TET1 Directs Chondrogenic Differentiation by Regulating SOX9 Dependent Activation of and In Vitro.

Skeletal development is a tightly orchestrated process in which cartilage and bone differentiation are intricately intertwined. Recent studies have highlighted the contribution of epigenetic modifications and their writers to skeletal development. Methylated cytosine (5mC) can be oxidized to 5-hydroxymethylcytosine (5hmC) by the Ten-eleven-translocation (TET) enzymes leading to demethylation. We have previously demonstrated that 5hmC is stably accumulated on lineage-specific genes that are activated during in vitro chondrogenesis in the ATDC5 chondroprogenitors. Knockdown (KD) of via short-hairpin RNAs blocked ATDC5 chondrogenic differentiation. Here, we aimed to provide the mechanistic basis for TET1 function during ATDC5 differentiation. Transcriptomic analysis of KD cells demonstrated that 54% of downregulated genes were SOX9 targets, suggesting a role for TET1 in mediating activation of a subset of the SOX9 target genes. Using genome-wide mapping of 5hmC during ATDC5 differentiation, we found that 5hmC is preferentially accumulated at chondrocyte-specific class II binding sites for SOX9, as compared with the tissue-agnostic class I sites. Specifically, we find that SOX9 is unable to bind to and after KD, despite no changes in SOX9 levels. Finally, we compared this KD scenario with the genetic loss of TET1 in the growth plate using embryos, which are approximately 10% smaller than their WT counterparts. In E17.5 embryos, loss of SOX9 target gene expression is more modest than upon KD in vitro Overall, our data suggest a role for TET1-mediated 5hmC deposition in partly shaping an epigenome conducive for SOX9 function. © 2020 The Authors. published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.