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

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.

Figure 1. DSETDB1 preferentially tri-methylates lysine 9 in nucleosomal H3.

(A) Schematic representation of dSETDB1 and dSETDB1(H775L), which contains a single amino acid exchange mutation of histidine (H) to leucine (L) at amino acid position 775. Rectangles mark the positions of the methyl cytosine binding (MBD)-like domain (MBDL), Pre-SET domain (Pre), SET-domain (SET), and Post-SET domain (Post). The dark box in the SET domain indicates the peptide insertion present in bifurcated SET domain of dSETDB1. (B) Coomassie-blue stained SDS-PAGE gel (left) and corresponding fluorogram (right) of histone methyltransferase (HMT) assays containing polynucleosomes, [³H]-S-adenosyl-methionine (SAM), and recombinant, immunopurified Flag-epitope–tagged dSETDB1 (lanes 1,3) or Flag-epitope–tagged dSETDB1(H775L) (lanes 2, 4). Asterisks mark the position of anti-Flag antibody light and heavy chain. (C) Microsequencing of radiolabeled nucleosomal H3. Polynucleosomes were incubated with recombinant dSETDB1 in the presence of [³H]-SAM. H3 was subjected to Edman degradation, and resulting amino acid fractions were analyzed by scintillation counting. The × axis shows amino acids 1–29 of H3. The y axis shows [³H]-labeling of amino acids in decays per minutes (D.p.m.). (D) Matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) spectra of a high-performance liquid chromatography (HPLC) fraction containing the peptide ⁹K(me_0–3)STGGKAPR of H3. Polynucleosomes were incubated with Flag-dSETDB1 and SAM. H3 was purified and proteolytically digested with trypsin. The resulting peptides were separated by HPLC. Fraction 4 containing ⁹K(me_0–3)STGGKAPR was subjected to MALDI-TOF mass spectrometry. The × axis indicates the mass:charge ratio. The y axis indicates the abundance of peptides. The positions and m:z ratios of KSTGGKAPR peptides containing non-methylated lysine 9 in H3 (H3-K9), mono-methylated H3-K9 [(me₁)H3-K9], di-methylated H3-K9 [(me₂)H3-K9], and tri-methylated H3-K9 [(me₃)H3-K9] are indicated. (E) Western blot analysis of HMT reactions programmed with recombinant Flag-dSETDB1, polynucleosomes, and SAM. Reaction products were separated by SDS-PAGE, electrophoretically transferred onto PVDF membrane, and probed with antibodies to (me₁)H3-K9, (me₂)H3-K9, (me₃)H3-K9, and H3.

Figure 2. 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.

Figure 3. The MBDL of dSETDB1 binds methylated CpA motifs.

(A) Schematic representation of dSETDB1 and the mutant dSETDB1(R436C), which contains the single amino acid exchange mutation arginine (R) to cysteine (C) at position 436. Rectangles mark the positions of the MBDL, Pre-SET domain (Pre), SET domain (SET), and Post-SET domain (Post). (B) Coomassie blue-stained SDS-PAGE gel detecting affinity-purified GST, the fusion protein consisting of GST, the MBDL of dSETDB1 (MBDL), and the GST-MBDL(R436C) [MBDL(R436C)] fusion protein. Recombinant proteins were expressed in Escherichia coli, purified with glutathione-agarose and subjected to SDS-PAGE. The positions and relative molecular weights of protein standards are indicated. The arrow marks the position of GST-MBDL and GST-MBDL(R436C), the asterisk the position of GST. The protein samples and amounts shown were used for the binding assays shown in (C-E). (C) Autoradiogram of in vitro protein–DNA interactions detecting the association of [³²P]-radiolabeled, non-methylated (-) and methylated (+) DNA oligonucleotides containing 1 symmetrically methylated CpG-, CpA- or CpT motif. After proteins were incubated with DNA, retained DNA was purified, separated on native PAGE, and detected by autoradiography. (D) Autoradiogram of in vitro protein–DNA interaction assays as described in (C) except that methylated DNA oligonucleotides contained 3 symmetrically methylated CpA-, CpT-, or CpG motifs. (E) Autoradiogram of in vitro protein–DNA interaction assays as described in (C) except that GST, MBDL, and MBDL(R436C) and a DNA oligonucleotide containing 3 symmetrically methylated CpA motifs [(^5mCpA)₃] and the corresponding non-methylated DNA oligonucleotide [(CpA)₃] were used. (C-E) The arrowhead marks the position of retained, radiolabeled DNA.

Figure 4. DSETDB1-mediated tri-methylation of H3-K9 initiates DNA methylation and silencing.

(A) Digital images of ethidium bromide-stained agarose gels showing the reaction products of RT-PCR assays monitoring the transcription of Rb, Antp, CG2316, Rt1b/gag, dSETDB1, Dnmt2, Su(var)205 and actin5C in total RNA pools isolated from S2 cells, S2 cells transiently expressing GFP (control), and S2 cells co-expressing GFP and dSETDB1, dSETDB1(H775L), or dSETDB1(R436C). (B) Digital images of ethidium bromide-stained agarose gels showing the products of PCR assays detecting the dSETDB1 target DNA sequence in Antp, CG2316, and Rt1b{}799 (see Figure S9) in DNA pools obtained by ChIP. Chromatin was isolated from cells described in (A) and immunoprecipitated with antibodies and controls as described in Figure 2C. 1% of the chromatin used for ChIP.

Figure 5. DSETDB1-mediated tri-methylation of H3-K9 propagates spreading of DNA methylation and silencing of Rb.

(A) Digital images of ethidium bromide-stained agarose gels showing reaction products for the PDE and Exon-I of Rb indicated in (A) in DNA pools obtained by ChIP. Chromatin was isolated from S2 cells transiently expressing GFP (control) and S2 cells co-expressing GFP and dSETDB1, dSETDB1(H775L), or dSETDB1(R436C). Chromatin was immunoprecipitated with antibodies and agarose beads described in Figure 2C. PCR assays detected the PDE, PPE and Exon-I in immunoprecipitated DNA pools. (A,C) Input represents the amount of target DNA present in 1% of the chromatin used for ChIP. (B) Digital images of ethidium bromide-stained agarose gels detecting the target DNA sequences for dSETDB1 in Antp, CG2136 and Rt1b{}779 (see Figure S9) in DNA pools obtained by ChIP. Chromatin was isolated from S2 cells transiently expressing GFP and dSETDB1(R436C). Chromatin was immunoprecipitated with antibodies and agarose beads described in Figure 2C. (C) Schematic representation of the Rb locus. Boxes mark the position of exons I (Exon-I), II, and VIII. The positions of the promoter distal enhancer element (PDE), promoter proximal enhancer element (PPE), and Exon-I fragments detected in ChIP assays are indicated. (D) Digital images of ethidium bromide stained agarose gels showing the reaction products of methylation-sensitive restriction analyses of genomic DNA isolated from cells described in (B). 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 PDE, Exon-I, and the promoter region of Peepsqueak (Psq) in treated DNA pools.

Figure 6. Dnmt2 and Su(var)205 cooperate with dSETDB1 in DNA methylation.

(A) Digital images of ethidium bromide-stained agarose gels showing reaction products of PCR assays detecting Rb 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 RNA targeting human GAPDH (mock-RNAi). (B) PCR assays as in (A) except that S2 cells were treated with siRNA targeting Su(var)205 [Su(var)205 RNAi]. (C) Digital images of ethidium bromide-stained agarose gels showing the presence of the PDE of Rb (Figure 5A) in DNA pools generated by ChIP with chromatin isolated from cells described in (a; left panel) and (b; right panel). Chromatin was immunoprecipitated with the antibodies and controls described in Figure 2C. (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). PCR assays detected the presence of PDE (Figure 5B) in treated DNA pools.

Figure 7. 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.

Figure 8. dSETDB1 represses Rb expression in the developing eye.

(A, B) In situ hybridization assays detecting the transcription of (A) Rb and (B) proliferating-cell nuclear antigen (PCNA) in eye imaginal discs prepared from control 3^rd-instar larvae containing the lz^Gal4 driver, the reporter UAS-dSETDB1.IR or UAS-Dnmt2, and lz^Gal4 with UAS-dSETDB1.IR (lz^Gal4;UAS-dSETDB1.IR) or lz^Gal4 with UAS-Dnmt2 (lz^Gal4;UAS-Dnmt2). (C) Immunostaining assays detecting the mitotic marker phosphorylated H3 (serine 10) in eye imaginal discs described in (A,B). The mitotic index is38+3 for lz^Gal4 eye discs, 37+4 for UAS-dSETDB1.IR discs, 36+2 for UAS-Dnmt2, 9+3 for lz^Gal4;UAS-dSETDB1.IR and 11+2 for lz^Gal4;UAS-Dnmt2 discs. (A-C) The white-filled arrowheads mark the position of the morphogenetic furrow (MF). (A) Blue arrowheads indicate areas of ectopic Rb transcription in the posterior region (rectangle) of eye imaginal discs lacking dSETDB1 or Dnmt2, as compared to controls (see area marked by rectangle in lz^Gal4 discs). (B) The rectangle marks the position of the posterior PCNA transcription domain. The transcription of the posterior PCNA domain (rectangle) is reduced in dSETDB1 or Dnmt2 deficient eye imaginal discs as compared to controls. (C) The dark arrowhead marks the position of mitotic cells in the second mitotic wave posterior to the morphogenetic furrow. Note that the number of mitotic cells in regions posterior to the morphogenetic furrow (rectangle) is significantly reduced in eye discs lacking dSETDB1 or Dnmt2 through RNAi.

Figure 9. DSETDB1-mediated DNA methylation mediates silencing of Rb in the developing eye.

(A) Scanning electron micrographs showing the adult eye phenotype of lz^GAl4, UAS-dSETDB1.IR and lz^GAL4;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 lz^GAl4, UAS-dSETDB1.IR, and lz^GAL4;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.

Figure 10. 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.