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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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Methylation of H3-K9 by dSETDB1 mediates transcriptional silencing.(A) Western blot analysis detecting dSETDB1 (black triangle) and tetracycline repressor (TetR)–SETDB1 (TetRSETDB1) fusion proteins (open triangle) in S2 cells transiently expressing dSETDB1, TetR, or fusion proteins of TetR with dSETDB1 or dSETDB1(H775L). Forty-eight hours after transfection, total cell extracts were prepared, separated by SDS-PAGE, and electrophoretically transferred onto nitrocellulose membrane for probing with antibody to dSETDB1. (B) Schematic representation of luciferase assays with whole-cell extracts prepared from cells described in (A). The tetO-tk-luc reporter gene contains tet operator (tetO) sequences, which are fused to the human thymidine kinase (tk) promoter driving the expression of luciferase (luc). The diagram shows the mean values calculated from data from 5 different experiments. Error bars indicate the standard error of the mean (SEM). (C) Digital images of ethidium bromide-stained agarose gels showing the reaction products of PCR assays monitoring the presence of the promoter region of the tetO-tk-luc reporter gene in DNA pools obtained by chromatin immunoprecipitation (ChIP). Chromatin was isolated from (tetO-tk-luc)-S2 cells expressing TetR, TetRdSETDB1, or TetRdSETDB1(H775L) and immunoprecipitated with antibodies to dSETDB1, Su(var)205, Dnmt2, (me3)H3-K9, and methylated cytosine (5mC), or protein-A agarose (control-A) and protein-G agarose (control-G). Input indicates the amount of target DNA present in 1% of the input chromatin.
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pone-0010581-g002: Methylation of H3-K9 by dSETDB1 mediates transcriptional silencing.(A) Western blot analysis detecting dSETDB1 (black triangle) and tetracycline repressor (TetR)–SETDB1 (TetRSETDB1) fusion proteins (open triangle) in S2 cells transiently expressing dSETDB1, TetR, or fusion proteins of TetR with dSETDB1 or dSETDB1(H775L). Forty-eight hours after transfection, total cell extracts were prepared, separated by SDS-PAGE, and electrophoretically transferred onto nitrocellulose membrane for probing with antibody to dSETDB1. (B) Schematic representation of luciferase assays with whole-cell extracts prepared from cells described in (A). The tetO-tk-luc reporter gene contains tet operator (tetO) sequences, which are fused to the human thymidine kinase (tk) promoter driving the expression of luciferase (luc). The diagram shows the mean values calculated from data from 5 different experiments. Error bars indicate the standard error of the mean (SEM). (C) Digital images of ethidium bromide-stained agarose gels showing the reaction products of PCR assays monitoring the presence of the promoter region of the tetO-tk-luc reporter gene in DNA pools obtained by chromatin immunoprecipitation (ChIP). Chromatin was isolated from (tetO-tk-luc)-S2 cells expressing TetR, TetRdSETDB1, or TetRdSETDB1(H775L) and immunoprecipitated with antibodies to dSETDB1, Su(var)205, Dnmt2, (me3)H3-K9, and methylated cytosine (5mC), or protein-A agarose (control-A) and protein-G agarose (control-G). Input indicates the amount of target DNA present in 1% of the input chromatin.

Mentions: Tri-methylated H3-K9 is one hallmark component of heterochromatin [34]; however, dSETDB1-mediated methylation of H3-K9 has been associated with activation and repression of transcription [13]–[14], [33]. To assess the role of dSETDB1-mediated tri-methylation of H3-K9 in gene expression, we performed transient transfection assays in S2 cells. Because dSETDB1 lacks a “classical” DNA binding domain, we used fusion proteins consisting of dSETDB1 and the DNA binding domain of the bacterial tetracycline repressor (TetR) [35]. TetRdSETDB1 fusion proteins repressed the expression of a chromosomal integrated TetR-dependent reporter gene, whereas the HMT-inactive TetRdSETDB1(H775L) and TetR did not (Figure 2A,B; Figure S5). Chromatin immunoprecipitation (ChIP) assays revealed that recruitment of TetRdSETDB1 but not TetRdSETDB1(H775L) mediated tri-methylation of H3-K9 at the reporter gene, which indicates that dSETDB1-mediated tri-methylation of H3-K9, mediates gene silencing (Figure 2C; Figure S6; Table S1).


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)

Methylation of H3-K9 by dSETDB1 mediates transcriptional silencing.(A) Western blot analysis detecting dSETDB1 (black triangle) and tetracycline repressor (TetR)–SETDB1 (TetRSETDB1) fusion proteins (open triangle) in S2 cells transiently expressing dSETDB1, TetR, or fusion proteins of TetR with dSETDB1 or dSETDB1(H775L). Forty-eight hours after transfection, total cell extracts were prepared, separated by SDS-PAGE, and electrophoretically transferred onto nitrocellulose membrane for probing with antibody to dSETDB1. (B) Schematic representation of luciferase assays with whole-cell extracts prepared from cells described in (A). The tetO-tk-luc reporter gene contains tet operator (tetO) sequences, which are fused to the human thymidine kinase (tk) promoter driving the expression of luciferase (luc). The diagram shows the mean values calculated from data from 5 different experiments. Error bars indicate the standard error of the mean (SEM). (C) Digital images of ethidium bromide-stained agarose gels showing the reaction products of PCR assays monitoring the presence of the promoter region of the tetO-tk-luc reporter gene in DNA pools obtained by chromatin immunoprecipitation (ChIP). Chromatin was isolated from (tetO-tk-luc)-S2 cells expressing TetR, TetRdSETDB1, or TetRdSETDB1(H775L) and immunoprecipitated with antibodies to dSETDB1, Su(var)205, Dnmt2, (me3)H3-K9, and methylated cytosine (5mC), or protein-A agarose (control-A) and protein-G agarose (control-G). Input indicates the amount of target DNA present in 1% of the input chromatin.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2871795&req=5

pone-0010581-g002: Methylation of H3-K9 by dSETDB1 mediates transcriptional silencing.(A) Western blot analysis detecting dSETDB1 (black triangle) and tetracycline repressor (TetR)–SETDB1 (TetRSETDB1) fusion proteins (open triangle) in S2 cells transiently expressing dSETDB1, TetR, or fusion proteins of TetR with dSETDB1 or dSETDB1(H775L). Forty-eight hours after transfection, total cell extracts were prepared, separated by SDS-PAGE, and electrophoretically transferred onto nitrocellulose membrane for probing with antibody to dSETDB1. (B) Schematic representation of luciferase assays with whole-cell extracts prepared from cells described in (A). The tetO-tk-luc reporter gene contains tet operator (tetO) sequences, which are fused to the human thymidine kinase (tk) promoter driving the expression of luciferase (luc). The diagram shows the mean values calculated from data from 5 different experiments. Error bars indicate the standard error of the mean (SEM). (C) Digital images of ethidium bromide-stained agarose gels showing the reaction products of PCR assays monitoring the presence of the promoter region of the tetO-tk-luc reporter gene in DNA pools obtained by chromatin immunoprecipitation (ChIP). Chromatin was isolated from (tetO-tk-luc)-S2 cells expressing TetR, TetRdSETDB1, or TetRdSETDB1(H775L) and immunoprecipitated with antibodies to dSETDB1, Su(var)205, Dnmt2, (me3)H3-K9, and methylated cytosine (5mC), or protein-A agarose (control-A) and protein-G agarose (control-G). Input indicates the amount of target DNA present in 1% of the input chromatin.
Mentions: Tri-methylated H3-K9 is one hallmark component of heterochromatin [34]; however, dSETDB1-mediated methylation of H3-K9 has been associated with activation and repression of transcription [13]–[14], [33]. To assess the role of dSETDB1-mediated tri-methylation of H3-K9 in gene expression, we performed transient transfection assays in S2 cells. Because dSETDB1 lacks a “classical” DNA binding domain, we used fusion proteins consisting of dSETDB1 and the DNA binding domain of the bacterial tetracycline repressor (TetR) [35]. TetRdSETDB1 fusion proteins repressed the expression of a chromosomal integrated TetR-dependent reporter gene, whereas the HMT-inactive TetRdSETDB1(H775L) and TetR did not (Figure 2A,B; Figure S5). Chromatin immunoprecipitation (ChIP) assays revealed that recruitment of TetRdSETDB1 but not TetRdSETDB1(H775L) mediated tri-methylation of H3-K9 at the reporter gene, which indicates that dSETDB1-mediated tri-methylation of H3-K9, mediates gene silencing (Figure 2C; Figure S6; Table S1).

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