Abstract
DNA methylation predominantly occurs at CG dinucleotides in vertebrate genomes; however, non-CG methylation (mCH) is also detectable in vertebrate tissues, most notably in the nervous system. In mammals it is well established that mCH is targeted to CAC trinucleotides by DNMT3A during nervous system development where it is enriched in gene bodies and associated with transcriptional repression. Nevertheless, the conservation of developmental mCH accumulation and its deposition by DNMT3A is largely unexplored and has yet to be functionally demonstrated in other vertebrates. In this study, by analyzing DNA methylomes and transcriptomes of zebrafish brains, we identified enrichment of mCH at CAC trinucleotides (mCAC) at defined transposon motifs as well as in developmentally downregulated genes associated with developmental and neural functions. We further generated and analyzed DNA methylomes and transcriptomes of developing zebrafish larvae and demonstrated that, like in mammals, mCH accumulates during post-embryonic brain development. Finally, by employing CRISPR/Cas9 technology, we unraveled a conserved role for Dnmt3a enzymes in developmental mCAC deposition. Overall, this work demonstrates the evolutionary conservation of developmental mCH dynamics and highlights the potential of zebrafish as a model to study mCH regulation and function during normal and perturbed development.
Highlights
In genomes of vertebrate adult somatic cells, the majority of CpG sites are methylated (>80%) with the exception of CpG-rich promoters and distal regulatory elements (Bird, 2002; Jones, 2012; Schübeler, 2015)
We investigated the genomic distribution of mCH at CAC trinucleotides (mCAC) in zebrafish brains to assess if the depletion in regulatory elements and enrichment in gene bodies previously described in mammals (Lister et al, 2013; Guo et al, 2014a; He and Ecker, 2015) is evolutionarily conserved
MCH has been established as an important base modification with likely biological functions during mammalian brain development (Lister et al, 2013), and links to Rett syndrome pathogenesis (Chen et al, 2015; Gabel et al, 2015; Boxer et al, 2020; Lavery et al, 2020), there are still many unknowns related to its regulation and function
Summary
In genomes of vertebrate adult somatic cells, the majority of CpG sites are methylated (>80%) with the exception of CpG-rich promoters and distal regulatory elements (Bird, 2002; Jones, 2012; Schübeler, 2015). Several studies have demonstrated that Methyl-CpG Binding Protein 2 (MeCP2) is able to bind to and regulate genes marked by mCH, which was evident at long genes (Guo et al, 2014a; Chen et al, 2015; Gabel et al, 2015; Boxer et al, 2020; Clemens et al, 2020). Whether this is due to biological or technical reasons is currently debated (Raman et al, 2018). MeCP2 is conserved across vertebrates, such as zebrafish, where depletion of MeCP2 results in similar pathologies to Rett syndrome including altered motor behavior, improper synapse formation and acute inflammation (Pietri et al, 2013; Gao et al, 2015; Nozawa et al, 2017; van der Vaart et al, 2017)
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