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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2009;29:1638.)
© 2009 American Heart Association, Inc.

Cell Biology/Signaling

Identification and Functional Analyses of Molecular Haplotypes of the Human Osteoprotegerin Gene Promoter

Claudia Hagedorn; Ralph Telgmann; Corinna Dördelmann; Boris Schmitz; Sandra Hasenkamp; François Cambien; Martin Paul; Eva Brand; Stefan-Martin Brand-Herrmann

From the Leibniz-Institute for Arteriosclerosis Research, Department of Molecular Genetics of Cardiovascular Disease (C.H., R.T., C.D., B.S., S.-M.B.-H.), University of Münster, Germany; University Hospital Münster, Internal Medicine D (S.H., E.B.), Münster, Germany; INSERM, UMR S937 (F.C.), Université Pierre et Marie Curie, Paris, France; Maastricht University, Faculty of Health, Medicine, and Life Science (M.P.), Maastricht, The Netherlands.

Correspondence to Univ-Prof Dr med Stefan-Martin Brand-Herrmann, MD, PhD, Leibniz-Institute for Arteriosclerosis Research at the University of Münster, Department of Molecular Genetics of Cardiovascular Disease, Domagkstrasse 3, D-48149 Münster, Germany. E-mail brandher{at}uni-muenster.de

Objective— Osteoprotegerin (OPG) has been reported to be involved in the development of atherosclerotic disease, and OPG gene variation has been associated with plasma OPG levels and different cardiovascular disease phenotypes. However, the genetic architecture of the OPG promoter and its transcriptional regulation are poorly characterized.

Methods and Results— We identified 1008 bp of the OPG 5'-flanking region to be sufficiently transcriptionally active in osteosarcoma cell lines and generated serial promoter deletion constructs. Individual subcloning revealed the existence of 3 molecular haplotypes (MolHaps): [T^–960-A^–946-G^–900-T^–864; MolHap1, wild type], [T^–960-G^–946-G^–900-T^–864; MolHap2], [C^–960-G^–946-A^–900-G^–864; MolHap4]. Compared to MolHap1, transcriptional activities of MolHaps 2 and 4 were significantly reduced (P=0.0018). Whereas introduction of the –159C allele reduced transcriptional activities of the full-length constructs (P=0.0014), it significantly increased activities of the deletion constructs (P=0.0005). Electrophoretic mobility shift, competition, and chromatin immunoprecipitation assays revealed specific DNA:protein interactions for the MolHaps with Sp1 and NF-1, and identified Egr1 interacting exclusively with the –159T allele.

Conclusions— We propose new structural and transcriptional features within the OPG promoter region and identified MolHaps being differentially transcriptionally active and allele-dependently interacting with a proximal polymorphic site.

Using osteosarcoma cell lines, we (1) assessed transcriptionally active portions of the osteoprotegerin gene promoter, (2) assessed differential DNA-protein interactions involving Sp1, NF-1, and Egr1 depending on molecular haplotypes, promoter truncation, and interaction with proximal gene sites, and (3) evidenced differential transcription start site usage.

Key Words: osteoprotegerin • genetic variants • functional promoter analyses • deletion constructs • transcriptional control