Localization of silencer and enhancer elements in the human type X collagen gene
Localization of silencer and enhancer elements in the human type X collagen gene
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DOI：
10.1002/(sici)1097-4644(19970801)66:2
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发表时间：
1997-08
影响因子：
4
通讯作者：
F. Beier;Silvia Vornehm;E. Pöschl;K. von der Mark;M. Lammi
中科院分区：
文献类型：
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作者：
F. Beier;Silvia Vornehm;E. Pöschl;K. von der Mark;M. Lammi
X型胶原蛋白是一种短的、形成网络的胶原蛋白，在软骨内骨化过程中，其在肥大软骨细胞中的表达在时间和空间上受到严格控制。对鸡软骨细胞的研究表明，X型胶原蛋白基因表达的调控在转录水平进行。在本研究中，我们通过报告基因构建体以及在软骨形成细胞和非软骨形成细胞中进行瞬时转染，分析了人X型胶原蛋白（Col10a1）的调控元件。覆盖多达2864bp的5′侧翼序列的四个不同启动子片段，包括或不包括第一内含子，都与荧光素酶报告基因相连，并转染到3T3成纤维细胞、HT1080纤维肉瘤细胞、来自静止区的肥大前软骨细胞、肥大软骨细胞以及软骨形成细胞系中。结果表明，除了近端启动子外，人Col10a1基因中还存在三个调控元件。首先，位于转录起始位点上游2.4 - 2.8kb之间的一个负调控元件在所有非软骨形成细胞和肥大前软骨细胞中都有活性。其次，在第一内含子中存在一个正向但非组织特异性的正调控元件。第三，一个仅在肥大软骨细胞中有活性的细胞类型特异性增强子元件位于 - 2.4 - - 0.9kb之间，这证实了Thomas等人先前的一份报告[（1995）：《基因》160：291 - 296]。然而，只有当磷酸钙用于转染或在脂质体转染后包含在培养基中时，才能观察到增强效应。这些发现表明，人Col10a1基因表达的严格控制是通过基因中的正负调控元件实现的，并为鉴定与这些元件结合的因子提供了基础。《细胞生物化学杂志》66：210 - 218，1997。© 1997威利 - 利斯公司
Collagen type X is a short, network‐forming collagen expressed temporally and spatially tightly controlled in hypertrophic chondrocytes during endochondral ossification. Studies on chicken chondrocytes indicate that the regulation of type X collagen gene expression is regulated at the transcriptional level. In this study, we have analyzed the regulatory elements of the human type X collagen (Col10a1) by reporter gene constructs and transient transfections in chondrogenic and nonchondrogenic cells. Four different promoter fragments covering up to 2,864 bp of 5′‐flanking sequences, either including or lacking the first intron, were linked to luciferase reporter gene and transfected into 3T3 fibroblasts, HT1080 fibrosarcoma cells, prehypertrophic chondrocytes from the resting zone, hypertrophic chondrocytes, and chondrogenic cell lines. The results indicated the presence of three regulatory elements in the human Col10a1 gene besides the proximal promoter. First, a negative regulatory element located between 2.4 and 2.8 kb upstream of the transcription initiation site was active in all nonchondrogenic cells and in prehypertrophic chondrocytes. Second, a positive, but also non‐tissue‐specific positive regulatory element was present in the first intron. Third, a cell‐type‐specific enhancer element active only in hypertrophic chondrocytes was located between −2.4 and −0.9 kb confirming a previous report by Thomas et al. [(1995): Gene 160:291‐296]. The enhancing effect, however, was observed only when calcium phosphate was either used for transfection or included in the culture medium after lipofection. These findings demonstrate that the rigid control of human Col10a1 gene expression is achieved by both positive and negative regulatory elements in the gene and provide the basis for the identification of factors binding to those elements. J. Cell. Biochem. 66:210‐218, 1997. © 1997 Wiley‐Liss, Inc.