Targeting DNA Demethylation Regulators in Osteoarthritis

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Abstract Osteoarthritis (OA) is a complex age-associated disorder with an unidentified etiology. Our long-term goal is to understand the epigenetic mechanisms underlying OA pathology especially the role of DNA methylation and demethylation. Conversion of methyl cytosine (5mC) to its hydroxylated form (5hmC), catalyzed by the ten- eleven translocation (TET) enzymes acts as an intermediate in active DNA demethylation. We have recently reported that OA patients have a dysregulation of the 5hmC homeostasis in cartilage, leading to increased 5hmC levels that are associated with activated OA genes. Our new unpublished findings show that TET1 knockout mice are resistant to OA development and that the key OA genes MMPs 3 and 13 are potential targets of TET1 and TET2. Based on these findings, we propose to test the central hypothesis that TET proteins-mediated DNA modifications activate OA-associated genes and that loss of TET function can be therapeutic in OA. Firstly, we will determine how expression of OA-associated genes is activated by 5hmC enrichment and DNA demethylation in human OA chondrocytes. We will study the global distribution of 5hmC and 5mC as well as gene expression in a cohort of non-OA and OA chondrocytes, using state-of-the-art chemical labeling and enrichment techniques followed by high-throughput sequencing (hme-Seal, MBD-seq and RNA-seq respectively). Secondly, we will directly effect a specific loss of TET1/2 in human chondrocytes and in post-natal cartilage in `conditional' KO mice. Treatment with inflammatory cytokines will be utilized in vitro in the presence or absence of TET function to reveal direct TET targets and their precise mode of regulation. For the in vivo studies, we will utilize OA induction in TET1 and TET2 conditional knockout mice to determine the effect of TET loss in the early and late stages of OA pathology. The major outcomes of these studies will be to (a) identify OA-associated target genes regulated by TET1 and 2, (b) identify 5mC and 5hmC dependent gene expression changes in early and late stages of OA and (c) and most importantly identify how TET inhibition can be utilized to modulate OA pathogenesis. Collectively, these studies have the potential to shed light on a new facet of OA pathogenesis and to identify new therapeutic strategies for modifying OA.