SP1 AND SP3 REGULATE BASAL TRANSCRIPTION OF THE HUMAN CYP2F1 GENE

• Published in: Drug metabolism and disposition Vol. 33; no. 8; pp. 1244 - 1253
• Main Authors: JIE WAN, CARR, Brian A, CUTLER, N. Shane, LANZA, Diane L, HINES, Ronald N, YOST, Garold S
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Pharmacology and Experimental Therapeutics 01-08-2005
• Subjects: Base Sequence; Binding Sites - genetics; Biological and medical sciences; Cell Line, Tumor; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Cytochrome P450 Family 2; DNA - chemistry; DNA - metabolism; Drosophila; Gene Expression Regulation; Genes, Reporter; Humans; Luciferases - genetics; Lung; Medical sciences; Molecular Sequence Data; Pharmacology. Drug treatments; Promoter Regions, Genetic; Repressor Proteins; Sp1 Transcription Factor - genetics; Sp1 Transcription Factor - metabolism; Sp3 Transcription Factor - genetics; Sp3 Transcription Factor - metabolism; Transcription, Genetic; Transcriptional Activation; Transfection; Human; Regulation(control); Gene; Transcription
• ISSN: 0090-9556; 1521-009X
• DOI: 10.1124/dmd.105.004069

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.