TRANSCRIPTIONAL CONTROL OF CHONDROGENIC DIFFERENTIATION

只有少数转录因子的表达是特定的软骨和骨骼形态发生在发展过程中已被发现。 目前对骨骼发生过程中基因表达的转录调控和模式知之甚少，这主要是由于体内研究前体细胞谱系进展的固有困难。 在初步的研究中，我们已经证明了可行性的识别相关基因的软骨分化使用一种新的体外软骨形成模型系统。 在这项研究中，我们将利用这个模型系统的潜力来表征从间充质祖细胞（MPC）到软骨细胞的谱系进展过程中表达的转录因子，并确定在此过程中受到调控的基因。 我们将集中我们的努力，锌指蛋白类的转录因子，因为它们很容易从软骨细胞的cDNA文库中分离，使用寡核苷酸编码的氨基酸序列基序常见的大多数锌指蛋白家族成员。 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起始事件。 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：鉴定和表征软骨形成分化第一阶段特异性的锌指蛋白。这将通过用对锌指蛋白共有的高度保守序列特异的简并寡核苷酸探针筛选消减cDNA文库来实现。 锌指蛋白表达将通过序列分析、遗传作图和组织表达分析来表征。具体目标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融合体将在CHO细胞或MPC中瞬时表达。将来自锌指融合蛋白转染细胞的总cDNA探针与cDNA阵列杂交，以鉴定由这些推定的转录因子的过表达调节的基因。 将鉴定基因组锌指结合位点。具体目标3：以确定是否阶段特异性锌指蛋白作为转录因子在软骨形成。将锌指结合位点-荧光素酶报告基因构建体转染到MPC中，以监测软骨形成期间的荧光素酶表达。 个别锌指蛋白的作用将通过分化MPC中的过表达或通过表达反义锌指蛋白构建体来确定。

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.