Zfp28 and Mesenchymal Stem Cell Differentiation

描述（由申请人提供）：尽管间充质干细胞在骨组织修复和再生中的临床应用很有兴趣，但控制分化的分子机制尚不清楚。间充质细胞向软骨细胞的分化需要特定调控基因的协调表达，其中很少被表征。Zfp28是Cys2His2锌指蛋白（ZFP），具有n端kraba和B结构域，是一种候选的软骨生成调节因子。大多数KRAB锌指蛋白已被证明具有转录抑制因子的功能。Zfp28在有限的成年组织中表达，并在骨骼形成开始时在小鼠胚胎中表达。Zfp28以分化阶段特异性的方式在成软骨小鼠ATDC5细胞系中表达。Zfp28过表达导致核易位、Col2a1 mRNA下调和软骨分化抑制。Zfp28的共表达导致ATDC5细胞中Col2a1启动子活性下调。Zfp28基因位于小鼠7号染色体（Mm7）上的一组锌指印迹基因中，其中一些基因在肢体发育中表达。ZFP28， ZFP28的人类同源基因在19q13.4上，在一个与含有ZFP28的Mm7区域重合的区域。由于锌指蛋白在不同物种之间存在高度差异，Zfp28序列在人类和小鼠之间的保守性表明其具有重要的功能。我们假设Zfp28作为软骨形成过程中表达的基因的转录抑制因子。验证这一假说的具体目的有：(1)确定体内软骨形成是否需要Zfp28。我们将产生Zfp28条件敲除小鼠，以确定该基因与骨骼形态发生的关系。胚胎的骨骼表型将分析大体形态，组织学和基因表达。(2)研究Zfp28在体外成软骨分化中的作用。我们将分析在ATDC5细胞或条件永生化间充质祖细胞系BMC9中操纵Zfp28表达后的软骨分化。我们将研究Zfp28表达对II型和XI型胶原转录的影响，以及聚集蛋白启动子-报告子结构。我们将通过染色质免疫沉淀和小鼠启动子芯片分析来鉴定Zfp28靶基因。该项目将有助于更好地了解间充质干细胞向软骨细胞分化的分子调控事件。这些知识可能会加速修复或再生受损或骨关节炎软骨的新疗法的进展。

DESCRIPTION (provided by applicant): Although there is much interest in the clinical use of mesenchymal stem cells for repair and regeneration of skeletal tissues, the molecular mechanisms controlling differentiation are not well understood. Differentiation of mesenchymal cells into chondrocytes requires the coordinated expression of specific regulatory genes, of which few have been characterized. Zfp28, a candidate chondrogenic regulator, is a Cys2His2 zinc finger protein (ZFP) having N-terminal KRAB-A and B domains. Most KRAB zinc finger proteins have been shown to function as transcriptional repressors. Zfp28 is expressed in a limited subset of adult tissues, and is expressed in mouse embryos at the onset of skeletogenesis. Zfp28 is expressed in the chondrogenic murine ATDC5 cell line in a differentiation stage-specific manner. Overexpression of Zfp28 results in nuclear translocation, downregulation of Col2a1 mRNA, and inhibition of chondrogenic differentiation. Coexpression of Zfp28 results in downregulation of Col2a1 promoter activity in ATDC5 cells. The Zfp28 gene resides on mouse chromosome 7 (Mm7) within a cluster of imprinted zinc-finger genes, some of which are expressed in limb development. ZFP28, the human ortholog of Zfp28 is on 19q13.4, in a region having synteny with the region of Mm7 containing Zfp28. As zinc finger proteins have been shown to be highly divergent between species, conservation of the Zfp28 sequence between human and mouse suggests an important function. We hypothesize that Zfp28 functions as a transcriptional repressor of genes expressed during chondrogenesis. Specific aims to test this hypothesis are: (1) To determine whether Zfp28 is required for chondrogenesis in vivo. We will produce Zfp28 conditional knockout mice to determine the relationship of this gene to skeletal morphogenesis. The skeletal phenotype of the embryos will be analyzed for gross morphology, histology and gene expression. (2) to examine the role of Zfp28 in chondrogenic differentiation in vitro. We will analyze chondrogenic differentiation following manipulation of Zfp28 expression in ATDC5 cells or in the conditionally immortalized mesenchymal progenitor cell line BMC9. We will study the effect of Zfp28 expression upon transcription from collagen II and XI, and aggrecan promoter-reporter constructs. We will identify Zfp28 target genes by coupled chromatin immunoprecipitation and mouse promoter microarray analysis. This project will result in a better understanding of the molecular events regulating the differentiation of mesenchymal stem cells toward chondrocytes. This knowledge may accellerate progress toward novel therapies to repair or regenerate damaged or osteoarthritic cartilage.