Tissue-specific in vivo protein-DNA interactions at the promoter region of the Xenopus 63 kDa keratin gene during metamorphosis.
Tissue-specific in vivo protein-DNA interactions at the promoter region of the Xenopus 63 kDa keratin gene during metamorphosis.
复制标题
变态过程中爪蟾 63 kDa 角蛋白基因启动子区域的组织特异性体内蛋白质-DNA 相互作用。
DOI：
10.1093/nar/23.21.4502
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发表时间：
1995
影响因子：
14.9
通讯作者：
Miller,L
中科院分区：
文献类型：
--
作者：
Warshawsky,D;Miller,L
xenopus63 kDa角蛋白基因受发育调控，仅在表皮中表达。63 kDa角蛋白基因的完全激活需要两个调控步骤，第一个独立的，第二个依赖于甲状腺激素三碘甲状腺素（T3）。对63 kDa角蛋白基因基因组克隆的序列分析发现了转录起始位点上游潜在的AP2和SP1位点。使用纯化或富集蛋白以及HeLa核提取物结合AP2-和SP1特异性抗体进行的电泳迁移转移试验已被用于证明人类AP2和SP1结合转录起始位点上游的元件。在体内用连接介导的PCR方法发现了几个印迹，位于转录起始位点上游350 bp内，包括AP2和SP1位点，这些印迹是表达角蛋白基因的表皮细胞所特有的。这些足迹在不表达该基因的血细胞和XL177细胞中不存在。比较低水平或高水平表达63 kDa角蛋白基因的细胞之间的足迹显示，相同的结合位点被占据，表明这些位点是63 kDa角蛋白基因基础表达和t3诱导表达所必需的。
TheXenopus63 kDa keratin gene is developmentally regulated and is expressed only in the epidermis. Full activation of the 63 kDa keratin gene requires two regulatory steps, the first Independent and the second dependent on the thyroid hormone triiodothyronlne (T3). Sequence analysis of a genomic clone of the 63 kDa keratin gene identified potential AP2 and SP1 bihding sites upstream of the transcription Initiation site. Electrophoretic mobility shift assays using purified or enriched proteins, as well as HeLa nuclear extract in conjunction with AP2- and SP1-specific antibodies, have been used to demonstrate that human AP2 and SP1 bind elements upstream of the transcription initiation site.In vivofootprinting with ligation mediated PCR revealed several footprints, within 350 bp upstream of the transcription initiation site, Including those at the AP2 and SP1 sites, that are unique to epidermal cells which express the keratin gene. These footprints were absent In blood cells and XL177 cells which do not express the gene. Comparison of footprints between cells which express the 63 kDa keratin gene at low or high levels showed that the same binding sites are occupied, indicating that these sites are required for basal as well as T3-induced expression of the 63 kDa keratin gene.