PMC

The Dnmt3a-associated HMTases methylate primarily H3-K9 and to a lesser extent H3-K4. Histone H3 labelled by Dnmt3a-associated HMTase activities were subjected to Edman degradation and each fraction was then counted by liquid scintillation counting.

Endogenous Dnmt3a purifies histone methyltransferase activity specific for H3. (A) Endogenous Dnmt3a associates with H3 methyltransferase activity. HeLa nuclear extracts were immunoprecipitated with either antibodies against Dnmt3a (164a or 164b; lanes 1 and 3) or their respective preimmune sera (lanes 2 and 4). After washing, the immune complexes where tested for histone methyltransferase activity using bulk histones as substrate. The reaction products were then analysed by SDS–PAGE followed by western blotting and autoradiography. The radiolabelled H3 is indicated by an arrow on the right. (B) Dnmt3a binds H3 methylase activity through its conserved PDH-like motif. On top is depicted a schematic representation of Dnmt3a with the different domains that were fused to GST. Numbers indicate amino acid residues. HeLa nuclear extracts and equivalent amounts of the indicated GST fusions bound to Sepharose beads were incubated with bulk histones as substrate. The beads were then washed and assayed for histone methyltransferase activity.

HP1β binds directly to Dnmt1 and Dnmt3a and interacts in vitro and in vivo with DNA methyltransferase activity. (A) HP1β directly contacts Dnmt1. GST, GST–Rb or the indicated GST–Dnmt1 fusions were incubated with histidine-tagged full-length HP1β. Direct binding was visualised by western blotting for HP1β using anti-His antibody (Roche). Input is 10% of total His-tagged HP1β in each assay. (B) HP1β binds directly to Dnmt3a. GST, GST–Rb or GST–Dnmt3a containing its PHD-like motif were incubated with histidine-tagged HP1β chromo-shadow domain (His-HP1β csd, residues 104–185). Western blotting was then performed using anti-His antibody. Input is 10% of total His-tagged HP1β chromo-shadow domain in each assay. (C) HP1β binds in vitro with DNA methyltransferase activity. Equivalent amounts of GST (lane 1) or GST–HP1β fusion proteins (lane 2) were incubated with HeLa nuclear extract. After extensive washing, the beads were 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. (D) Endogenous HP1β purifies DNA methyltransferase activity from nuclear extracts. HeLa nuclear extracts were immunoprecipitated with preimmune serum (lane 1) or an anti-HP1β antibody (lane 2). Immunoprecipitates were then assayed for DNA methyltransferase activity.

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.