The Ciona intestinalis cleavage clock is independent of DNA methylation.

The initiation of embryonic gene expression in ascidian embryos appears to be tightly regulated by the number of DNA replication cycles. DNA methylation is thought to contribute to the clock mechanism that counts the rounds of DNA replication. We used mass spectrometry and whole genome bisulfite sequencing to characterize DNA methylation changes that occur in early developmental stages of the ascidian, Ciona intestinalis. We found that global DNA methylation in early Ciona development was static, and a base-wise comparison between the genomes of consecutive developmental stages found no DNA demethylation that was related to zygotic gene activation. Additionally, 5hmC was hardly detected by mass spectrometry in the developmental samples, suggesting a lack of demethylation mediated by ten eleven translocation (TET) methylcytosine dioxygenase in C. intestinalis. We conclude that DNA methylation is not involved in regulating DNA replication-dependent transcriptional activation.