Identification of cis-acting elements in the proximal promoter region for brain-specific exon 1 of the mouse aromatase gene.
Among multiple exons 1 of the mouse aromatase gene, brain-specific exon 1 is only utilized in the hypothalamus and amygdala regions. In this study, identification of the promoter region necessary for basal transcription of the aromatase gene in the brain was undertaken. Deletions of various lengths were introduced into the overall promoter region, which was fused to the chloramphenicol acetyltransferase gene. The resulting reporters were transfected into cultured neurons from the diencephala of fetal mouse brains on embryonic day 13 and then their CAT mRNA levels were determined. The reporter plasmid containing the promoter region 202 bp upstream from the transcriptional initiation site gave the greatest expression. Then binding of trans-acting factors in a nuclear extract of the diencephala to the -202 bp promoter region was investigated by DNase I footprint analysis, multiple protected areas, referred to as Arom-Aalpha, Abeta, Agamma, B and C, being found. Gel shift assays, performed with oligonucleotides corresponding to the protected areas, showed that nuclear DNA binding factors form specific complexes exhibiting different mobilities. Substitution in the Arom-Aalpha or -B sequence in the promoter region in the CAT reporters decreased the CAT mRNA expression levels to about one-fifth the wild type one. These results suggest that multiple nuclear factors bound to the core promoter region participate in the expression of the aromatase gene in mouse brain neurons.
Honda
S
S
Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan. shonda@fujita-hu.ac.jp
Harada
N
N
Abe-Dohmae
S
S
Takagi
Y
Y
D000418:Albumins / Q000235:genetics; D000818:Animals; D001141:Aromatase / Q000235:genetics*; D015139:Blotting, Southern; D015153:Blotting, Western; D001923:Brain Chemistry / Q000235:genetics*; D002478:Cells, Cultured; D002795:Choline O-Acetyltransferase / Q000235:genetics; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics; D005091:Exons; D005260:Female; D005333:Fetus / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D005816:Genetic Complementation Test; D051379:Mice; D008813:Mice, Inbred ICR; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000201:enzymology*; D015345:Oligonucleotide Probes; D010957:Plasmids; D011247:Pregnancy; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D015533:Transcriptional Activation / Q000235:genetics
D000418:Albumins; D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D001141:Aromatase; D002795:Choline O-Acetyltransferase