Fig. 5. MLL1 phosphorylation by CSNK2-A2 is required for its competitive association with LEDGF/p75 and recruitment to the proviral LTR.

(A) TNFα stimulation decreases PAF1 but enhances MLL’s association with LEDGF/p75 in E4 cells, assessed by immunopreciptation and immunoblotting with the indicated antibodies. (B) PAF1 and MLL1 compete for LEDGF/p75 binding in 293 T cells. (C) The IBD of LEDGF/p75 mediates the increased association of LEDGF/p75 with MLL1 in E4 cells stimulated with TNFα for 4 hours. (D) PAF1 occupancy at the LTR in E4 cells depleted for indicated protein and treated with or without TNFα for 4 hours was normalized to input and shown as means + SD (n = 3). (E) MLL1 occupancy in E4 cells depleted for indicated protein and treated with or without TNFα for 4 hours was determined and shown as in (D). (F) Association of CSNK2-A2 with MLL1 in E4 cells stimulated with or without TNFα for 4 hours. (G) Association of LEDGF/p75 with MLL1 and PAF1 in E4 cells depleted for CSNK2-A2. (H) Association of LEDGF/p75 with MLL1 and PAF1 in E4 cells overexpressing exogenous CSNK2-A2. (I) Association of LEDGF/p75 with MLL1 and PAF1 in control- and CSNK2-A2–depleted E4 cells treated with or without TNFα for 4 hours. (J) Association of LEDGF/p75 with MLL1 and PAF1 in control- and CSNK2-A2– depleted 293 T cells overexpressing Halo-MLL1. (K and L) Lentiviral constructs encoding wild-type, nonphosphomimetic (S136, S142, and S153A), or phosphomimetic (S136, S142, and S153D) deletion mutant (1 to 1406) of MLL1 were transduced into E4 cells that had been depleted for CSNK2-A2 for 3 days. The transduced cells were either untreated (K) or stimulated with TNFα (L) for 4 hours and then used for immunoprecipitation and immunobloting with the indicated antibodies. (M) E4 cells were treated as in (K) and (L), and the level of N-terminal wild-type or mutant MLL1 fragment on HIV LTR was determined by Flag ChIP and shown as means + SD (n = 3). **P < 0.01 and ***P < 0.001. SE, short exposure; LE, long exposure.