© 1999 American Heart Association, Inc.
Molecular Medicine |
Species-Specific Differences in Positive and Negative Regulatory Elements in the Renin Gene Enhancer
From the Departments of Internal Medicine and Physiology & Biophysics (Q.S., C.D.S.), University of Iowa College of Medicine, Iowa City, Iowa, and Department of Molecular and Cellular Biology (T.A.B., K.W.G.), Roswell Park Cancer Institute, Buffalo, NY.
Correspondence to Curt D. Sigmund, PhD, Director, Transgenic Animal Facility, Departments of Internal Medicine and Physiology & Biophysics, The University of Iowa College of Medicine, 2191 Medical Laboratory (ML), Iowa City, IA 52242. E-mail curt-sigmund{at}uiowa.edu
Abstract—A distal transcriptional enhancer has been previously reported upstream of the mouse renin gene. A homologous sequence is also present upstream of the human renin gene, but the mouse and human renin enhancers differ markedly in their ability to activate transcription of a renin promoter. Although the 2 enhancers share high homology in their 202-bp promoter distal portions, their 40-bp proximal portions are heterogeneous. Chimeric enhancers were used to test the role of the 40-bp segment (m40) of the enhancer by using transient transfection analysis in mouse kidney renin-expressing As4.1 cells. Deletion of m40 from the mouse renin enhancer or its addition to the human renin enhancer did not significantly change transcriptional activity when placed close to a mouse or human renin promoter. However, when placed further upstream of a renin promoter, the same deletion and substitution markedly altered enhancer activity. Electrophoretic gel mobility shift analysis identified 2 elements, a and b, in m40 that specifically bound nuclear proteins from As4.1 cells. Mutagenesis and transient transfection analysis revealed that element b accounts for the function of m40 and that element a antagonizes the positive influence of element b. Gel competition and supershift analysis revealed that nuclear factor-Y, a ubiquitous CAAT-box binding protein, binds to element a. Sequence analysis revealed that the human renin enhancer has a natural loss-of-function mutation in element b that affects its ability to transactivate when placed far upstream of a promoter. Reversion of the human renin element b to match the mouse sequence restored transactivation of the enhancer in mouse As4.1 cells. These data suggest that element b cooperates with the rest of the enhancer to maintain full enhancer activity, whereas element a may regulate enhancer activity. Sequence differences in these elements may explain the functional differences in the mouse and human renin enhancer sequences.
Key Words: transfection • transcription factor • kidney • juxtaglomerular cell • electrophoretic mobility shift assay
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