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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 mutant salivary glands exhibit loss of mdg1-1403 and increases in retrotransposon copy number at other loci.FISH experiments using probes hybridized to the mdg1-1403 locus (green) and to mdg1 retrotransposon sequence (red) demonstrate that wild type glands retain the mdg1 retrotransposon sequence at the mdg1-1403 locus (middle panel and smaller panels on right in A) while dCap-D3 mutants do not (middle panel and smaller panels on right in B). Yellow arrows indicate co-localization of probes in wild type preparations and absence of co-localization in dCap-D3 mutant preparations. The average copy number for each genotype was determined for salivary glands from 5 separate larvae by counting total numbers of mdg1 bands. Salivary gland chromatin was stained with DAPI and is shown in white. Additional FISH experiments are shown in Figure S7.
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pgen-1003879-g007: dCap-D3 mutant salivary glands exhibit loss of mdg1-1403 and increases in retrotransposon copy number at other loci.FISH experiments using probes hybridized to the mdg1-1403 locus (green) and to mdg1 retrotransposon sequence (red) demonstrate that wild type glands retain the mdg1 retrotransposon sequence at the mdg1-1403 locus (middle panel and smaller panels on right in A) while dCap-D3 mutants do not (middle panel and smaller panels on right in B). Yellow arrows indicate co-localization of probes in wild type preparations and absence of co-localization in dCap-D3 mutant preparations. The average copy number for each genotype was determined for salivary glands from 5 separate larvae by counting total numbers of mdg1 bands. Salivary gland chromatin was stained with DAPI and is shown in white. Additional FISH experiments are shown in Figure S7.

Mentions: To confirm the specificity of our FISH probes and to visualize the chromatin at dCAP-D3-regulated retrotransposon containing loci in vivo, FISH was also performed on salivary gland squashes from wild type and dCAP-D3 mutant larvae. Drosophila salivary glands contain polytene chromatin which is formed by continuous endoreduplication of chromatids which then pair together. Homologous chromosomes (estimated to each contain over 500 copies of DNA) also pair, creating the beautiful banding pattern that polytene chromosomes are famous for. In the FISH experiments presented in Figure 7, two probes were used: an Alexa 555 (red) labeled probe which hybridized to the multi-copy mdg1 retrotransposon sequence and an Alexa 488 (green) labeled probe which hybridized to the single copy region just upstream of the mdg1-1403 retrotransposon. In agreement with previous PCR results, the mdg1 and mdg1-1403 probes co-localized in wild type larvae, indicating presence of the mdg1-1403 retrotransposon on both homologs (Figure 7A and Figure S7A). FISH analyses performed on dCAP-D3 mutant salivary gland squashes showed that the mdg1 and mdg1-1403 probes did not co-localize, confirming that a local loss of mdg1-1403 retrotransposon sequence had indeed occurred (Figure 7B and Figure S7B). The average mdg1 copy number (5 larvae examined per genotype) was also determined by counting the number of bands that the mdg1 probe hybridized to. The average copy number in wild type larvae was 16.2 and in dCAP-D3 mutants was 18.8. Therefore, FISH analyses suggest a 1.16 fold increase in mdg1 copy number in dCAP-D3 mutants and this is very close to the 1.1 fold increase seen by qPCR (Figure 3). Together, the FISH results in Drosophila somatic tissue culture cells and tissues support the idea that dCAP-D3/Condensin II prevents pairing of homologous chromosomes and restricts the movement of retrotransposons within the genome.


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 mutant salivary glands exhibit loss of mdg1-1403 and increases in retrotransposon copy number at other loci.FISH experiments using probes hybridized to the mdg1-1403 locus (green) and to mdg1 retrotransposon sequence (red) demonstrate that wild type glands retain the mdg1 retrotransposon sequence at the mdg1-1403 locus (middle panel and smaller panels on right in A) while dCap-D3 mutants do not (middle panel and smaller panels on right in B). Yellow arrows indicate co-localization of probes in wild type preparations and absence of co-localization in dCap-D3 mutant preparations. The average copy number for each genotype was determined for salivary glands from 5 separate larvae by counting total numbers of mdg1 bands. Salivary gland chromatin was stained with DAPI and is shown in white. Additional FISH experiments are shown in Figure S7.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003879-g007: dCap-D3 mutant salivary glands exhibit loss of mdg1-1403 and increases in retrotransposon copy number at other loci.FISH experiments using probes hybridized to the mdg1-1403 locus (green) and to mdg1 retrotransposon sequence (red) demonstrate that wild type glands retain the mdg1 retrotransposon sequence at the mdg1-1403 locus (middle panel and smaller panels on right in A) while dCap-D3 mutants do not (middle panel and smaller panels on right in B). Yellow arrows indicate co-localization of probes in wild type preparations and absence of co-localization in dCap-D3 mutant preparations. The average copy number for each genotype was determined for salivary glands from 5 separate larvae by counting total numbers of mdg1 bands. Salivary gland chromatin was stained with DAPI and is shown in white. Additional FISH experiments are shown in Figure S7.
Mentions: To confirm the specificity of our FISH probes and to visualize the chromatin at dCAP-D3-regulated retrotransposon containing loci in vivo, FISH was also performed on salivary gland squashes from wild type and dCAP-D3 mutant larvae. Drosophila salivary glands contain polytene chromatin which is formed by continuous endoreduplication of chromatids which then pair together. Homologous chromosomes (estimated to each contain over 500 copies of DNA) also pair, creating the beautiful banding pattern that polytene chromosomes are famous for. In the FISH experiments presented in Figure 7, two probes were used: an Alexa 555 (red) labeled probe which hybridized to the multi-copy mdg1 retrotransposon sequence and an Alexa 488 (green) labeled probe which hybridized to the single copy region just upstream of the mdg1-1403 retrotransposon. In agreement with previous PCR results, the mdg1 and mdg1-1403 probes co-localized in wild type larvae, indicating presence of the mdg1-1403 retrotransposon on both homologs (Figure 7A and Figure S7A). FISH analyses performed on dCAP-D3 mutant salivary gland squashes showed that the mdg1 and mdg1-1403 probes did not co-localize, confirming that a local loss of mdg1-1403 retrotransposon sequence had indeed occurred (Figure 7B and Figure S7B). The average mdg1 copy number (5 larvae examined per genotype) was also determined by counting the number of bands that the mdg1 probe hybridized to. The average copy number in wild type larvae was 16.2 and in dCAP-D3 mutants was 18.8. Therefore, FISH analyses suggest a 1.16 fold increase in mdg1 copy number in dCAP-D3 mutants and this is very close to the 1.1 fold increase seen by qPCR (Figure 3). Together, the FISH results in Drosophila somatic tissue culture cells and tissues support the idea that dCAP-D3/Condensin II prevents pairing of homologous chromosomes and restricts the movement of retrotransposons within the genome.

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
Related in: MedlinePlus