Epigenetic Regulation of Chondrogenesis and Cartilage Development

软骨形成和软骨发育的表观遗传调控

• 批准号: 10316343
• 负责人: Chia-Lung Wu
• 金额: $ 38.35万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-05 至 2024-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10316343
• 关键词: Acetylation; Adult; Binding; Binding Sites; Cartilage; Cell Line; Cells; ChIP-seq; Chondrocytes; Chondrogenesis; Chromatin; Chromosome 1; Complex; Coupling; DNA Binding; DNA analysis; Data; Deacetylation; Defect; Development; Disease; Embryo; Enhancers; Epigenetic Process; Exhibits; Family; Foundations; Gene Expression; Genes; Genetic Transcription; Goals; Harvest; Histones; Homeostasis; Human; In Vitro; Injury; Joints; Knock-out; Knockout Mice; Limb Bud; Limb Development; Limb structure; Link; Maps; Mesenchymal Stem Cells; Mesoderm Cell; Methylation; Modeling; Modification; Molecular; Mus; Pathogenesis; Pathway interactions; Pattern; Phase; Phenotype; Play; Pluripotent Stem Cells; Population; Process; RUNX1 gene; Regulation; Regulator Genes; Reporting; Research; Role; SOX6 gene; Testing; Time; Tissue Engineering; Work; adult stem cell; base; cartilage development; cartilage regeneration; cartilage repair; cofactor; conditional knockout; demethylation; epigenetic regulation; genome-wide; histone methyltransferase; histone modification; in vivo; induced pluripotent stem cell; insight; knock-down; microdeletion; mouse model; novel; osteogenic; overexpression; postnatal; promoter; recruit; single-cell RNA sequencing; skeletal; tissue regeneration; transcription factor; transcriptome; transcriptome sequencing

Project summary/abstract Recent evidence indicates that epigenetics plays an essential role in modulating tightly-coordinated transcription factors involved in cartilage development. Epigenetic modifications such as acetylation/deacetylation or methylation/demethylation have been intensively investigated in the chondrogenesis of adult stem cells such as mesenchymal stem cells (MSCs). However, there is a significant gap in our understanding of how epigenetics regulates chondrocyte specification from pluripotent stem cells (PSCs) in vitro or during cartilage development in vivo. Thus, the long-term goal of our proposed work is to unravel novel epigenetic mechanisms governing chondrocyte specification and cartilage development at the cellular and molecular levels. In our recent work of identifying gene regulatory networks of chondrogenesis of human induced pluripotent stem cells (hiPSCs) via single-cell RNA-Seq (scRNA-Seq), we reveled that PRDM16, a histone methyltransferase, was significantly up- regulated at the stage of chondrocyte specification from mesodermal cells, and its expression level was positively correlated with several key chondrogenic transcription factors. These results imply that PRDM16 may play an essential role in regulation chondrocyte cell fate decision. Thus, we hypothesize that PRDM16 is a critical epigenetic regulator in chondrogenic lineage specification and cartilage development. To test this hypothesis, our approaches in the K99 phase are to: 1) Elucidate the functional role and gene regulatory networks of PRDM16 in hiPSC chondrogenesis in vitro by scRNA-Seq of the cells with overexpression or knockdown of PRDM16 during chondrogenic differentiation. 2) Identify PRDM16 DNA binding sites and histone modification pattern within PRDM16-enriched regions in chondrogenesis using chromatin immunoprecipitation-sequencing (ChIP-Seq). With the results and insights obtained from the K99 phase, we will then be able to 3) Unravel the function of PRDM16 in embryonic limb development and postnatal cartilage homeostasis in vivo using cartilage- specific conditional knock-out mouse models in R00 phase. This work will extend our understanding of the epigenetic regulation of chondrogenesis and limb development, and could provide important insights into the homeostasis of cartilage, as well as the refinement of new strategies for cartilage repair and regeneration.

项目概要/摘要 最近的证据表明，表观遗传学在调节紧密协调的转录中起着重要作用 参与软骨发育的因素。表观遗传修饰，如乙酰化/脱乙酰化或 甲基化/去甲基化在成体干细胞的软骨形成中已经被深入研究， 间充质干细胞（MSC）。然而，在我们对表观遗传学如何 在体外或软骨发育过程中调节来自多能干细胞（PSC）的软骨细胞特化 in vivo.因此，我们提出的工作的长期目标是解开新的表观遗传机制， 在细胞和分子水平上的软骨细胞特化和软骨发育。在我们最近的工作中， 鉴定人诱导多能干细胞（hiPSC）的软骨形成的基因调控网络， 单细胞RNA-Seq（scRNA-Seq），我们发现PRDM 16，一种组蛋白甲基转移酶， 中胚层细胞在软骨细胞特化阶段进行调节，其表达水平呈阳性 与几个关键的软骨形成转录因子相关。这些结果暗示PRDM 16可能在细胞内起作用。 在调节软骨细胞命运决定中起重要作用。因此，我们假设PRDM 16是一个关键的 表观遗传调节因子在软骨形成谱系特化和软骨发育中的作用。为了检验这一假设， 我们在K99阶段的方法是：1）阐明功能作用和基因调控网络， PRDM 16在hiPSC软骨形成中的体外表达，通过scRNA-Seq对具有PRDM 16过表达或敲低的细胞进行检测。 PRDM 16在软骨形成分化过程中的作用。2)识别PRDM 16 DNA结合位点和组蛋白修饰 使用染色质免疫沉淀-测序在软骨形成中PRDM 16富集区域内的模式 （ChIP-Seq）。有了K99阶段的结果和见解，我们将能够3）解开 PRDM 16在胚胎肢体发育和出生后软骨体内稳态中的功能， R 00期特异性条件性基因敲除小鼠模型。这项工作将扩大我们对 软骨形成和肢体发育的表观遗传调控，并可以提供重要的见解， 软骨的动态平衡，以及软骨修复和再生新策略的完善。