Only a few transcription factors whose expression is specific to chondrogenesis and skeletal morphogenesis during development have been discovered. Transcriptional regulation and patterns of gene expression during skeletogenesis are poorly understood at present, largely due to the difficulty inherent in studying lineage progression of precursor cells in-vivo. In preliminary studies, we have demonstrated the feasability of identifying genes relevant to chondrogenic differentiation using a novel in-vitro chondrogenesis model system. In this study we will exploit the potential of this model system to characterize transcription factors expressed during lineage progression from mesenchymal progenitor cells (MPCs) to chondrocytes, and to identify genes which are regulated during this process. We will concentrate our efforts on transcription factors of the zinc-finger protein class because they are easily isolated from a chondrocyte cDNA library using oligonucleotides coding for an amino acid sequence motif common to most zinc-finger protein family members. Our CENTRAL HYPOTHESIS is that a specific temporal pattern of zinc-finger transcription factor expression determines the initiating events of chondrogenesis. We will test this hypothesis by accomplishing the following SPECIFIC AIMS.
Specific Aim 1 : To identify and characterize zinc-finger proteins specific to the first stage of chondrogenic differentiation. This will be accomplished by screening of subtracted cDNA libraries with a degenerate oligonucleotide probe specific to a highly conserved sequence common to zinc-finger proteins. Zinc finger protein expression will be characterized by sequence analysis, genetic mapping, and analysis of tissue expression.
Specific Aim 2 : To identify genes containing binding sites for stage-specific zinc-finger proteins. Zinc-finger protein/EGFP fusions will be transiently expressed in CHO cells or MPCs. Total cDNA probes from zinc-finger fusion protein transfected cells will be hybridized to cDNA arrays to identify genes regulated by overexpression of these putative transcription factors. Genomic zinc-finger binding sites will be identified.
Specific Aim 3 : To determine whether stage-specific zinc-finger proteins act as transcription factors during chondrogenesis. Zinc-finger binding site-Luciferase reporter gene constructs will be transfected into MPCs to monitor luciferase expression during chondrogenesis. The role of individual zinc-finger proteins will be determined by overexpression in differentiating MPCs, or by expressing anti-sense zinc-finger protein constructs.
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