Zebrafish Unga Affects Zygotic Gene Activation Through Regulating DNA Demethylation

Methylation of cytosines on DNA affects chromatin structure and gene transcription,thereby controlling cell behavior and embryonic development. Controversal results havebeen reported about DNA methylation level changes during early zebrafish development,and mechanisms controlling possible DNA demethylation remain elusive. In this study, theinspection of the genomic DNA methylation (5mC) level during early developmental stagesof zebrafish indicates that the5mC level gradually decreases from fertilization to4-cellstage, maintains stable until32-or64-cell stages, and starts to increase thereafter. Basedon high-throughput RNA seq, a uracil-DNA glycosylase (Ung) gene, unga, is found to beabundant in the egg. Unga’s known functions in DNA repair process implies itsinvolvement in DNA demethylation. We demonstrate that Unga protein is located in nucleiof blastomeres during the cleavage period and it is capable of removing mis-pairing uracilson DNA. When unga is knocked down, embryos show an increase of DNA methylationlevel at the premidlastula stages, delayed and decreased transcription of many zygoticgenes and slower epibolic process, and start to die at the onset of segmentation. In contrast,ovexpression of unga mRNA leads to a decrease of DNA methylation level and earlierexpression of zygotic genes, but no obvious morphological changes. These results indicatethat Unga plays an import role in postfertilization demethylation and zygotic genomeactivation and is required for normal embryonic development in zebrafish. Mutationalanalyses reveal that the activity of Unga to cleave mispairing uracils is indispensable for itsfunction in DNA demethylation, and this might cooperate with deaminase. Furthermore,unga overexpression results in an increase of H3K4me3and H3K27me3, suggesting thatUnga-mediated DNA demethylation facilitates chromatin remodeling for regulation ofzygotic gene transcription. Taken together, these results indicate that the zebrafish embryoundergoes Unga-mediated DNA demethylation after fertilization, which differs from themechanisms used by mammalian embryos. However, further studies are needed to elucidatethe molecular mechanism of Unga-mediated DNA demethylation in the zebrafish embryos.