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A proximal direct repeat motif characterized as a negative regulatory element in the human renin gene
Author(s) -
Konoshita Tadashi,
Fuchs Sébastien,
Makino Yasukazu,
Wakahara Shigeyuki,
Miyamori Isamu
Publication year - 2007
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.21341
Subject(s) - mutant , reporter gene , electrophoretic mobility shift assay , gene , biology , microbiology and biotechnology , regulatory sequence , sequence motif , oligonucleotide , transcription factor , gene expression , genetics
The regulation of renin gene expression is thought to be fundamental to regulation of the total renin–angiotensin system. The human renin gene contains a direct repeat (DR) motif AGGGGTCAC–AGGGCCA in the proximal region (−259/−245 bp), which contains similar sequence for nuclear receptor superfamily binding core motif, AGGTCA, and is the most similar to COUP‐TFII consensus. The DR motif was evaluated as a functional cis ‐element with renal cortex and chorio‐decidual cells by footprint assay, electromobility shift assay (EMSA) and reporter assay. The DR motif site was protected by footprint analysis with a clear hypersensitive and a minor hypersensitive region in good accordance with the DR of the consensus. One of the binding proteins was strongly suspected to be COUP‐TFII‐consensus‐specific by EMSA. The DNA/protein complexes obtained with nuclear extract of renin producing cells could be completely blocked by homologous competitor and strongly blocked by the second‐half mutant oligonucleotide of the DR motif but not by the first‐half mutant oligonucleotide. Finally, the transcriptional activity of second‐half mutant construct is slightly elevated and that first‐half mutant construct is significantly stronger by twofold compared with wild type construct in reporter assay. These findings suggest that the DR motif site of the human renin gene functions as a negative regulatory element involved in a twofold repression of transcription and that member(s) of nucleic receptor superfamily bind the site and play important roles in the human renin gene expression with a possibility that one of the binding protein is COUP‐TFII. J. Cell. Biochem. 102: 1043–1050, 2007. © 2007 Wiley‐Liss, Inc.