1. Volume 22, Issue 7, Pages 1861-1874 (February 2018)
Mutations in Cytosine-5 tRNA Methyltransferases Impact Mobile Element Expression and Genome Stability at Specific DNA Repeats 
Bianca Genenncher, Zeljko Durdevic, Katharina Hanna, Daniela Zinkl, Mehrpouya Balaghy Mobin, Nevcin Senturk, Bruno Da Silva, Carine Legrand, Clément Carré, Frank Lyko, Matthias Schaefer 
Cell Reports 
Volume 22, Issue 7, Pages (February 2018)
DOI: /j.celrep
Copyright © 2018 The Author(s) Terms and Conditions

2. Cell Reports 2018 22, 1861-1874DOI: (10.1016/j.celrep.2018.01.061)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1 Inefficient Silencing of Stress-Induced TEs in Dnmt2 Mutants
(A) qRT-PCR analysis for Hsp70A and TE sequences in w1118 and Dnmt2 mutant flies during a single and multiple heat shock experiment. Three independent experiments were performed and normalized to 18S rRNA expression for quantification (mean ± SD).
(B) qRT-PCR analysis for Hsp70A and TE sequences in w1118 and Dnmt2 mutant flies during the long-term recovery from a single heat shock. Three independent experiments were performed and normalized to 18S rRNA expression for quantification (mean ± SD).
(C) Cartoon summarizing the observed transient derepression/transcriptional activation of LTR-TEs in control flies and the failure to downregulate these sequences in Dnmt2 mutant flies during the recovery from heat shock.
(D) Schematic illustration of the long terminal repeat (LTR)-TE Invader4 (Flybase: FBte ), with primers used for DNA bisulfite sequencing analysis.
(E) DNA bisulfite sequencing on Invader4 LTR sequences in w1118 and Dnmt2 mutant flies during a single heat shock experiment. Heatmap representation: each row shows one sequence read; each column shows one cytosine in the analyzed region; red, unconverted cytosine (C); green, converted cytosine (T); white, sequencing gaps (n.d.); sequencing depth indicated by numbers; bc, barcodes of forward 454 sequencing primers. For PCR primers and deamination conditions, see the Supplemental Experimental Procedures.
See also Figure S1.
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4. Figure 2
Global Gene Expression Changes in Dnmt2 Mutants after Heat Shock
(A) Ethidium bromide-stained agarose gel containing total RNA from w1118 and Dnmt2 mutant flies from a heat shock experiment. Bracket indicates size range of extracted RNAs from Dnmt2 mutant lanes; black arrowhead, 28S and 18S rRNA; gray arrowhead, 5S rRNA.
(B) Pie chart depicting the identification of sequencing reads (%) obtained from extracted RNAs.
(C) Bar chart depicting the distribution of all Drosophila sequencing reads (%) in four main categories: LSU, large subunit-rRNA; SSU, small subunit-rRNA; cRNA, coding RNA; non-cRNA, non-coding RNA; TEs, transposable elements.
(D) Pie chart depicting the distribution of sequencing reads (%) that mapped to annotated TEs in the Drosophila genome.
(E) Bar charts depicting the distribution of sequencing reads (%) among TE families that belong to the three main classes (LTR-TE, non-LTR-TE, and DNA-TE).
See also Figure S2 and Tables S1–S3.
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5. Figure 3
An NSun2 Mutant Allele Affects TE Expression Heat Shock Independently
(A) Cartoon depicting the targeting of the NSun2 locus with guide RNA sequence (gRNA, underlined) and PAM domain (bold). Recovered NSun2 mutant (NSun2Δ21.5) locus is shown below.
(B) RNA bisulfite sequencing of tRNAs in w1118 and NSun2 mutant flies. Heatmap representation: each row shows one sequence read; each column shows one cytosine in the analyzed region; blue, unconverted cytosine (C); yellow, converted cytosine (T); red, mismatch; sequencing depth indicated by numbers; black arrowheads, NSun2 target sites; gray arrowheads, Dnmt2 target sites. For PCR primers and deamination conditions, see the Supplemental Experimental Procedures.
(C) Cartoon depicting the targeting of the Dnmt2 locus with gRNA and PAM as depicted in (A). Recovered Dnmt2 mutant (Dnmt2Δ5.4) locus is shown below.
(D) RNA bisulfite sequencing of tRNAs in w1118 and Dnmt2 mutant flies. Heatmap representation as in (C). Black arrowheads, Dnmt2 target sites; gray arrowheads, NSun2 target sites.
(E) Western blotting on NSun2Δ and Dnmt2Δ5.4-null mutant alleles using antibodies against endogenous proteins.
(F) qRT-PCR analysis for TE sequences in w1118, Dnmt2Δ5.4, and NSun2Δ21.5 mutant larval brain tissues at standard culture conditions. Data were normalized to RpL32 expression (mean ± SEM).
(G) qRT-PCR analysis for Hsp70A and TE sequences in w1118 and Dnmt2Δ5.4 mutant flies during the recovery from a single heat shock. Data were normalized to Actin5C expression (mean ± SEM).
(H) qRT-PCR analysis for TE sequences in w1118 and NSun2Δ21.5 mutant flies during a heat shock experiment. Two independent experiments were performed and normalized to RpL32 RNA expression for quantification (mean ± SD).
(I) qRT-PCR analysis for TE sequences in female and male soma from w1118, NSun2Δ21.5, Dnmt2Δ5.4, and NSun2Δ21.5; Dnmt2Δ5.4 homozygous double mutants at standard culture conditions. For Gypsy spliced see below (Figure S4). Data were normalized to RpL32 RNA expression for quantification (mean ± SEM).
See also Figure S3.
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6. Figure 4 Somatic TE Expression Changes in RCMT Mutants
(A) qRT-PCR analysis for Hsp70A and LTR-TEs during a heat shock experiment in dissected tissues (ovaries and remaining soma) from w1118 and Dnmt2 mutant females.
(B) qRT-PCR analysis for Hsp70A and LTR-TEs during a heat shock experiment in dissected tissues (testes and remaining soma) from w1118 and Dnmt2 mutant males.
Three independent experiments were performed and normalized to 18S rRNA expression (mean ± SD).
(C) Expression of a Copia::LTR-lacZ construct (blue) in testes tips and regions of sperm maturation in w1118 and Dnmt2 mutant males.
(D) Somatic and germline TE sensor activation (β-Gal staining, blue) in ovaries after GAL4-mediated knockdown of the indicated genes (indicated promoters: traffic jam, tj; and nanos, nos). Magnification, 20×.
(E) Somatic TE sensor activation in Dnmt2 and Nsun2 mutant ovaries. Magnification, 20×.
(F) Germline TE sensor activation before and after a single heat shock in ovaries after NGT > GAL4-mediated knockdown of piwi (positive control) and in RCMT mutants. Magnification, 20×.
See also Figure S4.
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7. Figure 5 RCMT Mutants Display Instability at a Y Chromosome Locus
(A) Eye pigmentation variation in F1 progeny of Tag-Inv4; Dnmt2Δ149 males after a single heat shock. Possible genotypes, white expression (P-element), and sex of F1-scored flies are depicted (for scoring scheme, see the Supplemental Experimental Procedures). Y, Y chromosome; X, X chromosome; A, autosome; w+, Tag-Inv4; F, female; M, male.
(B) Quantification of pigmentation changes in F1 originating from single Tag-Inv4; w1118 and Tag-Inv4; Dnmt2 mutant males under standard conditions.
(C) Quantification of pigmentation changes in F1 originating from single heat-shocked Tag-Inv4 adult males crossed to females under standard conditions. Genotypes as in (B). The p values were determined by the test of hypothesis for two populations.
(D) Quantification of pigmentation changes in flies with genotypes as in (B) origination from single Tag-Inv4 males that were heat-shocked as third instar larvae and crossed to females under standard conditions.
(E) Quantification of male fertility after a single heat shock, and mating with non-heat-shocked females under standard conditions (male) or in the F1 progeny from males that were heat-shocked as third instar larvae and crossed to females under standard conditions (3∗).
(F) Eye pigmentation of Tag-Inv4 in w1118, Dnmt2Δ5.4 homozygous and NSun2Δ21.5 hemizygous mutant male flies under standard conditions.
(G) Quantification of pigmentation changes in w1118 or hemizygous male and heterozygous female NSun2Δ21.5 mutant progeny from single Tag-Inv4 male crosses under standard conditions.
(H) Quantification of pigmentation changes in w1118 or hemizygous male and heterozygous female NSun2Δ21.5 mutant progeny from single Tag-Inv4 males that were heat-shocked and crossed to females under standard conditions.
(I) Cartoon of the mapping approach by inverse PCR. Restriction (RE) within the P-element reverted repeat at the 5′ end and at unknown genomic sites close to the Invader4 LTR creates genomic DNA fragments suitable for ligation and inverse PCR mapping. PCR was carried out with primers from within the 5′ inverted repeat.
(J) Inverse PCR on single F1 progeny from experiments as described in (C). Paternal Tag-Inv4 insertion: PCR product (a); males with darker eyes than their fathers and females with red eyes yielded additional PCR products (b and c). Fertility of individual F1 progeny was tested by crossing to control flies.
(K) Cartoon depicting Tag-Inv4 insertion sites as determined by inverse PCR and sequencing (see also Table S5).
See also Figure S5 and Tables S4 and S5.
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8. Figure 6
RCMT Mutants Display Reduced tRNA Stability and tRNA Abundance
(A) Northern blots of RNA from adult flies using a 5′ probe against the common RCMT substrates (tRNA-GlyGCC and tRNA-AspGUC) and two NSun2-specific substrates tRNA-Leu and tRNA-MetCAU. Black arrowhead, mature tRNAs; bracket, region containing tRNA fragments (tRFs); gray arrowhead, loading control.
(B) Northern blots on RNA from w1118 and Dnmt2 mutant flies during a heat shock experiment using 5′ probes against common substrates for Dnmt2 and NSun2 (tRNA-AspGUC and tRNA-GlyGCC) and an NSun2-substrate (tRNA-MetCAU). Black arrowhead and brackets, depiction as in (A); SYBR indicates loading.
(C) Northern blots on RNA from Dnmt2 mutants and Dnmt2Δ5.4 mutants carrying an UASp-controlled Dnmt2 (wild-type or C78A-mutated) and expressed with tubulin-Gal4 during a heat shock experiment. Blots were analyzed with a combination of 5′ probes against common substrates for Dnmt2. Black arrowhead and brackets, depiction as in (A).
(D) Northern blots on RNA from w1118 and NSun2 mutant flies during a heat shock experiment. Probes and depictions are as in (B).
(E) Northern blot on RNA from w1118 and NSun2 mutant flies during the recovery from a single heat shock using probes against tRNA-Leu (all except LeuAAG). Arrowheads, depiction as in (A).
(F) RNA bisulfite sequencing of the intron-containing tRNA-LeuCAA_2.3 in control and NSun2 mutant flies. Heatmap representation: each row shows one sequence read; each column shows one cytosine in the analyzed region; blue, unconverted cytosine (C); yellow, converted cytosine (T); red, mismatch; sequencing depth indicated by numbers; black arrowhead, NSun2 target site at C34; gray arrowhead, C48.
(G) RNA bisulfite sequencing of the isoacceptor tRNA-LeuCAG in w1118 and NSun2 mutant flies. Heatmap representation is as in (F); black arrowhead, NSun2 target site at C48; gray arrowhead, C34.
(H) Northern blot on RNA from w1118 and NSun2 mutant flies during the recovery from a single heat shock using probes against a specific isoacceptor of tRNA-Leu (LeuCAA). Black and gray arrowheads, depiction as in (E). The blot was stripped and re-analyzed (black vertical arrowhead) with a probe against the intron of one isodecoder of tRNA-LeuCAA (CAA_2.3); blue arrowhead, size of non-spliced tRNA precursor; red arrowhead, size of intron.
See also Figure S6.
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