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. 2008 May 21;105(21):7472–7477. doi: 10.1073/pnas.0711896105

Fig. 4.

Fig. 4.

Ap2δ forms a complex with endogenous ASH2L and ALR that methylates histone H3K4. (A) Endogenous ASH2L and ALR coimmunoprecipitate with transfected V5-Ap2δ in K562 cells. The V5-Ap2δ construct was transiently expressed in K562 cells. Levels of expression were documented as comparable in nuclear lysates, using anti-V5 antibodies (Upper). Immunoprecipitations were performed with those lysates using antibodies as indicated, and complexes were analyzed by Western blot, using anti-ASH2L or -ALR antibodies. (B) Complexes containing Ap2δ, ASH2L, ALR, and Su(z)12 methylate recombinant histone H3 in vitro. Nuclear lysates from K562 cells expressing V5-Ap2δ were immunoprecipitated with the indicated antibodies. Immunoprecipitates and GST fusion proteins containing G9a or ALR SET domain were assayed for HMT activity, using histone H3 (Upper). The Coomassie blue staining shows the histone H3 input (Lower). (C) Complexes containing Ap2δ, ASH2L, ALR, and Su(z)12 methylate native histone H3 in vitro. Immunocomplexes and GST fusion proteins as described in B were used for HMT assays with native histone H3. Quantities of TCA-precipitable radioactivity (cpm) are shown as the average of three experiments with the whiskers indicating standard error of the mean. (D) Complexes immunoprecipitated with Ap2δ-V5, but not an unrelated V5-tagged protein (SHP2-V5), methylate H3K4 in vitro. HMT activity is diminished upon incubation with an H3K4 (K4R) mutant but not with H3K9 (K9R) and H3K27 (K27R) mutants.