Regional epigenetic differentiation of the Z Chromosome between sexes in a female heterogametic system

Abstract

In male heterogametic systems, the X Chromosome is epigenetically differentiated between males and females, to facilitate dosage compensation. For example, the X Chromosome in female mammals is largely inactivated. Relative to well-studied male heterogametic systems, the extent of epigenetic differentiation between male and female Z Chromosomes in female heterogametic species, which often lack complete dosage compensation, is poorly understood. Here, we examined the chromosomal DNA methylation landscapes of male and female Z Chromosomes in two distantly related avian species, namely chicken and white-throated sparrow. We show that, in contrast to the pattern in mammals, male and female Z Chromosomes in these species exhibit highly similar patterns of DNA methylation, which is consistent with weak or absent dosage compensation. We further demonstrate that the epigenetic differences between male and female chicken Z Chromosomes are localized to a few regions, including a previously identified male hypermethylated region 1 (MHM1; CGNC: 80601). We discovered a novel region with elevated male-to-female methylation ratios on the chicken Z Chromosome (male hypermethylated region 2 [MHM2]; CGNC: 80602). The MHM1 and MHM2, despite little sequence similarity between them, bear similar molecular features that are likely associated with their functions. We present evidence consistent with female hypomethylation of MHMs and up-regulation of nearby genes. Therefore, despite little methylation differentiation between sexes, extremely localized DNA methylation differences between male and female chicken Z Chromosomes have evolved and affect expression of nearby regions. Our findings offer new insights into epigenetic regulation of gene expression between sexes in female heterogametic systems.

Figure 1.

DNA methylation and transcription in avian genomes. (A) Density of data points as a function of CpG methylation (5mC [%]) and gene expression (log₂ [TPM]) in female chickens and a male great tit (for male chickens, see Supplemental Fig. S1). The relationship between methylation and gene expression was smoothed with cubic splines (black lines). Number of genes with CpG methylation data: chicken: N = 11,662 for promoters and N = 11,723 for gene bodies; great tit: N = 14,694 for promoters and N = 14,721 for gene bodies. (B) Comparison of DNA methylation between aligned CpGs of Z and W gametologs. N depicts the number of Z-W-aligned CpGs with at least three mapped reads in each sample. Statistical significance was evaluated using paired Mann–Whitney U tests. For A and B, the promoter of a gene was defined as upstream 1.5 kb to downstream 500 bp of its transcription start site. (***) P < 0.001, (**) P < 0.01, (*) P < 0.05, (NS) not significant.

Figure 2.

Sex differences in DNA methylation on sex chromosomes and chromatin accessibility patterns of MHMs. (A) Differences in DNA methylation between male and female X Chromosomes in human brains (5mC [%] [M-F]). (B) Differences in DNA methylation between male and female Z Chromosomes in the white-throated sparrow. (C) Differences in DNA methylation between male and female Z Chromosomes in chicken. Two regions with extreme sex differences in DNA methylation (MHM1 and MHM2) are highlighted. For A–C, methylation values were plotted using a 10-kb window size with a 2-kb step size. (D) A zoomed-in view of the MHM loci. Methylation levels (5mC [%]) for males (blue) and females (red) are shown in the upper lines. Lower lines (orange) depict sex differences in DNA methylation. (E) Both MHMs (shaded areas) display increased chromatin accessibility in females compared with males. ATAC-seq reads were merged per sex and normalized to fragment pileup per million reads for direct comparison between sexes. For either CD4⁺ T cells (Foissac et al. 2019) or forelimb (E4.5) (Sackton et al. 2019), the two loci contained significantly female-biased peaks, tested using bdgdiff from the MACS2 program (Zhang et al. 2008). The vast majority of tissue/cell types with available data show similar patterns (Supplemental Fig. S5).

Figure 3.

Molecular characteristics of MHM1 and MHM2 loci. (A) Both loci are highly repetitive. Each rectangular area filled with matches in dot plots depicts a block of tandem repeats. (B) Expression differences between males and females for lncRNAs transcribed from MHMs. For each locus, one example lncRNA gene with the highest average expression is shown. Significant expression differences between males and females were detected using DESeq2 with raw counts generated from StringTie. (***) Q < 0.001, (**) Q < 0.01, (*) Q < 0.05, (NS) not significant.

Figure 4.

Sex differences in gene expression across the Z Chromosome. The log₂ (Male/Female) values are plotted using a 10-gene window. Genes with average log₂(TPM) lower than 1 in either males or females were filtered out. The dashed lines depict the borders of potential MHM1- or MHM2-affected protein-coding genes identified by a changepoint analysis (Methods). For MHM1, the border is 25–32 Mb; and for MHM2, the border is 72.5–73.5 Mb. One-tailed Mann–Whitney U tests were used to test whether the MHM1- or MHM2-neighboring regions consist of genes with lower Male/Female ratios than the Z background. (***) P < 0.001, (**) P < 0.01, (*) P < 0.05, (NS) not significant.

Figure 5.

Alternative models for the evolution of MHM1 region in chicken. Reduced male-to-female expression ratios near MHM1 in chicken could be due to female up-regulation (A) or male down-regulation (B). In scenario A, low expression in females is the ancestral state, and up-regulation in females has evolved recently in chicken. An expression pattern similar to that shown in A, in other words, higher expression in female chickens than in female tit and ostrich, would suggest that the reduced male-to-female expression ratios seen in chicken evolved due to up-regulation in females. In scenario B, high expression in males is ancestral and the depicted pattern of expression would suggest recently evolved down-regulation in males. For A and B, the time for the split of avian lineages is from Jarvis et al. (2014) and Zhou et al. (2014). (C) Pairwise species differences in expression of the MHM1-neighboring genes (adult brain). Expression was averaged across samples per species, and the pairwise expression difference (the ratio of expression of species 1 to the expression of species 2) was log₂-scaled. Only orthologs present in all species were used (numbers of genes compared are in parentheses). The statistical significance was evaluated using paired Mann–Whitney U tests. (**) P < 0.01, (*) P < 0.05, (NS) not significant.

Figure 6.

Hypomethylation of MHM loci relative to the entire Z Chromosome or repeats on the Z Chromosome in females. In the graphs, DNA methylation of MHM1 and MHM2 is compared with that of the Z Chromosome or repeats on the Z Chromosome for males and females. Coordinates of repetitive sequences were obtained from RepeatMasker track (last update: 2016-04-14) of the UCSC Table Browser. Statistical significance was evaluated using Mann–Whitney U tests. (***) P < 0.001, (NS) not significant.