Targeting DNA Demethylation Regulators in Osteoarthritis

摘要 骨关节炎是一种复杂的与年龄相关的疾病，病因不明。我们的长期目标是 了解骨性关节炎病理基础的表观遗传学机制，特别是DNA甲基化和 去甲基化。甲基胞嘧啶(5mC)到其羟基化形式(5hmC)的转化，由10- 11种易位(Tet)酶作为DNA活性去甲基化的中间产物。我们最近做了 报道称，骨性关节炎患者存在软骨内5hmC动态平衡失调，导致软骨细胞数量增加 与激活的OA基因相关的5hmC水平。我们未发表的新发现表明，TET1 基因敲除的小鼠对骨质疏松症的发育有抵抗力，关键的骨质疏松症基因MMP3和13有潜力 TET1和TET2的靶点。基于这些发现，我们建议检验TET的中心假设 蛋白质介导的DNA修饰激活了OA相关基因，Tet功能的丧失可能是 骨关节炎的治疗性治疗。首先，我们将确定5hmC是如何激活OA相关基因的表达的 人骨关节炎软骨细胞中DNA的浓缩和去甲基化。我们将研究5hmC在全球的分布 以及在一组非骨性关节炎和骨性关节炎软骨细胞中的基因表达，使用最先进的 高通量测序(hme-Seal，MBD-seq)之后的化学标记和浓缩技术 和RNA-seq)。其次，我们将直接影响人类软骨细胞中TET1/2的特异性丢失 在“条件性”KO小鼠的出生后软骨中。炎性细胞因子的治疗将用于 在体外有无Tet功能的情况下揭示Tet的直接靶点及其精确模式 监管。对于体内研究，我们将利用TET1和TET2条件基因敲除小鼠的OA诱导来 确定粉防己碱丢失在骨性关节炎病理早期和晚期的作用。这些措施的主要成果是 研究将：(A)确定TET1和TET2调节的OA相关靶基因，(B)确定5mC和5hmC 骨关节炎早期和晚期的依赖基因表达变化和(C)，最重要的是确定如何 Tet抑制可用于调节OA的发病机制。总的来说，这些研究有可能 阐明了骨性关节炎发病机制的一个新方面，并确定了修改骨性关节炎的新治疗策略。

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 Our new unpublished findings show that TET1 knockout mice are resistant to OA development and that the key OA genes MMPs 3 and 13 are potential targets of TET1 and TET2.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.