Mapping the zebrafish brain methylome using reduced representation bisulfite sequencing

Abstract

Reduced representation bisulfite sequencing (RRBS) has been used to profile DNA methylation patterns in mammalian genomes such as human, mouse and rat. The methylome of the zebrafish, an important animal model, has not yet been characterized at base-pair resolution using RRBS. Therefore, we evaluated the technique of RRBS in this model organism by generating four single-nucleotide resolution DNA methylomes of adult zebrafish brain. We performed several simulations to show the distribution of fragments and enrichment of CpGs in different in silico reduced representation genomes of zebrafish. Four RRBS brain libraries generated 98 million sequenced reads and had higher frequencies of multiple mapping than equivalent human RRBS libraries. The zebrafish methylome indicates there is higher global DNA methylation in the zebrafish genome compared with its equivalent human methylome. This observation was confirmed by RRBS of zebrafish liver. High coverage CpG dinucleotides are enriched in CpG island shores more than in the CpG island core. We found that 45% of the mapped CpGs reside in gene bodies, and 7% in gene promoters. This analysis provides a roadmap for generating reproducible base-pair level methylomes for zebrafish using RRBS and our results provide the first evidence that RRBS is a suitable technique for global methylation analysis in zebrafish.

Sex differences in DNA methylation and expression in zebrafish brain: a test of an extended ‘male sex drive’ hypothesis

“…In summary, the zebrafish RRBS genome contains higher levels of CpG methylation than mammalian RRBS genome and that high level of global CpG methylation is not exclusive to the zebrafish brain but is also found in other tissues (such as liver). Furthermore, consistent with recent base-resolution studies in zebrafish we found low levels (< 3.0%) of non-CpG methylation in zebrafish brain [25]. Further, the pooled samples (Male1 vs. Male2 and Female1 vs. Female2)…”

“…Further, the male and female methylomes also showed high positive correlation (Pearson's correlation coefficient r = 0.98) with each other [25]. Taken together, these results indicate that, overall, the DNA methylation patterns between female and male adult wild type zebrafish brains are very similar.…”

“…This strategy lowers the possibility of inter-individual variation in methylation to a large extent. We found very high positive correlation between the male (Pearson's correlation coefficient between Male1 vs. Male2 = 0.98) and female (Pearson's correlation coefficient between Female1 vs. Female2 = 0.97) replicate samples [25] demonstrating minimal technical variation between the pooled samples. Further, These data was derived using high coverage methylation analysis using RRBS.…”

“…In contrast, Numata et al found 5% of autosomal loci had significantly sex-biased methylation levels in the human prefrontal cortex [18]. However, amongst the gene body DMCs, 69% mapped to intronic regions, but only 59% of CpGs in zebrafish are located in introns [25], indicating enrichment of sexspecific DMCs in introns.…”

“…First, to determine global DNA methylation patterns of male and female brains we performed reduced representation bisulfite sequencing (RRBS). The characteristics of genome-wide methylation of the zebrafish brains, without differentiating between male and female samples, are presented in [25,26]. In summary, the zebrafish RRBS genome contains higher levels of CpG methylation than mammalian RRBS genome and that high level of global CpG methylation is not exclusive to the zebrafish brain but is also found in other tissues (such as liver).…”