- Email: [email protected]
Identification of methylated sequences in genomic DNA of adult Drosophila melanogaster
BBRC Biochemical and Biophysical Research Communications 322 (2004) 465–469 www.elsevier.com/locate/ybbrc
Identification of methylated sequences in genomic DNA of adult Drosophila melanogaster Adi Salzberga, Ohad Fisherb, Rama Siman-Tovb, Serge Ankrib,* a
Department of Genetics and the Rappaport Family Institute for Research in the Medical Sciences, Technion, Israel b Department of Molecular Microbiology, The Bruce Rappaport Faculty of Medicine, Technion, Israel Received 18 July 2004 Available online 6 August 2004
Abstract The genome of Drosophila melanogaster contains methylated cytosines. Recent studies indicate that DNA methylation in the fruit fly depends on one DNA methyltransferase, dDNMT2. No obvious phenotype is associated with the downregulation of this DNA methyltransferase. Thus, identifying the target sequences methylated by dDNMT2 may constitute the first step towards understanding the biological functions of this enzyme. We used anti-5-methylcytosine antibodies as affinity column to identify the methylated sequences in the genome of adult flies. Our analysis demonstrates that components of retrotransposons and repetitive DNA sequences are putative substrates for dDNMT2. The methylation status of DNA encoding Gag, a protein involved in delivering the transposition template to its DNA target, was confirmed by sodium bisulfite sequencing. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Drosophila melanogaster; DNA methylation; 5-Methylcytosine; Retrotransposons
In higher eukaryotes, DNA methylation regulates a number of important biological functions including chromatin structure [1], silencing of gene expression [2], parental imprinting, chromosome X inactivation in females [3], and development and protection from selfish genetic elements [4]. Methylation occurs in cytosine C5 at CG sequences and 60–90% of CG sequences are methylated. Methylation of CG sites in the promoter regions of genes usually leads to a reduction of gene expression [5–8]. In contrast to vertebrates, relatively little is known about DNA methylation in invertebrates such as the fruit fly Drosophila melanogaster. For a long time, the methylation status of D. melanogaster genome has been controversial. Recently, some evidences have provided a strong support for the existence of 5-methylcytosine (m5C) in DNA preparations from all stages of D. melanogaster development [9,10]. D. melanogaster
*
Corresponding author. Fax: +972 4 829 5225. E-mail address: [email protected] (S. Ankri).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.07.134
contains a single cytosine-5 DNA methyltransferase, called dDNMT2, that belongs to the DNMT2 family [9,11,12]. DNMT2-like genes are widely conserved during evolution, but their function is still elusive. The depletion of dDNMT2 in embryos of D. melanogaster [9] and in mouse embryonic stem cells [13] did not cause any apparent phenotypic effects. A first step towards understanding the function of DNMT2 is the identification of its DNA targets. The pattern of methylation in the fly where m5C residues are not only located in CpG sites but also in other dinucleotide sites [9,12] prevents the use of a methyl-binding domain (MBD) agarose chromatography as a method to fractionate methylated DNA. This MBD column which was used successfully to fractionate Neurospora DNA on the basis of methylation does not bind methylated non-CpG sites [14]. Recently, we used an anti-m5C affinity column to identify methylated sequences in the protozoan parasite Entamoeba histolytica [15]. This parasite like D. melanogaster expresses a single DNA methyltransferase homologous to DNMT2. Here we described the identification
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A. Salzberg et al. / Biochemical and Biophysical Research Communications 322 (2004) 465–469
of methylated sequences from the genome of adult flies using the same approach.
Materials and methods Preparation of D. melanogaster genomic DNA. Genomic DNA was prepared from adult yw flies. Approximately 200 flies were homogenized on ice in homogenization buffer (10 mM Tris, pH 8.0, 60 mM NaCl, 10 mM EDTA, 0.15 mM spermidine, and 0.5% Triton X-100). The homogenate was decanted through gauze and nuclei were collected by centrifugation (7 min, 7000g, 4 °C). The nuclear pellet was washed once with homogenization buffer and then resuspended in homogenization buffer plus 2% Sarkosyl. Proteinase K was added to a final concentration of 0.1 mg/ml and the sample was incubated overnight at 50 °C. The DNA was extracted by phenol/phenol chloroform and precipitated with 0.3 M sodium acetate and three volumes of 100% ethanol. Enrichment of methylated DNA on m5C antibody affinity column. Sheep polyclonal antibody to m5C (260 lg; Maine Biotechnology Service, USA) was cross-linked to a 0.2 ml column of immobilized protein A (Seize-X protein A immunoprecipitation kit; Pierce Biotechnology) in accordance with the manufacturerÕs instructions. D. melanogaster genomic DNA (2 lg) extracted from adult flies was cleaved with DpnII and ligated overnight with the adaptors R-Bgl-24 oligo and R-Bgl-12 oligo (5- 0 agcactctccagcctctcaccgca-3 0 and 5- 0 gatctgcggtga-3 0 ). The 12-mer adaptor was melted away by heating the reaction for 3 min at 72 °C and the ends were filled in with Taq DNA polymerase (5 U; Promega) for 5 min at 72 °C. The ligation was diluted by adding 300 ll binding/wash buffer (0.14 M NaCl, 0.008 M Na2PO4, 0.002 M potassium phosphate, and 0.01 M KCl, pH 7.4). The DNA was denatured by heat and incubated overnight at room temperature with the affinity column prepared above. The column was washed extensively with the binding/wash buffer, resuspended in 50 ll of the same buffer, and 5 ll of suspension was directly used for PCR. DNA bound to the column was amplified with the R-Bgl-24 oligo. A program of 1 min at 95 °C and 3 min at 72 °C for a total of 25 cycles was used. The PCR products were cloned into the pGEM-T Easy vector (Promega) and sequenced (DNA Facility, Faculty of Medicine, Technion, Haifa, Israel). Sodium bisulfite reaction and strand specific PCR. Sodium bisulfite treatment of D. melanogaster genomic DNA was performed according to the method described by [16]. The set of primers used to amplify Gag coding sequence of rover transposable element (Accession No. AF492764) after treatment with sodium bisulfite is 5 0 -ttattaatatttta taagaaaag-3 0 (from nucleotide 1820 to 1845) and 5 0 -agttggagggaagtta ttaggtg-3 0 (from nucleotide 2170 to 2193), respectively.
Results and discussion Identification of methylated sequences in genomic DNA of adult flies Purification of CpG islands using the methyl-binding domain of MeCP2 was previously described [17]. The pattern of methylation in the fly where m5C residues are not only located in CpG sites but also at other dinucleotide sites encouraged us to use a methylated DNAbinding column that contains m5C-specific antibodies as a ligand. We have previously used this method to identify methylated component of the E. histolytica genome [15].
Genomic DNA was extracted from adult flies, digested with DpnII, bound to adaptors, and loaded on the anti-5mC affinity chromatography column. DNA purified by the column was amplified using the adaptors as primers, cloned in pGEM-T-easy vector, and sequenced. Twenty-seven independent clones were sequenced (Table 1). To identify the natural targets of dDNMT2, we used BLAST to compare the sequences isolated by affinity chromatography to sequences in Flybase, the database of the D. melanogaster genome (http://flybase.bio.indiana.edu). Remarkably, 9 of 27 sequences analyzed were identified as retrotransposons, or retrotransposon-related sequences, including rover, R1Dm, and Pilger, and heterochromatin repetitive elements, including centromeric repeats and a dodeca satellite (Table 1, clones 1–9). Additional sequences isolated by this method include DNA that encodes for dusky, doublesex, and soxneuro and for a RNA helicase homologue. To confirm the specificity of the anti-m5C affinity chromatography, we focused our analysis on the DNA region that encodes for Gag coding sequence of rover LTR transposable element. We searched for methylated cytosines by sodium bisulfite reaction and strand-specific PCR. This procedure converts all cytosine residues to uracil, giving rise to thymine after amplification by PCR. Only methylated cytosines are refractory to the deamination. A fragment from the Gag coding sequence was amplified from sodium bisulfite-treated genomic DNA, cloned into pGEM-T easy vector, and sequenced. The inability of the bisulfite treatment to replace cytosines with thymines demonstrates the presence of methylated cytosines in Gag (Fig. 1). As previously described for D. melanogaster, methylation was not restricted to CG sites but was also detected in a CT site present in this cluster. To verify that the conservation of cytosines in the bisulfite-treated sequence is caused by their methylation, the Gag sequence was amplified from D. melanogaster genomic DNA, cloned into pGEM-T easy, and replicated in a methylation deficient strain of Escherichia coli (Gm2929). Sequence analysis of the Gag DNA after its replication in E. coli Gm2929 revealed that all cytosines present in the Gag DNA were converted to thymines following a sodium bisulfite treatment. This result confirms that our detection of methylated cytosines within the Gag sequence is not an artifact caused by the presence of a DNA region resistant to sodium bisulfite treatment. In this work, we have isolated a number of DNA sequences from the D. melanogaster genome using affinity chromatography with m5C antibody as a ligand. Remarkably, some of these DNA regions consist of repetitive elements including satellite repeats and retrotransposons. Transposons are considered intra-genomic parasites that threaten the structure and regulated expression of the genome. Many retrotransposons have
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Table 1 Identification of DNA sequences isolated by m5C antibody affinity column Clone number
BLAST results (Accession number and coordinates)
Cytology
(A) Transposable elements and related sequences 1 AF492764 (1814–2419) 85C, 12F–13A, 82D 2, 3
DMRER1Dm multiple hits
4
AC010915 (14309–14650)
(B) Satellite sequences 5 Multiple hits 6 AI947162 (39–407)
Type of identified sequence
Comments
Gag coding sequence of rover
Transposable element of the LTR family
101F–102F, 73A, 41C, 42A4–5 101F–102F
Type I retrotransposable element R1Dm. LINE family Retrotransposon-like sequence
Heterochromatin Heterochromatin
Centromeric repeat Bs35g08.y1 mRNA from adult testis library
7
Numerous hits
38A–B, 38C, 39D, 40A–C, 80B–D, 101F–102F
Heterochromatin repeat
8, 9
DROSATED
Centromeric
Dodeca satellite
(C) Unique sequences 10 AC009915 (137366–137747) 11 AC023729 (155647–156504) 12 AC010069 (130422–130620) 13 AC009384 (156597–156924) 14 AC093045 (67893–68299) 15 AY069604 (1391–1522) 16 AC007769 (150711–151119) 17 LD27659 (955–1208) 18 AE003487.2 (63594–63916)
94A6 8D 73E5 76F1 29F2 1B13–14 88F6 1D2–3 10E
CG5383 (850–1231) CG12106-PA (1–467) CG7724-PA (299–417) CG14186-PA (622–949) SoxNeuro CG18024-RA (1493–1899) CG13363 (1391–1522) CG5205-PA (5188–5596) CG11642-PA (751–1004) dusky M84606 (919–1385)
19
AE003676.3 (226602–226962)
84D–E
doublesex, CG11094 (2801–2972)
20 21 22 23 24 25 26 27
AL109630 (109842–110072) SD10289 (573–1146) AC092240 (17057–17391) AE003579.3 (126351–126791) AC008228 (38042–38318) AE003572.4 (57108–57487) AC010659 (125284–125528) AC069405 (121504–121777)
1D2 59F5 24C–D 24D 21E–22A 19A–C 3L 3L
CG3706-PA (13–243) tumorous imaginal discs X95241 (3706–3136) shaw CG2822-RB (1478–1803)
strong constitutive promoters, and chimeric mRNAs originating at the promoters of retrotransposons could lead to the production of proteins that have neomorphic or dominant-negative effects [18–20]. DNA methylation is a well-known mechanism of defense against transposable elements. In prokaryotes, Dam methylation controls the activity of Tn5 [21]. Most of the m5C in mammalian DNA resides in transposons. Transposon promoters are inactive when methylated and, over time, C–T transition mutations at methylated sites destroy many transposons [4]. The genome of Drosophila contains a number of resident transposons, to which it is very vulnerable [22–26]. From 50% to 80% of all spontaneous mutations in Drosophila are due to transposon
Shares 75% identity with non-LTR retrotransposon Pilger (gag and pol genes)
(TTCTC)n (AAGAG)n similar to DROSAT04 satellite fragment 1.705–1232 Shows similarity to DRODP1187X subtelomeric heterochromatin repeats (ACCAGTACGGG)n
GC content 62.5% (239/382) Oxireductase GC content 67.8% (222/328) GC content 64.1% (261/407) GC content 52.6% (109/207) RNA helicase GC content 62% (160/258) Contains two stretches of triple nucleotide repeat (CAG) 84 and 108 nt long GC content is 59.7% (120/201) within the mRNA sequence and 20.1% (34/169) 3 0 to it GC content 56.2% (132/235) GC content 63.9% (366/573) GC content 54.5% (240/440) GC content 50.3% (179/379) GC content 42.7% (105/246) GC content 49.8% (140/281)
insertion and rearrangements due to recombination between transposons [27,28]. The lack of DNA methylation in Drosophila was proposed to explain this high mutation rate induced by transposon [4]. Recently the methylation status of D. melanogaster has been reconsidered and the presence of m5C and of an active, unique DNA methyltransferase has been confirmed [9– 12]. The fact that a number of sequences isolated by m5C affinity chromatography encode transposable elements suggests that DNA methylation is also a mechanism of defense used by D. melanogaster to shield its genome. We demonstrated by m5C affinity chromatography and by bisulfite sequence analysis that a DNA encoding for the Gag protein of rover transposon was
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longs to the DNMT2 family. The data presented in this work show that in D. melanogaster, retrotransposons and repetitive elements are targeted by DNA methylation. This observation corroborates the idea that the function of DNMT2 and related proteins is the control of transposons and repetitive elements.
Acknowledgments This research was supported by Grants 370/00-1 and 219/00 from the Israel Science Foundation (to S.A. and A.S., respectively), by the Mars Pittsburg Foundation for Research, and by a ÔResearch Career Development AwardÕ to A.S. from the Israel Cancer Research Fund. Fig. 1. Genomic sodium bisulfite analysis sequencing of Gag coding sequence of rover LTR transposable element in D. melanogaster (Accession No: AF492764 from nucleotide 2150 to 2113). Bisulfite analysis of two independent clones from genomic DNA of D. melanogaster is shown in (A,B). Analysis of Gag following replication in a methylation deficient strain of E. coli Gm2929 is shown in (C). The methylated cytosine residues in D. melanogaster genomic DNA are resistant to the bisulfite treatment (designated by a star).
methylated in the genome of the fly. The gag and pol coding regions are typical of many retrotransposons. The pol sequence encodes reverse transcriptase activity which is necessary for transposition. Gag proteins from retroviruses are involved in packaging viral RNA and helping its export from the host cell. The role of Gag protein of retrotransposon is less understood. Gag encoded by the non-long repeat retrotransposons Het-A and TART which form telomeres of D. melanogaster by repeated transpositions onto the ends of chromosomes are involved in the targeting of the retrotransposon RNA to the telomeres [29–31]. The presence of m5C in this DNA region encoding Gag strengthens our hypothesis that DNA methylation may be one of the mechanisms that D. melanogaster uses to keep population of retrotransposable elements under control. Other DNA fragments selected by m5C affinity chromatography included DNA encoding dusky, doublesex, SoxNeuro, and RNA helicase. The explanation for their selection is less obvious. Some of these unique sequences like CG14186-PA and SoxNeuro have a high G + C content (>60%; Table 1). In mammals, preferred sites for DNA methylation include clusters of C–G pairs in regions of the DNA high in G + C content [32]. DNA methylation in Neurospora crassa is catalyzed by Dim-2. Interestingly, methylated component of N. crassa genome consists almost exclusively of relics of transposons [14]. In the protozoan parasite E. histolytica, repetitive elements like rDNA [15] and S/MAR containing DNA (Banerjee et al., unpublished) are targeted by Ehmeth, a DNA methyltransferase that be-
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Identification of methylated sequences in genomic DNA of adult Drosophila melanogaster Adi Salzberga, Ohad Fisherb, Rama Siman-Tovb, Serge Ankrib,* a
Department of Genetics and the Rappaport Family Institute for Research in the Medical Sciences, Technion, Israel b Department of Molecular Microbiology, The Bruce Rappaport Faculty of Medicine, Technion, Israel Received 18 July 2004 Available online 6 August 2004
Abstract The genome of Drosophila melanogaster contains methylated cytosines. Recent studies indicate that DNA methylation in the fruit fly depends on one DNA methyltransferase, dDNMT2. No obvious phenotype is associated with the downregulation of this DNA methyltransferase. Thus, identifying the target sequences methylated by dDNMT2 may constitute the first step towards understanding the biological functions of this enzyme. We used anti-5-methylcytosine antibodies as affinity column to identify the methylated sequences in the genome of adult flies. Our analysis demonstrates that components of retrotransposons and repetitive DNA sequences are putative substrates for dDNMT2. The methylation status of DNA encoding Gag, a protein involved in delivering the transposition template to its DNA target, was confirmed by sodium bisulfite sequencing. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Drosophila melanogaster; DNA methylation; 5-Methylcytosine; Retrotransposons
In higher eukaryotes, DNA methylation regulates a number of important biological functions including chromatin structure [1], silencing of gene expression [2], parental imprinting, chromosome X inactivation in females [3], and development and protection from selfish genetic elements [4]. Methylation occurs in cytosine C5 at CG sequences and 60–90% of CG sequences are methylated. Methylation of CG sites in the promoter regions of genes usually leads to a reduction of gene expression [5–8]. In contrast to vertebrates, relatively little is known about DNA methylation in invertebrates such as the fruit fly Drosophila melanogaster. For a long time, the methylation status of D. melanogaster genome has been controversial. Recently, some evidences have provided a strong support for the existence of 5-methylcytosine (m5C) in DNA preparations from all stages of D. melanogaster development [9,10]. D. melanogaster
*
Corresponding author. Fax: +972 4 829 5225. E-mail address: [email protected] (S. Ankri).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.07.134
contains a single cytosine-5 DNA methyltransferase, called dDNMT2, that belongs to the DNMT2 family [9,11,12]. DNMT2-like genes are widely conserved during evolution, but their function is still elusive. The depletion of dDNMT2 in embryos of D. melanogaster [9] and in mouse embryonic stem cells [13] did not cause any apparent phenotypic effects. A first step towards understanding the function of DNMT2 is the identification of its DNA targets. The pattern of methylation in the fly where m5C residues are not only located in CpG sites but also in other dinucleotide sites [9,12] prevents the use of a methyl-binding domain (MBD) agarose chromatography as a method to fractionate methylated DNA. This MBD column which was used successfully to fractionate Neurospora DNA on the basis of methylation does not bind methylated non-CpG sites [14]. Recently, we used an anti-m5C affinity column to identify methylated sequences in the protozoan parasite Entamoeba histolytica [15]. This parasite like D. melanogaster expresses a single DNA methyltransferase homologous to DNMT2. Here we described the identification
466
A. Salzberg et al. / Biochemical and Biophysical Research Communications 322 (2004) 465–469
of methylated sequences from the genome of adult flies using the same approach.
Materials and methods Preparation of D. melanogaster genomic DNA. Genomic DNA was prepared from adult yw flies. Approximately 200 flies were homogenized on ice in homogenization buffer (10 mM Tris, pH 8.0, 60 mM NaCl, 10 mM EDTA, 0.15 mM spermidine, and 0.5% Triton X-100). The homogenate was decanted through gauze and nuclei were collected by centrifugation (7 min, 7000g, 4 °C). The nuclear pellet was washed once with homogenization buffer and then resuspended in homogenization buffer plus 2% Sarkosyl. Proteinase K was added to a final concentration of 0.1 mg/ml and the sample was incubated overnight at 50 °C. The DNA was extracted by phenol/phenol chloroform and precipitated with 0.3 M sodium acetate and three volumes of 100% ethanol. Enrichment of methylated DNA on m5C antibody affinity column. Sheep polyclonal antibody to m5C (260 lg; Maine Biotechnology Service, USA) was cross-linked to a 0.2 ml column of immobilized protein A (Seize-X protein A immunoprecipitation kit; Pierce Biotechnology) in accordance with the manufacturerÕs instructions. D. melanogaster genomic DNA (2 lg) extracted from adult flies was cleaved with DpnII and ligated overnight with the adaptors R-Bgl-24 oligo and R-Bgl-12 oligo (5- 0 agcactctccagcctctcaccgca-3 0 and 5- 0 gatctgcggtga-3 0 ). The 12-mer adaptor was melted away by heating the reaction for 3 min at 72 °C and the ends were filled in with Taq DNA polymerase (5 U; Promega) for 5 min at 72 °C. The ligation was diluted by adding 300 ll binding/wash buffer (0.14 M NaCl, 0.008 M Na2PO4, 0.002 M potassium phosphate, and 0.01 M KCl, pH 7.4). The DNA was denatured by heat and incubated overnight at room temperature with the affinity column prepared above. The column was washed extensively with the binding/wash buffer, resuspended in 50 ll of the same buffer, and 5 ll of suspension was directly used for PCR. DNA bound to the column was amplified with the R-Bgl-24 oligo. A program of 1 min at 95 °C and 3 min at 72 °C for a total of 25 cycles was used. The PCR products were cloned into the pGEM-T Easy vector (Promega) and sequenced (DNA Facility, Faculty of Medicine, Technion, Haifa, Israel). Sodium bisulfite reaction and strand specific PCR. Sodium bisulfite treatment of D. melanogaster genomic DNA was performed according to the method described by [16]. The set of primers used to amplify Gag coding sequence of rover transposable element (Accession No. AF492764) after treatment with sodium bisulfite is 5 0 -ttattaatatttta taagaaaag-3 0 (from nucleotide 1820 to 1845) and 5 0 -agttggagggaagtta ttaggtg-3 0 (from nucleotide 2170 to 2193), respectively.
Results and discussion Identification of methylated sequences in genomic DNA of adult flies Purification of CpG islands using the methyl-binding domain of MeCP2 was previously described [17]. The pattern of methylation in the fly where m5C residues are not only located in CpG sites but also at other dinucleotide sites encouraged us to use a methylated DNAbinding column that contains m5C-specific antibodies as a ligand. We have previously used this method to identify methylated component of the E. histolytica genome [15].
Genomic DNA was extracted from adult flies, digested with DpnII, bound to adaptors, and loaded on the anti-5mC affinity chromatography column. DNA purified by the column was amplified using the adaptors as primers, cloned in pGEM-T-easy vector, and sequenced. Twenty-seven independent clones were sequenced (Table 1). To identify the natural targets of dDNMT2, we used BLAST to compare the sequences isolated by affinity chromatography to sequences in Flybase, the database of the D. melanogaster genome (http://flybase.bio.indiana.edu). Remarkably, 9 of 27 sequences analyzed were identified as retrotransposons, or retrotransposon-related sequences, including rover, R1Dm, and Pilger, and heterochromatin repetitive elements, including centromeric repeats and a dodeca satellite (Table 1, clones 1–9). Additional sequences isolated by this method include DNA that encodes for dusky, doublesex, and soxneuro and for a RNA helicase homologue. To confirm the specificity of the anti-m5C affinity chromatography, we focused our analysis on the DNA region that encodes for Gag coding sequence of rover LTR transposable element. We searched for methylated cytosines by sodium bisulfite reaction and strand-specific PCR. This procedure converts all cytosine residues to uracil, giving rise to thymine after amplification by PCR. Only methylated cytosines are refractory to the deamination. A fragment from the Gag coding sequence was amplified from sodium bisulfite-treated genomic DNA, cloned into pGEM-T easy vector, and sequenced. The inability of the bisulfite treatment to replace cytosines with thymines demonstrates the presence of methylated cytosines in Gag (Fig. 1). As previously described for D. melanogaster, methylation was not restricted to CG sites but was also detected in a CT site present in this cluster. To verify that the conservation of cytosines in the bisulfite-treated sequence is caused by their methylation, the Gag sequence was amplified from D. melanogaster genomic DNA, cloned into pGEM-T easy, and replicated in a methylation deficient strain of Escherichia coli (Gm2929). Sequence analysis of the Gag DNA after its replication in E. coli Gm2929 revealed that all cytosines present in the Gag DNA were converted to thymines following a sodium bisulfite treatment. This result confirms that our detection of methylated cytosines within the Gag sequence is not an artifact caused by the presence of a DNA region resistant to sodium bisulfite treatment. In this work, we have isolated a number of DNA sequences from the D. melanogaster genome using affinity chromatography with m5C antibody as a ligand. Remarkably, some of these DNA regions consist of repetitive elements including satellite repeats and retrotransposons. Transposons are considered intra-genomic parasites that threaten the structure and regulated expression of the genome. Many retrotransposons have
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Table 1 Identification of DNA sequences isolated by m5C antibody affinity column Clone number
BLAST results (Accession number and coordinates)
Cytology
(A) Transposable elements and related sequences 1 AF492764 (1814–2419) 85C, 12F–13A, 82D 2, 3
DMRER1Dm multiple hits
4
AC010915 (14309–14650)
(B) Satellite sequences 5 Multiple hits 6 AI947162 (39–407)
Type of identified sequence
Comments
Gag coding sequence of rover
Transposable element of the LTR family
101F–102F, 73A, 41C, 42A4–5 101F–102F
Type I retrotransposable element R1Dm. LINE family Retrotransposon-like sequence
Heterochromatin Heterochromatin
Centromeric repeat Bs35g08.y1 mRNA from adult testis library
7
Numerous hits
38A–B, 38C, 39D, 40A–C, 80B–D, 101F–102F
Heterochromatin repeat
8, 9
DROSATED
Centromeric
Dodeca satellite
(C) Unique sequences 10 AC009915 (137366–137747) 11 AC023729 (155647–156504) 12 AC010069 (130422–130620) 13 AC009384 (156597–156924) 14 AC093045 (67893–68299) 15 AY069604 (1391–1522) 16 AC007769 (150711–151119) 17 LD27659 (955–1208) 18 AE003487.2 (63594–63916)
94A6 8D 73E5 76F1 29F2 1B13–14 88F6 1D2–3 10E
CG5383 (850–1231) CG12106-PA (1–467) CG7724-PA (299–417) CG14186-PA (622–949) SoxNeuro CG18024-RA (1493–1899) CG13363 (1391–1522) CG5205-PA (5188–5596) CG11642-PA (751–1004) dusky M84606 (919–1385)
19
AE003676.3 (226602–226962)
84D–E
doublesex, CG11094 (2801–2972)
20 21 22 23 24 25 26 27
AL109630 (109842–110072) SD10289 (573–1146) AC092240 (17057–17391) AE003579.3 (126351–126791) AC008228 (38042–38318) AE003572.4 (57108–57487) AC010659 (125284–125528) AC069405 (121504–121777)
1D2 59F5 24C–D 24D 21E–22A 19A–C 3L 3L
CG3706-PA (13–243) tumorous imaginal discs X95241 (3706–3136) shaw CG2822-RB (1478–1803)
strong constitutive promoters, and chimeric mRNAs originating at the promoters of retrotransposons could lead to the production of proteins that have neomorphic or dominant-negative effects [18–20]. DNA methylation is a well-known mechanism of defense against transposable elements. In prokaryotes, Dam methylation controls the activity of Tn5 [21]. Most of the m5C in mammalian DNA resides in transposons. Transposon promoters are inactive when methylated and, over time, C–T transition mutations at methylated sites destroy many transposons [4]. The genome of Drosophila contains a number of resident transposons, to which it is very vulnerable [22–26]. From 50% to 80% of all spontaneous mutations in Drosophila are due to transposon
Shares 75% identity with non-LTR retrotransposon Pilger (gag and pol genes)
(TTCTC)n (AAGAG)n similar to DROSAT04 satellite fragment 1.705–1232 Shows similarity to DRODP1187X subtelomeric heterochromatin repeats (ACCAGTACGGG)n
GC content 62.5% (239/382) Oxireductase GC content 67.8% (222/328) GC content 64.1% (261/407) GC content 52.6% (109/207) RNA helicase GC content 62% (160/258) Contains two stretches of triple nucleotide repeat (CAG) 84 and 108 nt long GC content is 59.7% (120/201) within the mRNA sequence and 20.1% (34/169) 3 0 to it GC content 56.2% (132/235) GC content 63.9% (366/573) GC content 54.5% (240/440) GC content 50.3% (179/379) GC content 42.7% (105/246) GC content 49.8% (140/281)
insertion and rearrangements due to recombination between transposons [27,28]. The lack of DNA methylation in Drosophila was proposed to explain this high mutation rate induced by transposon [4]. Recently the methylation status of D. melanogaster has been reconsidered and the presence of m5C and of an active, unique DNA methyltransferase has been confirmed [9– 12]. The fact that a number of sequences isolated by m5C affinity chromatography encode transposable elements suggests that DNA methylation is also a mechanism of defense used by D. melanogaster to shield its genome. We demonstrated by m5C affinity chromatography and by bisulfite sequence analysis that a DNA encoding for the Gag protein of rover transposon was
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longs to the DNMT2 family. The data presented in this work show that in D. melanogaster, retrotransposons and repetitive elements are targeted by DNA methylation. This observation corroborates the idea that the function of DNMT2 and related proteins is the control of transposons and repetitive elements.
Acknowledgments This research was supported by Grants 370/00-1 and 219/00 from the Israel Science Foundation (to S.A. and A.S., respectively), by the Mars Pittsburg Foundation for Research, and by a ÔResearch Career Development AwardÕ to A.S. from the Israel Cancer Research Fund. Fig. 1. Genomic sodium bisulfite analysis sequencing of Gag coding sequence of rover LTR transposable element in D. melanogaster (Accession No: AF492764 from nucleotide 2150 to 2113). Bisulfite analysis of two independent clones from genomic DNA of D. melanogaster is shown in (A,B). Analysis of Gag following replication in a methylation deficient strain of E. coli Gm2929 is shown in (C). The methylated cytosine residues in D. melanogaster genomic DNA are resistant to the bisulfite treatment (designated by a star).
methylated in the genome of the fly. The gag and pol coding regions are typical of many retrotransposons. The pol sequence encodes reverse transcriptase activity which is necessary for transposition. Gag proteins from retroviruses are involved in packaging viral RNA and helping its export from the host cell. The role of Gag protein of retrotransposon is less understood. Gag encoded by the non-long repeat retrotransposons Het-A and TART which form telomeres of D. melanogaster by repeated transpositions onto the ends of chromosomes are involved in the targeting of the retrotransposon RNA to the telomeres [29–31]. The presence of m5C in this DNA region encoding Gag strengthens our hypothesis that DNA methylation may be one of the mechanisms that D. melanogaster uses to keep population of retrotransposable elements under control. Other DNA fragments selected by m5C affinity chromatography included DNA encoding dusky, doublesex, SoxNeuro, and RNA helicase. The explanation for their selection is less obvious. Some of these unique sequences like CG14186-PA and SoxNeuro have a high G + C content (>60%; Table 1). In mammals, preferred sites for DNA methylation include clusters of C–G pairs in regions of the DNA high in G + C content [32]. DNA methylation in Neurospora crassa is catalyzed by Dim-2. Interestingly, methylated component of N. crassa genome consists almost exclusively of relics of transposons [14]. In the protozoan parasite E. histolytica, repetitive elements like rDNA [15] and S/MAR containing DNA (Banerjee et al., unpublished) are targeted by Ehmeth, a DNA methyltransferase that be-
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