Epigenetic regulation of cartilage development by TET proteins and DNA hydroxymethylation

 描述（由申请人提供）：软骨再生和相关疾病（如骨关节炎）是一种未满足的医疗需求，治疗仅限于疼痛管理或最终的全关节置换术。为了释放干细胞在软骨再生中的潜力，重要的是要了解干细胞分化的基本过程。已知表观遗传调节因子如DNA甲基化标记在细胞分化中起关键作用;然而，它们在软骨细胞分化中的作用知之甚少。最近的发现导致我们对DNA甲基化和去甲基化调控网络的理解发生了范式转变，发现了甲基化胞嘧啶（5 mC）的新型氧化修饰，即5 hmC、5caC和5 fC，以及参与其产生和周转的酶，即10 - 11-易位（泰特）酶和碱基切除修复（BER）糖基化。已发现5 hmC稳定存在于DNA中，并且作为独立于5 mC的表观遗传标记影响基因表达。5 hmC和泰特酶在胚胎干细胞（ESC）分化过程中起着重要作用。我们的初步研究已经揭示了在软骨细胞分化过程中总体5 hmC水平的动态增加以及在不存在泰特1和2的情况下体外和体内软骨形成分化的损害。因此，本研究计划的重点是确定5 hmC和泰特酶家族在软骨细胞分化的表观遗传调控中的作用。在目标1中，我们将利用一个完善的小鼠软骨祖细胞系，以确定TET 1，2和3功能丧失对软骨细胞分化的影响。如初始数据所示，我们将检验泰特蛋白直接调节Sox trio-Sox 9，5和 6.最后，将进行全局基因表达分析，以确定软骨细胞分化中TET 1、2和3的共同和不同靶点。在目标2中，我们将在小鼠中验证我们的体外发现。将在野生型和TET 1突变小鼠中研究胚胎肢体发育期间的5 hmC动态。组织学、免疫染色、DNA和RNA分析将用于确定5 hmC变化对正常肢体发育中软骨形成标志物（包括Sox 9、5和6、col 2a 1、Runx 2和col 10a 1）的时间和影响，以及突变小鼠中5 hmC变化如何受损。这些研究将极大地推进对5 hmC和泰特蛋白对软骨细胞分化的表观遗传调节的理解，并将为测试这些新调节剂在软骨再生和骨关节炎中的治疗潜力奠定基础。

 DESCRIPTION (provided by applicant): Cartilage regeneration and related diseases like Osteoarthritis constitute an unmet medical need, with the treatments being limited to pain management or eventual total joint replacement. In order to unlock the potential of stem cells for cartilage regeneration, it is important to understand the fundamental processes governing stem cell differentiation. Epigenetic regulators like the DNA methylation marks are known to play key roles in cellular differentiation; however their role in chondrocyte differentiation is poorly understood. Recent discoveries have led to a paradigm shift in our understanding of the DNA methylation and demethylation regulatory network with the discovery of novel oxidative modifications of methylated cytosine (5mC) i.e. 5hmC, 5caC and 5fC as well as the enzymes involved in their generation and turnover i.e. Ten-eleven-translocation (TET) enzymes and Base-excision-repair (BER) glycosylates. 5hmC has been found to be stably present in DNA as well as influence gene expression as an epigenetic mark independent of 5mC. 5hmC and TET enzymes have been identified to be critical for embryonic stem cells (ESC) differentiation. Our initial studies have uncovered a dynamic increase in global 5hmC levels during chondrocyte differentiation as well as an impairment of chondrogenic differentiation both in vitro and in vivo n the absence of TET 1 and 2. This research proposal is therefore focused on defining the role of 5hmC and the TET family of enzymes in epigenetic regulation of chondrocyte differentiation. In Aim 1, we will utilize a well-established mouse chondroprogenitor cell line to determine the effects of TET1, 2 and 3 loss-of-functions on chondrocyte differentiation. As suggested by initial data, we will test the hypothesis that the TET proteins directly regulate the Sox trio-Sox 9, 5 and 6. Finally, global gene expression analyses will be performed to identify common and distinct targets of TET1, 2 and 3 in chondrocyte differentiation. In Aim 2, we will validate our in vitro findings in mice. 5hmC dynamics during embryonic limb development will be studied in both wild-type and TET1 mutant mice. Histology, immunostaining, DNA and RNA analyses will be used to define the timing and effect of 5hmC changes on chondrogenic markers including Sox9, 5 and 6, col2a1, Runx2 and col10a1 in normal limb development and how it is impaired in the mutant mice. These studies will greatly advance the understanding of the epigenetic regulation of chondrocyte differentiation by 5hmC and TET proteins, and will set the stage for testing the therapeutic potential of these novel regulators in cartilage regeneration and Osteoarthritis.