Wilms tumor–specific mutations in ENL are gain-of-function alterations. (A) Superimposition of somatic mutations identified in a subgroup of Wilms tumors onto the YEATS crystal structure of the ENL homolog AF9. These mutations affect a loop close to the histone-binding surface and either change a PPV peptide to a single leucine (ENL_del) or duplicate an immediately adjacent NHL tripeptide (ENL_ins). (B) Expression of Wilms tumor–specific mutations. Equal amounts of wt-ENL, ENL_del, and ENL_ins were transfected into 293T cells, and protein expression was detected by flag-specific western blot. (C) Derepressive activity of Wilms ENL mutants. Upon cotransfection with the elongation reporter plasmid and Rev-CBX8, the ability of ENL derivatives to counteract CBX8-induced repression was recorded in luciferase assays. Constructs were transfected as indicated with bars representing averages and standard deviation of triplicates. The figure is a typical example of a duplicate experiment. (D) Wilms-specific ENL mutations induce colony-forming cell activity. Primary hematopoietic precursor cells were isolated from bone marrow and transduced with vector wt-ENL, ENL_del, and ENL_ins, as indicated. Cells were replated twice into growth factor–supplemented methylcellulose. The figure shows stained colonies arising in third round of growth after 2 replatings (left; duplicate samples), and the bar chart depicts average colony numbers and standard deviations of an independent biological triplicate. (E) ENL Wilms mutants retard differentiation. FACS surface marker staining for the differentiation marker Gr1 (Ly6G) on cells transduced with ENL and derivatives as indicated. Cells recovered after the primary plating round were used before control cells exhausted their replicative potential. (F) Kaplan-Meier survival plot of animals transplanted with primary hematopoietic cells expressing either ENL or the Wilms tumor–derived mutant ENL_ins. Note that the experiment was terminated after 175 days with all ENL-injected animals still healthy. (G) Effect of ENL Wilms mutants on Hoxa9 and Meis1 loci. RNA and crosslinked chromatin was prepared from cells, as described for panel E, which were recovered after 1 round of replating. Hoxa9 and Meis1 expression was tested by qPCR (Gi) and H2Bub- specific ChIP was performed (Gii). Bar charts show averages and standard deviations of PCR triplicates and are representative of a duplicate experiment.