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. 2002 Sep 1;30(17):3831-8.
doi: 10.1093/nar/gkf509.

Dnmt3L is a transcriptional repressor that recruits histone deacetylase

Rachel Deplus  1 , Carmen Brenner, Wendy A Burgers, Pascale Putmans, Tony Kouzarides, Yvan de Launoit, François Fuks
Affiliations

Dnmt3L is a transcriptional repressor that recruits histone deacetylase

Rachel Deplus et al. Nucleic Acids Res. .

Abstract

The Dnmt3L protein belongs to the Dnmt3 family of DNA methyltransferases by virtue of its sequence homology in the plant homeodomain (PHD)-like motif. Dnmt3L is essential for the establishment of maternal genomic imprints and, given its lack of key methyltransferase motifs, is more likely to act as a regulator of methylation rather than as an enzyme that methylates DNA. Here, we show that Dnmt3L, like Dnmt3a and Dnmt3b, interacts both in vitro and in vivo with the histone deacetylase HDAC1. Consistent with the binding to a deacetylase, Dnmt3L purifies histone deacetylase activity from nuclear extracts. We find that Dnmt3L can repress transcription and that this repression is dependent on HDAC1 and is relieved by treatment with the HDAC inhibitor trichostatin A. Binding of Dnmt3L to HDAC1 as well as its repressive function require the PHD-like motif. Our results indicate that Dnmt3L plays a role in transcriptional regulation and that recruitment of the HDAC repressive machinery is a shared and conserved feature of the Dnmt3 family. The fact that, despite the absence of a methyltransferase domain, Dnmt3L retains the capacity to contact deacetylase further substantiates the notion that the Dnmts can repress transcription independently of their methylating activities.

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Figures

Figure 1
Figure 1
. Dnmt3L interacts with HDAC1 in vitro. (A) Schematic representation of the Dnmt3 family members. The conserved N-terminal cysteine-rich region, the PHD-like motif, characteristic of the Dnmt3 family is shown. The C-terminal domain is the catalytic methyltransferase domain present in Dnmt3a and Dnmt3b whereas the key catalytic motifs are absent in Dnmt3L. Numbers indicate amino acid residues. (B) Dnmt3L binds the histone deacetylase HDAC1 in GST pull-down experiments. Full-length HDAC1 was IVT and 35S-radiolabeled, incubated with equivalent amounts of GST (lane 2), GST fusion proteins of Dnmt3L 1–387 (full-length; lane 3), residues 53–145 (containing the PHD-like motif; lane 4) or Dnmt3L 1–387 Δ53–145 (a mutated version that lacks the PHD-like motif; lane 5). GST 1–387 Δ53–145 (lane 5) was tested in a separate experiment than the other constructs (lanes 1–4). Molecular weight in kDa is indicated on the right. The bound IVT HDAC1 is indicated by an arrow on the left. Lane 1, 35S-radiolabeled HDAC1 input (10%). (C) Dnmt3L 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 indicated GST–HDAC1 fusions were tested in GST pull-down experiments using IVT full-length Dnmt3L (lower panel, lanes 3–7). Lane 1, 35S-radiolabeled Dnmt3L input (10%).
Figure 1
Figure 1
. Dnmt3L interacts with HDAC1 in vitro. (A) Schematic representation of the Dnmt3 family members. The conserved N-terminal cysteine-rich region, the PHD-like motif, characteristic of the Dnmt3 family is shown. The C-terminal domain is the catalytic methyltransferase domain present in Dnmt3a and Dnmt3b whereas the key catalytic motifs are absent in Dnmt3L. Numbers indicate amino acid residues. (B) Dnmt3L binds the histone deacetylase HDAC1 in GST pull-down experiments. Full-length HDAC1 was IVT and 35S-radiolabeled, incubated with equivalent amounts of GST (lane 2), GST fusion proteins of Dnmt3L 1–387 (full-length; lane 3), residues 53–145 (containing the PHD-like motif; lane 4) or Dnmt3L 1–387 Δ53–145 (a mutated version that lacks the PHD-like motif; lane 5). GST 1–387 Δ53–145 (lane 5) was tested in a separate experiment than the other constructs (lanes 1–4). Molecular weight in kDa is indicated on the right. The bound IVT HDAC1 is indicated by an arrow on the left. Lane 1, 35S-radiolabeled HDAC1 input (10%). (C) Dnmt3L 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 indicated GST–HDAC1 fusions were tested in GST pull-down experiments using IVT full-length Dnmt3L (lower panel, lanes 3–7). Lane 1, 35S-radiolabeled Dnmt3L input (10%).
Figure 2
Figure 2
. Dnmt3L co-immunoprecipitates with HDAC1 and purifies HDAC activity in vitro and in vivo using its PHD-like finger. (A) 293 cells were transiently transfected as indicated (+) with 3 µg of either pcDNA3GAL4 Dnmt3L 1–387 or pcDNA3GAL4 Dnmt3L 53–145 (encompassing the PHD-like motif), together with 3 µg of pcDNA HDAC1-F (expressing Flag-tagged HDAC1). Whole cell extracts were then precipitated with anti-GAL4 antibody (5C1) and the presence of HDAC1-F in the immunoprecipitates was visualized by western blot analysis using anti-Flag antibody (M2). HDAC1-F is indicated by an arrow on the right. (B) Dnmt3L associates with deacetylase activity in vitro. Equivalent amounts of GST (lane 1) or GST fusion proteins (lanes 2–5) were incubated with HeLa nuclear extracts. After washing, the complexes were tested for histone deacetylase activity. Histone deacetylase activity is given as radioactivity (c.p.m.) released from an 3H-labeled acetylated histone H4 peptide. GST–Rb fusion protein (lane 5) was used as a positive control for the assay. The results shown are the average of at least two independent experiments with error bars displaying standard deviations. (C) Dnmt3L purifies deacetylase activity from transfected cells. Cells were transiently transfected with equal amounts of the indicated GAL4-tagged constructs. Whole cell extracts were immunoprecipited with anti-GAL4 antibody. After washing, the immune complexes were tested for histone deacetylase activity. Histone deacetylase activity is given as radioactivity (c.p.m.) released from an 3H-labeled acetylated histone H4 peptide. We used Gal4–Rb fusion protein as a positive control for the assay.
Figure 2
Figure 2
. Dnmt3L co-immunoprecipitates with HDAC1 and purifies HDAC activity in vitro and in vivo using its PHD-like finger. (A) 293 cells were transiently transfected as indicated (+) with 3 µg of either pcDNA3GAL4 Dnmt3L 1–387 or pcDNA3GAL4 Dnmt3L 53–145 (encompassing the PHD-like motif), together with 3 µg of pcDNA HDAC1-F (expressing Flag-tagged HDAC1). Whole cell extracts were then precipitated with anti-GAL4 antibody (5C1) and the presence of HDAC1-F in the immunoprecipitates was visualized by western blot analysis using anti-Flag antibody (M2). HDAC1-F is indicated by an arrow on the right. (B) Dnmt3L associates with deacetylase activity in vitro. Equivalent amounts of GST (lane 1) or GST fusion proteins (lanes 2–5) were incubated with HeLa nuclear extracts. After washing, the complexes were tested for histone deacetylase activity. Histone deacetylase activity is given as radioactivity (c.p.m.) released from an 3H-labeled acetylated histone H4 peptide. GST–Rb fusion protein (lane 5) was used as a positive control for the assay. The results shown are the average of at least two independent experiments with error bars displaying standard deviations. (C) Dnmt3L purifies deacetylase activity from transfected cells. Cells were transiently transfected with equal amounts of the indicated GAL4-tagged constructs. Whole cell extracts were immunoprecipited with anti-GAL4 antibody. After washing, the immune complexes were tested for histone deacetylase activity. Histone deacetylase activity is given as radioactivity (c.p.m.) released from an 3H-labeled acetylated histone H4 peptide. We used Gal4–Rb fusion protein as a positive control for the assay.
Figure 3
Figure 3
. Dnmt3L represses transcription through its PHD-like motif when fused to the GAL4 DBD. (A) Schematic representation of the reporter and the effector constructs used. The reporter construct 4XGAL4-TK-Luc contains four copies of GAL4 sites upstream of the thymidine kinase promoter driving expression of the luciferase gene. The effectors used are also indicated and consist of GAL4 DBD alone [GAL4(DBD)], GAL4 DBD fused to either full-length Dnmt3L (residues 1–387) or Dnmt3L PHD (residues 53–145). (B and C) U2OS cells were transiently transfected with 250 ng of 4XGAL4-TK-luc reporter with increasing amounts (50–250 ng) of GAL4-Dnmt3L 1–387 (B) or GAL4-Dnmt3L 53–145 (C). Whole cell extracts were used in luciferase assays. The basal activity of the reporter is normalized to a value of 100%. Transfection efficiencies were normalized using β-galactosidase activity. The results are the average of at least four independent transfections done in duplicate with error bars displaying standard deviations.
Figure 4
Figure 4
. Dnmt3L requires the deacetylase HDAC1 for transcriptional silencing and represses transcription in a TSA-sensitive manner. (A) Schematic representation of the reporter and the effector constructs used. The reporter construct 4XGAL4-TK-Luc contains four copies of GAL4 sites upstream of the thymidine kinase promoter driving expression of the luciferase gene. Also indicated are effector constructs expressing only the GAL4 DBD [GAL4(DBD)], the GAL4 DBD fused to the PHD of Dnmt3L (residues 53–145) and Flag-tagged full-length HDAC1 (HDAC1-F). (B) U2OS cells were transiently transfected with 250 ng of the 4XGAL4-TK-luc reporter and, as indicated, with 50 ng of GAL4 Dnmt3L 53–145 and/or 500 ng of HDAC1-F. Cells were then harvested and assayed for luciferase. The basal activity of the reporter is normalised to a value of 100%. Transfection efficiencies were normalized using β-galactosidase activity. The results shown are the average of at least two independent experiments with error bars displaying standard deviations. (C) Dnmt3L-mediated repression is sensitive to TSA. U2OS cells were transfected with 250 ng of the 5XGAL4-SV40-CAT reporter and 50 ng of GAL4 Dnmt3L 53–145. Sixteen hours after transfection, cells were treated (lanes 3 and 4) or not (lanes 1 and 2) with the HDAC inhibitor TSA (200 mM). The basal activity of the reporter is normalised to a value of 100%. Transfection efficiencies were normalized using β-galactosidase activity. The results shown are the average of at least two independent experiments with error bars displaying standard deviations.
Figure 4
Figure 4
. Dnmt3L requires the deacetylase HDAC1 for transcriptional silencing and represses transcription in a TSA-sensitive manner. (A) Schematic representation of the reporter and the effector constructs used. The reporter construct 4XGAL4-TK-Luc contains four copies of GAL4 sites upstream of the thymidine kinase promoter driving expression of the luciferase gene. Also indicated are effector constructs expressing only the GAL4 DBD [GAL4(DBD)], the GAL4 DBD fused to the PHD of Dnmt3L (residues 53–145) and Flag-tagged full-length HDAC1 (HDAC1-F). (B) U2OS cells were transiently transfected with 250 ng of the 4XGAL4-TK-luc reporter and, as indicated, with 50 ng of GAL4 Dnmt3L 53–145 and/or 500 ng of HDAC1-F. Cells were then harvested and assayed for luciferase. The basal activity of the reporter is normalised to a value of 100%. Transfection efficiencies were normalized using β-galactosidase activity. The results shown are the average of at least two independent experiments with error bars displaying standard deviations. (C) Dnmt3L-mediated repression is sensitive to TSA. U2OS cells were transfected with 250 ng of the 5XGAL4-SV40-CAT reporter and 50 ng of GAL4 Dnmt3L 53–145. Sixteen hours after transfection, cells were treated (lanes 3 and 4) or not (lanes 1 and 2) with the HDAC inhibitor TSA (200 mM). The basal activity of the reporter is normalised to a value of 100%. Transfection efficiencies were normalized using β-galactosidase activity. The results shown are the average of at least two independent experiments with error bars displaying standard deviations.
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References

    1. Jones P.A. and Takai,D. (2001) Cancer epigenetics comes of age. Science, 293, 1068–1070. - PubMed
    1. Bird A. (2002) DNA methylation patterns and epigenetic memory. Genes Dev., 16, 6–21. - PubMed
    1. Jaenisch R. (1997) DNA methylation and imprinting: why bother? Trends Genet., 13, 323–329. - PubMed
    1. Surani M.A. (2001) Reprogramming of genome function through epigenetic inheritance. Nature, 414, 122–128. - PubMed
    1. Bestor T.H. (2000) The DNA methyltransferases of mammals. Hum. Mol. Genet., 9, 2395–2402. - PubMed

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