Two helical domains of AF10 are necessary and sufficient for immortalization of myeloid progenitors by MLL-AF10.

(A) Schematic illustrations of AF10 sequences fused to MLL5′ are shown in the left panel. Amino acid numbering is based on that of wild-type AF10.35 The OM and LZ are indicated by filled boxes. Open box denotes LZ containing a site-directed missense T781P mutation. In the right panel, horizontal bars represent the number of colonies generated per 10⁴ cells plated in third-round cultures. Bars represent the mean ± SD of triplicate samples from a representative experiment. (B) Amino acid sequences of the OM and LZ of AF10 are conserved in orthologous proteins from Drosophilaand C elegans. Identical amino acids in 3 or more sequences are shown in bold. Asterisks indicate conserved heptad repeat leucine residues. Boxes indicate domains that have predicted α-helical secondary structures. Black horizontal bars denote OM and LZ, respectively. Downward arrow in AF10 schematic denotes most 3′ site of fusion with MLL in human acute myeloid leukemias. (C) Western blotting of whole cell extracts of transiently transfected COS7 cells. Lane 1, MLL-AF10; lane 2, MLL-OM+LZ; lane 3, MLL-ΔLZ; lane 4, MLL-T781P; lane 5, MLL-OM; lane 6, MLL-LZ; lane 7, MLL5′.