Identification and functional analyses of molecular haplotypes of the human osteoprotegerin gene promoter.
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.


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.
Hagedorn
Claudia
C
Leibniz-Institute for Arteriosclerosis Research, University of Münster, Münster, Germany.
Telgmann
Ralph
R
Dördelmann
Corinna
C
Schmitz
Boris
B
Hasenkamp
Sandra
S
Cambien
François
F
Paul
Martin
M
Brand
Eva
E
Brand-Herrmann
Stefan-Martin
SM
D001665:Binding Sites; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D006239:Haplotypes; D006801:Humans; D025542:Neurofibromin 1 / Q000378:metabolism; D053244:Osteoprotegerin / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014158:Transcription, Genetic
D025542:Neurofibromin 1; D053244:Osteoprotegerin; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor