Transcriptional Regulation of Chondrogenesis

描述（由申请人提供）：软骨发生是一个多步骤途径，在此过程中，多潜能间充质干细胞分化为软骨细胞，形成关节软骨。这个过程不仅对软骨发育至关重要，而且在关节修复过程中也是如此。对软骨发生的分子机制的研究将促进我们对骨骼发育的理解，并有可能确定治疗关节疾病的新方法。Sox 9高迁移率族（HMG）结构域转录因子是软骨细胞分化中的关键分子。Sox 9的表达几乎与Col 2a 1的表达平行，Col 2a 1在软骨形成过程中编码软骨特异性II型胶原。然而，在生殖嵴中，Col 2a 1不表达，尽管内源性Sox 9表达水平高，这表明存在软骨特异性Cola 1基因表达所需的Sox 9分子伴侣。在初步研究中，我们确定PGC-1作为Sox 9转录共激活因子。PGC-1以前被认为是过氧化物酶体增殖物激活受体γ共激活剂，并在脂肪生成和糖原生成中发挥作用;然而，其在软骨形成中的作用尚未被探索。第10-12天小鼠胚胎的整体原位杂交显示PGC-1基因仅在软骨形成部位的细胞中表达。PGC-1与Sox 9相互作用以增强Col 2a 1启动子活性并促进软骨形成。此外，Sox 9对Col 2a 1的活性受到乙酰化和脱乙酰化分子的严格调节。CBP（CREB binding protein）是染色质乙酰化酶之一，与Sox 9/PGC-1复合物相互作用，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Sox 9的共激活剂发挥作用，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：1.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Sox 9共激活剂的作用。2.确定Sox 9/PGC-1复合物与HAT活性和随后的染色质调节对组织特异性基因表达和软骨细胞分化的作用。3.研究软骨形成过程中HDAC和相关辅阻遏物复合物调控Sox 9/PGC-1复合物依赖性基因表达的机制。4.通过染色质体外重组和Col 2al启动子转录实验分析染色质依赖的Sox 9/PGC-1复合物转录机制。

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.