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）：该项目的目的是确定软骨细胞中SOX9表达的转录机制。 Sox9是主要的软骨转录因子，因此是软骨发育和关节变性疾病迫切需要疗法的有吸引力的靶标。由于SOX9主要在转录水平上控制，因此刺激其在这些疾病中活性的最佳方法是使用其自然转录机制。这些机制仍然难以捉摸，但是我们在初步研究中鉴定了两个高度保守的序列Sox9上游，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结构域，并在体外彼此协同作用。第一个增强子的核心区域具有多种类型的转录因子的推定位点，但是特定因素（称为“软骨蛋白”）介导其活性是未知的。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，并且具有多个SOX结合位点。这些数据表明，软骨蛋白通过与第一个增强剂结合而激活SOX9的表达，并且Sox9与其他增强子结合以与软骨蛋白协同作用并保持其自身的基因表达。我们提出了四个特定的目的来检验这一假设。 AIM 1是使用转基因小鼠方法来确定两个增强子是否足以在整个生命中激活软骨中的Sox9启动子。 AIM 2是使用条件灭活方法来测试增强子是否需要在软骨中激活内源性SOX9基因。 AIM 3是使用互补的遗传，细胞和分子方法来证明SOX9与软骨细胞中的第二增强子结合，通过特定位点进行，从而在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剂活性并确定其在软骨发生中的作用的软骨蛋白因子。我们预计这项工作的结果将产生很高的科学和技术影响。我们期望鉴定Sox9增强子和上游转录因子，这些因素对于软骨生成至关重要，因此将成为在软骨畸形和变性疾病的新处理中上调Sox9表达的相关靶标。 公共卫生相关性：该项目的目的是确定SOX9表达的机制，Sox9是软骨形成所需的转录因子的基因。我们已经确定了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这些序列在软骨中所需的sox9表达，并且它们结合了转录因子，并且共同称为软骨蛋白，这是激活SOX9基因并诱导软骨生成所必需的。预计这项研究的结果将增加对软骨发生的分子调节的了解，并提供新的DNA序列和因素，以开发出急需的软骨畸形和变性疾病的疗法，包括高度普遍的骨关节炎。