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SETDB1 is involved in postembryonic DNA methylation and gene silencing in Drosophila.

Gou D, Rubalcava M, Sauer S, Mora-Bermúdez F, Erdjument-Bromage H, Tempst P, Kremmer E, Sauer F - PLoS ONE (2010)

Bottom Line: We found that dSETDB1 tri-methylates H3-K9 and binds methylated CpA motifs.Tri-methylation of H3-K9 by dSETDB1 mediates recruitment of DNA methyltransferase 2 (Dnmt2) and Su(var)205, the Drosophila ortholog of mammalian "Heterochromatin Protein 1", to target genes for dSETDB1.By enlisting Dnmt2 and Su(var)205, dSETDB1 triggers DNA methylation and silencing of genes and retrotransposons in Drosophila cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of California Riverside, Riverside, California, United States of America.

ABSTRACT
DNA methylation is fundamental for the stability and activity of genomes. Drosophila melanogaster and vertebrates establish a global DNA methylation pattern of their genome during early embryogenesis. Large-scale analyses of DNA methylation patterns have uncovered revealed that DNA methylation patterns are dynamic rather than static and change in a gene-specific fashion during development and in diseased cells. However, the factors and mechanisms involved in dynamic, postembryonic DNA methylation remain unclear. Methylation of lysine 9 in histone H3 (H3-K9) by members of the Su(var)3-9 family of histone methyltransferases (HMTs) triggers embryonic DNA methylation in Arthropods and Chordates. Here, we demonstrate that Drosophila SETDB1 (dSETDB1) can mediate DNA methylation and silencing of genes and retrotransposons. We found that dSETDB1 tri-methylates H3-K9 and binds methylated CpA motifs. Tri-methylation of H3-K9 by dSETDB1 mediates recruitment of DNA methyltransferase 2 (Dnmt2) and Su(var)205, the Drosophila ortholog of mammalian "Heterochromatin Protein 1", to target genes for dSETDB1. By enlisting Dnmt2 and Su(var)205, dSETDB1 triggers DNA methylation and silencing of genes and retrotransposons in Drosophila cells. DSETDB1 is involved in postembryonic DNA methylation and silencing of Rt1b{} retrotransposons and the tumor suppressor gene retinoblastoma family protein 1 (Rb) in imaginal discs. Collectively, our findings implicate dSETDB1 in postembryonic DNA methylation, provide a model for silencing of the tumor suppressor Rb, and uncover a role for cell type-specific DNA methylation in Drosophila development.

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Dnmt2 and Su(var)205 mediate dSETDB1-dependent DNA methylation and silencing of Antp, CG2316 and Rt1b{}799.(A) (Left panel) Digital images of ethidium bromide-stained agarose gels showing reaction products of PCR assays detecting Antp, CG2316, and Rt1b{} transcription in total RNA isolated from S2 cells, S2 cells expressing dSETDB1, and S2 cells transiently expressing dSETDB1 in the presence of small interfering RNA (siRNA) targeting Dnmt2 (Dnmt2-RNAi) or control siRNA targeting human GAPDH (mock-RNAi). (Right panel) PCR assays as in (A) except that S2 cells were treated with siRNA targeting Su(var)205 [Su(var)205 RNAi]. (B) Digital images of ethidium bromide-stained agarose gels detecting the presence of Antp, CG2316, and Rt1b{} in DNA pools generated by ChIP with chromatin isolated from cells described in (A; left panel) Chromatin was immunoprecipitated with the antibodies and controls described in Figure 2C (C) ChIP assays as described in (C) except that chromatin was isolated from cells described in (A; right panel). (D) Digital images of ethidium bromide-stained agarose gels showing the reaction products of methylation-sensitive restriction analyses with genomic DNA isolated from cells described in (A,B). Assays were performed as described (Figure 5D) except that PCR assays detected the presence of Antp, CG2316, and Rt1b{'} in treated DNA pools.
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pone-0010581-g007: Dnmt2 and Su(var)205 mediate dSETDB1-dependent DNA methylation and silencing of Antp, CG2316 and Rt1b{}799.(A) (Left panel) Digital images of ethidium bromide-stained agarose gels showing reaction products of PCR assays detecting Antp, CG2316, and Rt1b{} transcription in total RNA isolated from S2 cells, S2 cells expressing dSETDB1, and S2 cells transiently expressing dSETDB1 in the presence of small interfering RNA (siRNA) targeting Dnmt2 (Dnmt2-RNAi) or control siRNA targeting human GAPDH (mock-RNAi). (Right panel) PCR assays as in (A) except that S2 cells were treated with siRNA targeting Su(var)205 [Su(var)205 RNAi]. (B) Digital images of ethidium bromide-stained agarose gels detecting the presence of Antp, CG2316, and Rt1b{} in DNA pools generated by ChIP with chromatin isolated from cells described in (A; left panel) Chromatin was immunoprecipitated with the antibodies and controls described in Figure 2C (C) ChIP assays as described in (C) except that chromatin was isolated from cells described in (A; right panel). (D) Digital images of ethidium bromide-stained agarose gels showing the reaction products of methylation-sensitive restriction analyses with genomic DNA isolated from cells described in (A,B). Assays were performed as described (Figure 5D) except that PCR assays detected the presence of Antp, CG2316, and Rt1b{'} in treated DNA pools.

Mentions: The association of HP1 with H3-K9 is a hallmark of silencing in heterochromatin and euchromatin [34]. The interaction of HP1 and Dnmts in vertebrates raised the possibility that Su(var)205 contributes to Rb silencing by recruiting Dnmt2 [45]. In ChIP assays, Su(var)205 was detected at the transcriptionally silent Antp, CG2316, and Rt1b{}779 (Figure 4B), the tetO-tk-luc reporter (Figure 2C) and Rb (Figure 5A). In contrast, Su(var)205 and Dnmt2 were not detected at dSETDB1 target genes in the absence of dSETDB1-mediated methylation of H3-K9, which suggests that the association of Su(var)205 with (me3)H3-K9 mediates recruitment of Dnmt2 to Rb (Figures 2C, 4B, and 5C). Su(var)205 interacted with Dnmt2 but not dSETDB1 in vitro and in vivo, which suggests that Su(var)205 is involved in recruiting Dnmt2 to target genes for dSETDB1 (Figure S22). To test this, we asked whether destruction of Dnmt2 and Su(var)205 through RNA interference (RNAi) affects dSETDB1-mediated silencing (Figure S23). Knockdown of Dnmt2 or Su(var)205 attenuated dSETDB1-mediated silencing of Rb (Figure 6A,B; Figure S24), Antp, CG2316, and Rt1b{} retrotransposons (Figure 7A; Figure S25). Knockdown of Dnmt2 attenuated DNA methylation of Rb (Figure 6C,D; Figures S26-S28), Antp, CG2316, and Rtb{}799 (Figure 7B,D; Figures S28-S30), whereas knockdown of Su(var)205 prevented recruitment of Dnmt2 and DNA methylation at the Rb locus (Figure 6C, right panel, Figure 6D; Figure S26-28), and the Antp, CG2316, and Rtb{}799 loci (Figure 7C; Figure S31). Collectively, these results reveal that dSETDB1 cooperates with Su(var)205 and Dnmt2 in DNA methylation and silencing of genes and Rt1b{} retrotransposons.


SETDB1 is involved in postembryonic DNA methylation and gene silencing in Drosophila.

Gou D, Rubalcava M, Sauer S, Mora-Bermúdez F, Erdjument-Bromage H, Tempst P, Kremmer E, Sauer F - PLoS ONE (2010)

Dnmt2 and Su(var)205 mediate dSETDB1-dependent DNA methylation and silencing of Antp, CG2316 and Rt1b{}799.(A) (Left panel) Digital images of ethidium bromide-stained agarose gels showing reaction products of PCR assays detecting Antp, CG2316, and Rt1b{} transcription in total RNA isolated from S2 cells, S2 cells expressing dSETDB1, and S2 cells transiently expressing dSETDB1 in the presence of small interfering RNA (siRNA) targeting Dnmt2 (Dnmt2-RNAi) or control siRNA targeting human GAPDH (mock-RNAi). (Right panel) PCR assays as in (A) except that S2 cells were treated with siRNA targeting Su(var)205 [Su(var)205 RNAi]. (B) Digital images of ethidium bromide-stained agarose gels detecting the presence of Antp, CG2316, and Rt1b{} in DNA pools generated by ChIP with chromatin isolated from cells described in (A; left panel) Chromatin was immunoprecipitated with the antibodies and controls described in Figure 2C (C) ChIP assays as described in (C) except that chromatin was isolated from cells described in (A; right panel). (D) Digital images of ethidium bromide-stained agarose gels showing the reaction products of methylation-sensitive restriction analyses with genomic DNA isolated from cells described in (A,B). Assays were performed as described (Figure 5D) except that PCR assays detected the presence of Antp, CG2316, and Rt1b{'} in treated DNA pools.
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pone-0010581-g007: Dnmt2 and Su(var)205 mediate dSETDB1-dependent DNA methylation and silencing of Antp, CG2316 and Rt1b{}799.(A) (Left panel) Digital images of ethidium bromide-stained agarose gels showing reaction products of PCR assays detecting Antp, CG2316, and Rt1b{} transcription in total RNA isolated from S2 cells, S2 cells expressing dSETDB1, and S2 cells transiently expressing dSETDB1 in the presence of small interfering RNA (siRNA) targeting Dnmt2 (Dnmt2-RNAi) or control siRNA targeting human GAPDH (mock-RNAi). (Right panel) PCR assays as in (A) except that S2 cells were treated with siRNA targeting Su(var)205 [Su(var)205 RNAi]. (B) Digital images of ethidium bromide-stained agarose gels detecting the presence of Antp, CG2316, and Rt1b{} in DNA pools generated by ChIP with chromatin isolated from cells described in (A; left panel) Chromatin was immunoprecipitated with the antibodies and controls described in Figure 2C (C) ChIP assays as described in (C) except that chromatin was isolated from cells described in (A; right panel). (D) Digital images of ethidium bromide-stained agarose gels showing the reaction products of methylation-sensitive restriction analyses with genomic DNA isolated from cells described in (A,B). Assays were performed as described (Figure 5D) except that PCR assays detected the presence of Antp, CG2316, and Rt1b{'} in treated DNA pools.
Mentions: The association of HP1 with H3-K9 is a hallmark of silencing in heterochromatin and euchromatin [34]. The interaction of HP1 and Dnmts in vertebrates raised the possibility that Su(var)205 contributes to Rb silencing by recruiting Dnmt2 [45]. In ChIP assays, Su(var)205 was detected at the transcriptionally silent Antp, CG2316, and Rt1b{}779 (Figure 4B), the tetO-tk-luc reporter (Figure 2C) and Rb (Figure 5A). In contrast, Su(var)205 and Dnmt2 were not detected at dSETDB1 target genes in the absence of dSETDB1-mediated methylation of H3-K9, which suggests that the association of Su(var)205 with (me3)H3-K9 mediates recruitment of Dnmt2 to Rb (Figures 2C, 4B, and 5C). Su(var)205 interacted with Dnmt2 but not dSETDB1 in vitro and in vivo, which suggests that Su(var)205 is involved in recruiting Dnmt2 to target genes for dSETDB1 (Figure S22). To test this, we asked whether destruction of Dnmt2 and Su(var)205 through RNA interference (RNAi) affects dSETDB1-mediated silencing (Figure S23). Knockdown of Dnmt2 or Su(var)205 attenuated dSETDB1-mediated silencing of Rb (Figure 6A,B; Figure S24), Antp, CG2316, and Rt1b{} retrotransposons (Figure 7A; Figure S25). Knockdown of Dnmt2 attenuated DNA methylation of Rb (Figure 6C,D; Figures S26-S28), Antp, CG2316, and Rtb{}799 (Figure 7B,D; Figures S28-S30), whereas knockdown of Su(var)205 prevented recruitment of Dnmt2 and DNA methylation at the Rb locus (Figure 6C, right panel, Figure 6D; Figure S26-28), and the Antp, CG2316, and Rtb{}799 loci (Figure 7C; Figure S31). Collectively, these results reveal that dSETDB1 cooperates with Su(var)205 and Dnmt2 in DNA methylation and silencing of genes and Rt1b{} retrotransposons.

Bottom Line: We found that dSETDB1 tri-methylates H3-K9 and binds methylated CpA motifs.Tri-methylation of H3-K9 by dSETDB1 mediates recruitment of DNA methyltransferase 2 (Dnmt2) and Su(var)205, the Drosophila ortholog of mammalian "Heterochromatin Protein 1", to target genes for dSETDB1.By enlisting Dnmt2 and Su(var)205, dSETDB1 triggers DNA methylation and silencing of genes and retrotransposons in Drosophila cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of California Riverside, Riverside, California, United States of America.

ABSTRACT
DNA methylation is fundamental for the stability and activity of genomes. Drosophila melanogaster and vertebrates establish a global DNA methylation pattern of their genome during early embryogenesis. Large-scale analyses of DNA methylation patterns have uncovered revealed that DNA methylation patterns are dynamic rather than static and change in a gene-specific fashion during development and in diseased cells. However, the factors and mechanisms involved in dynamic, postembryonic DNA methylation remain unclear. Methylation of lysine 9 in histone H3 (H3-K9) by members of the Su(var)3-9 family of histone methyltransferases (HMTs) triggers embryonic DNA methylation in Arthropods and Chordates. Here, we demonstrate that Drosophila SETDB1 (dSETDB1) can mediate DNA methylation and silencing of genes and retrotransposons. We found that dSETDB1 tri-methylates H3-K9 and binds methylated CpA motifs. Tri-methylation of H3-K9 by dSETDB1 mediates recruitment of DNA methyltransferase 2 (Dnmt2) and Su(var)205, the Drosophila ortholog of mammalian "Heterochromatin Protein 1", to target genes for dSETDB1. By enlisting Dnmt2 and Su(var)205, dSETDB1 triggers DNA methylation and silencing of genes and retrotransposons in Drosophila cells. DSETDB1 is involved in postembryonic DNA methylation and silencing of Rt1b{} retrotransposons and the tumor suppressor gene retinoblastoma family protein 1 (Rb) in imaginal discs. Collectively, our findings implicate dSETDB1 in postembryonic DNA methylation, provide a model for silencing of the tumor suppressor Rb, and uncover a role for cell type-specific DNA methylation in Drosophila development.

Show MeSH
Related in: MedlinePlus