Study On The Modification Mechanism And Function Of Phosphorylation Of Serine At 65^th Site Of LSD1 During Follicle Selection In Chicken

Follicle selection in chicken refers to the process of selecting a hierarchal follicle from a cohort of small yellow follicles（SY）with a diameter of 6-8mm.The follicle being selected develops rapidly,ovulates and an egg lays.Follicle selection is a key factor affecting the egg-laying performance of chickens.It is estimated that sexually mature hens have about12,000 oocytes,but only a few hundred of them（4%）mature and ovulate through follicle selection.Our previous studies found that the phosphorylation level of LSD1 serine 65^th was significantly increased in F6 follicles,but its function was unclear.In this study,we plan to analyze the mechanism of this phosphorylation modification,the effect of dephosphorylation on the gene expression of chicken hierarchal granulosa cells and the functiona of FGF9regulated by LSD1Ser65p by Western blotting,q RT-PCR,immunoprecipitation,transcriptome sequencing and other approaches.The results are as follows:（1）Mechanism of phosphorylation of LSD1Ser65p.Phosphorylation modification was mediated by some kinases.Therefore,we first predicted that LSD1Ser65p maybe catalyzed by phosphorylated kinases CDK5 and GSK3,and treated hierarchal granulosa cells and theca cells with two corresponding kinase inhibitors.We found that GSK3 inhibitor was able to significantly reduce phosphorylation in hierarchal theca cells（P<0.05）,however,not in granulosa cells;the level of phosphorylation in CDK5 inhibitor-treated hierarchal granulosa cells was significantly decreased（P<0.05）,but not in theca cells.Subsequently,it was determined that LSD1 was localized in the nucleus in hierarchal granulosa cells,and co-immunoprecipitation analysis demonstrated that the phosphorylation of LSD1 serine 65^thinteracted with CDK5,which proved that CDK5 mediated the phosphorylation of this site.（2）Effects of dephosphorylation of chicken LSD1 serine 65^th on gene expression in chicken hierarchal granulosa cells.In order to further study the role of LSD1Ser65p in chicken follicle selection,a mutation vector of this phosphorylation site was constructed and transfected into chicken hierarchal granulosa cells and pre-hierarchal granulosa cells.It was found that the phosphorylation level of serine 65^th of LSD1 was significantly reduced in hierarchal granulosa cells（P<0.05）,but there was no significant change in pre-hierarchal granulosa cells,so the transcriptome of hierarchal granulosa cells transfected with the mutant vector was analyzed.With|log2（Fold Change）|>1 and q value<0.05 as the criteria for screening differential genes,23 up-regulated genes and 7 down-regulated genes were screened.Bioinformatics analysis found that differential expressed genes enriched in biological processes of the binding of fibroblast factor receptors and the activity of growth factors.（3）The effect of dephosphorylation of LSD1Ser65p on the dimethylation of histone H3K4 in the promoter region of chicken follicle selection-related genes.In order to study whether the phosphorylation modification of serine 65^th of LSD1 regulates its own demethylation function,we analyzed the changes in the dimethylation level of histone H3K4in the promoter region of the two differentially expressed genes FGF9 and ACTN2,and found that when this site was dephosphorylated,the dimethylation level of H3K4me2 in the promoter region of FGF9 and ACTN2 was significantly increased（P<0.05）,suggesting that phosphorylation modification upregulates the demethylase function of LSD1.（4）The function of FGF9.To analyze the function of FGF9,we constructed FGF9overexpression vector and transfected chicken hierarchal granulosa cells.The results indicated that FGF9 promotes the proliferation of chicken hierarchal granulosa cells（P<0.05）.In conclusion,the phosphorylation of serine 65^th of LSD1 has different roles in chicken follicle selection,and its phosphorylation modification is mediated by CDK5 in chicken hierarchal granulosa cells.After dephosphorylation and transcriptome sequencing of hierarchal granulosa cells,the methylation level of H3K4 in the promoter region of differentially expressed genes related to follicle selection was increased,proving that this phosphorylation modification enhances the demethylation activity of LSD1.These results provide a molecular mechanism for understanding chicken follicle selection,and a theoretical basis for improving egg-laying performance and molecular breeding in chickens.