Epigenetic regulation of cartilage development by TET proteins and DNA hydroxymethylation

 描述(申请人提供)：软骨再生和骨关节炎等相关疾病构成了一个未得到满足的医疗需求，治疗仅限于疼痛控制或最终的全关节置换术。为了释放干细胞对软骨再生的潜力，了解干细胞分化的基本过程是很重要的。表观遗传调控因子如DNA甲基化标记在细胞分化中起关键作用，但对其在软骨细胞分化中的作用知之甚少。最近的发现使我们对DNA甲基化和去甲基化调控网络的理解发生了转变，发现了甲基化胞嘧啶(5mC)的新的氧化修饰，即5hmC，5caC和5fC，以及与它们的生成和周转有关的酶，即Ten-11易位(Tet)酶和碱基切除修复(BER)糖基化。5hmC已被发现稳定存在于DNA中，并作为一种独立于5mC的表观遗传标记影响基因表达。5hmC和Tet酶是胚胎干细胞(ESC)分化的关键酶。我们的初步研究发现，在软骨细胞分化过程中，全球5hmC水平动态增加，在缺乏Tet 1和Tet 2的情况下，体外和体内软骨细胞分化都受到损害。因此，本研究计划重点确定5hmC和Tet家族酶在软骨细胞分化的表观遗传调控中的作用。在目标1中，我们将利用成熟的小鼠软骨前体细胞系来确定TET1、2和3功能丧失对软骨细胞分化的影响。正如最初的数据所建议的那样，我们将检验Tet蛋白直接调节Sox trio-Sox 9，5和 6.最后，将进行整体基因表达分析，以确定TET1、TET2和TET3在软骨细胞分化中的共同和不同靶点。在目标2中，我们将在小鼠身上验证我们的体外发现。将在野生型和TET1突变小鼠身上研究胚胎肢体发育过程中的5hmC动力学。组织学、免疫染色、DNA和RNA分析将用于确定5hmC在正常肢体发育中对软骨形成标志物Sox9、5和6、col2a1、Runx2和col10a1的时间和影响，以及在突变小鼠中它是如何受损的。这些研究将极大地促进对5hmC和Tet蛋白对软骨细胞分化的表观遗传调控的理解，并将为测试这些新的调控因子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.