Transcriptional Control of Sox9

Sox9 的转录控制

• 批准号: 7985460
• 负责人: VERONIQUE M LEFEBVRE
• 金额: $ 47.36万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-20 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7985460
• 关键词: Adult; Affect; American; Binding; Binding Sites; Biochemical; Cartilage; Cartilage Diseases; Cell Maintenance; Cells; Chondrocytes; Chondrogenesis; Chromosomal Rearrangement; Commit; Complementary DNA; Complex; Conserved Sequence; Cultured Cells; DNA Sequence; DNA Sequence Rearrangement; Data; Degenerative polyarthritis; Disease; Elements; Embryo; Enhancers; Figs - dietary; Functional RNA; Gene Expression; Genes; Genetic; Genetic Enhancer Element; Goals; In Vitro; Joints; LacZ Genes; Life; Mediating; Mesenchymal; Mesenchymal Stem Cells; Molecular; Mouse, Founder, Transgenic; Mus; Pattern; Regulation; Repetitive Sequence; Reporter; Role; Screening procedure; Site; Skeleton; Structure; Syndrome; Testing; Therapeutic; Transact; Transcriptional Regulation; Transgenes; Transgenic Mice; Work; campomelic dysplasia; cartilage cell; chondrodysplasia; cis acting element; design; in vivo; malformation; promoter; public health relevance; skeletal dysplasia; skeletogenesis; therapeutic target; transcription factor; transcription factor USF

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. PUBLIC HEALTH RELEVANCE: The goal of this project is to identify mechanisms underlying expression of Sox9, the gene for a transcription factor required for cartilage formation. We have identified two sequences upstream of the gene that are sufficient to activate the Sox9 promoter in cartilage cells. This project will use complementary approaches in vitro and in vivo to test the hypothesis that these sequences are both needed and sufficient for Sox9 expression in cartilage and that they bind transcription factors, collectively referred to as chondrogenin, which are required to activate the Sox9 gene and thereby induce chondrogenesis. The results of this study are expected to increase understanding of the molecular regulation of chondrogenesis and to provide new DNA sequences and factors to develop urgently needed therapies for cartilage malformation and degeneration diseases, including the highly prevalent osteoarthritis disease.

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 是一种主要的软骨形成转录因子，因此是软骨发育不良和关节退行性疾病急需治疗的有吸引力的靶点。由于 Sox9 主要在转录水平上受到控制，因此在这些疾病中刺激其活性的最佳方法是利用其天然转录机制。这些机制仍然难以捉摸，但我们在初步研究中确定了位于 Sox9 上游的两个高度保守的序列，它们是有效的软骨细胞特异性增强子。这些增强剂在体内所有软骨结构中具有重叠的活性域，并且在体外相互协同。第一个增强子的核心区域具有多种转录因子的假定位点，但介导其活性的特定因子（称为“软骨原素”）尚不清楚。另一个增强子在体外被 Sox9 本身上调，内源性 Sox9 基因也是如此，它具有多个 Sox 结合位点。这些数据表明软骨原素通过与第一个增强子结合来激活Sox9表达，并且Sox9与另一个增强子结合以与软骨原素协同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。目标 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周期中激活软骨中的 Sox9 启动子。目标 2 是使用条件失活方法来测试增强子是否是激活软骨中内源 Sox9 基因所必需的。目标 3 是使用互补的遗传、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个增强子结合，从而上调其自身的基因表达。目标 4 是使用候选和生化方法来鉴定介导第一增强子活性的软骨生成素因子，并确定它们在软骨形成中的作用。我们预计这项工作的成果将产生很高的科学和技术影响。我们期望鉴定出对软骨形成至关重要的 Sox9 增强子和上游转录因子，因此将成为软骨畸形和退行性疾病新疗法中上调 Sox9 表达的相关靶标。 公共健康相关性：该项目的目标是确定 Sox9 表达的机制，Sox9 是软骨形成所需的转录因子的基因。我们已经鉴定出该基因上游的两个序列足以激活软骨细胞中的 Sox9 启动子。该项目将在体外和体内使用互补方法来测试以下假设：这些序列对于软骨中的 Sox9 表达来说是必要且充分的，并且它们结合转录因子（统称为软骨原素），这是激活 Sox9 基因和从而诱导软骨形成。这项研究的结果预计将增进对软骨形成分子调控的了解，并提供新的 DNA 序列和因子，以开发针对软骨畸形和退行性疾病（包括高度流行的骨关节炎疾病）急需的疗法。