Sp1 and Sp3 regulate basal transcription of the human CYP2F1 gene.
Selective transcription of the human CYP2F1 gene in lung tissues may control the susceptibilities of this organ to diverse pneumotoxicants and lung carcinogens. However, the mechanisms responsible for CYP2F1 organ-selective transcription have not been elucidated. The objectives of the current studies were to identify and characterize basal transcription elements within the TATA-less promoter region of CYP2F1. Four putative Sp1-like sites were identified in the CYP2F1 promoter. Competitive electrophoretic mobility shift assay analysis with mutated oligonucleotide probes and lung A549 cell nuclear extract, along with supershift studies using antibodies to either Sp1 or Sp3 proteins, demonstrated that all four sites formed three specific protein-DNA complexes. Mutations in any of the four core Sp1-like motifs abolished protein-DNA binding. Western blot analysis of both human tissues and cells showed that Sp1 was considerably higher in lung than liver and that Sp3 was much higher in liver than lung. Promoter activation of a luciferase reporter construct was sequentially increased by addition of each of the four Sp1-like motifs in lung A549 cells but not in liver HepG2 cells. Cotransfection of a Sp1 expression vector with the reporter construct dramatically increased luciferase activity in either A549 cells or Sp1-deficient Drosophila Schneider line 2 (SL-2) cells. However, similar cotransfections with an Sp3 expression vector failed to increase activity. Cotransfection of both the Sp1 and Sp3 expression vectors considerably decreased Sp1-mediated activity in A549 cells and abolished activity in SL-2 cells. Thus, these studies demonstrated that four Sp1-dependent proximal promoter elements drive organ-selective CYP2F1 gene transcription, and that Sp1 and Sp3 factors interact to modulate constitutive CYP2F1 transcription in lung cells.
Wan
Jie
J
Department of Pharmacology and Toxicology, 30 South 2000 East, Room 201, University of Utah, Salt Lake City, UT 84112-5820. gyost@pharm.utah.edu.
Carr
Brian A
BA
Cutler
N Shane
NS
Lanza
Diane L
DL
Hines
Ronald N
RN
Yost
Garold S
GS
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008168:Lung; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D051705:Sp3 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D012097:Repressor Proteins; D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D008156:Luciferases; C110332:CYP2F1 protein, human; D000072467:Cytochrome P450 Family 2