Our long term goal is to understand the transcriptional mechanisms of chondrocyte differentiation. Heterozygous mutations in the Sox9 gene are the cause of the rare human disease campomelic dysplasia, a generalized disease of cartilage characterized by hypoplasia of many endochondral bones. Genetic work in mice has demonstrated that Sox9 has a central role in the multistep pathway of chondrocyte differentiation and is needed at multiple steps. 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 multistep roles in the chondrocyte differentiation pathway, Sox9 is also needed for the differentiation of other cell lineages including progenitor cells of the pancreas and of the cranial neural crest and cells of the endocardial cushions, anlagen of the septa and valves of the heart. Sox9 also has a critical role in testis and kidney development. Our hypothesis is that different mechanisms are responsible for the essential roles of Sox9 at different stages of chondrocyte differentiation and in other cell lineages. We speculate that these different mechanisms are due to different proteins that interact with Sox9. In this application, which contains four Specific Aims, (1) we first hypothesize that in order to fulfill its role during chondrogenic mesenchymal condensations Sox9 has to control the expression of specific cell surface proteins. It is proposed, therefore, to identify genes that are downstream of Sox9 during mesenchymal condensations. (2) Polypeptides that interact with Sox9 in overtly differentiated chondrocytes will also be identified using both the yeast 2-hybrid and biochemical methods. (3) In order to identify polypeptides that are part of transcriptional complexes interacting with chondrocyte-specific enhancers in intact cells chromatin immunoprecipitation assays will be performed. (4) Based on the information gained about the nature of the polypeptides interacting with Sox9 and about the identity of proteins detected by chromatin IP, in vitro reconstituted nucleosomal templates will be used to begin to dissect the function of Sox9, L-Sox5 and other transcriptionally active polypeptides in chromatin disruption and transcription assays. Our experiments should provide novel insights about the mechanisms whereby Sox9 controls chondrocyte differentiation and may suggest new therapeutic approaches for cartilage diseases.
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