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Molecular and Cellular Biology, October 2000, p. 7319-7331, Vol. 20, No. 19
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Transcriptional Regulation of the CLC-K1 Promoter by myc-Associated Zinc Finger Protein and Kidney-Enriched Krüppel-Like Factor, a Novel Zinc Finger Repressor

Shinichi Uchida,1,* Yujiro Tanaka,1 Hiroshi Ito,1 Fumiko Saitoh-Ohara,2 Johji Inazawa,2 Kazunari K. Yokoyama,3 Sei Sasaki,1 and Fumiaki Marumo1

Second Department of Internal Medicine, School of Medicine,1 and Department of Molecular Cytogenetics, Medical Research Institute,2 Tokyo Medical and Dental University, Tokyo, and Tukuba Institute, RIKEN (The Institute of Physical and Chemical Research), Ibaraki,3 Japan

Received 15 May 2000/Accepted 21 June 2000

The expression of CLC-K1 and CLC-K2, two kidney-specific CLC chloride channels, is transcriptionally regulated on a tissue-specific basis. Previous studies have shown that a GA element near their transcriptional start sites is important for basal and cell-specific activities of the CLC-K1 and CLC-K2 gene promoters. To identify the GA-binding proteins, the human kidney cDNA library was screened by a yeast one-hybrid system. A novel member of the Cys2-His2 zinc finger gene designated KKLF (for "kidney-enriched Krüppel-like factor") and the previously isolated MAZ (for "myc-associated zinc finger protein") were cloned. KKLF was found to be abundantly expressed in the liver, kidneys, heart, and skeletal muscle, and immunohistochemistry revealed the nuclear localization of KKLF protein in interstitial cells in heart and skeletal muscle, stellate cells, and fibroblasts in the liver. In the kidneys, KKLF protein was localized in interstitial cells, mesangial cells, and nephron segments, where CLC-K1 and CLC-K2 were not expressed. A gel mobility shift assay revealed sequence-specific binding of recombinant KKLF and MAZ proteins to the CLC-K1 GA element, and the fine-mutation assay clarified that the consensus sequence for the KKLF binding site was GGGGNGGNG. In a transient-transfection experiment, MAZ had a strong activating effect on transcription of the CLC-K1-luciferase reporter gene. On the other hand, KKLF coexpression with MAZ appeared to block the activating effect of MAZ. These results suggest that a novel set of zinc finger proteins may help regulate the strict tissue- and nephron segment-specific expression of the CLC-K1 and CLC-K2 channel genes through their GA cis element.

* Corresponding author. Mailing address: Second Department of Internal Medicine, School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyo, Tokyo 113-8519, Japan. Phone: 81-3-5803-5216. Fax: 81-3-5803-0172. e-mail: suchida.med2{at}med.tmd.ac.jp.

Molecular and Cellular Biology, October 2000, p. 7319-7331, Vol. 20, No. 19
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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