Genetic and Epigenetic Fine‐Tuning of TGFB1 Expression Within the Human Osteoarthritic Joint

Abstract

Objective Osteoarthritis (OA) is an age‐related disease characterized by articular cartilage degeneration. It is largely heritable, and genetic screening has identified single‐nucleotide polymorphisms (SNPs) marking genomic risk loci. One such locus is marked by the G>A SNP rs75621460, downstream of TGFB1. This gene encodes transforming growth factor β1, the correct expression of which is essential for cartilage maintenance. This study investigated the regulatory activity of rs75621460 to characterize its impact on TGFB1 expression in disease‐relevant patient samples (n = 319) and in Tc28a2 immortalized chondrocytes. Methods Articular cartilage samples from human patients were genotyped, and DNA methylation levels were quantified using pyrosequencing. Gene reporter and electrophoretic mobility shift assays were used to determine differential nuclear protein binding to the region. The functional impact of DNA methylation on TGFB1 expression was tested using targeted epigenome editing. Results The analyses showed that SNP rs75621460 was located within a TGFB1 enhancer region, and the OA risk allele A altered transcription factor binding, with decreased enhancer activity. Protein complexes binding to A (but not G) induced DNA methylation at flanking CG dinucleotides. Strong correlations between patient DNA methylation levels and TGFB1 expression were observed, with directly opposing effects in the cartilage and the synovium at this locus. This demonstrated biologic pleiotropy in the impact of the SNP within different tissues of the articulating joint. Conclusion The OA risk SNP rs75621460 impacts TGFB1 expression by modulating the function of a gene enhancer. We propose a mechanism by which the SNP impacts enhancer function, providing novel biologic insight into one mechanism of OA genetic risk, which may facilitate the development of future pharmacologic therapies.

Identification of TMEM129, encoding a ubiquitin-protein ligase, as an effector gene of osteoarthritis genetic risk

“…Tc28a2 immortalised chondrocytes [ 22 ] were seeded onto a 96-well plate at 5000 cells/well and transfected 24h later with 100ng pCpG-free-promoter DNA and 10ng pGL3-promoter (Promega) with Lipofectamine 2000 (Invitrogen). Cells were lysed after 24h and luminescence was read using the Dual-Luciferase Reporter Assay System (Promega) [ 15 ]. Six biological replicates were performed for each plasmid construct.…”

Osteoarthritis year in review: genetics, genomics, epigenetics

“…TGFB1 of course encodes a ligand of the TGF-beta superfamily, with the pathway well described to regulate cartilage development and tissue homeostasis 24 . Rice et al demonstrated differential enhancer activity and protein binding per allele 25 . CRISPR-Cas9-mediated deletion of the locus implied regulation of only TGFB1, and correlation between DNA methylation of CpGs adjacent to the rs75621460 and TGFB1 expression were found, especially in cartilage tissue.…”

“…Rice et al. demonstrated differential enhancer activity and protein binding per allele 25 . Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated deletion of the locus implied regulation of only TGFB1 , and correlation between DNA methylation of CpGs adjacent to the rs75621460 and TGFB1 expression were found, especially in cartilage tissue.…”

Potential Methods of Targeting Cellular Aging Hallmarks to Reverse Osteoarthritic Phenotype of Chondrocytes

“…OA genetic susceptibility loci and SNPs have been largely superseded by hypothesis-free genome-wide association scans [ 67 ]. For example, a strong correlation between DNA methylation and transforming growth factor-β (TGF-β) expression was observed in OA patients [ 68 ]. An SNP located within the TGF-β enhancer region, rs75621460, impacted the activity of the enhancer, and showed protective effects in the cartilage and the synovium.…”