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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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DSETDB1-mediated DNA methylation mediates silencing of Rb in the developing eye.(A) Scanning electron micrographs showing the adult eye phenotype of lzGAl4, UAS-dSETDB1.IR and lzGAL4;UAS-dSETDB1.IR flies (50-fold magnification). (B) Magnification of the areas marked by white rectangles in (A) (1,000-fold total magnification). The arrowhead marks the position of fused ommatidia and the arrow the position of misshaped ommatidia. (C) (Top) Digital photograph showing Rb transcription in eye imaginal disc. The position of the posterior Rb transcription domain (PRbD) and posterior cell stripe (PCS) are indicated. (Bottom) Digital images of ethidium bromide-stained agarose gels detecting the PDE of Rb in DNA pools obtained by ChIP. Chromatin was isolated from cell stripes representing the PRbD and the PCS of imaginal discs isolated from third-instar lzGAl4, UAS-dSETDB1.IR, and lzGAL4;UAS-dSETDB1.IR larvae. Chromatin was immunoprecipitated with antibodies to dSETDB1, Dnmt2, 5mC or rabbit serum (mock). Input represents the amount of target DNA present in 4% of the chromatin used for ChIP.
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pone-0010581-g009: DSETDB1-mediated DNA methylation mediates silencing of Rb in the developing eye.(A) Scanning electron micrographs showing the adult eye phenotype of lzGAl4, UAS-dSETDB1.IR and lzGAL4;UAS-dSETDB1.IR flies (50-fold magnification). (B) Magnification of the areas marked by white rectangles in (A) (1,000-fold total magnification). The arrowhead marks the position of fused ommatidia and the arrow the position of misshaped ommatidia. (C) (Top) Digital photograph showing Rb transcription in eye imaginal disc. The position of the posterior Rb transcription domain (PRbD) and posterior cell stripe (PCS) are indicated. (Bottom) Digital images of ethidium bromide-stained agarose gels detecting the PDE of Rb in DNA pools obtained by ChIP. Chromatin was isolated from cell stripes representing the PRbD and the PCS of imaginal discs isolated from third-instar lzGAl4, UAS-dSETDB1.IR, and lzGAL4;UAS-dSETDB1.IR larvae. Chromatin was immunoprecipitated with antibodies to dSETDB1, Dnmt2, 5mC or rabbit serum (mock). Input represents the amount of target DNA present in 4% of the chromatin used for ChIP.

Mentions: Knockdown of dSETDB1 resulted in ectopic transcription of Rb in cells posterior to the MF (Figure 8A). As observed in imaginal discs expressing constitutively active Rb [47], ectopic Rb expression (Figure S35) suppressed PCNA transcription (Figure 8B; Figure S33) and mitosis during the second mitotic wave (Figure 8C) and resulted in defective eye development, as evidenced by the presence of misshaped and fused ommatidia and lack of bristles (Figure 9A,B). To assess the role of Dnmt2 in Rb transcription, we monitored Rb and PCNA expression in eye imaginal discs lacking Dnmt2 through RNAi. Knockdown of Dnmt2 resulted in ectopic expression of Rb in cells posterior to the MF (Figure 8A), repression of PCNA (Figure 8B), and attenuation of mitosis (Figure 8C). Collectively, these results indicate that dSETDB1 and Dnmt2 are involved in Rb expression in developing eye imaginal discs


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)

DSETDB1-mediated DNA methylation mediates silencing of Rb in the developing eye.(A) Scanning electron micrographs showing the adult eye phenotype of lzGAl4, UAS-dSETDB1.IR and lzGAL4;UAS-dSETDB1.IR flies (50-fold magnification). (B) Magnification of the areas marked by white rectangles in (A) (1,000-fold total magnification). The arrowhead marks the position of fused ommatidia and the arrow the position of misshaped ommatidia. (C) (Top) Digital photograph showing Rb transcription in eye imaginal disc. The position of the posterior Rb transcription domain (PRbD) and posterior cell stripe (PCS) are indicated. (Bottom) Digital images of ethidium bromide-stained agarose gels detecting the PDE of Rb in DNA pools obtained by ChIP. Chromatin was isolated from cell stripes representing the PRbD and the PCS of imaginal discs isolated from third-instar lzGAl4, UAS-dSETDB1.IR, and lzGAL4;UAS-dSETDB1.IR larvae. Chromatin was immunoprecipitated with antibodies to dSETDB1, Dnmt2, 5mC or rabbit serum (mock). Input represents the amount of target DNA present in 4% of the chromatin used for ChIP.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2871795&req=5

pone-0010581-g009: DSETDB1-mediated DNA methylation mediates silencing of Rb in the developing eye.(A) Scanning electron micrographs showing the adult eye phenotype of lzGAl4, UAS-dSETDB1.IR and lzGAL4;UAS-dSETDB1.IR flies (50-fold magnification). (B) Magnification of the areas marked by white rectangles in (A) (1,000-fold total magnification). The arrowhead marks the position of fused ommatidia and the arrow the position of misshaped ommatidia. (C) (Top) Digital photograph showing Rb transcription in eye imaginal disc. The position of the posterior Rb transcription domain (PRbD) and posterior cell stripe (PCS) are indicated. (Bottom) Digital images of ethidium bromide-stained agarose gels detecting the PDE of Rb in DNA pools obtained by ChIP. Chromatin was isolated from cell stripes representing the PRbD and the PCS of imaginal discs isolated from third-instar lzGAl4, UAS-dSETDB1.IR, and lzGAL4;UAS-dSETDB1.IR larvae. Chromatin was immunoprecipitated with antibodies to dSETDB1, Dnmt2, 5mC or rabbit serum (mock). Input represents the amount of target DNA present in 4% of the chromatin used for ChIP.
Mentions: Knockdown of dSETDB1 resulted in ectopic transcription of Rb in cells posterior to the MF (Figure 8A). As observed in imaginal discs expressing constitutively active Rb [47], ectopic Rb expression (Figure S35) suppressed PCNA transcription (Figure 8B; Figure S33) and mitosis during the second mitotic wave (Figure 8C) and resulted in defective eye development, as evidenced by the presence of misshaped and fused ommatidia and lack of bristles (Figure 9A,B). To assess the role of Dnmt2 in Rb transcription, we monitored Rb and PCNA expression in eye imaginal discs lacking Dnmt2 through RNAi. Knockdown of Dnmt2 resulted in ectopic expression of Rb in cells posterior to the MF (Figure 8A), repression of PCNA (Figure 8B), and attenuation of mitosis (Figure 8C). Collectively, these results indicate that dSETDB1 and Dnmt2 are involved in Rb expression in developing eye imaginal discs

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