Transcription factor PRDM15 binds to the promoter mutation locus of PPP2R5C promoted proliferation of goat granulosa cells

Abstract

Abstract Protein phosphatase 2 regulatory subunit B’gamma (PPP2R5C) is involved in a variety of physiological processes in mammals, such as growth and development, cellular metabolism, and immunity. Here, the PCR sequencing and KASP genotyping sequencing were used to detect the distribution of SNPs of PPP2R5C, and then a series of molecular biology technology were used to detect the regulation pathway between PPP2R5C and kidding number trait of goats. The sequencing results showed that there was a g.65977460A > G mutation locus in the promoter region of PPP2R5C, and there were 223 individuals with genotype GG, 91 with genotype GA, and 51 with genotype AA in the tested population of Yunshang black goats. Association analysis showed that the kidding number trait of second and third parity were significantly higher for the individuals with AA genotype than those with the AG and GG genotypes. Subsequently, the results of dual luciferase activity assay showed that the promoter activity of PPP2R5C-mutant-A was significantly higher than that of PPP2R5C-Wild-G (P < 0.05). The transcription factor PRDM15 was predicted to bind to a mutation in PPP2R5C (g.65977460A > G). The results of ChIP-qPCR showed that the transcription factor PRDM15 could bind to the promoter region of PPP2R5C. Moreover, the expression of PPP2R5C, proliferation factors CDK4 and Cyclin-D2 in goat GCs was significantly increased after PRDM15 (PR/SET domain 15) overexpression (P < 0.05). While the results of EdU and CCK-8 showed that the proliferation rate of goat GCs increased after PRDM15 overexpression and the proliferation efficiency of goat GCs was significantly reduced after PRDM15 inhibition (P < 0.05).Finally, the expression of the PI3K-Akt pathway (phosphatidylinositol-3-kinase/protein kinase B) marker AKT2 (AKT serine/threonine kinase 2) was significantly increased in granulosa cells overexpressing PRDM15, and the opposite was true after inhibition of PRDM15. Together, our results showed that PPP2R5C g.65977460A > G was significantly associated with the kidding number in Yunshang black goats. The transcription factor PRDM15 was identified to bind to the promoter region of PPP2R5C g.65977460A > G mutation locus promoted the proliferation of goat granulosa cells (GCs) by the PPP2R5C/PI3K-Akt pathway. These results provide a new insight into the molecular mechanisms underlying the phenotypic changes in goats.