Characterization of the testis-specific promoter region in the human pituitary adenylate cyclase-activating polypeptide (PACAP) gene.
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide localized in the testis at concentration comparable to that found in the brain, suggesting involvement in spermatogenesis. In this study, we identified the human PACAP testis-specific exon (TSE) 10.9 kb upstream from the translational start site and found that the testis-specific transcript of the human PACAP gene was found to be spliced from the TSE into a region of intron 2 without a frameshift. The resulting PACAP precursor has no signal peptide, suggesting that PACAP functions physiologically in an intracrine manner in the testis. The 5'-flanking region of the TSE contains an 80-bp fragment with potent promoter activity in testicular F9 cell. Electrophoresis mobility shift assays showed that proteins from the F9 nuclear extract interacted specifically with the 80-bp fragment. DNA affinity chromatography allowed isolation of the specific proteins bound to the 80-bp fragment, two of which were identified as Poly (ADP-ribose) polymerase-1 (PARP-1) and TIA-1-related protein (TIAR) by mass spectrometry. By using their siRNAs, the depletion of their proteins in F9 cells affected the potent promoter activity of the 80-bp fragment, suggesting that they might be involved in the testis-specific gene expression of PACAP.
Tominaga
Aiko
A
Department of Pharmacology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima-shi, Kagoshima 890-8544, Japan.
Sugawara
Hideki
H
Futagawa
Toshitaka
T
Inoue
Kazuhiko
K
Sasaki
Kazuki
K
Minamino
Naoto
N
Hatakeyama
Mamoru
M
Handa
Hiroshi
H
Miyata
Atsuro
A
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D005091:Exons / Q000235:genetics; D020869:Gene Expression Profiling; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000235:genetics* / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D041701:Swiss 3T3 Cells; D013737:Testis / Q000378:metabolism*; D014162:Transfection
D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D011065:Poly(ADP-ribose) Polymerases