Fig. 1. Co-expression of Mdm2 decreases Tip60 expression. (A) U2OS cells (1 × 10⁵) were transfected with 100 ng of pCMV luciferase reporter vector, 1 µg of pCMV 2N3T Tip60, 1 µg of pCMV 2N3T Ku80, in the presence (lane 1) or absence (lane 2) of 2 µg of pCMV NeoBam Mdm2. Ku80 is a component of the DNA end-binding Ku heterodimer. The amount of CMV promoter in the transfection was kept constant by the addition of empty vectors. Luciferase activity was measured 24 h after transfection,. After standardization for luciferase activity, total cell extracts (obtained by boiling cells in Laemmli sample buffer) were loaded directly and subjected to an anti-HA western blot to detect exogenous Tip60 and Ku80, as indicated. (B) Same experiment as in (A), except that we used pCDNA3 HA-Tip60 instead of pCMV 2N3T Tip60. Note that this exogenous Tip60 migrates as two bands in the absence of Mdm2, because of phosphorylation (unpublished results).

Fig. 2. Tip60 is subjected to proteasome-mediated degradation. (A) Jurkat cells (left panel) or HeLa cells (right panel) were treated or not with the proteasome inhibitor indicated (Lact., lactacystine). Total cell extracts were then tested for the presence of Tip60 by western blotting using either the anti-Tip60 DT antibody (left panel) or the anti-Tip60 SK antibody (right panel). The band indicated Tip60 co-migrates with the exogenous HA-Tip60 produced from pCDNA3 (data not shown). The asterisk (*) indicates a non-specific band recognized by the anti-Tip60 DT antibody. (B) U2OS cells were transfected as in Figure 1B. Twenty-four hours after transfection, the proteasome inhibitor ALLN was added where indicated (lanes 5–8). After 2 h, cycloheximide was added and cells were harvested after treatment for the length of time indicated. Total cell extracts were analysed as described in Figure 1A. (C) U2OS cells were transfected with 10 µg of pCMV 2N3T Tip60 and 10 µg of either pSG5 His-ubiquitin (lane 2) or pSG5 HA-ubiquitin (lane 1). Twenty-four hours after transfection, cells were lysed as described in Materials and methods and His-tagged proteins were purified by nickel chromatography. Nickel binding proteins were subjected to a western blot using the anti-HA antibody. The arrow points to the unmodified HA-Tip60 protein that harboured background binding to nickel beads.

Fig. 3. Mdm2 induces the ubiquitylation and proteasome-mediated degradation of exogenous Tip60. (A) U2OS cells were transfected as in Figure 1B with pCMV luciferase, pCDNA3 HA-Tip60, pCMV 2N3T Ku80 and with or without pCMVNeoBam Mdm2. Twenty-four hours after transfection, the proteasome inhibitor indicated (Lact., lactacystin) was added (lanes 1–4) or not (lanes 5–6). After 6 h, total cell extracts were analysed as described in Figure 1B. (B) U2OS cells were transfected with 100 ng of pCMV luciferase reporter vector, 1 µg of pCMV 2N3T RbAp48, 1 µg of pCMV 2N3T Tip60 where indicated and 2 µg of pCMV NeoBam Mdm2 as indicated. The amount of promoters in the transfection was kept constant using empty vectors. ALLN was added for 6 h, 24 h after transfection,. Total cell extracts were analysed by western blotting using the anti-HA antibody. The top panel shows a long exposure of the blot. Note the increase in the amount of HA-reactive more slowly migrating forms in the presence of Mdm2 (compare lane 2 with lane 4). The bottom panel shows a short exposure of the same blot. (C) U2OS cells transfected with 10 µg pCMV 2N3T Tip60, 10 µg pSG5 His-ubiquitin and 10 µg pCMV NeoBam Mdm2 where indicated, were analysed as in Figure 2C.

Fig. 4. Mdm2 interacts physically with Tip60. (A) GST pull-down with ³⁵S-labelled in vitro-translated Tip60 using beads harbouring bacterially produced GST–Mdm2 (lane 2) or control GST (lane 3) proteins. Bound proteins were analysed by SDS–PAGE followed by autoradiography. In lane 1, 2 µl of the in vitro translation reaction products were loaded directly. (B) U2OS cells were transfected with 10 µg of pCMV 2N3T Tip60 and/or 10 µg of pCMV NeoBam Mdm2, as indicated. ALLN was added 12 h after transfection. Whole-cell extracts were prepared 24 h after transfection, and subjected to immunoprecipitation with the anti-HA antibody. Immunoprecipitates were tested for the presence of Mdm2 by western blotting using the SMP14 antibody (left panel). In the right panel, 10 µl of whole-cell extracts were loaded directly and subjected to an anti-Mdm2 western blot. The asterisk indicates a band corresponding to the immunoglobulin light chain of the immunoprecipitating antibody. (C) As in (B), except that whole-cell extracts were immunoprecipitated with an anti-Mdm2 antibody (SMP14). Immuno precipitates were tested for the presence of exogenous Tip60 by western blotting using the anti-HA antibody (left panel). In the right panel, 10 µl of whole-cell extracts were loaded directly, and subjected to a western blotting with both the anti-HA and the anti-Mdm2 antibody. The asterisk indicates a band corresponding to the immunoglobulin light chain of the immunoprecipitating antibody. (D) HeLa nuclear extracts (Computer Cell Culture Center) were immunoprecipitated with the anti-Tip60 DT antibody (lane 2), or control pre-immune antibody (PI, lane 1) as a control. Immunoprecipitates were tested for the presence of Mdm2 by western blotting using the anti-Mdm2 antibody SMP14. In lane 3, 5 µl of extracts of U2OS transfected with the Mdm2 expression vector were loaded directly.

Fig. 5. Mdm2 induces Tip60 degradation through their physical interaction. (A) Schematic representation of the Tip60 protein. The hatched box represents the chromodomain, the grey box the putative Zn finger and the black box the AcCoA binding site. Also indicated is the HAT domain, which is conserved among proteins of the MYST family. (B) U2OS cells were transfected with 10 µg of pCMV NeoBam Mdm2 and either 10 µg of pCDNA3 HA-Tip60 fl, pcDNA3 HA-Tip60 1–258 or pCDNA3 HA-Tip60 1–364. Whole-cell extracts were prepared 24 h after transfection, and subjected to immunoprecipitation with either the anti-HA antibody (lanes 2, 5 and 8) or an irrelevant antibody (anti-myc 9E10, lanes 3, 6 and 9), as indicated. Immunoprecipitates were tested for the presence of Mdm2 by western blotting. In lanes labelled inp (input), 10 µl of whole-cell extracts were loaded directly. In lanes 10–12, 10 µl of whole-cell extracts were subjected to an anti-HA western blot, to monitor the quantity of the various mutants in the extracts. Note that Tip60 1–258 is present in far larger amounts than Tip60 fl or 1–364, because these latter proteins are very poorly extractible from cells (compare with C). The asterisks indicate two non-specific bands. HA-Tip60 1–364 migrates just below a non-specific band. (C) U2OS cells were transfected as in Figure 1B with expression vectors of the Tip60 deletion mutant indicated (in pCDNA3-HA, 1 µg), pCMV 2N3T Ku80 (1 µg), pCMV luciferase reporter vector (100 ng), in the presence or absence of pCMV NeoBam Mdm2 (2 µg), as indicated. The amount of promoters in the transfection was kept constant using empty vectors. Luciferase activity was measured 24 h after transfection. The steady-state levels of HA-tagged proteins were assessed by western blotting.

Fig. 6. Ubiquitylation of Tip60 by Mdm2 is involved in Tip60 degradation. (A) ³⁵S-labelled, in vitro-translated, full-length Tip60 (lanes 1–7) or Tip60 1–211 (lanes 8–11) were subjected to an in vitro ubiquitylation reaction by GST–Mdm2 or GST–Mdm2 Δ417, where indicated. Ubiquitin was omitted in reactions loaded in lanes 3, 6 and 10 whereas UbcH5 (E2) was omitted in lanes 7 and 11. Note the appearance in the presence of Mdm2 (lane 2) and Mdm2Δ417 (lane 5) of bands of lower mobility, which indicate mono- and polyubiquitylation, and the concomitant decrease in the amount of unmodified Tip60. (B) U2OS cells were transfected with 100 ng of pCMV luciferase reporter vector, 1 µg of pCDNA3 HA-Tip60, 1 µg of pCMV 2N3T Ku80, in the absence (lane 3) or presence of either 2 µg of pXJ Mdm2 (lane 1) or 2 µg of pXJ Mdm2 C462A (lane 2). The amount of promoters in the transfection was kept constant by the addition of empty vectors. Luciferase activity was measured 24 h after transfection. Total cell extracts were loaded directly and subjected to an anti-HA western blot to detect exogenous Tip60 and Ku80, as indicated (upper panel). In the lower panel, 10 µl of total cell extracts were loaded directly, and probed with the anti-Mdm2 antibody. (C) U2OS cells were transiently transfected with 100 ng of pCMV luciferase reporter vector, 1 µg of pCDNA3 Tip60 259–396, 1 µg of pCMV 2N3T Ku80 and increasing amounts of either pCMV Neo Bam Mdm2 or pCMV Mdm2 Δ101 (0.5, 1 or 2 µg as indicated by the height of the open triangle). Total cell extracts were loaded directly and subjected to an anti-HA western blot to detect exogenous Tip60 and Ku80, as indicated (upper panel). In the lower panel, 10 µl of total cell extracts were loaded directly, and probed with the anti-Mdm2 antibody.

Fig. 7. Tip60 expression is induced by UV irradiation. Jurkat cells (2 × 10⁶) were irradiated as indicated in Materials and methods and incubation was pursued for the period of time indicated. Cells were then collected, boiled in SDS loading buffer, and extracts were subjected to a western blot using the anti-Tip60 DT antibody (upper panel). The asterisk indicates a non-specific band. The membrane was also stained with Ponceau Red to check for loading differences (lower panel).