Identification of a negative cis-regulatory element and trans-acting protein that inhibit transcription of the angiotensin II type 1a receptor gene.
The rat angiotensin II type 1a receptor (AT1a-R) gene is expressed in a cell-specific manner. We demonstrated that the negative regulatory element (NRE) between -489 and -331 is active in PC12 cells (Murasawa, S., Matsubara, H., Urakami, M., and Inada, M. (1993) J. Biol. Chem. 268, 26996-27003). Gel retardation assays confirmed that PC12 cells have a trans-acting factor bound to the NRE. By means of a DNase I footprint assay we identified the core of the NRE as an (A+T)-rich sequence (TAATCTTTTATTTTA) located at nucleotides -456 to -442. Oligonucleotides corresponding to the NRE core sequence bound to nuclear protein. Site-directed mutagenesis at nucleotides -451 to -448 eliminated the specific protein/DNA binding and restored expression of the AT1a-R in transient transfection assays (2.7-fold increase). The NRE did not negatively affect the thymidine kinase promoter. No homology was found with known NREs, suggesting that this is a novel NRE. Southwestern blotting revealed a 53-kDa, specific binding protein in PC12 cells and the rat brain, but not in the liver, spleen, adrenal gland, and kidney. These findings demonstrate that the NRE of the rat AT1a-R is an (A+T)-rich sequence located at nucleotides -456 to -442 and the 53-kDa protein is a specific binding protein, and suggest that this protein may be a trans-acting factor which determines the neuron-specific down-regulation of the AT1a-R gene.
Murasawa
S
S
Second Department of Internal Medicine, Kansai Medical University, Osaka, Japan.
Matsubara
H
H
Mori
Y
Y
Kijima
K
K
Maruyama
K
K
Inada
M
M
D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D017931:DNA Primers; D005805:Genes, MHC Class I; D006801:Humans; D015151:Immunoblotting; D008156:Luciferases / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016716:PC12 Cells; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D051381:Rats; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000096:biosynthesis* / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection
D017931:DNA Primers; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D015534:Trans-Activators; D000804:Angiotensin II; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase