Transcriptional Control of Sox9

描述（由申请人提供）：该项目的目标是确定软骨细胞中Sox9表达的转录机制。Sox9是一种主要的软骨生成转录因子，因此是软骨发育不良和关节退行性疾病急需治疗的一个有吸引力的靶点。由于Sox9主要在转录水平受到控制，因此刺激其在这些疾病中的活性的最佳方法是利用其自然转录机制。这些机制仍然难以捉摸，但我们在初步研究中确定了Sox9上游的两个高度保守序列，它们是有效的软骨细胞特异性增强因子。这些增强子在体内所有软骨结构中具有重叠的活性域，并在体外相互协同作用。第一个增强子的核心区域具有多种转录因子的假定位点，但介导其活性的具体因子（称为“软骨素”）尚不清楚。另一个增强子在体外被Sox9自身上调，内源性Sox9基因也是如此，它具有多个Sox9结合位点。这些数据表明，软骨素通过结合第一个增强子激活Sox9的表达，而Sox9通过结合另一个增强子与软骨素协同作用并维持其自身的基因表达。我们提出了四个具体目标来检验这一假设。目的1是使用转基因小鼠方法来确定这两种增强子是否足以在整个生命过程中激活软骨中的Sox9启动子。目的2是使用条件失活方法来测试增强子是否需要激活软骨中内源性Sox9基因。目的3是利用互补的遗传、细胞和分子方法来证明Sox9通过特定位点与软骨细胞中的第二增强子结合，从而上调其自身的基因表达。目的4是使用候选和生化方法来确定介导第一增强子活性的软骨素因子，并确定它们在软骨形成中的作用。我们预期这项工作的成果将具有很高的科学和技术影响。我们期望找到对软骨形成至关重要的Sox9增强子和上游转录因子，从而在软骨畸形和退行性疾病的新治疗中成为上调Sox9表达的相关靶点。

DESCRIPTION (provided by applicant): The goal of this project is to identify transcriptional mechanisms underlying Sox9 expression in chondrocytes. Sox9 is a master chondrogenic transcription factor and thus an attractive target for urgently needed therapies for chondrodysplasias and joint degeneration diseases. Since Sox9 is primarily controlled at the transcriptional level, the best way to stimulate its activity in these diseases is to use its natural transcriptional mechanisms. These mechanisms remain elusive, but we identify in preliminary studies two highly conserved sequences far upstream of Sox9 that are potent chondrocyte-specific enhancers. These enhancers have overlapping domains of activity in all cartilage structures in vivo and synergize with each other in vitro. The core region of the first enhancer features putative sites for multiple types of transcription factors, but the specific factors, referred to as "chondrogenin", which mediate its activity are unknown. The other enhancer is upregulated by Sox9 itself in vitro, as is the endogenous Sox9 gene, and it features multiple Sox-binding sites. These data suggest that chondrogenin activates Sox9 expression by binding to the first enhancer and that Sox9 binds to the other enhancer to synergize with chondrogenin and to maintain its own gene expression. We propose four specific aims to test this hypothesis. Aim 1 is to use a transgenic mouse approach to determine whether the two enhancers may be sufficient to activate the Sox9 promoter in cartilage throughout life. Aim 2 is to use conditional inactivation approaches to test whether the enhancers are necessary to activate the endogenous Sox9 gene in cartilage. Aim 3 is to use complementary genetic, cellular and molecular approaches to demonstrate that Sox9 binds to the second enhancer in chondrocytes, does so through specific sites, and thereby upregulates its own gene expression. Aim 4 is to use candidate and biochemical approaches to identify the chondrogenin factors that mediate the activity of the first enhancer and to determine their roles in chondrogenesis. We anticipate that the results of this work will have high scientific and technological impact. We expect to identify Sox9 enhancers and upstream transcription factors that are essential for chondrogenesis and that will therefore become relevant targets to upregulate Sox9 expression in new treatments for cartilage malformation and degeneration diseases.