The transforming capability of an MLL-ENL fusion protein correlates with Dot1l binding. (A) Schematic depiction of alanine insertion mutations introduced into the ENL C-terminus. (B) Results of a serial replating assay with primary hematopoietic precursor cells isolated from bone marrow and transduced either with wild-type MLL-ENL, MLL-ENL mutant A, or MLL-ENL mutant B. Given are the average colony numbers including standard deviations in the third round of replating. Next to the bar graph, a representative example of stained colonies is shown. The insets show Western blot experiments detecting expression of the corresponding GAL4/MLL fusion proteins. (C) 2-hybrid test for interaction of ENL mutants A and B with full-length Dot1l and CBX8. Yeast was cotransformed with a bait plasmid containing mutants A and B as indicated and a Dot1l interaction target. Growth on adenine/histidine deficient selective medium is shown. (D) qRT-PCR analysis of MLL-ENL target gene expression. RNA was isolated from primary hematopoietic cells 1 week after transduction with retroviral constructs coding for wild-type MLL-ENL, mutant A, or mutant B, respectively. Relative RNA levels of the known MLL-ENL target genes Hoxa7, Hoxa9, and Meis1 were determined by qRT-PCR, normalized with respect to actin expression, and plotted with values for wild-type MLL-ENL transduced cells set arbitrarily to 1. Note that the results are displayed on a logarithmic scale. Average and standard deviation of triplicates are plotted.