Murray E J, Stott D, Rigby P W
Laboratory of Eukaryotic Molecular Genetics, MRC National Institute for Medical Research, Mill Hill, London, England.
Mol Cell Biol. 1991 Nov;11(11):5534-40. doi: 10.1128/mcb.11.11.5534-5540.1991.
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.
F9胚胎癌（EC）干细胞含有一种分化衍生物中所没有的E1a样活性。我们之前已对F9 EC细胞提取物中与E1a依赖的E2A启动子结合的蛋白质进行了表征，并表明其中两种蛋白TF68和DRTF1是体外高效转录所必需的（N. B. 拉唐格、B. 蒂马帕亚和P. W. J. 里格比，《核酸研究》18:2929 - 2938，1990）。我们现在表明，在瞬时转染实验中可检测到E1a样活性。缺失突变表明，一个包含ATF/CREB共有序列的远端序列元件在两种细胞类型中表达均是必需的，尽管它并不介导伴随分化的启动子活性下调。通过体外诱变产生的一系列点突变证实了这一点，并表明 - 60周围的序列对于干细胞中的高效表达是必需的，但对于分化衍生物则不是。这些序列与DRTF1结合，其活性在分化过程中被强烈下调。令人惊讶的是，启动子一个先前未被表征区域的突变使携带 - 60突变的启动子恢复活性，并导致形成一种新的DNA - 蛋白质复合物。
