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 mR...

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Bibliographic Details
Published in:Gene 2007-04, Vol.391 (1), p.63-75
Main Authors: Brûlé, Sophie, Sayasith, Khampoune, Sirois, Jean, Silversides, David W., Lussier, Jacques G.
Format: Article
Language:English
Subjects:
80K-H
Alternative Splicing
Amino Acid Sequence
Animals
Base Sequence
Beta-glucosidase II
Cattle
Cell Line
Electrophoretic Mobility Shift Assay
Exons
Female
Gene Expression Profiling
Granulosa Cells - metabolism
Hepatocystin
Introns
Luciferases - genetics
Luciferases - metabolism
Male
Membrane Proteins - genetics
Molecular Sequence Data
Ovarian Follicle - metabolism
PRKCSH
Promoter Regions, Genetic - genetics
Protein Binding
Reverse Transcriptase Polymerase Chain Reaction - methods
RNA, Messenger - genetics
RNA, Messenger - metabolism
Sequence Homology, Amino Acid
Sequence Homology, Nucleic Acid
Transcription Initiation Site
VASAP-60
YY1
YY1 Transcription Factor - metabolism
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Summary: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.
ISSN:0378-1119
1879-0038
DOI:10.1016/j.gene.2006.12.012