Wnt/β-catenin signaling modulates ETO transcriptional activity. (A) Genomic context of the human ETO gene on chromosome 8. Selected ETO mRNA variants (001: ENST00000523629; 002: ENST00000396218; 004: ENST00000422361; and 009: ENST00000360348) (top). Exons are depicted as bars numbered according to NCBI RefSeq: NC_000008.11. Numbers within square brackets indicate numbering as it has been reported.9 Green boxes (P1-P8) represent predicted genomic regions containing putative promoters.11 t(8,21): Chromosomal translocation sites RUNX1/ETO. The location of predicted TCF/LEF-binding elements (TBE; open circles) in the promoter region of variants 002, 004, and 009 (bottom). (B) Detection of alternative ETO mRNA variants 001, 002, 004, and 009 in CD34+ (top), KG1 (middle), and HEK293 cells (bottom) under control conditions or in the same cells treated with purified Wnt3a (200 ng/mL) for 4 hours. (C) Quantitative determination of mRNA levels for ETO-004 and ETO total (using primers designed in conserved exons 8 and 9 or determined by TaqMan assays; left and middle, respectively), as well as for Runx1 and Wnt/β-catenin target genes Axin2 and Myc (right). Cells were incubated under control conditions or stimulated with 200 ng/mL purified Wnt3a for 4 hours. Quantitative polymerase chain reaction results were normalized using GAPDH mRNA levels as a reference gene and are expressed as relative expression. Each figure corresponds to at least 5 independent experiments (with replicates). Ctrl, control conditions. Statistical significance was determined by a Mann-Whitney U test. (D) Dose-dependent effects of constitutively active β-catenin S33Y on the transcriptional activity of ETO-luciferase promoter constructs in HEK293 cells (24 hours). SuperTOPFLASH (STF) was measured as positive Wnt/β-catenin signaling readout. Each figure corresponds to at least 3 independent experiments. Statistical significance was determined by an ANOVA test (**P < .01). (E) Effect of β-catenin S33Y and increasing doses of dominant-negative ΔTCF4 on the promoter activity of pETO-004 deletion constructs, determined as in panel D. RLU, relative luciferase units.
