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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 levels result in spreading of repressive histone marks and an opening of the chromatin at a dCAP-D3 regulated gene cluster containing a retrotransposon.A) ChIP for trimethylated H3K9 (a' and b') and trimethylated H3K4 (c' and d') performed on the mdg1-1403 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 mdg1 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 mdg1-1403 (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 mdg1-1403. Results are the average of three experiments and (*) indicates p-value less than 0.05 as calculated by student unpaired t-test.
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pgen-1003879-g005: 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.A) ChIP for trimethylated H3K9 (a' and b') and trimethylated H3K4 (c' and d') performed on the mdg1-1403 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 mdg1 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 mdg1-1403 (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 mdg1-1403. Results are the average of three experiments and (*) indicates p-value less than 0.05 as calculated by student unpaired t-test.

Mentions: Increased levels of double strand breaks have been shown to lead to an opening of the chromatin structure in order to facilitate repair [49]–[51]. To examine whether the increased levels of double strand breaks within retrotransposon sequence in dCAP-D3 dsRNA treated cells also resulted in an opening of the chromatin structure, ChIP assays to detect histone modifications were performed. In order to better understand the timing of changes in histone marks in reference to the precise loss of retrotransposon sequence, these assays were done in the context of the SG4 time course experiments presented in Figure S2. ChIP was performed on the mdg1-1403 locus to examine levels of the repressive trimethylated H3K9 mark (Figure 5A, a' and b') and the activating trimethylated H3K4 mark (Figure 5A, c' and d'). Results of experiments performed in SG4 cells treated with control dsRNAs revealed significant levels of H3K9 trimethylation only within the retrotransposon sequence (Figure 5A, a' and b', black bars). High levels of H3K9 trimethylation have been reported previously at Drosophila retrotransposon sequences [40], [52]. No significant levels of H3K4 trimethylation were detected at the locus in cells treated with control dsRNAs (Figure 5A, c' and d', black bars). dCAP-D3 knockdown at a time point prior to local loss of sequence, significantly increased levels of H3K9me3 at the sequences surrounding the mdg1-1403 retrotransposon (Figure 5A, a' white bars). Transcription of the surrounding genes was correspondingly decreased (Figure 5B, top panel). qRT-PCR for the CG31343 gene, located approximately 12 kb upstream from the 3′ end of the mdg1-1403 retrotransposon, was performed as a negative control and demonstrates no significant change in transcription. Following loss of retrotransposon sequence in dCAP-D3 knockdown cells, H3K9me3 marks actually decreased within the retrotransposon sequence (Figure 5A, b', white bars). Recently, ChIP-seq experiments showed that repressive H3K9me3 marks found at mdg1 retrotransposons in Drosophila somatic cells are held within strict boundaries and, on average, do not extend into neighboring regions [40]. Therefore, this data suggests that decreases in dCAP-D3 expression may cause a loss of repressive boundary and a local spreading of H3K9me3 from within the retrotransposon sequence into the surrounding sequence. Also, prior to the local loss of retrotransposon sequence, significant increases in H3K4me3 levels were seen over the entire locus in dCAP-D3 dsRNA treated cells (Figure 5A, c', white bars). Transcription of the surrounding genes also increased and returned to basal levels on the day that the local loss of retrotransposon sequence occurred (Figure 5B, middle panel). The appearance of H3K4me3 marks in dCAP-D3 dsRNA treated cells prior to retrotransposon mobilization suggests that the increases in double strand breaks within mdg1 retrotransposon sequence may indeed lead to a local opening of chromatin. Finally, ChIP results show that the increase in H3K4me3 in areas surrounding the retrotransposon persisted following retrotransposon mobilization suggesting that dCAP-D3 knockdown may in fact cause a permanent change in chromatin structure.


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 levels result in spreading of repressive histone marks and an opening of the chromatin at a dCAP-D3 regulated gene cluster containing a retrotransposon.A) ChIP for trimethylated H3K9 (a' and b') and trimethylated H3K4 (c' and d') performed on the mdg1-1403 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 mdg1 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 mdg1-1403 (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 mdg1-1403. Results are the average of three experiments and (*) indicates p-value less than 0.05 as calculated by student unpaired t-test.
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pgen-1003879-g005: 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.A) ChIP for trimethylated H3K9 (a' and b') and trimethylated H3K4 (c' and d') performed on the mdg1-1403 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 mdg1 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 mdg1-1403 (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 mdg1-1403. Results are the average of three experiments and (*) indicates p-value less than 0.05 as calculated by student unpaired t-test.
Mentions: Increased levels of double strand breaks have been shown to lead to an opening of the chromatin structure in order to facilitate repair [49]–[51]. To examine whether the increased levels of double strand breaks within retrotransposon sequence in dCAP-D3 dsRNA treated cells also resulted in an opening of the chromatin structure, ChIP assays to detect histone modifications were performed. In order to better understand the timing of changes in histone marks in reference to the precise loss of retrotransposon sequence, these assays were done in the context of the SG4 time course experiments presented in Figure S2. ChIP was performed on the mdg1-1403 locus to examine levels of the repressive trimethylated H3K9 mark (Figure 5A, a' and b') and the activating trimethylated H3K4 mark (Figure 5A, c' and d'). Results of experiments performed in SG4 cells treated with control dsRNAs revealed significant levels of H3K9 trimethylation only within the retrotransposon sequence (Figure 5A, a' and b', black bars). High levels of H3K9 trimethylation have been reported previously at Drosophila retrotransposon sequences [40], [52]. No significant levels of H3K4 trimethylation were detected at the locus in cells treated with control dsRNAs (Figure 5A, c' and d', black bars). dCAP-D3 knockdown at a time point prior to local loss of sequence, significantly increased levels of H3K9me3 at the sequences surrounding the mdg1-1403 retrotransposon (Figure 5A, a' white bars). Transcription of the surrounding genes was correspondingly decreased (Figure 5B, top panel). qRT-PCR for the CG31343 gene, located approximately 12 kb upstream from the 3′ end of the mdg1-1403 retrotransposon, was performed as a negative control and demonstrates no significant change in transcription. Following loss of retrotransposon sequence in dCAP-D3 knockdown cells, H3K9me3 marks actually decreased within the retrotransposon sequence (Figure 5A, b', white bars). Recently, ChIP-seq experiments showed that repressive H3K9me3 marks found at mdg1 retrotransposons in Drosophila somatic cells are held within strict boundaries and, on average, do not extend into neighboring regions [40]. Therefore, this data suggests that decreases in dCAP-D3 expression may cause a loss of repressive boundary and a local spreading of H3K9me3 from within the retrotransposon sequence into the surrounding sequence. Also, prior to the local loss of retrotransposon sequence, significant increases in H3K4me3 levels were seen over the entire locus in dCAP-D3 dsRNA treated cells (Figure 5A, c', white bars). Transcription of the surrounding genes also increased and returned to basal levels on the day that the local loss of retrotransposon sequence occurred (Figure 5B, middle panel). The appearance of H3K4me3 marks in dCAP-D3 dsRNA treated cells prior to retrotransposon mobilization suggests that the increases in double strand breaks within mdg1 retrotransposon sequence may indeed lead to a local opening of chromatin. Finally, ChIP results show that the increase in H3K4me3 in areas surrounding the retrotransposon persisted following retrotransposon mobilization suggesting that dCAP-D3 knockdown may in fact cause a permanent change in chromatin structure.

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