Epigenetic modification and inheritance in sexual reversal of fish

Abstract

Environmental sex determination (ESD) occurs in divergent, phylogenetically unrelated taxa, and in some species, co-occurs with genetic sex determination (GSD) mechanisms. Although epigenetic regulation in response to environmental effects has long been proposed to be associated with ESD, a systemic analysis on epigenetic regulation of ESD is still lacking. Using half-smooth tongue sole (Cynoglossus semilaevis) as a model-a marine fish that has both ZW chromosomal GSD and temperature-dependent ESD-we investigated the role of DNA methylation in transition from GSD to ESD. Comparative analysis of the gonadal DNA methylomes of pseudomale, female, and normal male fish revealed that genes in the sex determination pathways are the major targets of substantial methylation modification during sexual reversal. Methylation modification in pseudomales is globally inherited in their ZW offspring, which can naturally develop into pseudomales without temperature incubation. Transcriptome analysis revealed that dosage compensation occurs in a restricted, methylated cytosine enriched Z chromosomal region in pseudomale testes, achieving equal expression level in normal male testes. In contrast, female-specific W chromosomal genes are suppressed in pseudomales by methylation regulation. We conclude that epigenetic regulation plays multiple crucial roles in sexual reversal of tongue sole fish. We also offer the first clues on the mechanisms behind gene dosage balancing in an organism that undergoes sexual reversal. Finally, we suggest a causal link between the bias sex chromosome assortment in the offspring of a pseudomale family and the transgenerational epigenetic inheritance of sexual reversal in tongue sole fish.

Figure 1.

Morphology, phylogeny, and DNA methylation of half-smooth tongue sole. (A) Photos of a female, a normal male, and a pseudomale at 2 yr of age. (B) Genome-based phylogenetic positions and sex chromosome systems of half-smooth tongue sole and other vertebrates. Red lines show that the sex chromosome pairs of tongue sole and chicken were derived from the same ancestral vertebrate proto-chromosome pairs in spite of their distant evolutionary relationship (Chen et al. 2014). (?) No sex chromosomes have been identified so far. (C) Experimental design: The offspring from a normal male (ZZ) and a female (ZW) were exposed to 28°C during the sensitive developmental period, which induced the development of genetic females (ZW) into pseudomales. One of these pseudomales was subsequently crossed with one normal female to produce F1 pseudomales and females. F1 offspring carrying WW sex chromosomes do not exist, as sperms with W instead of Z are not viable. (*) Samples used for both BS-seq and RNA-seq; (†) samples only used for BS-seq. Brown letters in parentheses indicate the symbols for corresponding samples used throughout this paper. (D) Percentage of mCs in the CG, CHG, and CHH contexts. (E) Fraction of CpG in low (methylation level less than 0.25), intermediate (between 0.25 and 0.75), and high (greater than 0.75) methylation levels in different genomic elements. (F) Methylation profile along transcript start sites (TSS) of genes in different expression quintiles. The first quintile is the lowest and the fifth is the highest. Dashed green line indicates the location of TSS. B and C are modified from Figures S16 and 3a in Chen et al. (2014), respectively.

Figure 2.

Genome-wide methylation level comparisons. (A) Methylation levels of different chromosomes. Numbers after the sample names represent methylation levels of the whole genome. Only CpGs with ≥10× coverage were used for analysis. (B) Total length (Mb) of DMRs identified in each pairwise comparison. (C) Venn diagrams for DMRs of P-ZWm/P-ZWf, F1-ZWm/P-ZWf, and ZZm/P-ZWf. Numbers represent the total length (Mb) of shared DMRs. (D) Percentage of testis/ovary DMRs on different chromosomes. Background expectation for each chromosome was calculated as the covered length (≥6× in all five samples) of each chromosome divided by the total covered length of all chromosomes. (E) Percentage of testis/ovary DMRs on different genomic elements. Background expectation for each element was calculated as the covered length of each element divided by the total covered length of the genome.

Figure 3.

Differential methylation and sex determination. (A) Differentially methylated and differentially expressed genes in the putative sex determination pathway of tongue sole. For each gene presented in the pathway, the methylation (left square) or expression (right square) changes when comparing testes with ovaries are shown by different colors. (B) DNA methylation and transcription of dmrt1 in different developmental stages after hatching. The methylation levels of different stages were estimated using bisulfite-PCR followed by TA-cloning with a pair of primers targeting the first exon, always using at least 10 randomly selected clones for sequencing for each stage. (C) DNA methylation profiles of gsdf in the five gonadal samples. Green vertical lines indicate the methylation level of cytosines. The light gray box indicates the DMR upstream of gsdf. Profiles of other DMGs in the pathway are presented in Supplemental Figure S8.

Figure 4.

Dosage compensation of the Z chromosome in pseudomale testes. (A) Methylated cytosine (mC) density (5-kb window), log2-transformed expression ratios (running averages of 20 genes), and DMR profiles of the Z chromosome. The light gray box indicates the outstanding dosage-compensated region where DMRs were concentrated ([red vertical lines] DMRs that were up-methylated in P-ZWm, [blue vertical lines] DMRs that were up-methylated in ZZm), and the green block indicates the pseudoautosomal region (PAR) where Z and W chromosomes still pair in meiosis. Only 22 genes were annotated in PAR. Z-chromosomal to autosomal gene expression ratios (Z:A) in P-ZWm (B), F1-ZWm (C), and ZZm (D). The dosage compensation region (light gray box in A) is plotted in red. For each Z interval, the expression level of each Z-gene was first divided by the median expression level of all autosomal genes, then the Z:A ratios in each interval were plotted.

Figure 5.

W-genes expression pattern in pseudomale testes. (A) Categories of W-genes based on gene expression levels in ovaries and pseudomale testes. (B) Frequency distribution of protein identities for the 272 W-Z paralogous pairs. (C) Pseudomale to normal-male expression ratios calculated from the 272 W-genes and their Z-counterparts. From left to right: The first box represents expression ratios of P-ZWm W-genes to ZZm Z-genes; the second box represents expression ratios of P-ZWm Z-genes to ZZm Z-genes; and the third box represents expression ratios of the sum of P-ZWm W- and Z-genes to ZZm Z-genes, indicating that the expression sum of W-Z paralogous genes in pseudomale testes was close to the dosage of Z-genes in normal males. (D) Alternative splicing and methylation profile of figla. The upper section indicates the gene model and the two splice forms of figla, with the dashed box indicating the position of the basic helix–loop–helix domain, which is coded by first and second exons of splice form1. The lower section shows the DNA methylation profiles in ovaries and pseudomale testes, with the light gray box indicating the position of DMR and green vertical lines indicating methylation levels of cytosines. (E,F) Expression of the two splice forms of figla in testes of normal and pseudomales and female ovaries as determined by RT-PCR. (AFE) Alternative first exon.