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Analysis transcriptional regulation of cartilage genes

软骨基因转录调控分析

基本信息

批准号：
15390458
负责人：
TSUMAKI Noriyuki
金额：
$ 7.94万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

"Insulator" is the name given to a class of DNA sequence that can block an enhancer of one gene from activating a promoter on another nearby gene. The a2(XI) collagen chain gene (Col11a2) is specifically expressed in cartilage. We previously isolated the Col11a2 gene and identified its first intron sequence as a tissue-specific enhancer. In addition, we disclosed that the Col11a2 gene resided very closely to the 3'-end of retinoid-X-receptor gene (Rxrb) which is ubiquitously expressed. This structure of the Rxrb / Col11a2 locus may raise the possibility of the existence of insulators between these two genes. Because almost all of the characterized enhancer-blocking elements identified in vertebrates bind the transcription factor CTCF, we here first searched for sequences that bound CTCF by electrophoretic mobility-shift assays (EMSA). The rat 2.1 kb stretch from termination codon of the Rxrb gene to the translational start codon of the Col11a2 gene was cloned. Twelve overlapping DNA fr … More agments 300 bp) covering this stretch were prepared, incubated with in vitro translated CTCF protein. One fragment located upstream of the transcriptional start site of the Col11a2gene specifically interacted with CTCF. To confirm the binding of CTCF to this locus in vivo, chromatin immunoprecipitation assays were performed on rat chondrosarcoma cells (RCS) using anti-CTCF antibody. Co-precipitating sequences were detected quantitatively by real-time PCR using 9 sets of primers spaced across 14 kb of the Rxrb / Col11a2 locus. The CTCF-binding site detected by EMSA was greatly enriched in anti-CTCF immunoprecipitates. Because it is reported that insulator acts as a block to spreading of acetylation of histones from the enhancer to the gene, we next performed chromatin immunoprecipitation assays using anti-acetylated histone H3 antibody. In RCS cells, the level of acetylation was relatively higher around the first intron of the Col11a2 gene than those of the rest of the locus. In contrast to the high level acetylation of histon H3 around the first intron, that of the CTCF binding site was dramatically low. These results suggest that the CTCF binding site in the Col11a2 promoter is involved in the regulation of histone H3 acetylation around the Rxrb/ Col11a2 locus. Less

“绝缘体”是一类 DNA 序列的名称，它可以阻止一个基因的增强子激活另一个附近基因的启动子。 a2(XI) 胶原链基因 (Col11a2) 在软骨中特异性表达。我们之前分离了 Col11a2 基因，并鉴定了其第一个内含子序列作为组织特异性增强子。此外，我们还发现 Col11a2 基因与普遍表达的视黄醇 X 受体基因 (Rxrb) 的 3' 端非常接近。 Rxrb / Col11a2 基因座的这种结构可能增加了这两个基因之间存在绝缘体的可能性。由于在脊椎动物中鉴定的几乎所有特征性增强子阻断元件都结合转录因子 CTCF，因此我们首先通过电泳迁移率变动测定 (EMSA) 搜索结合 CTCF 的序列。克隆了大鼠从 Rxrb 基因终止密码子到 Col11a2 基因翻译起始密码子的 2.1 kb 序列。制备覆盖该延伸段的 12 个重叠 DNA（300 bp），并与体外翻译的 CTCF 蛋白一起孵育。位于 Col11a2 基因转录起始位点上游的一个片段与 CTCF 特异性相互作用。为了确认 CTCF 在体内与该位点的结合，使用抗 CTCF 抗体对大鼠软骨肉瘤细胞 (RCS) 进行染色质免疫沉淀测定。使用间隔为 14 kb 的 Rxrb / Col11a2 基因座的 9 组引物，通过实时 PCR 定量检测共沉淀序列。 EMSA 检测到的 CTCF 结合位点在抗 CTCF 免疫沉淀物中显着富集。因为据报道，绝缘子起到了阻止组蛋白乙酰化从增强子向基因扩散的作用，所以我们接下来使用抗乙酰化组蛋白 H3 抗体进行了染色质免疫沉淀测定。在 RCS 细胞中，Col11a2 基因第一个内含子周围的乙酰化水平相对高于该基因座其余部分的乙酰化水平。与第一个内含子周围组蛋白 H3 的高水平乙酰化相反，CTCF 结合位点的乙酰化水平极低。这些结果表明 Col11a2 启动子中的 CTCF 结合位点参与 Rxrb/ Col11a2 基因座周围组蛋白 H3 乙酰化的调节。较少的