DNA methylation (5-methylcytosine; 5mC) is a repressive gene-regulatory mark required for numerous vertebrate developmental processes. Genomic 5mC is tightly regulated through the coordinated action of DNA methyltransferases, which deposit 5mC, and TET enzymes, which participate in its active removal through the formation of 5-hydroxymethylcytosine (5hmC). TET enzymes are essential for mammalian gastrulation and activation of vertebrate developmental enhancers, however, to date, a clear picture of 5hmC abundance, function and genomic distribution beyond invertebrate-vertebrate boundary, is lacking.
To address this outstanding question, we conducted a detailed analysis of genome-wide 5mC and 5hmC dynamics during development of two invertebrate species: purple sea urchin (Strongylocentrotus purpuratus), the closest known relative of chordates, and the invertebrate chordate European lancelet (Branchiostoma lanceolatum). Specifically, we employed whole genome bisulfite sequencing (WGBS) and APOBEC-coupled epigenetic sequencing (ACE-seq) to generate base-resolution maps of 5mC and 5hmC, at four developmental time points1.
Data analysis revealed 5hmC enrichment at both proximal (promoters) and distal (enhancers) regulatory regions, coinciding with the accessible chromatin, developmental loss of 5mC and activation of developmental genes. Furthermore, we have shown that the expression of invertebrate TET orthologues by and large resembles anamniote TET developmental expression profiles, which are characterised by peaks of TET expression during mid-development (phylotypic period). Finally, we have shown that 5hmC-regulated genes identified in the sea urchin also display 5hmC-mediated regulation in the zebrafish genome and that most of such 5hmC-enriched genes are either linked to actively demethylated phylotypic enhancers or regulatory regions that become demethylated in the adult brain.
Altogether, we demonstrated that active 5mC removal from regulatory regions, through TET-mediated formation of 5hmC, is a common feature of deuterostome embryogenesis. This points to an unexpected deep conservation of a major gene-regulatory module previously associated exclusively with vertebrate embryogenesis and human disease.