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Condensin II subunit dCAP-D3 restricts retrotransposon mobilization in Drosophila somatic cells.

Schuster AT, Sarvepalli K, Murphy EA, Longworth MS - PLoS Genet. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, United States of America.

ABSTRACT
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 Drosophila melanogaster 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 Drosophila 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.

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Decreased dCAP-D3 expression results in double strand break accumulation within retrotransposon sequence.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 mdg1-1403 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.
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pgen-1003879-g004: Decreased dCAP-D3 expression results in double strand break accumulation within retrotransposon sequence.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 mdg1-1403 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.

Mentions: Aside from retrotransposition, another mechanism by which retrotransposons mobilize is through homologous recombination with identical copies at allelic or non-allelic positions. The sequencing products shown in Figure S2 resemble products of single strand annealing events and/or unequal crossover between repeated sequences on the same chromosome or on homologous chromosomes, respectively [24], [46], [47]. Homologous recombination requires DNA double strand break formation for homologous sequences to recombine. γ-H2AV is a marker of DNA double strand breaks in Drosophila[48]. In order to determine if knock down of dCAP-D3 caused more cells to exhibit double strand breaks, we performed immunofluorescence analysis for γ-H2AV on SG4 cells treated with control or dCAP-D3 dsRNA for 4 days. Indeed, a significant increase in the percentage of cells exhibiting γ-H2AV foci was seen for cells treated with dCAP-D3 dsRNAs in comparison to cells treated with control dsRNAs (Figure 4A and 4B). Increases in double strand breaks can occur following stalling of replication forks and slowing of S phase. To determine whether acute knockdown of dCAP-D3 resulted in a change in the cell cycle distribution, FACS analysis of SG4 cells treated with control or dCAP-D3 dsRNAs was performed. Results showed that there were no dramatic changes (nothing more than 1.5% change) in cells treated with dCAP-D3 dsRNAs in comparison to control knockdown cells (Figure S5). ChIP for γ-H2AV indicated that in control dsRNA treated cells, double strand breaks at the mdg1-1403 locus occur more frequently outside the retrotransposon sequence (Figure 4C). Surprisingly, 4 days of dCAP-D3 dsRNA treatment (before local loss of retrotransposon sequence- Figure S2B) results in a shift in the distribution of γ-H2AV, causing fewer breaks to occur outside of the retrotransposon sequence and more to occur within. Results were similar for the γ-H2AV distribution at the locus containing the G2-1077 retrotransposon (Figure S6). These findings are surprising since the maximum level of dCAP-D3 knock-down achieved in multiple experiments was approximately 53%. This implies that even minimal decreases in dCAP-D3 levels result in major changes to the chromatin at these loci. Taken together, these data suggest that dCAP-D3 is involved in inhibiting DNA double strand break formation, especially within retrotransposon sequence.


Condensin II subunit dCAP-D3 restricts retrotransposon mobilization in Drosophila somatic cells.

Schuster AT, Sarvepalli K, Murphy EA, Longworth MS - PLoS Genet. (2013)

Decreased dCAP-D3 expression results in double strand break accumulation within retrotransposon sequence.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 mdg1-1403 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.
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Related In: Results  -  Collection

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Show All Figures
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pgen-1003879-g004: Decreased dCAP-D3 expression results in double strand break accumulation within retrotransposon sequence.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 mdg1-1403 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.
Mentions: Aside from retrotransposition, another mechanism by which retrotransposons mobilize is through homologous recombination with identical copies at allelic or non-allelic positions. The sequencing products shown in Figure S2 resemble products of single strand annealing events and/or unequal crossover between repeated sequences on the same chromosome or on homologous chromosomes, respectively [24], [46], [47]. Homologous recombination requires DNA double strand break formation for homologous sequences to recombine. γ-H2AV is a marker of DNA double strand breaks in Drosophila[48]. In order to determine if knock down of dCAP-D3 caused more cells to exhibit double strand breaks, we performed immunofluorescence analysis for γ-H2AV on SG4 cells treated with control or dCAP-D3 dsRNA for 4 days. Indeed, a significant increase in the percentage of cells exhibiting γ-H2AV foci was seen for cells treated with dCAP-D3 dsRNAs in comparison to cells treated with control dsRNAs (Figure 4A and 4B). Increases in double strand breaks can occur following stalling of replication forks and slowing of S phase. To determine whether acute knockdown of dCAP-D3 resulted in a change in the cell cycle distribution, FACS analysis of SG4 cells treated with control or dCAP-D3 dsRNAs was performed. Results showed that there were no dramatic changes (nothing more than 1.5% change) in cells treated with dCAP-D3 dsRNAs in comparison to control knockdown cells (Figure S5). ChIP for γ-H2AV indicated that in control dsRNA treated cells, double strand breaks at the mdg1-1403 locus occur more frequently outside the retrotransposon sequence (Figure 4C). Surprisingly, 4 days of dCAP-D3 dsRNA treatment (before local loss of retrotransposon sequence- Figure S2B) results in a shift in the distribution of γ-H2AV, causing fewer breaks to occur outside of the retrotransposon sequence and more to occur within. Results were similar for the γ-H2AV distribution at the locus containing the G2-1077 retrotransposon (Figure S6). These findings are surprising since the maximum level of dCAP-D3 knock-down achieved in multiple experiments was approximately 53%. This implies that even minimal decreases in dCAP-D3 levels result in major changes to the chromatin at these loci. Taken together, these data suggest that dCAP-D3 is involved in inhibiting DNA double strand break formation, especially within retrotransposon sequence.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, United States of America.

ABSTRACT
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 Drosophila melanogaster 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 Drosophila 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.

Show MeSH