Structure of the bovine VASAP-60/PRKCSH gene, functional analysis of the promoter, and gene expression analysis

Vacuolar system-associated protein-60 (VASAP-60) constitutes the bovine ortholog of the human “protein kinase C substrate 80K-H” (PRKCSH or 80K-H). We characterized the bovine VASAP-60/PRKCSH gene structure and promoter, identified cis-acting elements controlling VASAP-60 expression, searched for mRNA splice variants, and analyzed mRNA expression in ovarian follicles. Expression of VASAP-60 mRNA showed a 2.4-fold increase (P < 0.0001) in granulosa cells of dominant follicles compared to small follicles (2-4 mm) or ovulatory follicles, and no mRNA splice variant was identified. The bovine VASAP-60 gene encompasses 12.5 kb and is composed of 18 exons and 17 introns. Primer extension analysis revealed a single transcription initiation site, and the promoter lacks a TATA box. Promoter activity assays were performed with a series of deletion constructs in different bovine cell lines (endometrial epithelial glandular, kidney epithelial and aortic endothelial) to identify cis-acting elements. The − 53/+ 16 bp fragment (+ 1 = transcription start site) conferred minimal promoter activity whereas activator and repressor elements were located in the − 200/− 53 bp and − 653/− 200 bp fragments, respectively. Analysis of cis-acting elements in the − 200/− 53 bp activation domain revealed by gel shift assays and chromatin immunoprecipitation assay that transcription factor YY1 binds to VASAP-60 promoter. This study is the first to report that VASAP-60 is up-regulated in granulosa cells of dominant follicles, to document the primary structure of the bovine VASAP-60 gene and promoter, and to demonstrate that YY1 binds to the VASAP-60 proximal promoter and may act as a positive transcriptional regulator.