Taxman D J, Wojchowski D M
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802.
J Biol Chem. 1995 Mar 24;270(12):6619-27. doi: 10.1074/jbc.270.12.6619.
Using J2E cells and the murine beta maj-globin promoter as a model, we have performed the first direct analyses of erythropoietin (EPO)-activated transcription from defined templates. The -346 to +26 beta maj promoter was shown to comprise a target for maximal activation. This included a positive role for a -346 to -107-base pair (bp) domain in J2E cells, but not in F-MEL cells. Mutagenesis of a -215-bp AGATAA element within this domain showed that this effect did not require GATA-1 binding. In contrast, a critical role for GATA-1 at a -60-bp (G)GATAG element was defined by mutagenesis (GGg-TAG and TGATAG), complementation with a synthetic TGATAA element, and the demonstrated specific binding of GATA-1. Proximal CCAAT (-75) and CACCC (-90) elements also were shown to contribute to transcriptional activation in J2E cells, yet exerted quantitatively distinct effects in the F-MEL system. Based on these results, minimal [TGATAA]4-TATA and TGATAA-CACCC-TATA promoters were constructed and assayed in each system. Remarkably, the [TGATAA]4-TATA promoter, but not the TGATAA-CACCC-TATA promoter, was induced efficiently by EPO in J2E cells, whereas the TGATAA-CACCC-TATA promoter was highly induced by Me2SO in F-MEL cells. These findings suggest that mechanisms of EPO-induced transcription in J2E cells involve GATA-1 and differ from chemically activated mechanisms studied previously in F-MEL cells. Globin induction in J2E cells was not associated with effects of EPO on levels or nuclear translocation of GATA-1. However, hemoglobinization was induced by okadaic acid, 8-Br-cAMP, and forskolin, a finding consistent with induction mechanisms that may involve modulated serine/threonine phosphorylation.
利用J2E细胞和小鼠β珠蛋白maj启动子作为模型，我们首次对促红细胞生成素（EPO）激活特定模板转录进行了直接分析。研究表明，-346至+26的β珠蛋白maj启动子是最大激活的靶点。这包括在J2E细胞中-346至-107碱基对（bp）结构域的正向作用，但在F-MEL细胞中则没有。对该结构域内-215bp的AGATAA元件进行诱变表明，这种效应不需要GATA-1结合。相反，通过诱变（GGg-TAG和TGATAG）、与合成的TGATAA元件互补以及证明GATA-1的特异性结合，确定了GATA-1在-60bp（G）GATAG元件处的关键作用。近端CCAAT（-75）和CACCC（-90）元件也被证明有助于J2E细胞中的转录激活，但在F-MEL系统中发挥的定量作用不同。基于这些结果，构建了最小的[TGATAA]4-TATA和TGATAA-CACCC-TATA启动子，并在每个系统中进行检测。值得注意的是，[TGATAA]4-TATA启动子而非TGATAA-CACCC-TATA启动子在J2E细胞中被EPO有效诱导，而TGATAA-CACCC-TATA启动子在F-MEL细胞中被二甲基亚砜（Me2SO）高度诱导。这些发现表明，J2E细胞中EPO诱导转录的机制涉及GATA-1，并且与先前在F-MEL细胞中研究的化学激活机制不同。J2E细胞中的珠蛋白诱导与EPO对GATA-1水平或核转位的影响无关。然而，冈田酸、8-溴环磷腺苷（8-Br-cAMP）和福斯可林可诱导血红蛋白化，这一发现与可能涉及调节丝氨酸/苏氨酸磷酸化的诱导机制一致。
