Promoter analysis of the rat beta1-adrenergic receptor gene identifies sequences involved in basal expression.
The beta1-adrenergic receptor (beta1-AR) mediates several functions of catecholamines in the heart, including the stimulation of heart rate and contractility. The expression of the rat beta1-AR gene was assessed by transiently transfecting chimeric genes containing the beta1-AR promoter, driving the luciferase reporter gene into various cell lines. beta1-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 beta1-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 beta1-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 beta1-AR gene involves widely separated sequences 3' and 5' to the transcriptional start site.
Bahouth
S W
SW
Department of Pharmacology, College of Medicine, The University of Tennessee, Memphis 38163, USA. sbahouth@utmem1.utmem.edu
Cui
X
X
Beauchamp
M J
MJ
Shimomura
H
H
George
S T
ST
Park
E A
EA
D000818:Animals; D001483:Base Sequence; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D015870:Gene Expression; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018342:Receptors, Adrenergic, beta-1 / Q000096:biosynthesis* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection
D012333:RNA, Messenger; D018342:Receptors, Adrenergic, beta-1; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D003850:Deoxyribonuclease I