TRANSCRIPTIONAL CONTROL OF CHONDROGENIC DIFFERENTIATION

只有少数转录因子在发育过程中的软骨形成和骨骼形态发生中特异表达。目前对骨骼形成过程中的转录调控和基因表达模式知之甚少，这在很大程度上是因为在体内研究前体细胞的谱系进展存在固有的困难。在初步研究中，我们已经证明了使用一种新的体外软骨形成模型系统识别与软骨分化相关的基因是可行的。在这项研究中，我们将利用这个模型系统来表征从间充质祖细胞(MPC)到软骨细胞的谱系进化过程中表达的转录因子，并识别在这一过程中受到调控的基因。我们将集中精力研究锌指蛋白类的转录因子，因为它们很容易通过编码大多数锌指蛋白家族成员共同的氨基酸序列基序的寡核苷酸从软骨细胞cDNA文库中分离出来。我们的中心假设是，锌指转录因子表达的特定时间模式决定了软骨形成的启动事件。我们将通过实现以下具体目标来检验这一假设。具体目标1：鉴定和鉴定软骨分化第一阶段特异的锌指蛋白。这将通过用简并的寡核苷酸探针筛选消减的cDNA文库来完成，该探针针对锌指蛋白常见的高度保守的序列。锌指蛋白的表达将通过序列分析、遗传图谱和组织表达分析来表征。特定目的2：鉴定含有阶段特异性锌指蛋白结合位点的基因。锌指蛋白/EGFP融合蛋白将在CHO细胞或MPC中瞬时表达。来自锌指融合蛋白转基因细胞的总cDNA探针将与cDNA阵列杂交，以识别受这些假定转录因子过表达调控的基因。基因组锌指结合位点将被确定。具体目的3：确定阶段特异性锌指蛋白是否在软骨形成过程中起转录因子的作用。锌指结合部位荧光素酶报告基因的构建将被导入骨髓基质细胞，以监测软骨形成过程中荧光素酶的表达。单个锌指蛋白的作用将通过在分化MPC过程中的过度表达或通过表达反义锌指蛋白结构来确定。

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.