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首页> 外文期刊>Molecular and Cellular Biology >Sequences and factors required for the F9 embryonal carcinoma stem cell E1a-like activity.
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Sequences and factors required for the F9 embryonal carcinoma stem cell E1a-like activity.

机译：F9胚胎癌干细胞E1a样活性所需的序列和因子。

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摘要

F9 embryonal carcinoma (EC) stem cells contain an E1a-like activity that is absent from differentiated derivatives. We have previously characterized proteins present in F9 EC cell extracts that bind to the E1a-dependent E2A promoter and have shown that two of them, TF68 and DRTF1, are required for efficient transcription in vitro (N. B. La Thangue, B. Thimmapaya, and P. W. J. Rigby, Nucleic Acids Res. 18:2929-2938, 1990). We now show that the E1a-like activity is detectable in transient transfection assays. Deletion mutations show that a distal sequence element, which includes the ATF/CREB consensus, is required for expression in both cell types, although it does not mediate the down-regulation of promoter activity that accompanies differentiation. A series of point mutations generated by in vitro mutagenesis confirm this and show that sequences around -60 are necessary for efficient expression in stem cells but not in differentiated derivatives. These sequences bind DRTF1, the activity of which is strongly down-regulated during differentiation. Surprisingly, mutations in a previously uncharacterized region of the promoter restore activity to a promoter carrying the -60 mutation and lead to the formation of a new DNA-protein complex.
机译：F9胚胎癌（EC）干细胞具有E1a样活性，而分化衍生物没有这种活性。我们以前已经鉴定了存在于F9 EC细胞提取物中的蛋白质，该蛋白质与E1a依赖的E2A启动子结合，并显示其中两个TF68和DRTF1是体外有效转录所必需的（NB La Thangue，B。Thimmapaya和PWJ Rigby，Nucleic Acids Res.18：2929-2938，1990）。现在我们显示在瞬时转染测定中可检测到E1a样活性。缺失突变表明，在两种细胞类型中表达都需要包含ATF / CREB共有序列的远端序列元件，尽管它不介导伴随分化的启动子活性的下调。体外诱变产生的一系列点突变证实了这一点，并表明-60左右的序列对于在干细胞中有效表达是必需的，但在分化的衍生物中不是必需的。这些序列结合DRTF1，其活性在分化过程中被强烈下调。出人意料的是，启动子先前未表征的区域中的突变将活性恢复为携带-60突变的启动子，并导致形成新的DNA-蛋白质复合物。

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