The Effects Of Methylation Inhibitors On Genomic DNA Methylation Levels In Symbiodiniaceae Species And Changes In Methylation Levels With Methyltransferase And Photosystem Genes Expression Under Different Light Conditions

Epigenetics mechanism including DNA metylation,small RNA as well as Post translational modifications of histones(PTMs)can all regulate gene expression and phenotypic variation without changing DNA nucleotide sequences.DNA methylation is the most extensively studied epigenetic mechanism among them.Research on DNA methylation covers the animals,high plants and fungi,rarely dinoflagellates and other phytoplankton,particularly Symbiodiniaceae species that are crucial for coral reefs.Here we studied Fugacium kawagutii(The used name was Symbiodinium kawagutii)DNA methylation characteristics based on previous results from gene sequencing and annotation in F.kawagutii.I investigated gene methylation pattern in F.kawagutii and responses of gene body methylation as well as DNA methylase gene transcription to varied light conditions.The major experimental design and results are as summarized below:1.After treating.kawagutii cultures with different concentration of methylation inhibitors 5-AZA-CdR(0,5.M,20?M,100?M)and Zebularine(0,100?M,200?M,500?M),we found that at the higher concentrations of both inhibitors(e.g.5-AZA-CdR at 100?M),growth rates were signifiantly inhibited.Cell size,particularly DNA content and photochemical efficiency(Fv/Fm ratio)showed clear difference from control group 5-AZA-CdR.We chose the samples with high concentration of drugs treated 5-AZA-CdR(100p.M)and Zebularine(200?M,500?M)for bisulfate sequenceing but did not find difference between the control group and treated group in genomic DNA methylation level.This result suggests that the classical DNA methyltransferase(DNMT1,DNMT3A and DNMT3B)are absent and most cytosine methylation occurs in transposons.The physiological changes in response to the inhibitor treatments may have been caused by the cytotoxicity of the methylation inhibitors.2.Based on the unique methylation pattern and special DNA methytransferase at Retrotransposons SymbioDIRS-DNMT3 in F.kawagutii,we attempted to explore the responses of light harvesting complex gene(peridinin-chlorophyll a binding protein PCP,light-harvesting complex center protein LHC and peridinin-chlorophyll a/cbinding protein acpPC)methylation level to varied light conditions and correlate it to these genes transcription levels.Results showed that these genes were all significantly up-regulated under low light at the transcriptional level while their methylation levels either decreased,stayed unchanged,or increased.SymbioDIRS-DNMT3 gene expression increased under higher light intensities.These results led us to propose that light intensity likely affects the gene body methylation level of light harvesting complex genes,which in turns regulates the expression level of these genes through the change in the SymbioDIRS-DNMT3 transcription level in F.kawagutii.