autosomal region associated with childhood asthma

Mammalian males and females differ in their sex chromosome complements (XX in females and XY in males), gonadal hormones, anatomy and physiology. In humans, these differences include risks of developing particular diseases, such as asthma. DNA methylation levels of certain autosomal regulatory regions also differ between sexes, and therefore may contribute to differences in disease predisposition. Previous studies by our lab on the 17q12-q21 asthma-associated region have discovered sex-specific differences in methylation levels in the zona pellucida binding protein 2 (ZPBP2) promoter in peripheral blood cells that are consistent with the sex-specific bias we observed in genetic association in the Saguenay-Lac-Saint-Jean asthma familial collection (Naumova et al. 2013). Furthermore, we found that methylation levels at the ZPBP2 promoter were associated with asthma predisposition in females (Al Tuwaijri et al. 2016). In principle, sexual dimorphism in DNA methylation may be caused by differences in gonadal hormones or sex chromosome dosage. The goal of our study is to elucidate the role the sex chromosomes play in sex-specific DNA methylation of the asthma-associated ZPBP2 promoter region and map the region of the X that harbors the modifier(s) of ZPBP2 methylation. We therefore hypothesize that DNA methylation levels of the ZPBP2 promoter region are sex chromosome dose-dependent, and that X-linked genes play a role in establishing sex-specific epigenetic patterns.To test this hypothesis, we selected human fibroblast cell lines from individuals with different karyotypes and sex phenotype combinations, such as XX and XY females, XY and XX males, females with partial X deletions and duplications, and mutations of the X-linked candidate gene methyl-CpG-binding protein 2 (MECP2). DNA methylation levels in these cells were analyzed using sodium bisulfite sequencing and pyrosequencing assays. Two different regions of the ZPBP2 promoter were analyzed, referred to as the distal and proximal region. In previous experiments, both regions showed sex-specific methylation in blood cells. In fibroblasts, we report sex-specific DNA methylation in the proximal region but not the distal region of the ZPBP2 promoter. DNA methylation levels at 6 CGs in the proximal region of the ZPBP2 promoter were different between 46,XX females and 46,XY males. We focused on one representative CG of this group and compared its methylation in cell lines with different karyotypes. Our data show that presence of sex-determining region Y (SRY) is associated with lower methylation levels independent of the X chromosome dosage, whereas presence of more than one X in the absence of SRY is associated with higher methylation levels (18% vs 32%, for one X vs more than one X, respectively, ANOVA p = 0.003). Female carriers of MECP2 mutations had lower methylation levels compared to females without mutations (22% methylation in mutation carriers vs. 30% methylation in normal females, p = 0.047, Student's t-test), and similar methylation levels compared to males. We map the region of the X chromosome associated with female-type methylation in SRY- individuals to chromosomal region Xq10-Xq22.3. To determine if the mouse orthologous region may be used to study the mechanism of sex-specific methylation, Zpbp2 promoter methylation was analyzed in tails and lungs of C57BL/6J (B6) and B6.Zpbp2-/- mice carrying a deletion in the Zpbp2 gene. No sex differences in DNA methylation levels were detected except for a modest increase in the lungs of female mutant mice. We report an increase in DNA methylation in this region associated with mutant mice.Thus, in human cells, both the X chromosome complement and SRY influence sex-specific DNA methylation levels at the proximal region of the ZPBP2 promoter that may have a role in modulating predisposition to asthma. Additionally, B6 mice cannot be used to model the pattern observed in humans.

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