The emergence of the brain non-CpG methylation system in vertebrates

Abstract

Mammalian brains feature exceptionally high levels of non-CpG DNA methylation alongside the canonical form of CpG methylation. Non-CpG methylation plays a critical regulatory role in cognitive function, which is mediated by the binding of MeCP2, the transcriptional regulator that when mutated causes Rett Syndrome. However, it is unclear if the non-CpG neural methylation system is restricted to mammalian species with complex cognitive abilities or has deeper evolutionary origins. To test this, we investigated brain DNA methylation across 12 distant animal lineages, revealing that non-CpG methylation is restricted to vertebrates. We discovered that in vertebrates, non-CpG methylation is enriched within a highly conserved set of developmental genes transcriptionally repressed in adult brains, indicating that it demarcates a deeply conserved regulatory program. Concomitantly, we found that the writer of non-CpG methylation, DNMT3A, and the reader, MeCP2, originated at the onset of vertebrates as a result of the ancestral vertebrate whole genome duplication. Together, we demonstrate how this novel layer of epigenetic information assembled at the root of vertebrates and gained new regulatory roles independent of the ancestral form of the canonical CpG methylation. This suggests the emergence of non-CpG methylation may have fostered the evolution of sophisticated cognitive abilities found in the vertebrate lineage.

Developmental Accumulation of Gene Body and Transposon Non-CpG Methylation in the Zebrafish Brain

“…We found that intronic and intergenic regions are the only regions enriched in mCAC and mCH ( Supplementary Figures 1C-E), whereas a notable depletion, similar to the one described in mammals, was observed at H3K27ac peaks (Supplementary Figure 1F). These results support the previous observations of mCA presence in gene bodies of vertebrate brains (de Mendoza et al, 2021) and demonstrate a conserved depletion of mCH in active regulatory regions.…”

“…To better understand the sequence context of mCH deposition in the zebrafish brain and how it compares to non-neural tissues (liver), we performed a motif search on the 10,000 most highly methylated CH sites. Expectedly, we recovered the TGCT motif associated with mosaic satellite repeats in both brain and liver (Ross et al, 2020), and the previously characterized TACAC-containing motif (Lister et al, 2013;de Mendoza et al, 2021), which was specific to the brain sample ( Supplementary Figure 1A). Furthermore, the brain sample showed a notable enrichment in CAC trinucleotide methylation (∼1.5%) with a threefold increase compared to the unmethylated lambda genome spikein control (∼0.5%) (Supplementary Figure 1B).…”

“…A recent report described the conserved enrichment of mCH in vertebrate brains, which originated alongside MeCP2 and DNMT3A enzymes at the root of the vertebrate lineage (de Mendoza et al, 2021). This study also highlighted the anti-correlation between gene body mCH and expression in some, but not all, vertebrate brains.…”

“…To investigate mCH in the zebrafish nervous system, we analyzed WGBS data (bisulfite conversion > 99.5%) of adult brain (mean coverage = 9.9X), as well as of adult liver (mean coverage = 7.6X), to use as a non-neural control tissue (Bogdanovic et al, 2016;Skvortsova et al, 2019). We employed stringent genotype correction (de Mendoza et al, 2021) to allow for more sensitive interrogation of mCH patterns. To better understand the sequence context of mCH deposition in the zebrafish brain and how it compares to non-neural tissues (liver), we performed a motif search on the 10,000 most highly methylated CH sites.…”

“…RRBS data were processed as above with the additional option of: HEADCROP:5 CROP:140 added during trimming and without deduplication. BAM files were made FLAG-compatible and processed with CGmapTools (Chen et al, 2016;Guo et al, 2018) (convert bam2cgmap) to obtain ATCGmap files, which were corrected for CH positions that showed evidence of CG SNPs (de Mendoza et al, 2021). A summary of library statistics can be found in Supplementary Table 2.…”