Nuclear factor Y (NF-Y) regulates the proximal promoter activity of the mouse collagen α1(XI) gene (Col11a1) in chondrocytes
Nuclear factor Y (NF-Y) regulates the proximal promoter activity of the mouse collagen α1(XI) gene (Col11a1) in chondrocytes
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DOI：
10.1007/s11626-013-9692-3
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发表时间：
2014-04-01
影响因子：
2.1
通讯作者：
Matsuo, Noritaka
中科院分区：
文献类型：
--
作者：
Hida, Mariko;Hamanaka, Ryoji;Matsuo, Noritaka
XI型胶原是由α 1（XI）、α 2（XI）和α 3（XI）组成的异源三聚体，在软骨形成和骨骼形态发生中起关键作用。然而，α 1（XI）胶原基因（Col 11 a1）在软骨细胞中的转录调控的特点很差。在这项研究中，我们研究了小鼠Col 11 a1基因在软骨细胞中的近端启动子。主要转录起始位点位于翻译起始位点上游-299 bp处，并且近端启动子缺乏TATA序列，但具有高的鸟嘌呤-胞嘧啶（GC）含量。细胞转染实验表明，从-116到-256的片段是激活近端Col 11 a1启动子所必需的，并且电泳迁移率变动分析表明，核蛋白结合到该启动子中从-116到-176的片段。另外的比较和计算机模拟分析表明，对于与核因子Y（NF-Y）结合至关重要的ATTGG序列在-135至-145的高度保守区域内。使用野生型和突变体寡核苷酸或特异性抗体的干扰试验表明，NF-Y蛋白结合到该区域。在不存在NF-Y结合序列的情况下用报告构建体进行的细胞转染实验显示出启动子活性的抑制。染色质免疫沉淀实验表明，NF-Y蛋白在体内可直接与该区域结合，而显性负性NF-Y A突变体的过表达也抑制了近端启动子的活性。综上所述，这些结果表明，转录因子NF-Y调节小鼠Col 11 a1基因在软骨细胞中的近端启动子活性。
Type XI collagen, a heterotrimer composed of alpha 1(XI), alpha 2(XI), and alpha 3(XI), plays a critical role in cartilage formation and in skeletal morphogenesis. However, the transcriptional regulation of alpha 1(XI) collagen gene (Col11a1) in chondrocyte is poorly characterized. In this study, we investigated the proximal promoter of mouse Col11a1 gene in chondrocytes. Major transcription start site was located at -299 bp upstream of the translation start site, and the proximal promoter lacks a TATA sequence but has a high guanine-cytosine (GC) content. Cell transfection experiments demonstrated that the segment from -116 to -256 is necessary for activation of the proximal Col11a1 promoter, and an electrophoretic mobility shift assay showed that a nuclear protein is bound to the segment from -116 to -176 in this promoter. Additional comparative and in silico analyses demonstrated that an ATTGG sequence, which is critical for binding to nuclear factor Y (NF-Y), is within the highly conserved region from -135 to -145. Interference assays using wild-type and mutant oligonucleotide or with specific antibody revealed that NF-Y protein is bound to this region. Cell transfection experiments with reporter constructs in the absence of NF-Y binding sequence exhibited the suppression of the promoter activity. Furthermore, chromatin immunoprecipitation assay demonstrated that NF-Y protein is directly bound to this region in vivo, and overexpression of dominant-negative NF-Y A mutant also inhibited the proximal promoter activity. Taken together, these results indicate that the transcription factor NF-Y regulates the proximal promoter activity of mouse Col11a1 gene in chondrocytes.