Figure 3.

The SUV39H1 H3-K9 methylase binds to Dnmt3a and Dnmt1. (A) SUV39H1 associates with DNA methyltransferase activity from nuclear extracts. Equivalent amounts of MBP (lane 1) or MBP–SUV39H1 fusion proteins (lane 2) were incubated with HeLa nuclear extract, washed and assayed for DNA methyltransferase activity. Activity is given as c.p.m. of S-adenosyl-l-[methyl-³H]methionine incorporated into a hemi-methylated oligonucleotide substrate. Incorporation of radioactivity was determined by liquid scintillation counting. (B) SUV39H1 binds in vitro to Dnmt3a. GST or GST–SUV39H1 were incubated with in vitro translated [³⁵S]Dnmt3a and subjected to GST pull-downs. Lane 1 shows 10% of the Dnmt3a input. (C) The PHD-like motif of Dnmt3a mediates in vitro the association with SUV39H1. GST or GST–Dnmt3a (encompassing or containing its PHD-like motif) were incubated with in vitro translated [³⁵S]SUV39H1 and subjected to GST pull-downs. Lane 1 shows 30% of the SUV39H1 input. (D) SUV39H1 interacts in vitro with Dnmt1. GST pull-downs were done using GST or GST–SUV39H1 incubated with in vitro translated [³⁵S]Dnmt1. Lane 1, [³⁵S]Dnmt1 input (10%). (E) SUV39H1 co- immunoprecipitates with Dnmt3a and Dnmt1. U20S cells were transfected with the indicated plasmids. Whole cell extracts were precipitated with Myc antibody and the presence of GAL4–SUV39H1 in the immunoprecipitate was detected by western blot using anti-GAL4 antibody. The unrelated Myc–Axin was used as a negative control and was tested in a separate experiment than the other constructs (lanes 7 and 8). The input lane contains 10% of total extract. (F) Dnmt3a co-immunoprecipitates with HA–SUVAR39H1 using its PHD-like finger. GAL4–Dnmt3a was precipitated using a GAL4 antibody and the presence of HA–SUV39H1 was detected by western blot using anti-HA antibody. Lane 1, 20% of input.