Abstract
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.
Highlights
Osteoarthritis (OA) is a common disease of the synovial joints, affecting millions of people worldwide
GDF5 is an important growth factor that plays a vital role in the development and repair of articulating joints. rs143383 is a polymorphism within the regulatory region of the GDF5 gene and has two allelic forms, C and T
Genetic studies have demonstrated that the T allele is associated with an increased risk of osteoarthritis in a range of ethnic populations whilst previous functional studies revealed that this allele mediates its effect by producing less GDF5 transcript than the C allele
Summary
Osteoarthritis (OA) is a common disease of the synovial joints, affecting millions of people worldwide. It is a chronic, highly disabling disease, characterised by the progressive loss of articular cartilage, changes in the subchondral bone, and variable levels of synovial inflammation [1]. Non-steroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase 2 (COX-2) inhibitors are recommended for the pharmacological management of OA. These have proven to be effective for pain relief and suppression of inflammation, these treatments are failing to target the underlying cause and progression of disease. There has been limited success so far in trials of disease-modifying drugs, with arthroplasty remaining the basis for curative therapy [3]
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