Study On The DNA Methylation Of The Whole Genome And Its Formation Mechanism And Biological Function Of The Red Rice Pirate

DNA cytosine methylation is one of the most important and conserved epigenetic modifications in diverse eukaryotic organisms.This type of methylation is involved in various epigenetic processes,including X chromosome inactivation,gene imprinting and histone modification,and plays critical roles in cellular differentiation,regulation of gene expression,gene alternative splicing,suppression of transcriptional noise and tumourigenesis.In recent years,with the rapid development of next-generation sequencing technology,combining bisulphite-based detection of methylated cytosines with high-throughput whole-genome sequencing technology(Bisulphite Sequencing,BS-Seq)has enabled the determination of genome-wide methylomes in many eukaryotic organisms.These methylomes have significantly expanded the understanding of the distribution characteristics,functions and evolution of DNA methylation in eukaryotes.The red flour beetle Tribolium castaneum is an important model organism for the study of gene function.Its genome encodes two DNMTs,DNMT1 and DNMT2,but lacks DNMT3,which is regarded as the only de novo methyltransferase and a necessary component of the DNA methylation system.Therefore,the presence of DNA methylation in T.castaneum has long been controversial.Whether the DNA cytosine methylation is present in T.castaneum?if so,what is the distribution pattern of the DNA methylome?How is it established and how does the information encoded by DNA methylation interpret into functional output?More important,What is the biological functions of DNA methylation in T.castaneum?Here,by using bisulphite sequencing(BS-Seq),we first confirmed the existence of DNA methylation and described the methylation profiles of the four life stages of T.castaneum.Then we identified the DNA methyltransferases gene in T.castaneum and characterized its functional mechanisms,lastly,we identified the methyl-CpG-binding domain protein in T.castaneum and explored the functional mechanisms of DNA methylation.In the T.castaneum genome,both symmetrical CpG and non-CpG methylcytosines were observed which occupy about 0.1%-0.2%of the whole genome cytosines,the global methylation level showed an increased trend from the new born to the mature stage.Asymmetrical non-CpG methylation,which was predominant in the methylome,was strongly concentrated in intergenic regions and introns but absent from exons.Gene body methylation was negatively correlated with gene expression levels.While the symmetrical CpG methylation occupied a small part in T.castaneum methylome,was primarily enriched in gene bodies and was positively correlated with gene expression levels.The overall DNA methylation patterns of the four samples were highly correlated with a few different methylated regions(DMR),Gene Ontology(GO)analysis for the genes associated with the DMRs show,most of the genes were enriched for biological phase,enzyme regulator and transporter activity,which indicate DNA methylation play important roles during the transition between the different development stages.How does the novel DNA methylation pattern establish in T.castaneum?we next identified the DNA methyltransferases gene in T.castaneum and characterized its biological functions.There are two DNA methyltransferase genes in T.castaneum:Tcdnmtl which has two alternative spliced isoforms Tcdnmt1a and Tcdnmt1b and Tcdnmt2,while absent the dnmt3-de novo DNA methyltransferases.Tcdnmt1a shares conserved functional domains with other organisms,whereas Tcdnmt1b lacks the C-terminal DNA methylase domain.The DNA methylase domain is the catalytic domain of DNMTs responsible for the transfer of methyl groups from SAM to the corresponding 5-C in the pyrimidine ring of the DNA.The absence of the DNA methylase domain indicates loss of the basic function of DNMTs.Thus,Tcdnmt1b likely cannot methylate DNA.By using electrophoretic mobility shift assay,we analysed the catalytic activity of the two DNA methyltransferases,the TcDNMTla and TcDNMTlb can bind to the CpG sites while only the TcDNMTla can specifically methylate the hemi-methylated CpG sites,which act as the maintenance DNA methyltransferase in T.castaneum,the TcDNMTlb can not catalyze the formation of methylated CpG sites which may due to the loss of the catalytic domain.The TcDNMT2 can not bind to the DNA substrate which may suggest the TcDNMT2 can not catalyze the DNA methylation which may responsible for the tRNA methylation as in other eukaryotes.Knockdown the Tcdnmt1a using RNAi caused the metamorphosis deficiency and embryonic lethality,while knockdown the Tcdnmt1b and Tcdnmt2,there is no significant influence during the normal development of T.castaneum,which indicate DNA methylation play important roles in metamorphosis and embryogenesis of T.castaneum.Additionaly,the methylation level of CpG sites is significantly decreased after knocking the Tcdnmt1a but not Tcdnmt1b or Tcdnmt2.Which support the TcDNMT1a is responsible for the CpG methylation in vivo.How does the information encoded by the novel methylation pattern interpret into functional significance in T.castaneum?Methyl-GpG-binding domain protein(MBD)can specifically bind to methylated DNA and translate the chemical modification into appropriate cell states through interacting with diverse partners by altering chromain structure,interpreting gene imprinting,gene specific transcription activation or repression and controlling RNA splicing.We next identified the methyl-CpG-binding domain protein and characterized its functions.The T.castaneum genome encodes a single protein TcMBD2/3 which shows extensive homologous to the MBD2/3 of vertebrates.Knockdown the gene expression using RNAi in different developmental stages caused lethal phenotypes including ecdysial arrest in larvae,morphological deficiency in pupae,abnormal neuronal function and lost fecundity in adult.In addition,the band shift assay demonstrated the TcMBD2/3 lost the DNA binding activity either with the CpG or the nonCpG methylated sites,which may indicate the TcMBD2/3 lost the function as epigenetic regulators.In this study,we identified the first single-base resolution DNA methylome in different development stages of T.castaneum and characterized its distributionpatterns,then we identified the DNA methyltransferases and methyl-CpG-bindingdomain protein and clarify the establishment mechanisms,functionl methanisms and biological functions of DNA methylation in T.castaneum.