CONTROL OF CHONDROCYTE DIFFERENTIATION

描述（由申请人提供）：我们的长期目标是了解软骨细胞分化的转录机制。我们之前的工作已经证明Sox9在软骨形成中起核心作用，并且在软骨细胞分化途径的多个步骤中都需要Sox9。Sox9最初是建立骨软骨祖细胞所需要的；随后，它是软骨间质凝聚所必需的。因此，软骨细胞的显性分化需要Sox9，部分原因是Sox9是表达Sox5和Sox6所必需的，而Sox5和Sox6是软骨细胞显性分化所必需的。在该通路的后期，Sox9还具有另一个重要作用，因为它参与了软骨细胞向增生性软骨细胞成熟的生理抑制。除了在软骨细胞分化途径中发挥作用外，Sox9还需要用于少数其他细胞系的分化。提出了四个具体目标，以获得关于Sox9和L-Sox5控制软骨细胞分化的机制的新见解。首先将确定在软骨细胞分化程序的两个主要步骤中由Sox9控制的基因库。该假设还将被测试Sox9是否控制特定细胞表面相关蛋白或其他蛋白的表达，这些蛋白是软骨间充质凝聚所必需的。在软骨形成过程中，TIP60作为Sox9和L-Sox5的共激活因子的作用将被进一步表征，作为转录复合物的一部分，与Sox9相互作用的新多肽将被鉴定出来。完整软骨细胞中与软骨细胞特异性启动子和增强子相互作用的Sox9和L-Sox5结合位点以及多肽复合物的模式也将被确定。体外重组的Col2a1调控片段核小体模板将用于解剖Sox9， L-Sox5和其他转录活性多肽在染色质破坏和转录中的功能。这些实验将极大地增强我们对Sox9控制软骨细胞分化机制的理解，并可能为软骨疾病提供新的治疗方法。

DESCRIPTION (provided by applicant): Our long term goal is to understand the transcriptional mechanisms of chondrocyte differentiation. Our previous work has demonstrated that Sox9 has a central role in chondrogenesis and is needed at multiple steps in the pathway of chondrocyte differentiation. Sox9 is initially needed to establish an osteochondroprogenitor; subsequently it is required for chondrogenic mesenchymal condensations. Sox9 is then needed for overt differentiation of chondrocytes, in part because Sox9 is required for expression of Sox5 and Sox6, which are needed for overt chondrocyte differentiation. Later in the pathway Sox9 still has another important role because it participates in the physiological inhibition of the maturation of chondrocytes into hypertrophic chondrocytes. In addition to its roles in the chondrocyte differentiation pathway, Sox9 is also needed for the differentiation of a small number of other cell lineages. Four specific aims are proposed to gain new insights in the mechanisms by which Sox9 and L-Sox5 control chondrocyte differentiation. The repertoire of genes controlled by Sox9 at two major steps in the chondrocyte differentiation program will first be identified. The hypothesis will also be tested whether Sox9 controls the expression of specific cell surface associated proteins or other proteins, needed for chondrogenic mesenchymal condensations. The role of TIP60 as a coactivator of Sox9 and L-Sox5 during chondrogenesis will be further characterized and new polypeptides that are part of transcriptional complexes, which interact with Sox9 will be identified. The patterns of Sox9 and L-Sox5 binding sites and those of polypeptide complexes, which interact with chondrocyte-specific promoters and enhancers, in intact chondrocytes will also be determined. In vitro reconstituted nucleosomal templates of the Col2a1 regulatory segments will be used in order to dissect the function of Sox9, L-Sox5 and other transcriptionally active polypeptides in chromatin disruption and transcription. These experiments should greatly enhance our understanding of the mechanisms whereby Sox9 controls chondrocyte differentiation and may suggest new therapeutic approaches for cartilage diseases.