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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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dCAP-D3 binds to loci containing retrotransposons.ChIP performed for dCAP-D3 at the (A) mdg1-1403 locus or (B) G2-1077 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” (mdg1-1403 locus) and “4” (G2-1077 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.
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pgen-1003879-g002: dCAP-D3 binds to loci containing retrotransposons.ChIP performed for dCAP-D3 at the (A) mdg1-1403 locus or (B) G2-1077 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” (mdg1-1403 locus) and “4” (G2-1077 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.

Mentions: To understand whether dCAP-D3's ability to restrict the local loss of retrotransposon sequence was direct, Chromatin immunoprecipitation (ChIP) experiments were performed. ChIP for dCAP-D3 in cells treated with control dsRNA for 4 days demonstrated that dCAP-D3 does in fact bind to the mdg1-1403 locus. In the Figure, (*) indicates a quantitative comparison between the indicated ChIP signal in control dsRNA and dCAP-D3 dsRNA treated cells with a p-value less than 0.05 as calculated by a student unpaired t-test. In other words, (*) indicates that the dependency of the ChIP signal on the presence of dCAP-D3 is statistically significant. (+) 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. In other words, (+) indicates that the position of the ChIP signal relative to the rest of the locus is statistically significant. In Figure 2A, the peak of dCAP-D3 binding occurred at the junction of the retrotransposon and the CG42335 exon. Much of this binding was lost in cells treated with dCAP-D3 dsRNAs for 4 days (i.e. before loss of retrotransposon sequence, Figure S2B). dCAP-D3 binding was also seen within the retrotransposon sequence, but these results are harder to interpret, since the primers used detect all mdg1 sequences throughout the entire genome. Interestingly, ChIP for dCAP-D3 at the G2-1077 locus exhibited very similar results, with the peak of dCAP-D3 binding again occurring at the junction of the retrotransposon and flanking gene sequence (Figure 2B). These results suggest that dCAP-D3 does associate with different retrotransposon-containing loci and at similar places within the loci.


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

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

dCAP-D3 binds to loci containing retrotransposons.ChIP performed for dCAP-D3 at the (A) mdg1-1403 locus or (B) G2-1077 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” (mdg1-1403 locus) and “4” (G2-1077 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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3814330&req=5

pgen-1003879-g002: dCAP-D3 binds to loci containing retrotransposons.ChIP performed for dCAP-D3 at the (A) mdg1-1403 locus or (B) G2-1077 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” (mdg1-1403 locus) and “4” (G2-1077 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.
Mentions: To understand whether dCAP-D3's ability to restrict the local loss of retrotransposon sequence was direct, Chromatin immunoprecipitation (ChIP) experiments were performed. ChIP for dCAP-D3 in cells treated with control dsRNA for 4 days demonstrated that dCAP-D3 does in fact bind to the mdg1-1403 locus. In the Figure, (*) indicates a quantitative comparison between the indicated ChIP signal in control dsRNA and dCAP-D3 dsRNA treated cells with a p-value less than 0.05 as calculated by a student unpaired t-test. In other words, (*) indicates that the dependency of the ChIP signal on the presence of dCAP-D3 is statistically significant. (+) 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. In other words, (+) indicates that the position of the ChIP signal relative to the rest of the locus is statistically significant. In Figure 2A, the peak of dCAP-D3 binding occurred at the junction of the retrotransposon and the CG42335 exon. Much of this binding was lost in cells treated with dCAP-D3 dsRNAs for 4 days (i.e. before loss of retrotransposon sequence, Figure S2B). dCAP-D3 binding was also seen within the retrotransposon sequence, but these results are harder to interpret, since the primers used detect all mdg1 sequences throughout the entire genome. Interestingly, ChIP for dCAP-D3 at the G2-1077 locus exhibited very similar results, with the peak of dCAP-D3 binding again occurring at the junction of the retrotransposon and flanking gene sequence (Figure 2B). These results suggest that dCAP-D3 does associate with different retrotransposon-containing loci and at similar places within the loci.

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