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. 2005 Nov 10;33(19):6405-17.
doi: 10.1093/nar/gki952. Print 2005.

Silencing of retrotransposons in Dictyostelium by DNA methylation and RNAi

Markus Kuhlmann  1 , Branimira E Borisova, Markus Kaller, Pontus Larsson, Dirk Stach, Jianbo Na, Ludwig Eichinger, Frank Lyko, Victor Ambros, Fredrik Söderbom, Christian Hammann, Wolfgang Nellen
Affiliations

Silencing of retrotransposons in Dictyostelium by DNA methylation and RNAi

Markus Kuhlmann et al. Nucleic Acids Res. .

Abstract

We have identified a DNA methyltransferase of the Dnmt2 family in Dictyostelium that was denominated DnmA. Expression of the dnmA gene is downregulated during the developmental cycle. Overall DNA methylation in Dictyostelium is approximately 0.2% of the cytosine residues, which indicates its restriction to a limited set of genomic loci. Bisulfite sequencing of specific sites revealed that DnmA is responsible for methylation of mostly asymmetric C-residues in the retrotransposons DIRS-1 and Skipper. Disruption of the gene resulted in a loss of methylation and in increased transcription and mobilization of Skipper. Skipper transcription was also upregulated in strains that had genes encoding components of the RNA interference pathway disrupted. In contrast, DIRS-1 expression was not affected by a loss of DnmA but was strongly increased in strains that had the RNA-directed RNA polymerase gene rrpC disrupted. A large number of siRNAs were found that corresponded to the DIRS-1 sequence, suggesting concerted regulation of DIRS-1 expression by RNAi and DNA modification. No siRNAs corresponding to the standard Skipper element were found. The data show that DNA methylation plays a crucial role in epigenetic gene silencing in Dictyostelium but that different, partially overlapping mechanisms control transposon silencing.

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Figures

Figure 1
Figure 1
Amino acid sequence alignment of Dnmt2 homologues from D.discoideum, Arabidopsis thaliana, Homo sapiens and Danio rerio. The 10 characteristic Dnmt2 motifs (44) are indicated by roman numbers. TRD, target recognition domain.
Figure 2
Figure 2
Expression of dnmA. (A) Semi-quantitative RT–PCR was performed on total mRNA isolated from vegetatively grown cells (veg) and from cells after 6 and 16 h of development on filters. Expression of the thioredoxin (trx) gene family is shown as a comparison. (B) Expression of DnmA-c-Myc fusion protein in Dictyostelium visualized by staining with mAb 9E10 and a secondary TRITC coupled polyclonal antibody (top right). The DnmA-GFP fusion protein (bottom right) showed the same localization. DNA was stained with DAPI to localize the nucleus (left). Bars are 8 µm.
Figure 3
Figure 3
(A) Semi-quantitative RT–PCR on dnmA gene transcripts from wild-type Ax2 cells and the dnmA disruption mutant. Capillary electrophoresis on DNA from wild-type cells (B) and the dnmA disruption strain. (C) The position of 5-methyl cytosine is indicated.
Figure 4
Figure 4
(A) Bisulfite sequencing of 294 bp in the Skipper RT gene (bottom strand). The top part shows the organization of the retrotransposon with GAG, PRO and RT genes and LTRs. ESTs that were included in the microarray are indicated below. (B) Bisulfite sequencing of 214 bp in the Skipper LTR region (bottom strand). (C) Bisulfite sequencing of 128 bp in the right LTR of DIRS-1 (bottom strand). The top part shows the organization of the retrotransposon with the overlapping reading frames ORF I, III and II, LTRs and the E1 antisense transcript. Grey triangles indicate unmethylated C-residues; black squares methylated C-residues. Symbols above the sequence refer to DNA from Ax2 wild-type cells; symbols below the sequence refer to DNA from the dnmA− strain. No symbol is shown when the sequence could not be unambiguously determined.
Figure 4
Figure 4
(A) Bisulfite sequencing of 294 bp in the Skipper RT gene (bottom strand). The top part shows the organization of the retrotransposon with GAG, PRO and RT genes and LTRs. ESTs that were included in the microarray are indicated below. (B) Bisulfite sequencing of 214 bp in the Skipper LTR region (bottom strand). (C) Bisulfite sequencing of 128 bp in the right LTR of DIRS-1 (bottom strand). The top part shows the organization of the retrotransposon with the overlapping reading frames ORF I, III and II, LTRs and the E1 antisense transcript. Grey triangles indicate unmethylated C-residues; black squares methylated C-residues. Symbols above the sequence refer to DNA from Ax2 wild-type cells; symbols below the sequence refer to DNA from the dnmA− strain. No symbol is shown when the sequence could not be unambiguously determined.
Figure 4
Figure 4
(A) Bisulfite sequencing of 294 bp in the Skipper RT gene (bottom strand). The top part shows the organization of the retrotransposon with GAG, PRO and RT genes and LTRs. ESTs that were included in the microarray are indicated below. (B) Bisulfite sequencing of 214 bp in the Skipper LTR region (bottom strand). (C) Bisulfite sequencing of 128 bp in the right LTR of DIRS-1 (bottom strand). The top part shows the organization of the retrotransposon with the overlapping reading frames ORF I, III and II, LTRs and the E1 antisense transcript. Grey triangles indicate unmethylated C-residues; black squares methylated C-residues. Symbols above the sequence refer to DNA from Ax2 wild-type cells; symbols below the sequence refer to DNA from the dnmA− strain. No symbol is shown when the sequence could not be unambiguously determined.
Figure 5
Figure 5
(A) Northern blots with specific Skipper (top) and DIRS-1 (bottom) probes on RNA isolated from wild-type Ax2 and dnmtA− cells after a short period of growth in axenic medium and after 16 passages through the developmental cycle (indicated by asterisk). Ethidium bromide staining is shown for quantitative comparison. (B) Northern blot with a Skipper (top) and a DIRS-1 probe (bottom) on RNA isolated from axenically growing cells of different mutants. Ax2: wild-type control, rrpA−, rrpB−, rrpC−: knock outs of the RNA-directed RNA polymerase genes rrpA, B and C, respectively, helF−: knock out of the gene encoding the putative RNA helicase HelF, drnA: knock out of the gene encoding the putative dicer homologue A, dnmA−: knock out of the gene encoding the DNA methyltransferase A, dnmA−*: knock out of the gene encoding dnmA, RNA from cells after 16 rounds of the developmental cycle.
Figure 6
Figure 6
RNAs derived from the small RNA library are aligned on the schematic depiction of DIRS-1: sense strand (A) and antisense strand (B). The inverted repeats are marked in yellow. The ORFs I, II and III and the E1 antisense transcript are depicted as colored arrows. siRNAs are shown as red lines. Most of the small RNAs are in the range of 21 nt; some longer molecules have been retrieved and are also shown. Small RNA alignment in respect to the DIRS-1 sequence is shown in Supplementary Figure 1S.
Figure 7
Figure 7
(A) Southern blot with Skipper and DIRS-1 probes on wild-type Ax2 and dnmA− mutant cells after 0 and 16 rounds (indicated by asterisk) of the developmental cycle. From left: ethidium bromide staining, hybridization with an eriA probe, hybridization with a Skipper probe, hybridization with a DIRS-1 probe. (B) Relative copy numbers were calculated in respect to ethidium bromide staining and to hybridization to the single copy gene eriA (see Materials and Methods).
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