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首页> 外文期刊>Molecular and Cellular Biology >Isolation of a thyroid hormone-responsive gene by immunoprecipitation of thyroid hormone receptor-DNA complexes.
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Isolation of a thyroid hormone-responsive gene by immunoprecipitation of thyroid hormone receptor-DNA complexes.

机译：通过甲状腺激素受体-DNA复合物的免疫沉淀分离甲状腺激素反应性基因。

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Thyroid hormone (T3) receptor (TR) is a ligand-dependent transcription factor that acts through specific binding sites in the promoter region of target genes. In order to identify new genes that are regulated by T3, we used anti-TR antiserum to immunoprecipitate TR-DNA complexes from GH4 cell nuclei that had previously been treated with a restriction enzyme. Screening of the immunopurified, cloned DNA for TR binding sites by electrophoretic mobility shift assay yielded 53 positive clones. A subset of these clones was specifically immunoprecipitated with anti-TR antiserum and may therefore represent biologically significant binding sites. One of these clones, clone 122, was characterized in detail. It includes sequences highly related to the NICER long terminal repeat-like element and contains three TR binding sites as determined by DNase I footprinting. Two of the clone 122 TR binding sites are located upstream of the TATA box, and one is located downstream. The TR binding site downstream from the promoter was necessary and sufficient to confer T3-dependent regulation in transient transfection experiments. Expression of a reporter construct under the control of the clone 122 promoter region was activated by TR in the absence of ligand and returned to basal levels after T3 addition. Clone 122 sequences hybridize to at least two different mRNAs of approximately 6 and 10 kb from GH4 cells. The levels of both of these mRNAs increased upon removal of T3. Our studies suggest that specific immunoprecipitation of chromatin allows identification of binding sites and target genes for transcription factors.
机译：甲状腺激素（T3）受体（TR）是依赖配体的转录因子，其通过靶基因启动子区域中的特定结合位点起作用。为了鉴定受T3调控的新基因，我们使用了抗TR抗血清从先前已用限制性酶处理过的GH4细胞核中免疫沉淀出TR-DNA复合物。通过电泳迁移率漂移分析筛选免疫纯化的克隆DNA的TR结合位点，得到53个阳性克隆。这些克隆的子集用抗TR抗血清特异性免疫沉淀，因此可能代表生物学上重要的结合位点。这些克隆之一，克隆122，进行了详细描述。它包含与NICER长末端重复序列样元件高度相关的序列，并包含三个DNase I足迹确定的TR结合位点。克隆122 TR结合位点中的两个位于TATA盒的上游，一个位于下游。启动子下游的TR结合位点是必需的，并且足以在瞬时转染实验中赋予T3依赖性调控。在不存在配体的情况下，在克隆122启动子区域控制下的报告构建体的表达被TR激活，并在添加T3后恢复到基础水平。克隆122序列与来自GH4细胞的至少两个大约6和10 kb的不同mRNA杂交。去除T3后，这两个mRNA的水平均增加。我们的研究表明，染色质的特异性免疫沉淀可以识别结合位点和转录因子的靶基因。

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