Expression levels and DNA methylation profiles of the growth gene SHOX in cartilage tissues and chondrocytes

Abstract

All attempts to identify male-specific growth genes in humans have failed. This study aimed to clarify why men are taller than women. Microarray-based transcriptome analysis of the cartilage tissues of four adults and chondrocytes of 12 children showed that the median expression levels of SHOX, a growth gene in the pseudoautosomal region (PAR), were higher in male samples than in female samples. Male-dominant SHOX expression was confirmed by quantitative RT-PCR for 36 cartilage samples. Reduced representation bisulfite sequencing of four cartilage samples revealed sex-biased DNA methylation in the SHOX-flanking regions, and pyrosequencing of 22 cartilage samples confirmed male-dominant DNA methylation at the CpG sites in the SHOX upstream region and exon 6a. DNA methylation indexes of these regions were positively correlated with SHOX expression levels. These results, together with prior findings that PAR genes often exhibit male-dominant expression, imply that the relatively low SHOX expression in female cartilage tissues reflects the partial spread of X chromosome inactivation into PAR. Altogether, this study provides the first indication that sex differences in height are ascribed, at least in part, to the sex-dependent epigenetic regulation of SHOX. Our findings deserve further validation.

Figure 1

Representative results of mRNA quantification. (a) Microarray-based transcriptome analysis of X chromosomal genes. We analyzed cartilage tissues obtained from two women and two men and cultured chondrocytes established from six girls and six boys. Female/male expression ratios of X chromosome inactivation (XCI)-escape genes that passed the filtering criteria are shown. PAR1, the short arm pseudoautosomal region. (b) SHOX expression levels calculated from transcriptome data. The value was manually calculated by subtracting the median log2 value of SHOX expression levels of all samples from the value of each sample. The horizontal bars indicate the median values of the female and male groups. (c) RT-qPCR analysis of cartilage tissues. The results of knee cartilage tissues from 22 adolescent/adults and digit cartilage tissues from 14 children are shown. Relative SHOX expression levels were analyzed using two internal controls (TBP and GUSB). The diamond and triangle symbols indicate fresh frozen and RNAlater-treated tissues, respectively. The horizontal bars depict the median values of the female and male groups. Unfilled symbols indicate values lower than the detection limit.

Figure 2

Representative results of DNA methylation analyses. (a) Reduced representation bisulfite sequencing (RRBS) of the cartilage tissues obtained from two women and two men. A DNA methylation index is defined as the ratio of 5-methylcytosines to total cytosines. For each CpG site, we calculated the difference in the mean methylation index between the female and male samples (“F-M difference”). The white and black boxes depict non-coding and coding exons of SHOX, respectively. PAR1, pseudoautosomal region 1. (b) Pyrosequencing of knee cartilage tissues obtained from 11 women and 11 men. Methylation indexes of 16 CpG sites in the SHOX-flanking regions (SHOX-upstream region, intron 2, and exon 6a) are shown. The horizontal bars describe the mean value of each group. The P values regarding the sex difference in DNA methylation are described below. Upstream region: CpG1, 0.1223; CpG2, 0.06164; CpG3, 0.09916; CpG4, 0.1387; CpG5, 0.08258; CpG6, 0.1294; CpG7, 0.07291. Intron 2: CpG1, 0.4622; CpG2, 0.3061; CpG3, 0.314; CpG4, 0.3213; CpG5, 0.3822. Exon 6a: CpG1, 0.2769; CpG2, 0.3137; CpG3, 0.1179; CpG4, 0.3256. (c) Correlations between SHOX expression levels and DNA methylation indexes at CpG sites in the flanking regions. The X-axis represents the average DNA methylation index at CpG sites in each region: the SHOX upstream and exon 6a. The Y-axis represents the log2-transformed relative SHOX expression levels against TBP. Linear regression lines fitted to the data are shown. The correlation coefficients and P values for the relationship between DNA methylation index and SHOX expression in each region are listed below. Upstream region: coefficient = 0.61 and P value = 0.0090. Exon 6a: coefficient = 0.70 and P value = 0.0018.