Wilms mutations change the YEATS-binding properties. (A) Binding of PAF1 by wt-ENL and Wilms mutants. Flag-tagged versions of wt-ENL, ENL_del, and ENL_ins were coexpressed together with HA-labeled PAF1, and the interaction was probed by immunoblot. (B) Affinity of ENL and mutants for histone H3. Cell extracts expressing ENL and respective mutants were supplemented with identical amounts of recombinant histone H3, and flag-specific immunoprecipitates were analyzed for ENL and H3 content. Note that because H3 was added exogenously, there is no separate input sample. (C) Coimmunoprecipitation of endogenous ENL, PAF1, and H3. ENL was purified from native 293T cell extracts by anti-ENL precipitation, and coprecipitating material was analyzed for PAF1 and H3 by western blotting with antibodies recognizing the respective proteins. The histone blot was done with two different sample volumes of precipitated material. (D) Pulldown of PAF1 and ENL/ENL_ins. The ENL N-terminus was expressed and purified as GST fusion protein (left). After cleavage from the GST moiety, ENL was covalently attached to agarose beads and incubated with a purified GST-HA-PAF266 protein, excluding the intrinsic PAF1 histone- binding domain. Pulldown was done either in the absence or after addition of recombinant histone H3. Note that the immunoblot was simultaneously developed with antihistone H3 and anti-HA antibodies. Analogous experiment done with ENL (right upper) or ENL_ins protein bound to beads (right lower), including H3K27 acetylated histone peptides as competitors for PAF1 binding. (E) Schematic depiction of different ENL functions. In the environment of polycomb-repressed chromatin, ENL can bind to CBX8 and recruit PAF1 through the YEATS domain. Because silenced chromatin is devoid of acetylated histone H3, the YEATS domain is free to interact with PAF1. After chromatin opening, large quantities of acetylated H3 becomes available, occupying the YEATS surface, thus supporting transfer of PAF1 to other binding partners such as RNA polymerase II. Because no CBX8 is present on active chromatin, this will free the ENL C-terminus for interaction with either DOT1L or AFF1. Wilms-specific mutations weaken interaction with histone H3, shift the balance, and favor derepression. MLL fusions permanently tether PAF1 to DOT1/AFF1 through the constitutive PAF1-binding site in the MLL N-terminus. Cont, control; vect, vector.