GDF5 encodes an extracellular signalling molecule that is essential for normal skeletal development. The rs144383 C to T SNP located in the 5ʹUTR 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 5ʹUTR 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00439-014-1447-z) contains supplementary material, which is available to authorized users.

rs143383 is a C to T transition SNP located in the 5′untranslated region (5′UTR) of the growth differentiation factor 5 gene GDF5. The T allele of the SNP is associated with increased risk of osteoarthritis (OA) in Europeans and in Asians. This susceptibility is mediated by the T allele producing less GDF5 transcript relative to the C allele, a phenomenon known as differential allelic expression (DAE). The aim of this study was to identify trans-acting factors that bind to rs143383 and which regulate this GDF5 DAE. Protein binding to the gene was investigated by two experimental approaches: 1) competition and supershift electrophoretic mobility shift assays (EMSAs) and 2) an oligonucleotide pull down assay followed by quantitative mass spectrometry. Binding was then confirmed in vivo by chromatin immunoprecipitation (ChIP), and the functional effects of candidate proteins investigated by RNA interference (RNAi) and over expression. Using these approaches the trans-acting factors Sp1, Sp3, P15, and DEAF-1 were identified as interacting with the GDF5 5′UTR. Knockdown and over expression of the factors demonstrated that Sp1, Sp3, and DEAF-1 are repressors of GDF5 expression. Depletion of DEAF-1 modulated the DAE of GDF5 and this differential allelic effect was confirmed following over expression, with the rs143383 T allele being repressed to a significantly greater extent than the rs143383 C allele. In combination, Sp1 and DEAF-1 had the greatest repressive activity. In conclusion, we have identified four trans-acting factors that are binding to GDF5, three of which are modulating GDF5 expression via the OA susceptibility locus rs143383.

Osteoarthritis (OA) is a polygenic disease characterized by cartilage loss, with the single-nucleotide polymorphism (SNP) rs143383 (C/T) influencing OA susceptibility across a range of ethnic groups. The SNP resides within the 5′-UTR of the growth and differentiation factor 5 gene (GDF5), with the OA-associated T-allele mediating reduced GDF5 expression. As GDF5 codes for a cartilage anabolic protein, this reduced expression may explain why the T-allele of rs143383 is an OA risk factor. Our deep sequencing of GDF5 identified a C/A transversion located −41 bp relative to the gene's transcription start site. This promoter variant is predicted to affect transcription factor binding and it may therefore highlight a regulatory site that could be exploited to manipulate GDF5 expression and alleviate the detrimental effect mediated by the T-allele of rs143383. Here, we describe our functional assessment of the −41 bp variant. Using reporter constructs we demonstrated that the transversion leads to increased gene expression to such a degree that the A-allele is able to compensate for the reduced expression mediated by the T-allele of rs143383. Using electrophoretic mobility shift assays we identified YY1 as a trans-acting factor that differentially binds to the alleles of the −41 bp variant, with more avid binding to allele A. Knockdown of YY1 led to a significant reduction in GDF5 expression, supporting YY1 as a GDF5 activator. In conclusion, we demonstrated that the −41 bp variant is functional and we have identified a regulatory region of GDF5 that can be exploited to overcome the OA genetic deficit mediated by the T-allele of rs143383.

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