| Epigenetics is the study of potentially heritable changes in gene expression that does not involve changes in the sequence of nuclear DNA.DNA methylation is one of the most important epigenetic modification.Previous studies have shown that DNA methylation plays an important role in sex determination and differentiation of fish,but the role of DNA methyltransferases during the process remain unclear.Furthermore,the evolution,expression and function of dnmts in vertebrates have not been fully understood yet.In the present study,we explored the relationship between dnmts expansion and animal genome duplication,and analyzed the spatial and temporal expression profiles of dnmts in different tissues and development stages of gonads in Nile tilapia.The expression profiles of dnmts were validated by RT-PCR?qPCR and ISH.Furthmore,we evaluated the role of dnmts in sex differentiation by in vivo and in vitro experiments of dnmts inhibitor treatment.The main results were as follows:1)In this study,we identified seven dnmts genes(dnmt1,dnmt2,dnmt3 aa,dnmt3ab,dnmt3 ba,dnmt3bb.1 and dnmt3 bb.2)in Nile tilapia genome.Furthermore,we seperated all dnmts from the genome of ascidian(3),elephant shark(4),coelacanth(4),xenopus(4),chicken(4),lizard(5),mouse(5),human(5),spotted gar(5),stickleback(7),medaka(5),Fugu(7),zebrafish(8)and common carp(12).The increase of the dnmts genes number is mainly due to the replication of dnmt3 subfamily with one member in vertebrate under going 1R,2-3 members in vertebrate undergoing 2R,4-6 members in vertebrate undergoing 3R and 10 members in vertebrate undergoing 4R.These results revealed that the number of the dnmts genes have undergone an expansion following genome duplication in the teleost.Our results supported the 2R and 3R hypotheses.2)Phylogenetic analysis shown that the dnmts family divided into three branches: dnmt1,dnmt2 and dnmt3.Dnmt1 and dnmt2 were well conserved,while dnmt3 subfamily showed divergence.The result of synteny analysis showed during the 3R whole genome duplication,the dnmt3 a gave rise to dnmt3 aa and dnmt3 ab,and the dnmt3 b produced dnmt3 ba and dnmt3 bb.1.Furthermore,dnmt3 bb.1 underwent consecutive lineage-specific duplications to create dnmt3 bb.2.The results indicated that these dnmt3 genes might have duplicated in the common ancestor of teleosts and other vertebrates,and they expanded after the separation of the teleosts from other vertebrates but prior to the radiation of teleosts.3)We compared the divergence expression pattern of dnmts based on transcriptome data from eight adult tilapia tissues(brain,heart,head kidney,liver,ovary,testis,kidney and muscle).The results indicated that all dnmts genes were expressed in heart kidney and testis,and six of them were expressed in heart,ovary and kidney.Dnmt1 was expressed ubiquitously in all eight tissues with relatively high expression level,while dnmt2 presented low expression level.Dnmt3 paralogues were differentially expressed between different tissues.Especially,the various dnmt3 b paralogues notably were highly expressed in the gonads.4)We characterized the spatial and temporal expression pattern of dnmts during gonadal development.The gonadal transcriptome data indicated that all dnmts genes were expressed in tilapia gonads.Almost all dnmts displayed significant sexually dimorphic expression patterns,in which four of them(dnmt3aa,dnmt3 ab,dnmt3bb.1 and dnmt3 bb.2)exhibited the higher expression level in testis than that of ovary,while dnmt1,dnmt2 and dnmt3 ba exhibited the higher expression level in ovary than that of testis.Interestingly,four de novo dnmts(dnmt3aa,dnmt3 ab,dnmt3bb.1 and dnmt3 bb.2)were significantly up-regulated in sex-reversed gonad compared to normal ovary.The results indicated these de novo dnmts may play an important roles in gonadal development.We verified the cells expressing population of the dnmts family members in the developing gonads by in situ hybridization,the seven dnmts were found to be expressed mainly in phase I and II oocytes of the ovary and spermatocytes of the testis.5)We evaluated the expression pattern of dnmts and the key genes in sex differentiation after the treatment of DNA methyltransferase inhibitor(5-aza-dC).When gonads were incubated with 5-aza-dC in vitro,the expression of dnmts genes were down regulated significantly,while the expression of cyp19a1a(a key gene in female pathway)and dmrt1(a key gene in male pathway)increased significantly.Furthermore,when tilapia embryos were breeded in the water with different concentration of 5-aza-dC,deformities rate and mortality rate of embryos increased.The results of alizarin red staining showed that the spines of most fish in the treated group were deformed.When female tilapia larvae were intraperitoneally injected with a relatively high concentration of 5-aza-dC,25-34% of females were reversed into males.Immunohistochemical results showed that after 60 days of treatment,the gonads of the treated fish expressed the male specific gene dmrt1,and no longer expressed the female specific gene cyp19a1 a.Our results suggest that dnmts plays an important role in the development of Nile tilapia,especially in gonadal development,but the molecular mechanism still needs to be clarified... |
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