La Thangue N B, Thimmappaya B, Rigby P W
Laboratory of Eukaryotic Molecular Genetics, National Institute for Medical Research, Mill-Hill, London, UK.
Nucleic Acids Res. 1990 May 25;18(10):2929-38. doi: 10.1093/nar/18.10.2929.
Murine F9 embryonal carcinoma (EC) stem cells have an Ela-like transcription activity that is undetectable in F9 cells differentiated to parietal endoderm-like cells (F9-PE). The Ela-inducible adenovirus E2A promoter has been used to further define this activity and we show that in vitro the transcription of this promoter in F9 EC and F9-PE cell extracts reflects the regulation in vivo. In EC cell extracts several trans-acting protein factors bind to E2A promoter sequences. A distal domain containing a CRE binds proteins present in F9 EC, F9-PE and Hela cell extracts. Sequences between -71 and -50 define a multiplicity of binding activities, termed DRTF1, all of which are down regulated as EC stem cells differentiate. DRTF2, a low abundance, regulated binding activity requires DNA sequences that overlap those required by DRTF1. The CRE and the DRTF1 binding site compete for transcription in vitro, indicating that in EC cell extracts the respective proteins function as positively acting, binding site dependent transcription factors. Comparison of DRTF1 with the previously defined HeLa cell factor E2F, induced during adenovirus infection, indicates that although both factors recognise the same region of the promoter there are clear differences between them. These data indicate that multiple factors are necessary for efficient transcription of the E2A promoter in F9 EC cell extracts and suggest that DRTF1 is responsible, at least in part, for the developmental regulation of the cellular Ela-like activity.
小鼠F9胚胎癌细胞（EC）干细胞具有一种Ela样转录活性，而在分化为滋养层内胚层样细胞（F9-PE）的F9细胞中无法检测到这种活性。Ela诱导型腺病毒E2A启动子已被用于进一步确定这种活性，并且我们发现，在体外，该启动子在F9 EC和F9-PE细胞提取物中的转录反映了体内的调控情况。在EC细胞提取物中，几种反式作用蛋白因子与E2A启动子序列结合。一个包含CRE的远端结构域结合F9 EC、F9-PE和Hela细胞提取物中存在的蛋白质。-71至-50之间的序列定义了多种结合活性，称为DRTF1，随着EC干细胞的分化，所有这些活性都被下调。DRTF2是一种低丰度的、受调控的结合活性，需要与DRTF1所需的DNA序列重叠的序列。CRE和DRTF1结合位点在体外竞争转录，这表明在EC细胞提取物中，各自的蛋白质作为正向作用的、依赖结合位点的转录因子发挥作用。将DRTF1与先前定义的在腺病毒感染期间诱导的Hela细胞因子E2F进行比较，结果表明，尽管这两种因子识别启动子的同一区域，但它们之间存在明显差异。这些数据表明，在F9 EC细胞提取物中，E2A启动子的有效转录需要多种因子，并且表明DRTF1至少部分负责细胞Ela样活性的发育调控。
