Both the deposition and removal of DNA methylation (5mC) are crucial for governing gene regulatory programs during embryogenesis. Transcriptional enhancers, in particular, display robust 5mC remodeling during developmental processes. Enhancer DNA demethylation is a crucial feature of vertebrate embryonic development. Despite the current paradigm considering 5mC-mediated enhancer activity regulation as a vertebrate innovation, we have recently shown that the invertebrate chordate amphioxus (lancelet) also displays enhancer DNA demethylation during tissue differentiation. This finding suggests that 5mC-mediated genome regulation might be more evolutionary conserved than previously anticipated.
In vertebrates, enhancer demethylation occus via the 5-hydroxymethycytosine (5hmC) intermediate. Depletion of TET proteins, major DNA oxidases responsible for 5hmC formation, results in enhancer hypermethylation, reduced chromatin accessibility, delayed gene induction, and skewed lineage specification. However, it is currently underexplored, how widely is 5hmC-mediated enhancer regulation utilized in invertebrates. To address this outstanding question, we profiled 5mC, 5hmC, and open chromatin across consecutive developmental stages of sea urchin, sea star, and amphioxus. We found that during gastrulation and adult tissue differentiation, a number of intragenic enhancers undergo DNA demethylation coinciding with the formation of open chromatin state. Notably, at the blastula stage, 5hmC pre-marks enhancers deemed for demethylation during gastrulation, revealing that 5hmC-mediated genome regulation predates the evolutionary emergence of chordates. Synteny analysis identified that invertebrate 5hmC-regulated enhancers are enriched in metazoan-specific microsyntenic units. Significantly, analysis of 5mC dynamics in human embryo revealed that these ancient microsyntenic pairs harbour enhancers that undergo DNA demethylation during early human development.
Overall, our study unravels the ancient module of 5hmC-mediated enhancer regulation conserved for over > 540 million years. This work thus lays the foundation for more comprehensive understanding of 5hmC evolution and its functional significance in genome regulation in vertebrates.