1Department of Hematology, C14, BMC, S-221 84 Lund, Sweden.
BMC Mol Biol. 2010 May 20;11:38. doi: 10.1186/1471-2199-11-38.
The Eight-Twenty-One (ETO) nuclear co-repressor gene belongs to the ETO homologue family also containing Myeloid Translocation Gene on chromosome 16 (MTG16) and myeloid translocation Gene-Related protein 1 (MTGR1). By chromosomal translocations ETO and MTG16 become parts of fusion proteins characteristic of morphological variants of acute myeloid leukemia. Normal functions of ETO homologues have as yet not been examined. The goal of this work was to identify structural and functional promoter elements upstream of the coding sequence of the ETO gene in order to explore lineage-specific hematopoietic expression and get hints to function.
A putative proximal ETO promoter was identified within 411 bp upstream of the transcription start site. Strong ETO promoter activity was specifically observed upon transfection of a promoter reporter construct into erythroid/megakaryocytic cells, which have endogeneous ETO gene activity. An evolutionary conserved region of 228 bp revealed potential cis-elements involved in transcription of ETO. Disruption of the evolutionary conserved GATA -636 consensus binding site repressed transactivation and disruption of the ETS1 -705 consensus binding site enhanced activity of the ETO promoter. The promoter was stimulated by overexpression of GATA-1 into erythroid/megakaryocytic cells. Electrophoretic mobility shift assay with erythroid/megakaryocytic cells showed specific binding of GATA-1 to the GATA -636 site. Furthermore, results from chromatin immunoprecipitation showed GATA-1 binding in vivo to the conserved region of the ETO promoter containing the -636 site. The results suggest that the GATA -636 site may have a role in activation of the ETO gene activity in cells with erythroid/megakaryocytic potential. Leukemia associated AML1-ETO strongly suppressed an ETO promoter reporter in erythroid/megakaryocytic cells.
We demonstrate that the GATA-1 transcription factor binds and transactivates the ETO proximal promoter in an erythroid/megakaryocytic-specific manner. Thus, trans-acting factors that are essential in erythroid/megakaryocytic differentiation govern ETO expression.
Eto 核共抑制因子基因属于 Eto 同源家族，还包含 16 号染色体上的髓系易位基因（MTG16）和髓系易位基因相关蛋白 1（MTGR1）。通过染色体易位，ETO 和 MTG16 成为急性髓系白血病形态变异体融合蛋白的一部分。ETO 同源物的正常功能尚未被检测到。本工作的目的是鉴定 ETO 基因编码序列上游的结构和功能启动子元件，以探索谱系特异性造血表达，并获得功能的提示。
在转录起始位点上游 411bp 处鉴定出一个推定的近端 ETO 启动子。在将启动子报告基因构建体转染到具有内源性 ETO 基因活性的红系/巨核细胞中时，观察到强烈的 ETO 启动子活性。228bp 的进化保守区揭示了潜在的顺式元件，参与 ETO 的转录。破坏进化保守的 GATA-636 共识结合位点抑制了反式激活，破坏 ETS1-705 共识结合位点增强了 ETO 启动子的活性。过表达红系/巨核细胞中的 GATA-1 可刺激启动子。电泳迁移率变动分析显示，GATA-1 与 GATA-636 位点特异性结合。此外，染色质免疫沉淀的结果显示，GATA-1 在体内与含有 GATA-636 位点的 ETO 启动子保守区结合。结果表明，GATA-636 位点可能在具有红系/巨核细胞潜能的细胞中激活 ETO 基因活性中发挥作用。白血病相关的 AML1-ETO 强烈抑制红系/巨核细胞中的 ETO 启动子报告基因。
我们证明 GATA-1 转录因子以红系/巨核细胞特异性的方式结合并反式激活 ETO 近端启动子。因此，在红系/巨核细胞分化中起关键作用的反式作用因子控制 ETO 的表达。
