TRANSCRIPTIONAL CONTROL OF CHONDROGENIC DIFFERENTIATION

软骨分化的转录控制

• 批准号: 6375234
• 负责人: THOMAS Martin HERING
• 金额: $ 22.38万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-15 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6375234
• 关键词: CHO cells; DNA footprinting; binding sites; cartilage development; cell differentiation; chondrocytes; developmental genetics; fluorescence microscopy; fusion gene; gel electrophoresis; gene expression; genetic library; genetic mapping; human genetic material tag; human tissue; in situ hybridization; laboratory mouse; molecular cloning; northern blottings; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; protein structure function; stem cells; tissue /cell culture; transcription factor

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.

在发育过程中，只有少数转录因子的表达与软骨形成和骨骼形态发生有关。目前，人们对骨形成过程中的转录调控和基因表达模式知之甚少，这主要是由于在体内研究前体细胞谱系进展的困难。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基因的可行性。在这项研究中，我们将利用该模型系统的潜力来表征从间充质祖细胞（MPCs）到软骨细胞谱系进展过程中表达的转录因子，并鉴定在这一过程中受到调节的基因。我们将集中研究锌指蛋白类的转录因子，因为它们很容易从软骨细胞cDNA文库中分离出来，使用编码大多数锌指蛋白家族成员共有的氨基酸序列基序的寡核苷酸。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。特异性目的1：鉴定和表征软骨分化第一阶段特异性的锌指蛋白。这将通过对锌指蛋白高度保守序列特异性的退化寡核苷酸探针筛选减去的cDNA文库来完成。锌指蛋白的表达将通过序列分析、遗传作图和组织表达分析来表征。特异性目的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融合物将在CHO细胞或MPCs中短暂表达。从锌指融合蛋白转染的细胞中提取的cDNA探针将被杂交到cDNA阵列中，以鉴定受这些可能的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。特异性目的3：确定阶段特异性锌指蛋白在软骨形成过程中是否作为转录因子。锌指结合位点-荧光素酶报告基因构建体将被转染到MPCs中，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MPCs中的过表达或通过表达反义锌指蛋白结构来确定。