The Influence Of AC3 Deletion On DNA Methylation In The MOE Of Mouse

The type 3 adenylyl cyclase?AC3?plays an important role in the olfactory induction of mice.Compared with wild-type mice,AC3-deficient mice showed a decline in olfactory ability.The main olfactory epidermal tissue?MOE?gradually thinned with the growth and development of mice,the structure of the tissue cells is loose,and dendritic development damaged.It has been reported that AC3^-/-mice have altered the expression of a large number of genes,including olfactory receptor genes and epigenetic factors,compared with AC3^+/+ mice.It is well known that epigenetics plays an important role in animal development and regulation of gene expression.However,whether the methylation level and the hydroxymethylation level of the MOE genes in mice were changed after AC3 deletion,whether the change of methylation level of these genes and the change of hydroxymethylation level were related to the change of expression level,that problems have not been answered yet.In this study,we investigated the methylation level of the promoter region of mouse MOE by DNA methylation chip and bioinformatics analysis was performed.The results showed that the degree of methylation of the promoters of the 1978 genes in AC3^-/-mice was altered compared to AC3 wild-type mice.In this study,the 1978 genes were analyzed by Gene Ontology.The results shows that 1313 genes are involved in biological processes,1299 genes are involved in cell components and 1296 genes are involved in molecular functions.These differential methylation genes were found to be distributed on 36 channels by signal pathway analysis.In this study,ten genes associated with olfactory and cAMP,including Cngb1,Hcn4,Olfm1,Pde4 a,Olfr231,Olfr378,Olfr1153,Olfr651,Olfr691 and Olfr1394,were selected and the methylation level of the promoter region was verified by methylation-specific PCR.The results showed that the methylation level of the promoter regions of Hcn4,Olfm1,Olfr1153,Olfr231,Olfr378,Olfr651 and Olfr691 was higher in the AC3^-/-mice than that in the AC3^+/+ mice,while the methylation level of the promoter regions of Cngb1,Pde4 a and Olfr1394 decreased.Then the expression level of the ten genes was detected by qRT-PCR,and the correlation between the methylation level of the promoter region and its expression was investigated.The mRNA expression levels of Cngb1,Hcn4,Olfm1,Olfr1394,Olfr1153,Olfr231,Olfr378 and Olfr691 were decreased in the AC3^-/-mice compared with the AC3^+/+ mice,while the mRNA expression levels of the Pde4 a and Olfr651 were increased.In addition,we also used hydroxymethylated DNA immunoprecipitation combined quantitative real-time PCR to explore the hydroxymethylation level of the ten genes.The results showed that the hydroxymethylation level of Hcn4,Pde4 a and Olfr1153 were lower in the AC3^-/-mice than that in the AC3^+/+ mice,while the hydroxymethylation level of Olfm1,Olfr1394,Olfr231,Olfr378,Olfr651 and Olfr691 showed an upward trend.There was a significant difference in the hydroxymethylation level of Olfr231 and Olfr651,but there was no significant difference in other genes.This may be due to the fact that hydroxymethylation is an epigenetic marker,and the differences between individuals may be large,so the number of samples will be increased later to supplement the experimental data.This study further elucidates the mechanism of olfactory loss in AC3^-/-mice and provides a theoretical basis for the treatment of diseases caused by loss of olfaction.