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.
Brûlé
Sophie
S
Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, Université de Montréal, P.O. Box 5000, St-Hyacinthe, Québec, Canada J2S 7C6.
Sayasith
Khampoune
K
Sirois
Jean
J
Silversides
David W
DW
Lussier
Jacques G
JG
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D005091:Exons; D005260:Female; D020869:Gene Expression Profiling; D006107:Granulosa Cells / Q000378:metabolism; D007438:Introns; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D006080:Ovarian Follicle / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D024363:Transcription Initiation Site; D050986:YY1 Transcription Factor / Q000378:metabolism
D008565:Membrane Proteins; D012333:RNA, Messenger; C406303:VASAP-60 protein, Bos taurus; D050986:YY1 Transcription Factor; D008156:Luciferases