title: The lung enriched transcription factor TTF-1 and the ubiquitously expressed proteins Sp1 and Sp3 interact with elements located in the minimal promoter of the rat Clara cell secretory protein gene.
abstract: The mechanisms that direct expression of the Clara cell secretory protein (CCSP) gene to the bronchiolar epithelial cells of the lung remain to be elucidated. Previous studies have identified a number of proteins which bind to a functionally important region (Region 1) located -132 to -76 bp from the transcription start site in the rat CCSP gene. Subsequently we have shown that while Region 1 is an important positive regulator of CCSP gene expression, sequences 3' of this region (-75 to +38) are sufficient to confer tissue-specific expression of a reporter gene. In the present study we have used transient transfections with a deletion series of CCSP-CAT reporter plasmids (where CAT is chloramphenicol acetyltransferase) and gel mobility shift assays with a series of overlapping oligonucleotides covering the whole minimal promoter region to study protein-DNA interactions within this region. These studies have identified a conserved functional binding site for the lung and thyroid enriched homeodomain transcription factor TTF-1, located between positions -51 and -42 from the transcription start site. CCSP-CAT chimaeric reporters containing this region are specifically activated by TTF-1 in co-transfection assays, and nuclear extracts from cells which express TTF-1 bind to this region, as does in vitro translated rat TTF-1. Three additional conserved regions were identified, and in further gel mobility shift studies with an oligonucleotide spanning the conserved region immediately 5' to the TTF-1 site we identified a binding site for the ubiquitously expressed zinc-finger-containing proteins Sp1 and Sp3. These studies suggest that cell-type-restricted and ubiquitous nuclear proteins may play a combined role in the regulation of the CCSP gene within the bronchiolar epithelium by interacting with the minimal promoter region.
authors.affiliation: DH Department of Toxicology, Medical College of St. Bartholomew's Hospital, London, U.K.
authors.forename: R F
authors.initials: RF
authors.lastname: Toonen
authors.forename: S
authors.initials: S
authors.lastname: Gowan
authors.forename: C D
authors.initials: CD
authors.lastname: Bingle
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000737:chemistry; D017124:Conserved Sequence; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics*; D051381:Rats; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D014598:Uteroglobin
chemical_list: D015342:DNA Probes; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C000614412:NKX2-1 protein, human; C000614416:Nkx2-1 protein, rat; D009687:Nuclear Proteins; D011506:Proteins; C556389:SCGB1A1 protein, human; C497121:SP3 protein, human; C556391:Scgb1a1 protein, rat; D016329:Sp1 Transcription Factor; C497123:Sp3 protein, rat; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D014598:Uteroglobin