Species-specific differences in positive and negative regulatory elements in the renin gene enhancer.
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.
Shi
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Departments of Internal Medicine and Physiology & Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Black
T A
TA
Gross
K W
KW
Sigmund
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CD
D000818:Animals; D001483:Base Sequence / Q000235:genetics; D002460:Cell Line; D002678:Chimera; D004247:DNA / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000235:genetics; D017353:Gene Deletion; D005809:Genes, Regulator; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D010446:Peptide Fragments / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000502:physiology; D012083:Renin / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014158:Transcription, Genetic / Q000502:physiology
D010446:Peptide Fragments; D004247:DNA; D012083:Renin