Transcriptional regulation of the CLC-K1 promoter by myc-associated zinc finger protein and kidney-enriched Krüppel-like factor, a novel zinc finger repressor.
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.
Uchida
S
S
Second Department of Internal Medicine, School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan. suchida.med2@med.tmd.ac.jp
Tanaka
Y
Y
Ito
H
H
Saitoh-Ohara
F
F
Inazawa
J
J
Yokoyama
K K
KK
Sasaki
S
S
Marumo
F
F
D000595:Amino Acid Sequence; D000818:Animals; D027321:Anion Transport Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D018118:Chloride Channels / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins; D004195:Disease Models, Animal; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D005796:Genes; D017930:Genes, Reporter; D006801:Humans; D051741:Kruppel-Like Transcription Factors; D008565:Membrane Proteins; D051379:Mice; D008817:Mice, Mutant Strains; D008969:Molecular Sequence Data; D009395:Nephritis, Interstitial / Q000378:metabolism; D009399:Nephrons / Q000378:metabolism; D009687:Nuclear Proteins; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D016335:Zinc Fingers / Q000502:physiology*
D027321:Anion Transport Proteins; C489932:CLCNKA protein, human; C412733:CLCNKB protein, human; D002352:Carrier Proteins; D018118:Chloride Channels; C489933:Clcnka protein, mouse; C082118:Clcnka protein, rat; C486573:Clcnkb protein, mouse; C486572:Clcnkb protein, rat; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C414172:KLF15 protein, human; D051741:Kruppel-Like Transcription Factors; D008565:Membrane Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C076035:c-MYC-associated zinc finger protein; D003094:Collagen