Methylation Of The Promoter In TSH-Dio2/Dio3-TH System To Regulate The Seasonal Reproduction In Striped Hamster (Cricetulus Barabensis)

Cricetulus barabensis is a small rodent widely distributed in northern China.It has high fecundity and has two breeding peaks in spring and autumn.It is a typical seasonal breeding animal.Mammalian seasonal breeding activities are regulated by the photoperiod.The secretion of melatonin and the release of GnRH are the major factors responsible for photoperiodic regulation of seasonal reproductive activity in animals.The PT-TSH-TSHR-Dio2/Dio3-TH-GnRH pathway is involved in photoperiodic regulation of melatonin(MEL)in animals.The seasonal reproduction plays an important role,but the mechanisms and effects of photoperiodic regulation of the expression of the TSH-Dio2/Dio3-TH system have not been reported.In this study,the sequence of TSHB promoter region 1772 bp(MH005827)was cloned and the TSHR promoter sequence was 2054 bp(MH005826);Dio2 1479 bp(MH005824);Dio3 1550 bp(MH005825);TH 974 bp(MH005828).By bioinformatics analysis,the core promoter region was identified and CpG islands were successfully predicted.Based on the analysis results,the methylation analysis sequences with CpG island were predicted: Dio2 gene sequence is 216 bp and contains 11 CpG sites;Dio3 gene sequence is 159 bp and contains 11 CpG sites;TSHB gene sequence is 299 bp and contains 4 CpG sites;TSHR gene sequence is 233 bp and contains 12 CpG sites;TH gene sequence 216 bp,contains a total of 11 CpG sites.Secondly,high-breeding(autumn)and low-breeding(winter)Cricetulus barabensis were selected to study and the hypothalamus were used to extract genomic DNA.Bisulfite Genomic Sequence(BSP)was used to detect the methylation status of promoters of TSHB,TSHR,Dio2,Dio3,and TH genes.The promoter methylation results were analyzed using Biq Analyzer 2.0 and QUMA to obtain a methylation profile.The differences in gene methylation levels between different genes,seasons,and genders were statistically analyzed,and the correlation between gene methylation and relative expression lever was explored.The results show that:(1)There was different methylation level of each gene,in which the TSHB and Dio2 gene promoters were hypermethylated(about 80%),and the TSHR and Dio3 were hypomethylation state(about 10%),while the TH is moderately methylated(about 50%).The results suggest that the TSH-Dio2/Dio3-TH system may affect the reproductive activity through different levels of methylation in genes participating in the TSH-Dio2/Dio3-TH system,and by the comparative analysis of the promoter regions of the genes,the methylation level of genes may be associated with the sequence structure characteristics and the position of the CpG Island in the promoter.(2)The correlation analysis between the methylation rate of TSHB promoter and the relative expression level of TSHB mRNA in the high-and low-propagation Cricetulus barabensis revealed that: The correlation coefficient was r=-0.649,P=0.022.It was further clarified that there was a significant negative correlation between the expression level of TSHB mRNA and the methylation level of TSHB promoter(P<0.05).The same analysis of the correlation between TSHR,Dio2,and Dio3 mRNA expression levels and promoter methylation rates revealed negative correlations: The correlation coefficients were-0.912(P=0.000),-0.583(P=0.047),and-0.849(P=0.000),respectively.Therefore,we can infer that the methylation level of the promoter regulates the transcriptional expression of the gene,and that hypermethylation of the promoter reduces the transcriptional expression level of the gene and thus participates in influencing the seasonal breeding activity of the Cricetulus barabensis.(3)Differences in expression levels of genes between different seasons are inconsistent: There was a significant difference in the methylation level of Dio2 promoter between high and low breeding seasons(P=0.03).In addition,analysis of TSHR showed that there was a significant difference in methylation levels between different seasons(P<0.001).However,the methylation status of Dio3,TSHB and TH promoters was further analyzed and found that there was no significant difference in methylation rates between different seasons(P>0.05),suggesting that seasonal breeding of Cricetulus barabensis may be related to TSH-Dio2/Dio3-TH system gene promoter particularly related to seasonal changes in the methylation of Dio2 and TSHR.(4)There was a significant difference in the methylation level of Dio3 promoter region between genders(P=0.012,P<0.05).The methylation level of male individuals was significantly higher than that of females,suggesting that it may be related to the breeding activity of Cricetulus barabensis.There was no significant difference in the methylation level of Dio2 promoter between genders(P=0.226).There was no significant difference in the methylation levels of TSHB,TSHR,and TH promoters between genders(P>0.05).Analysis of seasonal and gender interactions of methylation levels of Dio2,Dio3,TSHB,TSHR,and TH promoters in the hypothalamus of the Cricetulus barabensis revealed a cross-over effect only in the Dio3 promoter methylation analysis.In summary,Cricetulus barabensis were sampled in natural seasons and the genomic DNA were treated by sulfite sequencing(BSP)to explore the methylation level exploring the mechanism of TSH-Dio2/Dio3-TH system to regulate the reproductive activity.Result reveals the seasonal differences in methylation of the promoter region of and participates in the regulation of expression.It provides new ideas and new experimental evidences for the further study of the regulatory mechanisms of mammalian seasonal reproductive rhythms.