Supplementary Figures. Legend to Supplementary Figure 1. Characterization of the t(X;6). i) Chromosome X bps: GATA1, at approximately 48.5Mb within Xp11.2, is represented by a blue horizontal line. Boxes indicate exons, arrows the 5’-3’ orientation. Green small horizontal lines represent X chromosomegenomic clones mapping to the derivative chromosome 6; red lines, X chromosomegenomic clones retained on the derivative chromosome X. Pink vertical arrows indicate the position of the 2 inversion bps (A); the green vertical arrow marks the translocation bp (B). ii) Chromosome 6 bp: MYB, at approximately 135.5Mb, is represented by a blue horizontal line: boxes indicate exons, arrows the 5’-3’ orientation. Green small horizontal lines represent chromosome 6-genomic clones retained on the derivative chromosome 6; red lines, chromosome 6-genomic clones mapping to the derivative X chromosome; the orange line indicates the chromosome 6-genomic clone spanning the bp. Green vertical arrows mark the position of the translocation bp (C). iii) FISH results with the RP11-32B1 clone, spanning the bp on chromosome 6. Involved chromosomes are indicated by number, as follows: 1= normal chromosome 6; 2= derivative chromosome 6; 3= derivative X chromosome. Legend to Supplementary Figure 2. Schematic representation of the rearrangement. MYB and GATA1 orientation along chromosome 6 and X, respectively, together with FISH and molecular data, revealed that the rearrangement was more complex than expected in our case, harboring an X chromosome internal inversion associated with the translocation to chromosome 6. Top: organization of the MYB and GATA1 loci on chromosomes 6 and X, respectively. Each rectangle represents a portion of the locus, ordered according to its position along the chromosome. Arrows indicate the 5’-3’ orientation of the genes. On the right is the modified orientation of the GATA1 locus, after the inversion. Bottom: organization of the bp regions on the derivative chromosomes 6 and X. On the derivative chromosome 6, the formation of the fusion transcript, resulting from the translocation and associated X chromosome inversion, is shown. As indicated, the rearrangement implicates also the possible formation of a truncated GATA1 transcript, including exons 1 to 4. On the derivative X, the rearrangement leaves MYB exons 9 to 15 without any evident transcription start site able to direct the formation of a corresponding truncated product. Legend to supplementary figure 3. MDS-like features induced by the MYB-GATA1 fusion product. Peripheral blood smears from one of the four mice (mMG2), reconstituted with GATA1low lin- cells transduced with PINCO-MYB-GATA1 and not developing leukemia. Anysocytosis and poikilocytosis are evident at both 13 and 15 months from transduction, as well as dysplastic cells. Legend to supplementary figure 4. Histopathologic analysis of the bone marrow of the leukemic mouse (mMG1). Representative images of hypercellular bone marrow and spleen of mMG1, showing overt acute leukemia. Legend to supplementary figure 5. Immunophenotypic characterization of a control mPev mouse. 7-AAD negative selection was performed in order to select for viable events. The analysis for Gr-1, CD8 and Sca-1 is shown. Legend to supplementary figure 6. Effects of the MYB-GATA1 fusion product. Overall survival (OS) of mice reconstituted with transduced GATA1low lin- cells (PINCO-MYB-GATA1, red line) versus controls (PINCO-ev, green line) as well as mice transplanted with MYBGATA1 leukemic cells, from spleen, in 3 consecutive passages (purple, blue, and orange lines). Survival was determined by Kaplan-Meier analysis, performed with the JMP statistical software. Numbers of mice for each type is indicated in the table.