H3K27me3 Inhibits The Transcription Of FoxO1 And Affects The Function Of Ovarian Granulosa Cells In Pigs

The mammalian ovary is the last effector organ of hypothalamic-pituitary-ovarian axis,and it is also the main site of follicular development,ovulation and hormone secretion.Granulosa cells,as the important component of follicles,play a crucial role in the growth and atresia of follicles.FoxO1(forkhead box O1)is an important member of Fox O family,which regulates granulosa cells growth,proliferation,differentiation and apoptosis in mammalian ovary.H3K27me3(Tri-methylation of Histone H3 lysine 27)mainly inhibits gene transcription by changing the chromatin states of gene promoter.In this study,q RT-PCR and Western blot were applied to explore the expression of FoxO1 in prepubertal and pubertal ovaries of pigs,as well as follicles of different sizes and their granulosa cells.The over-expression vector and si RNA of FoxO1 were constructed and transfected into granulosa cells.Then the effects of FoxO1 on granulosa cells proliferation,apoptosis,cell cycle,steroid hormone secretion and expression of genes related to steroid hormone synthesis were detected.We adopt bioinformatic prediction and Ch IP-q PCR to determine the binding site of H3K27me3 in FoxO1 promoter.Moreover,granulosa cells were treated with H3K27me3 agonist(GSK-J4)and inhibitor(GSK-126)in order to explore the regulatory mechanism of H3K27me3 on FoxO1 transcription.Then CYP1A1 was screened as the downstream target gene of FoxO1.The regulation of FoxO1 on CYP1A1 expression was analyzed by q RT-PCR.Finally,over-expression vector and si RNA of CYP1A1 were constructed to study the effect of CYP1A1 on granulosa cells proliferation and apoptosis.The results of this study are as follows:(1)The expression of FoxO1 was significantly higher in prepubertal ovaries than that in pubertal ovaries.The expression of FoxO1 in small follicles(<3 mm)was very high,but decreased gradually in medium follicles(3 mm-6 mm)and large follicles(>6 mm).Furthermore,the expression of FoxO1 in small follicle granulosa cells was extremely higher than that in medium and large follicle granulosa cells.(2)FoxO1 can inhibit granulosa cells cycle progression,proliferation and testosterone secretion,as well as stimulate granulosa cells apoptosis and progesterone secretion.(3)H3K27me3 binds to FoxO1 promoter region 15437948-15438065 and restrains the transcription of FoxO1.(4)FoxO1 can up-regulate the transcription level of CYP1A1.(5)CYP1A1 can promote granulosa cells proliferation.In summary,H3K27me3 inhibits the transcription of CYP1A1 by regulating FoxO1,thereby suppressing the cell cycle,proliferation and testosterone secretion of granulosa cells,stimulating the apoptosis and progesterone secretion of granulosa cells.These studies lay a foundation for further exploration of how H3K27me3 regulates ovarian granulosa cells function and follicular development.