Epigenetic regulation of cartilage development by TET proteins and DNA hydroxymethylation

TET 蛋白和 DNA 羟甲基化对软骨发育的表观遗传调控

基本信息

批准号：
9132165
负责人：
Nidhi Bhutani
金额：
$ 8.35万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-18 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9132165
关键词：
Affect Base Excision Repairs Binding Proteins Birth Bone Development Cartilage Cartilage Diseases Cartilage injury Cell Line Chondrocytes Coitus Cytosine DNA DNA Methylation DNA Repair Data Degenerative polyarthritis Disease Embryo Enzymes Epigenetic Process Event Exhibits Extracellular Matrix Family Gene Expression Gene Targeting Generations Genes Genetic Transcription Goals Growth Health Histology Hypertrophy Impairment In Vitro Individual Knowledge Lead Limb Bud Limb Development Mediating Medical Modification Molecular Monitor Mus Mutant Strains Mice Outcome Pain management Pathway interactions Play Process Protein Family Protein translocation Proteoglycan RNA Regulation Regulatory Pathway Replacement Arthroplasty Reporting Research Research Project Grants Research Proposals Rheumatoid Arthritis Role SOX9 protein Site Staging Staining method Stains Stem cells Testing Therapeutic Time base cartilage development cartilage regeneration chondrodysplasia chromatin immunoprecipitation demethylation developmental disease embryonic stem cell epigenetic regulation epigenome in vivo insight loss of function novel novel strategies research study stem cell differentiation transcription factor

项目摘要

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.

描述（由适用提供）：软骨再生和相关疾病（如骨关节炎）构成了未满足的医疗需求，这些治疗局限于疼痛管理或事件总关节置换。为了释放干细胞的软骨再生潜力，重要的是要了解有关干细胞分化的基本过程。已知表观遗传调节剂（例如DNA甲基化标记）在细胞分化中起关键作用。但是，它们在软骨细胞分化中的作用知之甚少。 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）糖基化。已经发现5HMC在DNA中稳定存在，并且影响基因表达是独立于5MC的表观遗传标记。 5HMC和TET酶已被确定为胚胎干细胞（ESC）分化至关重要。我们的最初研究已经发现了软骨细胞分化过程中全球5HMC水平的动态增加以及体外和体内缺乏TET 1和2的软骨分化的损害。因此，这项研究的重点是定义5HMC和TET家族在enzymes中的作用。在AIM 1中，我们将利用良好的小鼠软骨元基因细胞系来确定TET1、2和3功能丧失对软骨细胞分化的影响。正如初始数据所暗示的那样，我们将测试以下假设：TET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，以确定软骨成生成标记中TET1、2和3的共同靶标。在AIM 2中，我们将在小鼠中验证我们的体外发现。胚胎肢体发育过程中的5HMC动力学将在野生型和TET1突变小鼠中研究。组织学，免疫染色，DNA和RNA分析将用于定义5HMC变化对软骨源标记的时间和影响，包括SOX9、5和6，COL2A1，RUNX2和COL10A1在正常肢体发育中以及在突变小鼠中如何受损。这些研究将大大提高对5HMC和TET蛋白对软骨细胞分化的表观遗传调节的理解，并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了基础。