Involvement of nuclear factor I transcription/replication factor in the early stage of chondrocytic differentiation
Involvement of nuclear factor I transcription/replication factor in the early stage of chondrocytic differentiation
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DOI：
10.1016/j.bone.2007.08.028
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发表时间：
2007-12-01
期刊：
影响因子：
4.1
通讯作者：
Michigami, Toshimi
中科院分区：
文献类型：
--
作者：
Uchihashi, Takayuki;Kimata, Masaaki;Michigami, Toshimi
基因诱捕诱变基于诱捕载体的随机插入可能干扰插入基因的功能的概念。为了确定参与软骨细胞分化的基因，我们应用这种方法的小鼠间充质细胞系，ATDC 5，分化成成熟的软骨细胞在胰岛素的存在下，并分离出一个克隆，其中的基因编码的转录/复制因子，核因子I-B（NFIB），被困。在命名为#7-57的该特定克隆中，将诱捕载体pPT 1-geo插入等位基因之一的NFIB基因的内含子6中。结果，野生型NFIB和缺乏羧基末端反式激活/抑制结构域的突变蛋白都在克隆中表达。免疫沉淀/蛋白质印迹证实了野生型NFIB和截短的蛋白质之间的相互作用，从被困的等位基因，这表明突变蛋白形成了异源二聚体与野生型NFI蛋白。当在分化培养基中培养时，与亲本ATDC 5细胞相比，#7-57表现出受损的结节形成和较少的软骨基质积累。此外，增殖软骨细胞的标记基因的表达，包括II型胶原（Col 2a 1），matrillin-1，PTHrP，在克隆中减少。与亲本细胞相比，S 0X 9的表达在克隆#7-57中也略微降低。野生型NFIB在亲本ATDC 5细胞中的过表达导致Col 2a 1的表达增加，并且使用Col 2a 1启动子/增强子-荧光素酶构建体的一系列报告基因测定证明了NFIB对基因的转录调节和来自被捕获等位基因的截短突变体的显性负效应。有趣的是，位于内含子1中的48 bp顺式元件中的SOX 9结合位点的突变未能消除NFIB对Col 2a 1基因的反式激活，这表明NFI至少部分地独立于SOX 9调节Col 2a 1的反式激活。这些结果表明NFI家族转录/复制因子在软骨细胞分化的早期阶段的关键作用。(C)2007年爱思唯尔公司All rights reserved.
Gene-trap mutagenesis is based on the notion that the random insertion of a trapping vector may disturb the function of inserted genes. To identify the genes involved in chondrocytic differentiation, we applied this method to a murine mesenchymal cell line, ATDC5, which differentiate into mature chondrocytes in the presence of insulin, and isolated a clone in which the gene encoding a transcription/replication factor, nuclear factor I-B (NFIB), was trapped. In this particular clone, named #7-57, the trap vector pPT1-geo was inserted into intron 6 of the NFIB gene in one of the alleles. As a result, both wild-type NFIB and a mutant protein lacking the carboxyl-terminal transactivation/repression domain were expressed in the clone. Immunoprecipitation/Western blotting confirmed the interaction between wild-type NFIB and the truncated protein derived from the trapped allele, suggesting that the mutant protein formed a heterodimer with wild-type NFI proteins. When cultured in the differentiation medium, #7-57 exhibited impaired nodule formation and less accumulation of cartilageous matrices compared with the parental ATDC5 cells. In addition, the expression of marker genes for proliferating chondrocytes, including type II collagen (Col2a1), matrillin-1, and PTHrP, was reduced in the clone. The expression of SOX9 was also slightly decreased -in the clone #7-57 compared with the parental cells. The overexpression of wild-type NFIB in parental ATDC5 cells resulted in the increased expression of Col2a1, and a series of reporter assays using a Col2a1 promoter/enhancer-luciferase construct demonstrated the transcriptional regulation of the gene by NFIB and the dominant-negative effect of the truncated mutant derived from the trapped allele. Interestingly, mutation in the SOX9-binding site in the 48-bp cis-element located in intron 1 failed to abolish the transactivation of Col2a1 gene by NFIB, suggesting that NFI regulates the transactivation of Col2a1, at least in part, independently of SOX9. These results indicate the critical roles of NFI family transcription/replication factors in the early stage of chondrocytic differentiation. (C) 2007 Elsevier Inc. All rights reserved.