TET1 Directs Chondrogenic Differentiation by Regulating SOX9 Dependent Activation of Col2a1 and Acan In Vitro.
TET1 Directs Chondrogenic Differentiation by Regulating SOX9 Dependent Activation of Col2a1 and Acan In Vitro.
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作者：
Smeriglio P;Grandi FC;Taylor SEB;Zalc A;Bhutani N
骨骼发育是一个紧密协调的过程，软骨和骨的分化错综复杂地交织在一起。最近的研究强调了表观遗传修饰及其作者对骨骼发育的贡献。甲基化胞嘧啶(5mC)可被Ten-11易位(Tet)酶氧化为5-羟甲基胞嘧啶(5hmC)，导致去甲基化。我们先前已经证明，在ATDC5软骨前体细胞的体外软骨形成过程中，5hmC稳定地积聚在谱系特异性基因上，这些基因被激活。Tet1通过短发夹状RNA的敲除(KD)阻断了ATDC5的软骨分化。本研究旨在为TET1在ATDC5分化过程中的作用提供机制基础。对Tet1KD细胞的转录分析表明，54%的下调基因是SOX9靶基因，这表明TET1在介导SOX9靶基因亚集的激活中发挥了作用。利用ATDC5分化过程中5hmC的全基因组作图，我们发现5hmC优先聚集在SOX9的软骨细胞特异性II类结合部位，而不是组织不可知的I类结合部位。具体地说，我们发现SOX9在Tet1 kD后不能与Col2a1和Acan结合，尽管SOX9水平没有变化。最后，我们将这种KD方案与使用Tet1−/−胚胎的生长板中TET1的遗传丢失进行了比较，Tet1 Tet1胚胎比WT对应的胚胎小约10%。在体外，E17.5Tet1−/−胚胎中，SOX9靶基因表达的损失比Tet1KD中的要轻微。总体而言，我们的数据表明，TET1介导的5hmC沉积在部分形成有利于SOX9功能的表观基因组中发挥了作用。©2020作者。JBMR Plus由威利期刊公司代表美国骨与矿物研究学会出版。
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 Tet1 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 Tet1 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 Col2a1 and Acan after Tet1 KD, despite no changes in SOX9 levels. Finally, we compared this KD scenario with the genetic loss of TET1 in the growth plate using Tet1 −/− embryos, which are approximately 10% smaller than their WT counterparts. In E17.5 Tet1 −/− embryos, loss of SOX9 target gene expression is more modest than upon Tet1 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. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.