Figure 4.

Myc corepresses p21Cip1 promoter together with Dnmt3a through association with the DNA-binding protein Miz-1. (A) U2OS cells were transfected with GAL4-Dnmt3a and/or Miz-1. Cell extracts were then precipitated with anti-GAL4 antibody followed by Western blotting using a specific anti-Miz-1 antibody. Expression levels of the different proteins in the inputs were verified by Western blotting using anti-GAL4 or anti-HA antibodies (Input controls). (B) Miz-1 coimmunoprecipitates with Dnmt3a from untransfected cells. HeLa nuclear extracts were immunoprecipitated using anti-Dnmt3a, anti-CREB1 (used as negative control) or the beads only. Precipitated were blotted with a Miz-1 antibody. (C) Endogenous Miz-1 purifies DNA methyltransferase activity from HeLa nuclear extracts. (D) Myc binds directly to Dnmt3a, whereas Miz-1 only weakly binds directly to Dnmt3a. GST-Myc 1-204, GST-RP58, used as positive control (Fuks et al, 2001) or GST-Miz-1 were incubated with bacterially expressed His-Dnmt3a, followed by Western blotting using anti-His antibody. Coomassie stain gel show the input of GST fusion proteins used. (E) Myc mediates the interaction of Dnmt3a with Miz-1. U2OS were transfected with limited amount of GAL4-Dnmt3a and Miz-1 in the presence (lanes 3–6) or absence (lanes 2, 7 and 8) of increasing levels of Myc (1–4 μg). After immunoprecipitation of GAL4-Dnmt3a with anti-GAL4, Western blotting was performed using anti-Miz-1 antibody. (F) Myc increases the association of Miz-1 with Dnmt3a enzymatic activity. DNA methyltransferase assays were performed using immobilized-GST-Miz-1 and bacterially expressed and active Dnmt3a, in the presence or absence of bacterially expressed His-Myc. (G) Corepression of p21Cip1 by Myc and Dnmt3a requires binding of Myc to Miz-1. Upper panel: Representation of Myc wt or the Myc mutant (MycV394D; unable to bind to Miz-1 and that lost Miz-1-dependent repression) (Herold et al, 2002). GST pull-downs using GST-Dnmt3a 490–592 and IVT Myc wt or Myc mut show that the Myc mutant still retains binding to Dnmt3a. Lower panel: U2OS were transfected with p21Cip1-Luc reporter and, as indicated, with limited amount of Dnmt3a and either Myc wt or the Myc point mutant.