articleThe leukemia associated ETO nuclear repressor gene is regulated by the GATA-1 transcription factor in erythroid / megakaryocytic cells

Background: 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. Results: 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 AML1ETO strongly suppressed an ETO promoter reporter in erythroid/megakaryocytic cells. Conclusions: 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. Background The human ETO co-repressor family comprises the homologous nuclear proteins ETO (Eight-Twenty-One), MTG16 (Myeloid Translocation Gene on chromosome 16) and MTGR1 (Myeloid translocation Gene-Related protein1) evolutionary related to the Drosophila protein Nervy [1]. The ETO homologues do not interact directly with DNA but are recruited by transcription factors such as PLZF, BCL6, TAL1/SCL, Gfi1and Heb [2-7] to become partners of multi-protein complexes on a gene promoter [8,9]. The ETO homologues of the complexes recruit nuclear co-repressors such as N-CoR, [9-11] SIN3 [9,10,12] and SMRT [8,13], which in turn interact with histone deacetylase (HDAC) compelling transcriptional repression. Importantly, ETO homologue genes are commonly involved in reciprocal chromosomal translocation (t) characteristic of acute leukemia. For example, the ETO gene becomes fused to the AML1 (Runx1) transcription factor gene by t(8;21) resulting in the biosynthesis of the AML1-ETO fusion protein [14,15]. Similarly, the MTG16 gene becomes fused to the AML1 gene by t(16;21) resulting in the production of the AML1-MTG16 fusion protein [16]. The oncogenic fusion proteins interfere with * Correspondence: Rakesh.Singh@med.lu. se 2 Protista Biotechnology AB, IDEON, Ole Römers väg 12, SE 223 70 Lund,