Promoter Analysis of the Rat β1-Adrenergic Receptor Gene Identifies Sequences Involved in Basal Expression
- Suleiman W. Bahouth1,
- Xiaoli Cui1,1,
- Michael J. Beauchamp1,
- Hiromi Shimomura2,
- Shaji T. George3 and
- Edwards A. Park1
- 1Department of Pharmacology, College of Medicine, The University of Tennessee, Memphis, Tennessee 38163 (S.W.B., X.C., M.J.B., E.A.P.),2Department of Oral Biochemistry, Nippon Dental University, Niigata, Japan (H.S.), and 3Inovir Inc., New York, New York 10021 (S.T.G.)
Abstract
The β1-adrenergic receptor (β1-AR) mediates several functions of catecholamines in the heart, including the stimulation of heart rate and contractility. The expression of the rat β1-AR gene was assessed by transiently transfecting chimeric genes containing the β1-AR promoter, driving the luciferase reporter gene into various cell lines. β1-AR/luciferase vectors containing 3 kb of the 5′-flanking region and extending to −126 relative to the start site of translation were expressed at high levels in ventricular myocytes, SK-N-MC cells, and HepG2 cells. The addition of 26 nucleotides from −125 to −100 to the −3311 β1-AR/luciferase chimeric gene reduced expression in myocytes and SK-N-MC cells while eliminating expression in HepG2 cells. This element is located 125 base-pairs 3′ to the transcriptional start site. The mutation of four nucleotides between −121 and −118 diminished the inhibitory effect of this element. The inhibitory activity of the −125 to −100 sequence was completely dependent on promoter context and positioning. In addition to this 3′ element, sequences between −3311 and −2740 in the 5′-flanking region of the β1-AR gene were required for the full transcriptional suppression. Using DNase I footprinting and gel mobility assays, it was determined that within the 26-bp region, rat heart nuclear proteins bound to two sites between nucleotides −123 and −112 and −106 and −100. Therefore, appropriate basal expression of the β1-AR gene involves widely separated sequences 3′ and 5′ to the transcriptional start site.
Footnotes
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Send reprint requests to: Dr. S. W. Bahouth, Department of Pharmacology, College of Medicine, The University of Tennessee, 874 Union Avenue, Memphis, TN 38163. E-mail:sbahouth{at}utmem1.utmem.edu
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↵1 Current affiliation: St. Jude Children’s Research Hospital, Memphis, TN 38101.
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↵2 S. W. Bahouth, X. Cui, M. J. Beauchamp, E. A. Park. Thyroid hormone induces β1-adrenergic receptor gene transcription through a direct repeat separated by 5 nucleotides. Submitted for publication.
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This work was supported by National Institutes of Health Grant HL48169.
- Abbreviations:
- β1-AR
- β1-adrenergic receptor
- TSS
- transcriptional start site
- VM
- ventricular myocytes
- PEPCK
- phosphoenolpyruvate carboxykinase
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- TR
- thyroid hormone receptor
- SV40
- simian virus
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- Received August 12, 1996.
- Accepted January 2, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
