Figure 1.

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.