Transcriptional Control of JAM-A via the Interaction of Nuclear Factor-Y, Nuclear Factor-1, and GATA Proteins with NF-Y Motifs

Abstract

The expression of JAM-A, a key integral tight junction protein, is tightly regulated to ensure that the integrity of the seminiferous epithelium could be maintained during the translocation of spermatocytes at the blood-testis barrier. In this study, we have characterized the core promoter region for the basal transcription of JAM-A gene and elucidated how the NF-Y motifs within the core promoter of JAM-A gene are modulated by binding of various transcription factors. By transient transfection assay performed in a mouse Sertoli cell cell line, TM4 cells, we showed that the minimal JAMA promoter is located between nt -360 and -1 (relative to the translation start site). By the use of site-directed mutagenesis, NF-Y motifs within the minimal promoter region have found to be functionally cooperated with one another to regulate JAM-A gene transcription. Overexpression of NFYA, NF-1A, NF-1B, NF-1C, NF-1X and GATA-6 significantly increased the promoter activity whilst overexpression of mutant NY-YA, wild-type GATA-1 and GATA-4 showed no effect on JAM-A promoter activity. By EMSAs, NF-YA and GATA-6 formed DNA-protein complex with NY-F motifs. Work is now in progress to assess the in vitro and in vivo binding of NF-1 proteins by EMSA and chromatin immunoprecipitation (ChIP) assay. Taken together, nuclear factor-Y, nuclear factor-1 and GATA proteins play an important role in regulating JAM-A gene expression in Sertoli cells. This work was supported by Hong Kong Research Grant Council grant (HKU7609/06M and HKU7715/07M) and HKU/CRCG Seed Funding to WYL.