Mason M M, Grasso J A, Gavrilova O, Reitman M
Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1996 Oct 11;271(41):25459-67. doi: 10.1074/jbc.271.41.25459.
Expression of the chicken globin genes is regulated in part by competition between the betaA-globin and epsilon-globin promoters for the enhancer found between the genes. To understand the determinants of the enhancer-promoter interaction in stage-specific regulation, the functional elements of the embryonic chicken epsilon-globin promoter were characterized. In vitro assays demonstrated that: (a) the TATA motif at -30 bound GATA-1, (b) Sp1 bound to an element centered at -54, and (c) both Sp1 and another factor, designated CACCC (which appears related to erythroid Krüppel-like factor, EKLF) bound in the -120 to -128 region. The functions of these motifs were tested using transient expression in embryonic erythroid cells. In the absence of the enhancer, promoter point mutants showed that the TATA, Sp1, and CCAAT motifs (but not the CACCC motif) contributed to promoter activity. In contrast, in the presence of the enhancer, all four motifs (including the CACCC motif) contributed to transcription. Developmental regulation of the enhancer activity was observed, with enhancement decreasing sharply from 185-fold at 4 days (cells expressing epsilon-globin) to 16-fold at 10 days (when epsilon-globin is no longer expressed). Taken together, the data suggest that multiple transcription factors contribute to promoter-enhancer interaction and the developmental regulation of epsilon-globin expression, with EKLF-like factors having an especially important role. Regulation of stage specificity occurs at the level of enhancer/epsilon-promoter interaction, even in the absence of competition, and is not simply a property of the enhancer or promoter in isolation.
鸡珠蛋白基因的表达部分受βA -珠蛋白和ε -珠蛋白启动子之间对位于这两个基因之间的增强子的竞争调控。为了了解在阶段特异性调控中增强子 -启动子相互作用的决定因素，对胚胎鸡ε -珠蛋白启动子的功能元件进行了表征。体外实验表明：(a) -30处的TATA基序结合GATA -1，(b) Sp1结合到以 -54为中心的元件上，(c) Sp1和另一个名为CACCC的因子（其似乎与红系Krüppel样因子EKLF相关）都结合在 -120至 -128区域。使用胚胎红细胞中的瞬时表达对这些基序的功能进行了测试。在没有增强子的情况下，启动子点突变表明TATA、Sp1和CCAAT基序（但不是CACCC基序）对启动子活性有贡献。相反，在有增强子的情况下，所有四个基序（包括CACCC基序）都对转录有贡献。观察到增强子活性的发育调控，增强作用从4天时的185倍急剧下降到10天时的16倍（此时ε -珠蛋白不再表达）。综上所述，数据表明多种转录因子参与启动子 -增强子相互作用以及ε -珠蛋白表达的发育调控，其中EKLF样因子发挥着特别重要的作用。阶段特异性调控发生在增强子/ε -启动子相互作用的水平，即使在没有竞争的情况下也是如此，而不仅仅是增强子或启动子单独的特性。
