Fig. 1. Dnmt3a binds the sequence-specific transcriptional repressor RP58 in a yeast two-hybrid screen. (A) Schematic representation of Dnmt3a. The conserved cysteine-rich region that is closely related to the PHD-like motif of ATRX (residues 490–582), used as bait in the yeast two-hybrid screen, is indicated. The C-terminal methyltransferase domain is shown (numbers indicate amino acid residues). (B) RP58 is a specific Dnmt3a-binding protein in yeast. L40 yeast cells were co-transformed with the plasmids indicated and plated on to –THULL plates containing 35 mM 3-AT in order to examine the specificity of the interaction between bait and prey proteins. (C) Schematic representation of RP58. The N-terminal POZ domain and C-terminal Kruppel-type zinc finger motifs are shown. The region found to interact with Dnmt3a in the yeast two-hybrid screen (residues 310–427) is indicated.

Fig. 5. Dnmt3 proteins associate with HDAC activity. (A) Endogenous Dnmt3a and Dnmt3b associate with HDAC activity. HeLa nuclear extracts were immunoprecipitated with either preimmune serum to Dnmt3a or Dnmt3b [lanes 1, P.I. (3a), and 3, P.I. (3b)], anti-Dnmt3a and anti-Dnmt3b sera (lanes 2 and 4) or irrelevant antibodies for ER and GAL4 (lanes 5 and 6, respectively). After washing, the immune complexes were tested for HDAC activity. HDAC activity is given as radioactivity (c.p.m.) of [³H]acetate released from an acetylated histone H4 peptide. IP ab, immunoprecipitation antibody. (B) Dnmt3a 490–582 associates with HDAC activity. Equivalent amounts of GST (lane 1) or GST–Dnmt3a 490–582 (lane 2) bound to Sepharose beads were incubated with HeLa nuclear. The beads were then washed and assayed for HDAC activity as described above.

Fig. 3. Dnmt3a cooperates with RP58 to repress transcription in a methylation-independent manner. (A) A schematic representation of the reporter and effector constructs used. The reporter construct BS–RP58/SV40 contains 10 copies of the RP58 DNA-binding site upstream of the SV40 promoter driving expression of the luciferase gene. Constructs expressing full-length RP58, full-length wild-type Dnmt3a (Dnmt3a-wt) and catalytically inactive Dnmt3a (Dnmt3a-mut) are indicated. Dnmt3a-mut contains a single mutation in its methyltransferase catalytic domain that has been shown in vivo to abolish its enzymatic activity (Hsieh, 1999). (B) Dnmt3a is recruited to a promoter via RP58 to repress transcription independently of its methyl transferase activity. 293T cells were transiently transfected with 1 µg of the BS–RP58/SV40 reporter and 50 ng of RP58 in the absence (–) or presence (+) of increasing amounts of Dnmt3a-wt (wt) or Dnmt3a-mut (mut) (50 ng, lanes 3 and 5, and 200 ng, lanes 4 and 6). Absolute luciferase activity is given in rlu. (C and D) RP58 represses transcription independently of methylation. Increasing amounts of RP58 (100, 500 and 1000 ng) were transfected into 293T cells together with 1 µg of unmethylated (C) or SssI-in vitro methylated (D) BS–RP58/SV40 reporter.

Fig. 6. The ATRX-homology region of Dnmt3a actively represses transcription in a TSA-sensitive manner. (A) A schematic representation of the reporter and effector constructs used. The reporter construct 5×GAL4/SV40-CAT contains five binding sites for the yeast transcriptional activator GAL4 upstream of the SV40 early promoter driving the CAT gene. Dnmt3a 490–582, encompassing the ATRX-homology region, is fused to the GAL4 DNA-binding domain. (B) Dnmt3a represses transcription when fused to the GAL4 DNA-binding domain. U2OS cells were transiently transfected with 3 µg of the 5×GAL4/SV40-CAT reporter along with increasing amounts (5 and 10 µg) of GAL4–Dnmt3a 490–582. Whole-cell extracts were used in CAT assays and the results quantified on a phosphoimager. The activity of the reporter in the absence of GAL4 fusion is normalized to a value of 100. (C) Inhibition of HDAC activity relieves repression by Dnmt3a 490–582. U2OS cells were transiently transfected with 3 µg of the 5×GAL4/SV40-CAT reporter along with 5 µg of GAL4–Dnmt3a 490–582. About 10 h after transfection, the cells were treated (+) with the deacetylase inhibitor TSA (100 nM) for 24 h before harvesting, or left untreated (–). CAT assays were performed as above. The activity of the reporter, in the absence of TSA and GAL4 fusion, is normalized to a value of 100.

Fig. 4. Dnmt3a interacts with the histone deacetylase HDAC1 in vitro and in vivo. (A) Dnmt3a binds HDAC1 through its ATRX-homology domain in vitro. GST fusion proteins of Dnmt3a (residues 490–582 and residues 615–908) were tested for interaction with HDAC1 in a GST pull-down assay. In vitro translated and ³⁵S-labelled full-length HDAC1 (IVT HDAC1) was incubated with equivalent amounts of bacterially expressed GST (lane 2) or GST fusions of Dnmt3a (lanes 3 and 4) or, as a positive control, GST–Rb (lane 5), and analysed by SDS–PAGE. Lane 1, IVT [³⁵S]HDAC1 input (25%). (B) Dnmt3a binds specific regions of HDAC1 in vitro. The upper panel is a schematic representation of the HDAC1 deacetylase with its catalytic domain depicted by a grey box. The GST–HDAC1 fusions indicated were tested in GST pull-down experiments (lower panel, lanes 3–7), using in vitro translated full-length Dnmt3a (IVT Dnmt3a). Lane 1, IVT [³⁵S]Dnmt3a input (20%). (C) Dnmt3a co-immunoprecipitates with HDAC1 from transfected cell extracts. 293T cells were transiently transfected with 30 µg of pMT3aMyc (expressing Myc-tagged full-length mouse Dnmt3a, Myc–Dnmt3a) and/or 10 µg of pcDNA3-HDAC1-F (expressing Flag-tagged full-length HDAC1, HDAC1-F) as indicated. Whole-cell extracts were then precipitated with anti-Myc antibody (9E10) and the presence of HDAC1-F in the immuno precipitates was visualized by western blot analysis using anti-Flag antibody (M2). (D) Dnmt3a 490–582 co-immunoprecipitates with HDAC1 from transfected cell extracts. 293T cells were transiently transfected with 30 µg of pcDNA3GAL4–Dnmt3a 490–582 (expressing GAL4-tagged Dnmt3a residues 490–582) and/or 10 µg of HDAC1-F as indicated. Whole-cell extracts were then precipitated with anti-GAL4 antibody (5C1) and the presence of HDAC1-F in the immuno precipitates was visualized by western blot analysis using anti-Flag antibody (M2).

Fig. 2. Dnmt3a interacts with RP58 in vitro and in vivo. (A) Dnmt3a binds RP58 in vitro. The upper panel is a schematic representation of the GST–RP58 fusion proteins, RP58 full length (residues 1–522) and the region of RP58 isolated from the yeast two-hybrid screen (residues 310–427). The indicated GST–RP58 fusion proteins or GST alone were tested in GST pull-down experiments for binding to in vitro translated and ³⁵S-radio labelled full-length Dnmt3a (IVT Dnmt3a). The bound IVT Dnmt3a is indicated by an arrow on the left. Lane 1, IVT [³⁵S]Dnmt3a input (20%). Molecular weight in kDa is indicated on the right. (B) RP58 associates with DNA methyltransferase activity. Equivalent amounts of GST or GST–RP58 fusion proteins bound to Sepharose beads were incubated with HeLa nuclear extract, washed and assayed for methyltransferase activity. Activity is read as c.p.m. of S-adenosyl-l-[methyl-³H]methionine incorporated into an oligonucleotide substrate. Values are normalized to background controls lacking substrate. (C) Dnmt3a binds RP58 directly. GST (lane 2) or the indicated GST fusion proteins of Dnmt3a (residues 490–582, the ATRX-homology region; residues 615–908, encompassing the methyltransferase domain) or GST–Dnmt3b (residues 440–532, the conserved ATRX-homology region) (lanes 3–5) were incubated with histidine-tagged RP58 (His–RP58). Direct binding of RP58 was visualized by western blot analysis using anti-His antibody. Lane 1, His–RP58 input (50%). (D) Dnmt3a co-immunoprecipitates with RP58 310–427 from transfected cell extracts. 293T cells were transiently transfected with 25 µg of pMT3aMyc (expressing Myc-tagged full-length mouse Dnmt3a, Myc–Dnmt3a) and/or 25 µg of pcDNA3GAL4-RP58 310–427 (expressing GAL4-tagged RP58 310–427) as indicated. Whole-cell extracts were then precipitated with anti-GAL4 antibody (5C1) and the presence of Dnmt3a in the immunoprecipitates was visualized by western blot analysis using anti-Myc antibody (9E10). (E) Dnmt3a co-immunoprecipitates with full-length RP58 from transfected cell extracts. 293T cells were transiently transfected as in (D) with Myc–Dnmt3a, and/or pcDNA3GAL4-RP58 (expressing GAL4-tagged RP58 full length) as indicated. Precipitation with the anti-GAL4 antibody was followed by western blot analysis using anti-Myc antibody to detect Dnmt3a.