A negative regulatory element required for light-dependent pinopsin gene expression.
In vertebrates, a variety of light-stimulated genes are distributed in the retina, the pineal gland, and the suprachiasmatic nucleus, but a cis-element(s) responsible for the light-dependent transcriptional regulation is left unexplored. Focusing on the pinopsin gene, a light-stimulated gene in the chick pineal gland, we performed a transcriptional analysis in the primary culture of the chick pineal cells that were transiently transfected with a luciferase reporter gene fused with various lengths of the 5' upstream region of the pinopsin gene. Light-dependent enhancer activity was detectable in the construct with the upstream region between -1156 and +31. Introduction of mutations within the 18 bp sequence at positions -1103 to -1086 (TGGCACGTGGGGTTCCTC), including a CACGTG E-box sequence, elevated the transcriptional activity in the dark and thereby abrogated the light dependency, suggesting that the 18 bp sequence is essential for a reduction of the transcriptional activity in the dark. In an electrophoretic mobility-shift assay, we identified a pineal nuclear factor(s) capable of binding to the 18 bp element in a sequence-specific manner. When a 49 bp fragment (-1122 to -1074) including the 18 bp sequence was placed upstream of the simian virus 40 promoter, the transcriptional activity was dramatically suppressed regardless of light conditions in the chick pineal cells, and a more pronounced repression was observed in nonpineal/nonphotosensory LMH and NIH 3T3 cells. These results suggest that the 18 bp element in the pinopsin promoter constitutes the binding site of a ubiquitous factor that serves for the transcriptional repression that is required, although not sufficient, for the light-dependent expression of pinopsin gene in the chick pinealocytes.
Takanaka
Yoko
Y
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Japan.
Okano
Toshiyuki
T
Yamamoto
Kazuyuki
K
Fukada
Yoshitaka
Y
D016475:3T3 Cells; D024506:5' Flanking Region / Q000502:physiology; D000818:Animals; D030161:Avian Proteins; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive / Q000187:drug effects; D002478:Cells, Cultured; D002645:Chickens; D003459:Crystallins / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000528:radiation effects; D020868:Gene Silencing / Q000502:physiology; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000378:metabolism; D008027:Light; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D010870:Pineal Gland / Q000166:cytology / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology; D012045:Regulatory Sequences, Nucleic Acid / Q000502:physiology*; D012097:Repressor Proteins / Q000378:metabolism; D017299:Rod Opsins / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors
D030161:Avian Proteins; D003459:Crystallins; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D009841:Oligonucleotides; D012097:Repressor Proteins; D017299:Rod Opsins; D014157:Transcription Factors; C090139:OPNP protein, chicken