MYC binds TIP60 and recruits it to chromatin in vivo. (A) 293T cells were transfected with vectors expressing human MYC and FLAG–haemagglutinin (HA)-tagged TIP60. Whole-cell lysates (400 μg) were immunoprecipitated (IP) with antibodies against TIP60 (CLHF antiserum; upper panel) or MYC (C33 monoclonal; lower panel), followed by immunoblotting as indicated. 'Total' indicates that the whole-cell lysate (40 μg) was directly loaded for immunoblotting. 'Ctrl' indicates that control IPs were performed with pre-immune serum or with a monoclonal antibody of the same isotype as C33. (B) Quiescent Rat1 cells expressing a MYC–oestrogen-receptor (ER) chimaera were analysed by chromatin precipitation with MYC and TIP60 antibodies before or after 4-hydroxytamoxifem (OHT) treatment. PCR analysis was performed with primers that recognise MYC-bound E-boxes in NUC intron 1 or with primers that amplify the ACHR promoter, which was used as a control. The right panel shows representative PCR amplification curves from TIP60 immunoprecipitations, which were performed either before treatment with OHT (−OHT) or 1 h after treatment (+OHT). (C) P493-6 cells were growth-arrested by the addition of tetracycline (Tet) to serum-containing medium. To induce MYC expression, cells were washed and resuspended in fresh serum-containing medium without Tet (−Tet). Control cells were treated in an identical manner, but in the continuous presence of Tet (+Tet). Chromatin immunoprecipitation analysis was performed with antibodies against MYC, TRRAP, TIP60 or acetylated histone H4 (AcH4), as indicated.

TIP60 associates with MYC target-loci. (A) Chromatin immunoprecipitation (ChIP) analysis of quiescent and serum-stimulated Rat1 cells using antibodies against acetylated histone H4 (AcH4; upper panels) and TIP60 (lower panels). The MYC binding sites (NUC, HSP60, GPAT, NM23-H2, α-PT and CAD) and the control site (ACHR) are the same as were used previously (Frank et al., 2001). (B) MYC, TIP60 and TRRAP colocalize on the NUC gene in Rat1 cells stimulated with serum for 4 h. DNA that was recovered by ChIP using the antibodies indicated was amplified with different primer pairs to give amplicons 1–5 in NUC, as indicated in the map. Numerical positions on the map are relative to the cap site, and correspond to the 5′ ends of the amplicons. (C) Kinetics of MYC binding, TIP60 recruitment and H4 acetylation at NUC intron 1 after serum stimulation of Rat1 cells. (D) Serumstimulated MYC^−/− cells were infected with either a control adenovirus that expresses green fluorescent protein (AdGFP; black bars) or a MYC adenovirus (AdMYC, stippled bars). ChIP analysis showed that AdMYC restored binding of TIP60 and TRRAP and H4 acetylation at NUC intron 1.

MYC recruits the TIP60 complex subunits TIP48, TIP49 and p400 to chromatin. (A) MYC expression was induced in P493-6 cells by the removal of tetracycline (Tet), as described in Fig. 1C. Chromatin immunoprcipitation (ChIP) analysis was performed with antibodies against TIP48, TIP49 or p400, as indicated. (B) Quiescent and serumstimulated Rat1 cells were analysed by ChIP using the same antibodies as in (A). Amplicons 1–5 located along the rat NUC gene are the same as those shown in Fig. 2B.

Expression of catalytically inactive TIP60 delays MYC-dependent H4 acetylation. MYC^−/− cells were stimulated with serum and infected with a MYC-expressing adenovirus (AdMyc) in combination with viruses that express either green fluorescent protein (AdGFP), wild-type TIP60 (AdTIP60wt) or catalytically inactive TIP60 (AdTIP60mut). Chromatin immunopreciptiation analysis was performed at the indicated timepoints, using antibodies against acetylated H4 (AcH4; upper panel) or MYC (lower panel). PCR amplification was performed on the same sites as for Fig. 2A.