Transcriptional Regulation of Chondrogenesis

描述（由申请人提供）：时间发生是多能间充质干细胞向软骨细胞分化形成关节软骨的多步骤途径。这一过程不仅对软骨发育至关重要，而且在关节修复过程中也很重要。对时间发生的分子机制的研究将促进我们对骨骼发育的理解，并有可能找到治疗关节疾病的新方法。Sox9高迁移率基团（HMG）结构域转录因子是软骨细胞分化的关键分子。Sox9的表达几乎与Col2a1相似，Col2a1在软骨形成过程中编码软骨特异性II型胶原蛋白。然而，在生殖器嵴中，尽管内源性Sox9表达水平很高，但Col2a1不表达，这表明Sox9的分子伴侣的存在是软骨特异性Cola1基因表达所必需的。在初步研究中，我们发现PGC-1是Sox9转录共激活因子。PGC-1先前被称为PPAR γ共激活因子，在脂肪生成和糖生成中起作用；然而，其在软骨形成中的作用尚未被探索。第10-12天的小鼠胚胎全座原位杂交显示PGC-1基因仅在软骨形成部位的细胞中表达。PGC-1与Sox9相互作用，增强Col2a1启动子活性，促进软骨形成。此外，Sox9在Col2a1上的活性受到乙酰化和去乙酰化分子的严格调控。CBP （CREB结合蛋白）是染色质乙酰化酶之一，也被证明与Sox9/PGC-1复合物相互作用，并参与了这一机制。这些初步数据为我们的假设提供了基础，即PGC-1在软骨形成过程中作为组织和发育阶段特异性的Sox9共激活因子，其机制涉及特异性染色质调节。为此，我们提出以下具体目标：确定PGC-1在软骨形成和软骨细胞特异性基因表达过程中作为Sox9共激活因子的作用。2. 确定Sox9/PGC-1复合物相关HAT活性和随后的染色质调控在组织特异性基因表达和软骨细胞分化中的作用。3. 研究在软骨形成过程中，hdac和相关的协同抑制因子复合物调控Sox9/PGC-1复合体依赖基因表达的机制。4. 利用体外染色质重构和Col2al启动子转录分析染色质依赖性Sox9/PGC-1复合体转录机制。

DESCRIPTION (provided by applicant): Chrondrogenesis is a multi-step pathway during which multipotential mesenchymals stem cells differentiate into chondrocytes to form articular cartilage. This process is not only essential for cartilage development but also during joint repair. Research on molecular mechanisms of chrondrogenesis will advance our understanding of skeletal development and has the potential to identify new approaches to the treatment of joint diseases. The Sox9 high-mobility group (HMG) domain transcription factor is a key molecule in chondrocyte differentiation. Expression of Sox9 almost parallels that of Col2a1, which encodes cartilage-specific type II collagen during chondrogenesis. In the genital ridge, however, Col2a1 is not expressed, despite high level of endogenous Sox9 expression, suggesting the existence of molecular partner(s) of Sox9 that are required for cartilage-specific Cola1 gene expression. In preliminary studies, we identified PGC-1 as a Sox9 transcriptional co-activator. PGC-1 was previously known as PPAR gamma co-activator and plays a role in adipogenesis and glycogenesis; however, its role in chondrogensis has been unexplored. Whole mount in situ hybridization of day 10-12 mice embryos revealed PGC-1 gene expression only in cells at chondrogenic sites. PGC-1 interacts with Sox9 to enhance Col2a1 promoter activity and promotes chondrogenesis. Furthermore, Sox9 activity on the Col2a1 is tightly regulated by acetylation and deacetylation molecules. CBP (CREB binding protein), one of the chromatin acetylation enzymes, was also shown to interact with Sox9/PGC-1 complex and is involved in this mechanism. These preliminary data provide the basis for our hypothesis that PGC-1 functions as a tissue and developmental stage specific co-activator of Sox9 during chondrogenesis in a mechanism that involves specific chromatin regulation. Toward this end, we propose following Specific Aims: 1. Determine the role of PGC-1 as a co-activator of Sox9 during chondrogenesis and chondrocyte-specific gene expression. 2. Determine the role of Sox9/PGC-1 complex associating HAT activity and subsequent chromatin regulation for tissue specific gene expression and chondrocyte differentiation. 3. Examine mechanisms by which Sox9/PGC-1 complex dependent gene expression is regulated by HDACs and related co-repressor complex during chondrogenesis. 4. Analyze chromatin dependent Sox9/PGC-1 complex transcription mechanism by using in vitro reconstitution of chromatin and transcription assay of the Col2al promoter.