title: CCAAT binding transcription factor binds and regulates human COL1A1 promoter activity in human dermal fibroblasts: demonstration of increased binding in systemic sclerosis fibroblasts.
abstract: OBJECTIVE
  To determine the binding factors that interact with the proximal promoter region of the human type I collagen gene, COL1A1, and to examine their involvement in its transcriptional regulation in normal and systemic sclerosis (SSc) dermal fibroblasts.
  
  
  METHODS
  Nuclear extracts from dermal fibroblasts from 4 patients with SSc and 4 age- and sex-matched control individuals were examined by electrophoresis mobility shift assays with a COL1A1 promoter fragment encompassing nucleotides -174 to -50 bp. Supershift assays with antibodies specific to various transcription factors, and competition experiments using consensus, wild-type, or mutated oligonucleotides corresponding to their specific binding sites, were performed. The effects of specific oligonucleotides as "intracellular competitors" were examined by transient transfection experiments in SSc fibroblasts using a COL1A1 construct containing -174 bp of the promoter.
  
  
  RESULTS
  The findings demonstrate that the CCAAT binding transcription factor (CBF) binds the proximal CCAAT box located at -100 to -96 bp, but not the distal CCAAT box at -125 to -121 bp, of the human COL1A1 promoter in both SSc and normal fibroblasts. CBF binding activity was 3-5-fold higher in the SSc fibroblasts. Moreover, the promoter activity of the -174-bp COL1A1 construct was decreased by up to 50% when specific oligonucleotides were used as "intracellular competitors." In addition, Sp1 and Sp3 were other transcription factors found to be involved in the formation of the DNA-protein complexes within this region of the COL1A1 promoter.
  
  
  CONCLUSIONS
  These results indicate that the transcription factor CBF binds the human COL1A1 proximal promoter region in human dermal fibroblasts, and its binding activity is higher in SSc fibroblasts.
authors.affiliation: Istituto di Biologia dello Sviluppo, Palermo, Italy.
authors.forename: B
authors.initials: B
authors.lastname: Saitta
authors.forename: S
authors.initials: S
authors.lastname: Gaidarova
authors.forename: L
authors.initials: L
authors.lastname: Cicchillitti
authors.forename: S A
authors.initials: SA
authors.lastname: Jimenez
mesh_terms: D001483:Base Sequence; D001667:Binding, Competitive / Q000187:drug effects; D023081:CCAAT-Binding Factor / Q000378:metabolism* / Q000494:pharmacology*; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D012595:Scleroderma, Systemic / Q000378:metabolism* / Q000473:pathology*; D012867:Skin / Q000473:pathology*; D016329:Sp1 Transcription Factor / Q000494:pharmacology; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D023081:CCAAT-Binding Factor; D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D003094:Collagen