Molecular mechanism of ATF6 regulating the reproduction related gene HSPA1L

Objective To explore the effect of endoplasmic reticulum stress activating transcription factor 6 (ATF6) on the expression of reproduction related gene heat shock protein family A member 1 like (HSPA1L) and preliminarily clarify its regulatory molecular mechanism. Methods The ATF6 over-expression plasmid was transfected into HEK-293T cells and the over-expression efficiency was verified by RT-qPCR and Western blot. The transcriptome sequencing information of testis tissue of male ATF6 knockout mice was used to screen five reproduction related genes downstream of ATF6. The dual luciferase reporter assay selected the downstream genes with high promoter activity and detected the effect of over-expression of ATF6 on the promoter activity of downstream genes.The possible binding sites of ATF6 and downstream gene promoters were predicted by gene-regulation. RT-qPCR and Western blot were used to detect the effect of over-expression of ATF6 on downstream gene expression in HEK-293T cells. Whether ATF6 binds to downstream gene promoters was determined by electrophoretic mobility shift assay (EMSA). Results The expression of ATF6 mRNA (P＜0.001) and protein (P＜0.001 and P＜0.05) in HEK-293T cells was significantly increased after transfection. HSPA1L(P＜0.001 and P＜0.05), a reproductive related gene downstream of ATF6 was screened by transcriptome sequencing and dual luciferase reporter assay. ATF6 promoted the truncated promoter activity of HSPA1L(P＜0.001). After over-expression of ATF6, the expression of HSPA1L was significantly increased(P＜0.001). The differences were statistically significant. ATF6 protein could bind to the aagtcgtcac DNA sequence of HSPA1L promoter. Conclusions ATF6, a key molecule of endoplasmic reticulum stress, regulates the expression of reproduction related gene HSPA1L by binding to the promoter of HSPA1L.This result will lay a foundation for further research on the prevention or treatment of endoplasmic reticulum stress (ERS) related male infertility.