SF-1,Foxl2 Expression Patterns And CpG Methylation Regulate The Promoter Of Aromatase In Cheilinus Undulates

Hermaphroditic fish shows a plastic sex and exhibited natural sex reversal during their lifespans.However,the underlying mechanisms for the sex reversal remaine unclear.Cheilinus undulates,a hermaphrodite fish,whose aromatase is a synthesis switch from androgen to estrogen.Accordingly,it is important work to figure out the regulatory mechanism of the Cheilinus undulates cyp19a1a and cyp19a1b during sex reverse.In the present thesis,tissue distribution levels of steroidogenic factor 1(SF-1)and Forkhead box L2(Foxl2)in Cheilinus undulates were evaluated by RT-q PCR.Possible methylation patterns of the promoter were analyzed.A luciferase reporter analysis revealed that effect of DNA methylation on the transcriptional activity of cyp19a1a and cyp19a1b promoter.The electrophoretic mobility shift assay(EMSA)was established to identify the exact bingding site of SF-1 and Foxl2 on the cyp19a1a and cyp19a1b promoter.The current study provides important basis for revealing the molecular mechanism of sex reversal in Cheilinus undulates.1.Methylation profile of cyp19a1a and cyp19a1b promoter at different CpG sites in immature,female,and male individuals were determined by Sequenom Mass Array.The results showed that the methylation rate of the same CpG site in different individuals was different.According to the key binding sites of SF-1 and Foxl2 on cyp19a1a and cyp19a1b obtained in our previous study,cyp19a1a(+114bp to-462bp)and cyp19a1b(-633bp to-1144bp)were selected for the expression vector construction.The methylated promoter constructed into luciferase reporter was co-transfected with eukaryotic expression vectors of SF-1 or Foxl2 into HEK293T cells to examine the effect of methylation on promoter transcriptional activity.The results showed that induced hypermethylation of the Cheilinus undulates cyp19a1a and cyp19a1b promoter completely suppressed promoter transcription stimulation in vitro by Foxl2 and SF-1 in combination.2.The transcripts of SF-1 genes were detected in Gil,Kidney,Spleen,Brain,Gonad,Pituitary,Liver,Cerebellum,Mesencephalon and Hypothalamu.SF-1 transcript was mainly expressed in Liver,Pituitary,Cerebellum and Head-kidney.The transcripts of Foxl2genes were detected in Gil,Kidney,Spleen,Brain,Gonad,Pituitary,Liver,Cerebellum,Mesencephalon and Hypothalamu.Foxl2 transcript was highly expressed in Pituitary and Liver.3.Prokaryotic expression vectors coding SF-1 and Foxl2 were constructed by double digestion.The expression of both proteins wereinduced at 16℃,30℃,37℃ with different concentration of IPTG(final concentrations of 0.1mmol/L,0.5mmol/L,1mmol/L).However,the products were only expressed in inclusion bodies.Instead,eukaryotic expression was employed to obtain both proteins.The eukaryotic expression vectors coding SF-1 and Foxl2 were transfected into HEK293T cells,while,only Foxl2 was detected by Western Blot,but SF-1.The conditions for EMSA were explored,and an experimental platform was established for EMSA.