Condensin II Subunit dCAP-D3 Restricts Retrotransposon Mobilization in <i>Drosophila</i> Somatic Cells

<div><p>Retrotransposon sequences are positioned throughout the genome of almost every eukaryote that has been sequenced. As mobilization of these elements can have detrimental effects on the transcriptional regulation and stability of an organism's genome, most organisms have evolved mechanisms to repress their movement. Here, we identify a novel role for the <i>Drosophila melanogaster</i> Condensin II subunit, dCAP-D3 in preventing the mobilization of retrotransposons located in somatic cell euchromatin. dCAP-D3 regulates transcription of euchromatic gene clusters which contain or are proximal to retrotransposon sequence. ChIP experiments demonstrate that dCAP-D3 binds to these loci and is important for maintaining a repressed chromatin structure within the boundaries of the retrotransposon and for repressing retrotransposon transcription. We show that dCAP-D3 prevents accumulation of double stranded DNA breaks within retrotransposon sequence, and decreased dCAP-D3 levels leads to a precise loss of retrotransposon sequence at some dCAP-D3 regulated gene clusters and a gain of sequence elsewhere in the genome. Homologous chromosomes exhibit high levels of pairing in <i>Drosophila</i> somatic cells, and our FISH analyses demonstrate that retrotransposon-containing euchromatic loci are regions which are actually less paired than euchromatic regions devoid of retrotransposon sequences. Decreased dCAP-D3 expression increases pairing of homologous retrotransposon-containing loci in tissue culture cells. We propose that the combined effects of dCAP-D3 deficiency on double strand break levels, chromatin structure, transcription and pairing at retrotransposon-containing loci may lead to 1) higher levels of homologous recombination between repeats flanking retrotransposons in dCAP-D3 deficient cells and 2) increased retrotransposition. These findings identify a novel role for the anti-pairing activities of dCAP-D3/Condensin II and uncover a new way in which dCAP-D3/Condensin II influences local chromatin structure to help maintain genome stability.</p></div

dCAP-D3 binds to loci containing retrotransposons.

<p>ChIP performed for dCAP-D3 at the (A) <i>mdg1-1403</i> locus or (B) <i>G2-1077</i> locus demonstrate binding over the entire region with the peak of binding occurring at the region encompassing both retrotransposon sequence and neighboring DNA sequence. Black bars indicate ChIP signal from SG4 cells treated with control dsRNA for 4 days and white bars indicate ChIP signal from cells treated with dCAP-D3 dsRNA for 4 days. Primer sets used are depicted above the charts. Primer sets “LTR” and “5” (<i>mdg1-1403</i> locus) and “4” (<i>G2-1077</i> locus) are not specific for each of the loci but instead prime global retrotransposon sequence. Results are the averages of 2 experiments involving duplicate IPs and are presented as a percentage of the IP with control IgG ChIP signal subtracted. (*) indicates a quantitative comparison between dCAP-D3 signal in control dsRNA and dCAP-D3 dsRNA treated cells with a p-value less than 0.05 as calculated by student unpaired t-test. (+) indicates a quantitative comparison of specific dCAP-D3 signal to the average over the entire locus with a p-value less than 0.05 as calculated by student unpaired t-test.</p

Decreased dCAP-D3 expression results in double strand break accumulation within retrotransposon sequence.

<p>A) Immunofluorescence analysis shows increased numbers of SG4 cells exhibiting γ-H2AV foci following 4 days of treatment with dCAP-D3 dsRNAs compared to cells treated with T7 control dsRNA. γ-H2AV is shown in green and DAPI stained nuclei in white. Two representative panels are shown for each dsRNA treatment. The average percentage of cells in each of 10 random frames (n≥1000 cells) harboring γ-H2AV foci is quantified in (B). C) ChIP for γ-H2AV performed on the <i>mdg1-1403</i> locus in SG4 cells treated with control dsRNA (black bars) demonstrates higher levels of binding in the regions flanking retrotransposon sequence. ChIP in cells treated with dCAP-D3 dsRNA (white bars) show a shift in γ-H2AV distribution out of retrotransposon flanking regions and into retrotransposon sequence. Primer sets used are depicted above the charts. Primer sets “LTR” and “5” are not specific for each of the loci but instead prime global retrotransposon sequence. Results are the averages of 2 experiments involving duplicate IPs and are presented as a percentage of the IP with control IgG ChIP signal subtracted. (*) indicates a quantitative comparison between γ-H2AV signal in control dsRNA and dCAP-D3 dsRNA treated cells with a p-value less than 0.05 as calculated by student unpaired t-test. (+) indicates a quantitative comparison of specific γ-H2AV signal to the average over the entire locus with a p-value less than 0.05 as calculated by student unpaired t-test.</p

Decreased dCAP-D3 levels result in spreading of repressive histone marks and an opening of the chromatin at a dCAP-D3 regulated gene cluster containing a retrotransposon.

<p>A) ChIP for trimethylated H3K9 (a' and b') and trimethylated H3K4 (c' and d') performed on the <i>mdg1-1403</i> locus in SG4 cells treated with control dsRNA (black bars) demonstrates absent or low levels of the marks in the areas surrounding the retrotransposon but high levels of H3K9me3 within retrotransposon sequence. ChIP for trimethylated H3K9 in cells treated with dCAP-D3 dsRNA demonstrates a dCAP-D3 dependent increase of the mark in the areas surrounding the retrotransposon (a') and a dCAP-D3 dependent decrease of the mark in within retrotransposon sequence following retrotransposon mobilization (b'). ChIP for trimethylated H3K4 in cells treated with dCAP-D3 dsRNAs shows a dCAP-D3 dependent increase over the entire locus prior to mobilization (c') which persist following mobilization. Primer sets used are depicted above the charts. Primer sets “LTR” and “5” are not specific for each of the loci but instead prime global <i>mdg1</i> sequence. Results are the averages of 2 experiments involving duplicate IPs and are presented as a percentage of the IP with control IgG ChIP signal subtracted. (*) indicates a quantitative comparison between indicated ChIP signal in control dsRNA and dCAP-D3 dsRNA treated cells with a p-value less than 0.05 as calculated by student unpaired t-test. (+) indicates a quantitative comparison of specific ChIP signal to the average over the entire locus with a p-value less than 0.05 as calculated by student unpaired t-test. (B) qRT-PCR for transcript levels of genes surrounding <i>mdg1-1403</i> (as depicted in diagram in A) indicates that compared to control dsRNA treated cells, transcription is decreased in dCAP-D3 dsRNA treated cells prior to retrotransposon mobilization (day 4) and increases to almost normal levels on the day of retrotransposon mobilization (day 5). Following retrotransposon mobilization (day 6), transcription increases more than 2-fold in dCAP-D3 dsRNA treated cells. CG31343 is positioned approximately 12 kb upstream of <i>mdg1-1403</i>. Results are the average of three experiments and (*) indicates p-value less than 0.05 as calculated by student unpaired t-test.</p

Decreased levels of dCAP-D3/Condensin II result in a local loss of retrotransposon sequence <i>in vivo</i> and <i>in vitro</i>.

<p>A) PCR performed on DNA from wild type (<i>w¹¹¹⁸</i>) adults and dCAP-D3 transheterozygous mutant adults (d<i>Cap-D3^c07081</i>d<i>Cap-D3^/Δ25</i>) to detect whether three different retrotransposon sequences (<i>mdg1-1403, G2-1077, X-978</i>) were present (P) or absent (A) in dCAP-D3 regulated gene clusters indicates absence of the retrotransposon sequence only in <i>dCap-D3</i> mutants. In the diagram for each locus, transposon positions are illustrated with pink brackets. Single black boxes represent the entire coding sequence for each gene, except in the case of CG42335 where two black boxes are used to represent the coding sequence upstream and downstream of the <i>mdg1-1403</i> retrotransposon. Primers sets used are depicted in the diagrams above the gels and their sequences can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003879#s4" target="_blank">Materials and Methods</a>. Tubulin23C (Tub) was used as a control for each reaction. In the PCRs performed on the <i>mdg1-1403</i> locus, an asterisk denotes the band for presence. The miscellaneous band seen in the wild type absence reaction was confirmed to be a mispriming event off of tubulin (data not shown). B) PCR performed to detect presence or absence of <i>mdg1-1403</i> in different <i>dCap-D3</i> mutant genotypes shows that events which cause local loss of retrotransposon sequence increase as dCAP-D3 expression levels decrease. PCR was performed in individual, female, wild type flies and flies expressing different <i>dCap-D3</i> mutant alleles. <i>dCap-D3</i> mutants were heterozygous or homozygous for a hypomorphic allele of <i>dCap-D3</i> (<i>c07081</i>) which expresses about 10% the levels of wild type protein <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003879#pgen.1003879-Longworth2" target="_blank">[8]</a>, or transheterozygous for a combination of the hypomorphic allele and a deletion (<i>c07081/Δ25</i>). <i>rp49</i> was used as a control for each reaction. C) PCRs performed as described in (A) on DNA from flies expressing two mutant alleles of a second Condensin II subunit, <i>dCap-H2</i> (<i>dCap-H2^Z3-0019/dCap-H2^Z3-</i>⁵¹⁶³), demonstrates identical results seen for <i>dCap-D3</i> mutants. D) PCR for <i>mdg1-1403</i> presence or absence in Condensin I subunit, <i>dCap-D2</i>, heterozygous mutants <i>(dCap-D2^f03381/+)</i> shows only presence of the retrotransposon. PCRs were performed on 1) wild type adults to test for presence, 2) wild type adults to test for absence, 3) <i>dCap-D2</i> mutants to test for presence and 4) <i>dCap-D2</i> mutants to test for absence. E) PCR for presence and absence of <i>mdg1-1403</i> (left) and <i>G2-1077</i> (right) in SG4 cells treated with control or dCAP-D3 dsRNAs for 6 days shows the appearance of an absence band (pink arrows) in dCAP-D3 dsRNA treated cells but not in control dsRNA treated cells.</p

Possible model for how dCAP-D3/Condensin II might restrict retrotransposon mobilization in <i>Drosophila</i> somatic cells.

<p>(A) In this model, dCAP-D3/Condensin II (green circles) organizes gene clusters that it transcriptionally regulates (orange) into a rigid and possibly looped structure (shown on the left). This would serve to position retrotransposon sequences within the cluster (purple) in a manner that is inhibitory to recombination with the homologous chromosome. A decrease in dCAP-D3 levels would result in increased DNA double strand breaks (DSBs) within retrotransposon sequence and an opening of the chromatin structure. This would then allow retrotransposon sequences to more frequently contact the homologous chromosome (magnified image of dCAP-D3 regulated gene cluster shown on the right). While having only minor effects on regions that normally pair at high frequencies (blue), the increased frequency of contacts between repeats flanking retrotransposon sequences on homologous chromosomes combined with increased double strand breaks could lead to unequal crossover events and/or repair by single strand annealing. This would then result in loss of locus-specific retrotransposon sequence and gain of sequence in a separate place. (B) It is also possible that, at some retrotransposon containing dCAP-D3 regulated gene clusters, the opening of chromatin and slight increase in retrotransposon transcription seen following decreased dCAP-D3 expression could lead to generation of RNA intermediates and retrotransposition of new copies to other places in the genome.</p

Pairing of retrotransposon loci on homologous chromosomes is increased in cells treated with dCAP-D3 dsRNAs.

<p>A) Quantification of the percentage of cells harboring single FISH dots representing paired loci in SG4 cells treated with control dsRNAs for 4 days shows that the control 28B euchromatic locus (black bar) which is not proximal to retrotransposon sequence is paired at a significantly higher frequency than the <i>mdg1-1403</i> locus (white bar) or the <i>G2-1077</i> locus (grey bar). B) Quantification of the percentage of cells harboring single FISH dots representing paired loci demonstrates a slight but not significant increase in pairing of the 28B locus in dCAP-D3 dsRNA treated SG4 cells (white bar) compared to T7 control dsRNA treated cells (black bar). C) FISH experiments using probes hybridized to the <i>mdg1-1403</i> locus demonstrate a significantly higher frequency of single FISH dots/pairing in cells treated with dCAP-D3 dsRNA for 4 days, as compared to cells treated with control dsRNA for 4 days. Probes were labeled with Alexa-555 and DAPI-stained nuclei are shown in blue. Inset boxes are magnifications of a single cell within the larger image. D) Quantification of the percentage of cells harboring single FISH dots from FISH experiments using probes hybridized to the <i>mdg1-1403</i> locus (left) and <i>G2-1077</i> locus (right) demonstrate significantly higher frequencies of pairing in cells treated with dCAP-D3 dsRNA (white bars) as compared to cells treated with control dsRNA (black bars). E) The distance between unpaired homologous <i>mdg1-1403</i> loci in control dsRNA (green, n = 124) and dCAP-D3 dsRNA (blue, n = 135) treated SG4 cells was measured by manually planing through z stacks of images taking on a confocal microscope. The mean of each sample set is shown as a black horizontal line. For each FISH experiment/quantification in (A–D), over 200 cells were counted using the maximum projections and experiments were repeated three times. Only non-mitotic cells were counted. F) Nuclear volumes of cells treated with control or dCAP-D3 dsRNAs were measured using the Volocity software and changes were found to be not statistically different between the two populations. 5 separate fields of view containing at least 50 cells (two independent experiments) were counted for each population. Only non-mitotic cells were counted.</p

Global retrotransposon transcript levels and copy numbers increase as a result of decreased dCAP-D3 expression in somatic cells.

<p>A) qRT-PCR for 6 different retrotransposon transcripts in SG4 cells demonstrates that after 4 days of dCAP-D3 dsRNA treatment (white bars), there is a slight increase in global transcript levels as compared to cells treated with control dsRNA (black bars). B) qRT-PCR performed on cDNA from 20 wild type (<i>w¹¹¹⁸</i>-black bar) or 20 <i>dCap-D3</i> mutant (<i>dCap-D3^c07081dCap-D3^/Δ25</i>-white bar) larval brains indicates that a significant decrease in dCAP-D3 transcripts <i>in vivo</i> results in a slight increase in <i>mdg1</i> transcripts. All qRT-PCR results were normalized to housekeeping gene <i>rp49</i> and experiments are the average of three biological replicates. C) qPCR to determine relative global copy numbers of mdg1, G2, and X element in wild type and <i>dCap-D3</i> mutant larvae indicates that copy numbers increase slightly in the mutants. Copy numbers for each retrotransposon were normalized to single copy regions located upstream. Analyses were performed on pooled samples of DNA from 10 larvae for each genotype. (*) indicates p-value less than 0.05 as calculated by student unpaired t-test.</p

RBF1 and dCAP-D3 regulate many of the same transcripts in the fly.

<p>RNA was isolated from <i>rbf1</i> mutant and <i>dCap-D3</i> mutant female third instar larvae and adult flies. cDNA was hybridized to Nimblegen 385 k whole genome arrays. A) Venn diagrams show the numbers of RBF1, dCAP-D3 or RBF1/dCAP-D3 shared target genes which exhibited at least a 2 fold log change in expression with a p value of ≤0.15. Genes significantly upregulated in the mutant flies are shown in red while genes significantly downregulated are shown in green. B) P values for shared RBF1 and dCAP-D3 target genes indicate that RBF1 and dCAP-D3 regulate a significant number of the same genes in both adults and larvae. The numbers above the diagonal represent p-values for upregulated shared subsets and are colored red while the numbers below the diagonal represent p-values for downregulated shared genes and are colored green. C) qRT-PCR analyses of 12 E2F targets shows that the majority of RBF1/dCAP-D3 shared targets are not E2F targets. The one target that was significantly upregulated in dCAP-D3 and RBF1 mutant flies, <i>CG5250</i>, is highlighted in red. Results are the average of three independent experiments involving 10 female flies per genotype. D) Significant (p≤0.05) Gene Ontology (GO) groupings for shared target genes include defense response genes in the adult fly. The top box lists GO categories for upregulated shared genes in mutant larvae only, and the bottom box lists selected GO categories for downregulated shared genes in adults only. There were no significant GO groupings for upregulated shared target genes in adults or for downregulated shared target genes in the larvae.</p

RBF1 is necessary for a proper immune response to Gram positive bacterial infection.

<p>Adult female flies expressing RBF1 (purple) dsRNAs under the control of <i>yolk-GAL4</i> were infected with the Gram positive bacterium, <i>Staphylococcus aureus</i> (A) or the Gram negative bacterium, <i>Pseudomonas aeruginosa</i> (B). Flies expressing GFP dsRNAs under the control of <i>yolk-GAL4</i> (green) were used as “wild-type” controls. Eater mutants which are defective in phagocytosis (blue) or flies expressing IMD dsRNAs which are compromised in a major innate immune signaling pathway (yellow) were used as positive controls. Results demonstrate that flies expressing reduced levels of RBF1 in the fat body cells are more susceptible to infection with Gram positive bacteria (A) than wild type controls. Three independent experiments are depicted with results of each experiment shown as the average of three sets of 10 infected adults per genotype. Results are presented as cox regression models with statistical significance (p≤0.05) represented as shaded areas above and below the curves. In the third experiment in (A), which is highlighted by a star, the survival endpoint becomes significant when the confidence level is changed to 90% (p≤0.10) instead of 95% (p≤0.05). These experiments were also performed using a sterile needle dipped in PBS to rule out death as a result of wounding and survival curves matched those of yolk-GAL4 expressing flies (data not shown).</p