New Functional Promoter Polymorphism, CETP/−629, in Cholesteryl Ester Transfer Protein (CETP) Gene Related to CETP Mass and High Density Lipoprotein Cholesterol Levels: Role of Sp1/Sp3 in Transcriptional Regulation

• Published in: Arteriosclerosis, thrombosis, and vascular biology Vol. 20; no. 2; p. 507
• Main Authors: Dachet, Christiane, Poirier, Odette, Cambien, François, Chapman, John, Rouis, Mustapha
• Format: Journal Article
• Language: English
• Published: Philadelphia, PA American Heart Association, Inc 01.02.2000; Hagerstown, MD Lippincott
• Subjects: Adult; Binding Sites - physiology; Biological and medical sciences; Cardiology. Vascular system; Carrier Proteins - blood; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cholesterol Ester Transfer Proteins; Cholesterol, HDL - blood; Coronary heart disease; DNA-Binding Proteins - genetics; Electrophoresis, Gel, Two-Dimensional; Gene Frequency; Glycoproteins; Heart; Humans; Medical sciences; Middle Aged; Peptide Fragments - metabolism; Polymorphism, Genetic - physiology; Promoter Regions, Genetic - genetics; Sp1 Transcription Factor - genetics; Sp1 Transcription Factor - metabolism; Sp2 Transcription Factor; Transcription Factors - genetics; Transcription, Genetic; Human; Phospholipid transfer protein; Ester; Gene; Cholesterol HDL; Risk factor; Cardiovascular disease; Coronary heart disease; Genetic determinism; Cholesterol; Polymorphism
• ISSN: 1079-5642; 1524-4636
• DOI: 10.1161/01.ATV.20.2.507

ABSTRACTA new polymorphism located at position −629 (CETP/−629A/C) in the promoter of the cholesteryl ester transfer protein (CETP) gene is described. The −629A allele was associated with lower CETP mass (P <0.0001) and higher high density lipoprotein cholesterol (P <0.001) than the C allele in a sample of 536 control subjects from the ECTIM study. Transfection studies in HepG2 cells with a luciferase expression vector incorporating a 777-bp fragment of the CETP promoter and containing either A or C at position −629 showed significantly lower luciferase activity with the promoter fragment of the A allele (−25%, P <0.05). By gel-shift assay, DNA-protein interactions were evaluated in nuclear extracts of HepG2 cells with the use of 2 probes (A or C probe) composed of 20 bp of the promoter sequence surrounding the polymorphic site. Two specific complexes of distinct migration rate were identified with the A and the C probe. Competition with an excess of oligonucleotide containing the Sp1 consensus binding site showed that a protein(s) of the Sp transcription factor family was implicated in complex formation with the A probe but not with the C probe. Incubation with specific antibodies indicated that Sp1 and Sp3 bound specifically to the A probe. We introduced mutations in the −629-Sp1 binding site to test its functionality and to define the characteristics of transcription factor binding. We showed, by gel-shift assay, that no nuclear proteins bound to the mutated sequence. Transient transfection of HepG2 cells revealed that the expression of the mutated fragment was significantly increased compared with that of the A promoter fragment (25%, P <0.05). The mutated fragment displayed the same activity as that of the C promoter. These results indicate that Sp1 and/or Sp3 repress CETP promoter activity, whereas nuclear factors binding the C allele are without effect on promoter expression.