title: CpG methylation regulates allelic expression of GDF5 by modulating binding of SP1 and SP3 repressor proteins to the osteoarthritis susceptibility SNP rs143383.
abstract: GDF5 encodes an extracellular signalling molecule that is essential for normal skeletal development. The rs144383 C to T SNP located in the 5'UTR of this gene is functional and has a pleiotropic effect on the musculoskeletal system, being a risk factor for knee-osteoarthritis (OA), congenital hip dysplasia, lumbar disc degeneration and Achilles tendon pathology. rs143383 exerts a joint-wide effect on GDF5 expression, with expression of the OA-associated T allele being significantly reduced relative to the C allele, termed allelic expression imbalance. We have previously reported that the GDF5 locus is subject to DNA methylation and that allelic imbalance of rs143383 is mediated by SP1, SP3 and DEAF1 transcriptional repressors. In this study, we have assayed GDF5 methylation in normal and osteoarthritic cartilage, and investigated the effect of methylation on the allelic imbalance of rs143383. We observed demethylation of the GDF5 5'UTR in OA knee cartilage relative to both OA (p = 0.009) and non-OA (p = 0.001) hip cartilage, with the most significant demethylation observed at the highly conserved +37 CpG site located 4 bp upstream of rs143383. Methylation modulates the level and direction of allelic imbalance of rs143383, with methylation of the +37 CpG dinucleotide within the SP1/SP3 binding site having an allele-specific effect on SP1 and SP3 binding. Furthermore, methylation attenuated the repressive effects of SP1, SP3 and DEAF1 on GDF5 promoter activity. This data suggest that the differential methylation of the +37 CpG site between osteoarthritic hip and knee cartilage may be responsible for the knee-specific effect of rs143383 on OA susceptibility.
authors.affiliation: Musculoskeletal Research Group, Institute of Cellular Medicine, 4th Floor Catherine Cookson Building, The Medical School, Framlington Place, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK, louise.reynard@newcastle.ac.uk.
authors.forename: Louise N
authors.initials: LN
authors.lastname: Reynard
authors.forename: Catherine
authors.initials: C
authors.lastname: Bui
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Syddall
authors.forename: John
authors.initials: J
authors.lastname: Loughlin
mesh_terms: D000483:Alleles; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism; D016022:Case-Control Studies; D002478:Cells, Cultured; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation; D024202:Electrophoretic Mobility Shift Assay; D020022:Genetic Predisposition to Disease; D055428:Growth Differentiation Factor 5 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D010003:Osteoarthritis / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor / Q000378:metabolism*
chemical_list: C525893:GDF5 protein, human; D055428:Growth Differentiation Factor 5; D012333:RNA, Messenger; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor