Critical roles of a cyclic AMP responsive element and an E-box in regulation of mouse renin gene expression.
Mouse As4.1 cells, obtained after transgene-targeted oncogenesis to induce neoplasia in renal renin expressing cells, express high levels of renin mRNA from their endogenous Ren-1(c) gene. We have previously identified a 242-base pair enhancer (coordinates -2866 to -2625 relative to the CAP site) upstream of the mouse Ren-1(c) gene. This enhancer, in combination with the proximal promoter (-117 to +6), activates transcription nearly 2 orders of magnitude in an orientation independent fashion. To further delimit sequences necessary for transcriptional activation, renin promoter-luciferase reporter gene constructs containing selected regions of the Ren-1(c) enhancer were analyzed after transfection into As4.1 cells. These results demonstrate that several regions are required for full enhancer activity. Sequences from -2699 to -2672, which are critical for the enhancer activity, contain a cyclic AMP responsive element (CRE) and an E-box. Electrophoretic mobility shift assays demonstrated that transcription factors CREB/CREM and USF1/USF2 in As4.1 cell nuclear extracts bind to oligonucleotides containing the Ren-1(c) CRE and E-box, respectively. These two elements are capable of synergistically activating transcription from the Ren-1(c) promoter. Moreover, mutation of either the CRE or E-box results in almost complete loss of enhancer activity, suggesting the critical roles these two elements play in regulating mouse Ren-1(c) gene expression. Although the Ren-1(c) gene contains a CRE, its expression is not induced by cAMP in As4.1 cells. This appears to reflect constitutive activation of protein kinase A in As4.1 cells since treatment with the protein kinase A inhibitor, H-89, caused a significant reduction in Ren-1(c) gene expression and this reduction is mediated through the CRE at -2699 to -2688.
Pan
L
L
Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Black
T A
TA
Shi
Q
Q
Jones
C A
CA
Petrovic
N
N
Loudon
J
J
Kane
C
C
Sigmund
C D
CD
Gross
K W
KW
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004247:DNA; D004268:DNA-Binding Proteins; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D012083:Renin / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C497183:Usf1 protein, mouse; C497186:Usf2 protein, mouse; D004247:DNA; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D012083:Renin