Epigenetic Regulation of Chondrogenesis and Cartilage Development

项目摘要/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至关重要的作用。 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进行了深入的研究，例如 间充质干细胞。然而，我们对表观遗传学的理解存在很大差距。 在体外或软骨发育过程中调节来自多能干细胞(PSCs)的软骨细胞规格 在活体内。因此，我们提议的工作的长期目标是揭开新的表观遗传机制 细胞和分子水平上的软骨细胞规格和软骨发育。在我们最近的工作中 鉴定人诱导多能干细胞软骨形成的基因调控网络 单细胞RNA-Seq(scRNA-Seq)，我们发现组蛋白甲基转移酶PRDM16显著上调。 在软骨细胞分化阶段由中胚层细胞调控，其表达水平为正 与几个关键的成软骨转录因子相关。这些结果表明PRDM16可能起到了 在调控软骨细胞命运决定中的重要作用。因此，我们假设PRDM16是一个关键的 表观遗传调控因子在软骨形成谱系规范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K99的功能作用和基因调控网络 PRDM16基因过表达或下调细胞的scRNA-Seq在HiPSC软骨形成中的作用 PRDM16在软骨细胞分化过程中的表达。2)鉴定PRDM16DNA结合部位和组蛋白修饰 用染色质免疫沉淀-测序法研究软骨形成中PRDM16富集区的模式 (芯片序列)。有了从K99阶段获得的结果和见解，我们将能够揭开 PRDM16在胚胎肢体发育和出生后软骨体内动态平衡中的作用 R00期特定条件性基因敲除小鼠模型。这项工作将扩大我们对 表观遗传学对软骨发生和肢体发育的调控，并可能为 软骨的动态平衡，以及软骨修复和再生的新策略的改进。

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.