Wnt3a induces spatial proximity and translocation of RUNX1 and ETO in human hematopoietic progenitors. (A) Localization of β-catenin into active RNAPII transcription factories. Immunodetection of β-catenin and active RNAPII-Ser5P through confocal microscopy in CD34⁺ cells incubated under control conditions or in the presence of purified Wnt3a (200 ng/mL; 4 hours). White boxes indicate selected regions of interest that are shown at higher magnification at the right panel. Scale bar represents 5 μm. (B) Surface-rendered CD34⁺ cells (as depicted in panel A) and zoom in (4×; white boxes) showing colocalization of β-catenin and RNAPII-Ser5P in Wnt3a-treated cells. (C) Representative confocal images of RNA-FISH experiments in CD34⁺ cells upon 4-hour treatment with 200 ng/mL purified Wnt3a (number of cells and other parameters are given in supplemental Table 2). RUNX1 alleles are shown in red and ETO alleles in green. Scale bar represents 4 μm. Selected region of interest (white box) is shown at higher magnification below as a maximum intensity projection reconstruction for 4,6 diamidino-2-phenylindole (DAPI) and signal rendering for RUNX1 (red) and ETO (green). Scale bar represents 4 μm. Zoom in (4× and 8×; middle and right, respectively) corresponds to region of interest defined above. (D) Representative confocal images of DNA-FISH experiments in control and Wnt3a-treated CD34⁺ cells (4 hours) (top). White arrowheads in Wnt3a-treated cells indicate RUNX1 (red) and ETO (green) alleles with a distance of less than 1 μm. Scale bar represents 10 μm. Scatterplot showing shortest distances between RUNX1 and ETO alleles in control and Wnt3a-treated cells for 4 and 48 hours (bottom) (number of cells and other parameters are given in supplemental Table 3). The median in each group is depicted as a red line. Statistical significance was determined by a Mann-Whitney U test (***P < .0001; NS, nonsignificant). (E) Wnt/β-catenin signaling induces a common RUNX1-ETO translocation event. Diagram representing a recurrent RUNX1-ETO translocation event (left). Detection of RUNX1-ETO fusion gene and GAPDH products in CD34⁺ cells following long-term treatment with Wnt3a for 48 hours (middle). Chromatogram of the band shown for CD34⁺ cells treated with Wnt3a (48 hours) (right). no RT, no reverse transcription; pAML1-ETO, amplification of RUNX1-ETO from chimeric plasmid pCMV5-AML1-ETO.