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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 Rt1b{} and HeT-A retrotransposons in the developing wing.(A) In situ hybridization assays detecting Rt1b and HeT-A transcription in wing (W), haltere (H), and third-instar leg (L) imaginal discs isolated from third-instar larvae, which ubiquitously express Gal4 [Gal4(71B)], contain the reporter construct USA-Dnmt2 and UAS-dSETDB1.IR or lack dSETDB1 [Gal4(71B);UAS-dSETDB1.IR], Dnmt2 [Gal4(71B);USA-Dnmt2], or both [Gal4(71B);UAS-dSETDB1.IRGal4(71B);USA-Dnmt2] by RNAi. (B,C) RvT-PCR assays monitoring the transcription of Rt1b{} and HeT-A retrotransposons in wing imaginal discs described in (A). RNA was isolated from 50 wing discs and reverse transcribed. PCR detected the presence of (A) Rt1b{} and (D) HeT-A transcription. (D,E) Digital images of ethidium bromide-stained agarose gels showing the reaction products of PCR assays detecting the presence of (B) Rt1b{} and (E) HeT-A in DNA pools generated by ChIP. Chromatin was isolated from in vivo cross-linked wing imaginal discs isolated from larvae of the genotype described in (A,D). Chromatin was immunoprecipitated with antibodies to dSETDB1, 5mC, Dnmt2, Su(var)205, and rabbit serum (control). Input represents the amount of retrotransposons detectable in 2.5% of the input material. (F,G) Digital images of ethidium bromide-stained agarose gels showing the reaction products of methylation-sensitive restriction analyses with genomic DNA isolated from wing imaginal discs described in (A). Genomic DNA was isolated, incubated with bovine serum albumin (BSA) (mock), the methylation-sensitive restriction endonuclease HpaII, or the methylation-insensitive restriction enzyme MspI. PCR assays monitored the presence of the (C) Rt1b{} and (F) HeT-A in treated DNA pools.
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pone-0010581-g010: DSETDB1-mediated DNA methylation mediates silencing of Rt1b{} and HeT-A retrotransposons in the developing wing.(A) In situ hybridization assays detecting Rt1b and HeT-A transcription in wing (W), haltere (H), and third-instar leg (L) imaginal discs isolated from third-instar larvae, which ubiquitously express Gal4 [Gal4(71B)], contain the reporter construct USA-Dnmt2 and UAS-dSETDB1.IR or lack dSETDB1 [Gal4(71B);UAS-dSETDB1.IR], Dnmt2 [Gal4(71B);USA-Dnmt2], or both [Gal4(71B);UAS-dSETDB1.IRGal4(71B);USA-Dnmt2] by RNAi. (B,C) RvT-PCR assays monitoring the transcription of Rt1b{} and HeT-A retrotransposons in wing imaginal discs described in (A). RNA was isolated from 50 wing discs and reverse transcribed. PCR detected the presence of (A) Rt1b{} and (D) HeT-A transcription. (D,E) Digital images of ethidium bromide-stained agarose gels showing the reaction products of PCR assays detecting the presence of (B) Rt1b{} and (E) HeT-A in DNA pools generated by ChIP. Chromatin was isolated from in vivo cross-linked wing imaginal discs isolated from larvae of the genotype described in (A,D). Chromatin was immunoprecipitated with antibodies to dSETDB1, 5mC, Dnmt2, Su(var)205, and rabbit serum (control). Input represents the amount of retrotransposons detectable in 2.5% of the input material. (F,G) Digital images of ethidium bromide-stained agarose gels showing the reaction products of methylation-sensitive restriction analyses with genomic DNA isolated from wing imaginal discs described in (A). Genomic DNA was isolated, incubated with bovine serum albumin (BSA) (mock), the methylation-sensitive restriction endonuclease HpaII, or the methylation-insensitive restriction enzyme MspI. PCR assays monitored the presence of the (C) Rt1b{} and (F) HeT-A in treated DNA pools.

Mentions: Because dSETDB1 mediates silencing of Rt1b{} retrotransposons in S2 cells (Figures 4 and 7). To test whether dSETDB1 is involved in silencing of Rt1b[] retrotransposons in Drosophila, we monitored the transcriptional activity and DNA methylation status of Rt1b{} and HeT-A retrotransposons in developing wing imaginal discs, which lack dSETDB1 and/or Dnmt2 expression by RNAi [11]. HeT-A retrotransposons are integral components of Drosophila telomers and silencing of HeT-A retrotransposons involves dSETDB1-dependent DNA methylation [10]. Wing imaginal discs were isolated from Gal4(71B);UAS-dSETDB1.IR;UAS-Dnmt2 and control third-instar larvae. The Gal4-dependent reporter gene UAS-Dnmt2 transcribes an interfering double-stranded RNA targeting the Dnmt2 mRNA. The Gal4(71B) driver expresses Gal4 ubiquitously in imaginal discs. Knockdown of dSETDB1, Dnmt2, or both, resulted in ectopic transcription of Rt1b{} and HeT-A retrotransposons in wing imaginal discs (Figure 10A-C; Figure S35).


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 Rt1b{} and HeT-A retrotransposons in the developing wing.(A) In situ hybridization assays detecting Rt1b and HeT-A transcription in wing (W), haltere (H), and third-instar leg (L) imaginal discs isolated from third-instar larvae, which ubiquitously express Gal4 [Gal4(71B)], contain the reporter construct USA-Dnmt2 and UAS-dSETDB1.IR or lack dSETDB1 [Gal4(71B);UAS-dSETDB1.IR], Dnmt2 [Gal4(71B);USA-Dnmt2], or both [Gal4(71B);UAS-dSETDB1.IRGal4(71B);USA-Dnmt2] by RNAi. (B,C) RvT-PCR assays monitoring the transcription of Rt1b{} and HeT-A retrotransposons in wing imaginal discs described in (A). RNA was isolated from 50 wing discs and reverse transcribed. PCR detected the presence of (A) Rt1b{} and (D) HeT-A transcription. (D,E) Digital images of ethidium bromide-stained agarose gels showing the reaction products of PCR assays detecting the presence of (B) Rt1b{} and (E) HeT-A in DNA pools generated by ChIP. Chromatin was isolated from in vivo cross-linked wing imaginal discs isolated from larvae of the genotype described in (A,D). Chromatin was immunoprecipitated with antibodies to dSETDB1, 5mC, Dnmt2, Su(var)205, and rabbit serum (control). Input represents the amount of retrotransposons detectable in 2.5% of the input material. (F,G) Digital images of ethidium bromide-stained agarose gels showing the reaction products of methylation-sensitive restriction analyses with genomic DNA isolated from wing imaginal discs described in (A). Genomic DNA was isolated, incubated with bovine serum albumin (BSA) (mock), the methylation-sensitive restriction endonuclease HpaII, or the methylation-insensitive restriction enzyme MspI. PCR assays monitored the presence of the (C) Rt1b{} and (F) HeT-A in treated DNA pools.
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getmorefigures.php?uid=PMC2871795&req=5

pone-0010581-g010: DSETDB1-mediated DNA methylation mediates silencing of Rt1b{} and HeT-A retrotransposons in the developing wing.(A) In situ hybridization assays detecting Rt1b and HeT-A transcription in wing (W), haltere (H), and third-instar leg (L) imaginal discs isolated from third-instar larvae, which ubiquitously express Gal4 [Gal4(71B)], contain the reporter construct USA-Dnmt2 and UAS-dSETDB1.IR or lack dSETDB1 [Gal4(71B);UAS-dSETDB1.IR], Dnmt2 [Gal4(71B);USA-Dnmt2], or both [Gal4(71B);UAS-dSETDB1.IRGal4(71B);USA-Dnmt2] by RNAi. (B,C) RvT-PCR assays monitoring the transcription of Rt1b{} and HeT-A retrotransposons in wing imaginal discs described in (A). RNA was isolated from 50 wing discs and reverse transcribed. PCR detected the presence of (A) Rt1b{} and (D) HeT-A transcription. (D,E) Digital images of ethidium bromide-stained agarose gels showing the reaction products of PCR assays detecting the presence of (B) Rt1b{} and (E) HeT-A in DNA pools generated by ChIP. Chromatin was isolated from in vivo cross-linked wing imaginal discs isolated from larvae of the genotype described in (A,D). Chromatin was immunoprecipitated with antibodies to dSETDB1, 5mC, Dnmt2, Su(var)205, and rabbit serum (control). Input represents the amount of retrotransposons detectable in 2.5% of the input material. (F,G) Digital images of ethidium bromide-stained agarose gels showing the reaction products of methylation-sensitive restriction analyses with genomic DNA isolated from wing imaginal discs described in (A). Genomic DNA was isolated, incubated with bovine serum albumin (BSA) (mock), the methylation-sensitive restriction endonuclease HpaII, or the methylation-insensitive restriction enzyme MspI. PCR assays monitored the presence of the (C) Rt1b{} and (F) HeT-A in treated DNA pools.
Mentions: Because dSETDB1 mediates silencing of Rt1b{} retrotransposons in S2 cells (Figures 4 and 7). To test whether dSETDB1 is involved in silencing of Rt1b[] retrotransposons in Drosophila, we monitored the transcriptional activity and DNA methylation status of Rt1b{} and HeT-A retrotransposons in developing wing imaginal discs, which lack dSETDB1 and/or Dnmt2 expression by RNAi [11]. HeT-A retrotransposons are integral components of Drosophila telomers and silencing of HeT-A retrotransposons involves dSETDB1-dependent DNA methylation [10]. Wing imaginal discs were isolated from Gal4(71B);UAS-dSETDB1.IR;UAS-Dnmt2 and control third-instar larvae. The Gal4-dependent reporter gene UAS-Dnmt2 transcribes an interfering double-stranded RNA targeting the Dnmt2 mRNA. The Gal4(71B) driver expresses Gal4 ubiquitously in imaginal discs. Knockdown of dSETDB1, Dnmt2, or both, resulted in ectopic transcription of Rt1b{} and HeT-A retrotransposons in wing imaginal discs (Figure 10A-C; Figure S35).

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