The analysis of genome evolution in the MHC class III region and function of extracellular matrix tenascin-X

为了识别与外链环境中的外链矩阵Tenascin-X （TNX）相互作用的蛋白，我们用一个简单的双混合系统搜寻TNX-结合蛋白。We used mouse TNX-specific fibronectin type III repeats （mTNX/FNIII D213-25） as a bait。We found that vas？endothelial growth factor B （VEGF-B） binds to mTNX/FNIII D213-25这一互动是由下拉式分析和共同容忍性分析证实的。mTNX/FNIII型D213 - 25型D2 specifically interacted with both altenative splice isoforms， VEGF-B型D2186型D2 and VEGF-B型D2167型D2，但不与其他成员。Likewise，全长度TNX也可以绑定到VEGF-B同位素。TNX that interacts with VEGF-B was mapped to the FNIII repeats （mTNX/FNIII D213-25） that were used for a bait but not to other？teristic domains of TNX。VECJF-B的TNX绑定站点位于N-终端115-氨基酸区域。mTNX/FNIII型D213-25型D2未预防t ... More VEGF-B与VEGFR-1（VEGF受体1）的相互作用，VEGF-B可以完全绑定到mTNX/FNIII D213-25和VEGFR-1。从转染的293 T细胞中提取的条件介质可以协同表达全长度TNX和VEGF-B可以增强ECV 304细胞中的DNA合成。这些发现表明，TNX对细胞的增加负有责任，从而使TNX基因表达的分子基础成为小鼠TNX基因（mTnx）的推动者区域已被表征。两个adjacent transcription initiation sites were identified at 68 and 67 bp upstream of the previously known 5'- untranslated exon。L和293 T细胞的瞬态转染与5'-缺失结构链接到该区域（-141至-136）揭示的荧光酶报告器包含转录因子Sp1-绑定元素与mTnx表达式的关系。Sp1-绑定区域的站点指向突变确认了此结果。利用核提取物从细胞中获得的电波移动性移位分析，证明其分离的Sp1-DNA复合体形成在元素上。我们的结果显示了Sp1在mTnx基因表达中扮演了关键角色。Less（低）

To identify proteins that interact with extracellular matrix tenascin-X (TNX) in the extracellular environment, we searched for TNX-binding proteins using a yeast two-hybrid system. We used mouse TNX-specific fibronectin type III repeats (mTNX/FNIIIィイD213-25ィエD2) as a bait. We found that vascular endothelial growth factor B (VEGF-B) binds to mTNX/FNIIIィイD213-25ィエD2. This interaction was confirmed by pull-down assays and coimmunoprecipitation assays. mTNX/FNIIIィイD213 -25ィエD2 specifically interacted with both altenative splice isoforms, VEGF-BィイD2186ィエD2 and VEGF-BィイD2167ィエD2, but not with other family members. Likewise, the full-length TNX could also bind to both VEGF-B isoforms. The minimal region of TNX that interacts with VEGF-B was mapped to the FNIII repeats (mTNX/FNIIIィイD213-25ィエD2) that were used for a bait but not to other characteristic domains of TNX. The TNX-binding site of VECJF-B was located in the N-terminal 115-amino-acids region. mTNX/FNIIIィイD213-25ィエD2 did not prevent t … More he interaction of VEGF-B with VEGFR-1 (VEGF receptor 1), and VEGF-B could simultaneously bind to both mTNX/FNIIIィイD213-25ィエD2 and VEGFR-1. A conditioned medium from transfected 293T cells coexpressing full-length TNX and VEGF-B can enhance DNA synthesis in ECV304 cells. These findings suggest that TNX is responsible for proliferation of the cells.To elucidate the molecular basis of the TNX gene expression, the promoter region of the mouse TNX gene (mTnx) has been characterized. The two adjacent transcription initiation sites were identified at 68 and 67 bp upstream of the previously known 5'- untranslated exon. Transient transfection of L and 293T cells with 5'-deletion constructs of the promoter region linked to the luciferase reporter revealed that the region (-141 to -136) containing a transcription factor Sp1-binding element contributes to the expression of mTnx. Site-directed mutagenesis of the Sp1-binding region confirmed this result. Electrophoretic mobility shift analysis using nuclear extracts obtained from the cells demonstrated that a distinct Sp1-DNA complex is formed at the element. Our results show that Sp1 plays a critical role in the gene expression of mTnx. Less