Sp1 site is crucial for the mouse claudin-19 gene expression in the kidney cells.
Members of the claudin family play important roles in the formation of tight junctions in the kidneys, liver and intestine. Claudin-19 (Cldn19), a newly identified member of this family, is highly expressed in the kidney of the mouse. To have a better understanding on mouse claudin-19 gene expression, a 0.9-kb DNA fragment containing the 5'-flanking region of the Cldn19 gene was isolated. DNA sequence comparison between the mouse and human Cldn19 promoter regions exhibited little homology. One transcription initiation site was located at 104 nucleotides upstream of the start codon (ATG) of the Cldn19 gene. The mouse claudin-19 promoter lacked typical CAAT or GC-box. Deletion constructs of the 0.9-kb DNA fragment were generated and fused to a promoterless luciferase (Luc) reporter plasmid. Transfection studies using various kidney cell lines (MDCK, mIMCD3 and HEK293) revealed that the minimal promoter fragment resided in the -39 to -108 region, which contained a number of binding sites for transcription factors including Sp1. Site-directed mutagenesis using specific oligo probes confirmed that Sp1 was crucial for Cldn19 transactivation in the three cell lines studied. Electromobility shift assay confirmed that the nuclear extracts of these cells bound to the Sp1 oligo derived from Cldn19 promoter, but not to the mutated Sp1 oligo probe. Moreover, this DNA-protein complex would be recognized by Sp1 antibody, indicating specific Sp1 binding. Collectively, our data suggest that Sp1 binds to the claudin-19 promoter region and is responsible for its expression in the kidney cell lines in vitro.
Luk
John M
JM
Department of Surgery, Hong Kong Jockey Club Clinical Research Centre, Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China. jmluk@hkucc.hku.hk
Tong
Man-Kit
MK
Mok
Bobo W
BW
Tam
Po-Chor
PC
Yeung
William S B
WS
Lee
Kai-Fai
KF
D024506:5' Flanking Region; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D018387:Codon, Initiator; D004285:Dogs; D024202:Electrophoretic Mobility Shift Assay; D005091:Exons; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D007668:Kidney / Q000166:cytology*; D008156:Luciferases / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D024363:Transcription Initiation Site; D015533:Transcriptional Activation
D000911:Antibodies, Monoclonal; D018387:Codon, Initiator; D008565:Membrane Proteins; D016329:Sp1 Transcription Factor; D008156:Luciferases