Neural-restrictive silencers in the regulatory mechanism of pituitary adenylate cyclase-activating polypeptide gene expression.
Pituitary adenylate cyclase-activating polypeptide (PACAP) is known as a pleiotropic neuropeptide and is present abundantly in central nervous system. During a detailed analysis of the 5'-flanking region of the mouse PACAP gene, we found and characterized two negative regulatory elements, which are homologous to the neural-restrictive silencer element, and are termed neural-restrictive silencer-like elements 1 and 2 (NRSLE1 and NRSLE2). Their sequence and position were significantly conserved among mouse, human, and rat PACAP genes. In the electrophoretic mobility shift assay (EMSA) with nuclear extracts of Swiss-3T3 cells and individual oligonucleotide probes for NRSLE1 and NRSLE2, a specific complex was observed to have the same migration as compared with the NRSE probe of rat type II sodium channel gene (NaII). Furthermore, these complexes were efficiently competed by the unlabeled NaII probe. In the luciferase reporter assay, the reporter gene constructs containing NRSLEs, driven by heterologous SV40 promoter, exhibited repression of luciferase activity almost equal to basal level in Swiss-3T3 cells. In contrast, the repression was not observed in differentiated PC12 cells with NGF. These results suggested that the neural-restrictive silencer system might be involved in the regulatory mechanism of neuron-specific PACAP gene expression.
Sugawara
Hideki
H
Department of Pharmacology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan.
Inoue
Kazuhiko
K
Iwata
Shin-ichi
S
Shimizu
Takao
T
Yamada
Katsushi
K
Mori
Nozomu
N
Miyata
Atsuro
A
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D020868:Gene Silencing; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics; D051379:Mice; D009414:Nerve Growth Factors / Q000235:genetics*; D009420:Nervous System / Q000254:growth & development / Q000378:metabolism; D009479:Neuropeptides / Q000235:genetics*; D018377:Neurotransmitter Agents / Q000235:genetics*; D016716:PC12 Cells; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D051381:Rats; D041701:Swiss 3T3 Cells
C495402:Adcyap1 protein, mouse; C495403:Adcyap1 protein, rat; D009414:Nerve Growth Factors; D009479:Neuropeptides; D018377:Neurotransmitter Agents; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D004247:DNA; D008156:Luciferases