Analysis transcriptional regulation of cartilage genes

“绝缘子”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。α 2（Xi）胶原链基因（Col 11 a2）在软骨中特异性表达。我们以前分离的Col 11 a2基因，并确定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Col 11 a2基因位于广泛表达的类维生素A X受体基因（Rxrb）的3 '端附近。Rxrb /Col 11 a2基因座的这种结构可能提高了这两个基因之间存在绝缘子的可能性。因为几乎所有的特征增强子阻断元件在脊椎动物中确定的结合转录因子CTCF，我们在这里首先搜索的序列结合CTCF电泳迁移率变动分析（EMSA）。克隆了大鼠Rxrb基因终止密码子至Col 11 a2基因翻译起始密码子的2.1kb片段。12个重叠DNA框架 关于我们 制备覆盖该片段的片段（300 bp），与体外翻译的CTCF蛋白一起温育。位于Col 11 a2基因转录起始位点上游的一个片段与CTCF特异性相互作用。为了证实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。共沉淀序列通过实时PCR定量检测，使用9套引物间隔14 kb的Rxrb /Col 11 a2基因座。通过EMSA检测的CTCF结合位点在抗CTCF免疫沉淀物中大大富集。因为据报道，绝缘子作为一个块组蛋白的乙酰化从增强子到基因的传播，我们接下来进行染色质免疫沉淀试验，使用抗乙酰化组蛋白H3抗体。在RCS细胞中，乙酰化水平相对较高的第一内含子的Col 11 a2基因比其余的位点。与第一内含子周围的希斯顿H3的高水平乙酰化相反，CTCF结合位点的乙酰化水平非常低。这些结果表明，CTCF结合位点的Col 11 a2启动子参与调控组蛋白H3乙酰化周围的Rxrb/Col 11 a2基因座。少

"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