The Myc-repressed p21Cip1 gene is silenced by Dnmt3a in vivo. (A) Left panel: U2OS cells were transfected with the reporter construct p21Cip1 promoter-Luc together with increasing amounts of Myc full length (1–100 ng). The activity of the reporter in the absence of Myc is normalized to a value of 100. Right panel: Myc-mediated repression of p21Cip1 promoter is enhanced by Dnmt3a wild type. U2OS cells were transfected with the reporter p21Cip1 promoter-Luc together with combinations of limiting amount of expression vectors for Myc and Dnmt3a wild-type (Dnmt3a wt) or catalytic mutant (Dnmt3a mut). Bottom: Western blotting using anti-Myc shows that enhancement of Myc-mediated p21Cip1 silencing by Dnmt3a (Figure 2A, right panel, lane 5) is not simply due to increased expression of Myc protein levels after cotransfection of Dnmt3a. (B) Depletion of Dnmt3a from cells reactivates endogenous p21Cip1 expression. U2OS were treated either with Dnmt3a antisense inhibitor or with mismatch control. Left panel: Dnmt3a antisense specifically depletes Dnmt3a but not Dnmt1 or Dnmt3b. Quantitative real-time PCR analysis of Dnmt3a mRNA is shown, as well as Western blotting of the Dnmts. Actin serves as a loading control for Western blotting. Right panel: mRNA expression of the indicated genes was determined by quantitative real-time PCR and normalized against that of β-actin. Expression of each mRNA with mismatch control was set to a value of 100. Error bars represent standard deviations. Statistical significant reactivation of p21Cip1 was observed with Dnmt3a antisense compared to control (^*P<0.05).

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.

Myc binds the corepressor Dnmt3a and associates with DNA methyltransferase activity in vivo. (A) Representation of GST fusions of Dnmt3a (left panel) or Myc (right panel). The indicated GST fusions were tested in GST pull-down experiments using IVT full-length Dnmt3a (left) or Myc (right). (B) Myc coimmunoprecipitates with Dnmt3a. U2OS were transiently transfected as indicated (+) with expression vectors for HA-tagged Myc full-length and/or GAL4-tagged Dnmt3a full length. Left panel: cell extracts were precipitated with anti-GAL4 antibody and the presence of HA-Myc in the immunoprecipitates was visualized by Western blot analysis using anti-HA antibody. Right panel: anti-HA was used to immunoprecipitate HA-Myc and anti-GAL4 was used to immunoblot GAL4-Dnmt3a. Expression levels of the different proteins in the inputs were verified by Western blotting using anti-GAL4 or anti-HA antibodies (Input controls). (C) Myc coimmunoprecipitates with Dnmt3a from untransfected cells. Left panel: HeLa nuclear extracts were immunoprecipitated using anti-Dnmt3a, anti-CREB1 (used as negative control) or the beads only. Precipitated were blotted with anti-Myc antibody. Bottom: the presence of Dnmt3a or CREB1 in immunoprecipitates was visualized by Western blotting using anti-Dnmt3a or anti-CREB1, respectively. Right panel: reverse endogenous coimmunoprecipitation of Dnmt3a with Myc. (D) Endogenous Myc purifies DNA methyltransferase activity from nuclear extracts. HeLa nuclear extracts were immunoprecipitated with either a specific antibody against Myc (lane 2) or an irrelevant antibody (PLZF; lane 1). After washing, the immune complexes were tested for DNA methyltransferase activity. Activity is given as c.p.m. of radiolabelled methyl groups from S-adenosyl-L-[methyl-³H]-methionine incorporated into an oligonucleotide substrate. (E) Myc-associated Dnmt activity is provided by Dnmt3a. DNA methyltransferase assay was performed as in (D) using immobilized-GST-Myc 1–204 and bacterially expressed and active Dnmt3a.

Myc recruits Dnmt3a to the p21Cip1 promoter. (A) ChIPs from overexpressed cells show that binding of Dnmt3a to the p21Cip1 promoter depends on Myc. 293 cells (one 6 cm dish) were transfected with p21Cip1 promoter-Luc and Dnmt3a in the presence (lanes 1–4) or absence of HA-tagged Myc (lanes 5–8). The crosslinked chromatin was then immunoprecipitated as indicated. (The irrelevant antibody is against GFP.) The purified DNA was then amplified by PCR. Note that given the low amount of transfected 293 cells used, endogenous Myc is not detectable on p21Cip1.(B) ChIPs in untransfected c-myc+/+ and c-myc−/− show Myc dependence for Dnmt3a recruitment to p21Cip1 promoter in vivo. Crosslinked chromatin from one 15 cm dish of c-myc+/+ (lanes 1–4) or c-myc−/− (lanes 5–8) were immunoprecipitated with the indicated antibodies (the irrelevant antibody was against HA) or the beads only. (C) ChIPs performed in c-myc+/+ cells show that Dnmt3a does not bind the Myc-activated E-box genes ODC and NM23-H2.

DNA methylation is required for Myc-mediated repression of p21Cip1. (A) Inhibition of DNA methyltransferase activity relieves repression by Myc. U2OS were transfected with p21Cip1-Luc reporter and Myc as indicated and treated or not with the demethylating agent 5-AZC (2 μM). (B) Inhibition of p21Cip1 by DNA methylation depends on the presence of Myc. c-myc+/+, c-myc−/− and ‘c-myc−/− +Myc' cells were treated or not with the inhibitor 5-AZC (5 μM). RNA was then isolated and equal amounts were analysed by RT–PCR for the expression of p21Cip1 and GAPDH (used as a negative control). (C) Bisulphite genomic sequencing shows that the p21Cip1 proximal promoter is differentially methylated at specific CpGs in c-myc+/+ versus c-myc−/− cells. Left: several CpGs (numbered 1–5) showed consistent differential methylation in c-myc+/+ versus c-myc−/− cells (CpGs situated outside this cluster region did not show significant difference in methylation status; data not shown). Reintroduction of Myc into c-myc−/− cells led to a gain in p21Cip1 methylation. Each row of circles represents a single sequenced cloned and each circle represents a single CpG. Right: Chromatogram of one sequenced clone derived from bisulphite-treated c-myc+/+, c-myc−/− or ‘c-myc−/− +Myc' cells. Underlined sequences show differential methylation between the three cell types; unmethylated C converted to T (italic) upon bisulphite treatment. (D) U2OS cells show methylation of the p21Cip1 proximal promoter after bisulphite sequencing. Top: representation of the methylated CpG sites in human p21 that are homologous in rat p21 promoter.