Identification and characterization of a silencer regulatory element in the 3 « -flanking region of the murine CD46 gene
Identification and characterization of a silencer regulatory element in the 3 « -flanking region of the murine CD46 gene
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鼠 CD46 基因 3 « 侧翼区域沉默子调控元件的鉴定和表征
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通讯作者：
J. C. Stoof
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作者：
B. Drukarch;J. C. Stoof
鼠膜辅因子蛋白（CD 46）基因仅在睾丸中表达，而人CD 46基因则广泛表达。为了阐明物种间差异表达CD 46基因的机制，我们克隆了整个小鼠CD 46基因组DNA，并将可能的调控区域置于荧光素酶报告基因的测序区域。报告基因分析显示，沉默活性不在启动子中，而是在基因的3个碱基区中，并且在终止密码子下游0.6和0.8 kb之间的0.2 kb区域内鉴定出沉默子样元件艾德。这种沉默器样元件与猪MHC I类基因的沉默器样元件高度相似。将突变引入小鼠CD 46的这种假定的沉默元件导致沉默效应的消除。电泳迁移率变化试验表明，在小鼠细胞系和组织中存在该引言沉默子序列的结合分子。根据用于制备核提取物的增溶剂，观察到蛋白质-沉默物-元件复合物的大小差异。突变的沉默序列未能与结合分子相互作用。与其他器官相比，睾丸生殖细胞中的结合因子水平较低。因此，沉默元件及其结合因子可能在小鼠CD 46基因表达的转录调控中发挥作用。这些结果意味着CD 46沉默元件的影响涵盖先天免疫和生殖系统，并且在小鼠中可能决定CD 46的睾丸生殖细胞主导表达。
The murine membrane cofactor protein (CD46) gene is expressed exclusively in testis, in contrast to human CD46, which is expressed ubiquitously. To elucidate the mechanism of differential CD46 gene expression among species, we cloned entire murine CD46 genomic DNA and possible regulatory regions were placed in the flanking region of the luciferase reporter gene. The reporter gene assay revealed a silencing activity not in the promoter, but in the 3 « -flanking region of the gene and the silencer-like element was identified within a 0.2-kb region between 0.6 and 0.8 kb downstream of the stop codon. This silencer-like element was highly similar to that of the pig MHC class-I gene. The introduction of a mutation into this putative silencer element of murine CD46 resulted in an ab-rogation of the silencing effect. Electrophoretic mobility-shift assay indicated the presence of the binding molecule(s) for this INTRODUCTION silencer sequence in murine cell lines and tissues. A size difference of the protein–silencer-element complex was observed depending upon the solubilizers used for preparation of the nuclear extracts. A mutated silencer sequence failed to interact with the binding molecules. The level of the binding factor was lower in the testicular germ cells compared with other organs. Thus the silencer element and its binding factor may play a role in transcriptional regulation of murine CD46 gene expression. These results imply that the effects of the CD46 silencer element encompass the innate immune and reproductive systems, and in mice may determine the testicular germ-cell-dominant expression of CD46.