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Meiotic recombination initiation in and around retrotransposable elements in Saccharomyces cerevisiae.

Sasaki M, Tischfield SE, van Overbeek M, Keeney S - PLoS Genet. (2013)

Bottom Line: When they do, they create a risk for deleterious genome rearrangements in the germ line via recombination between non-allelic repeats.From whole-genome DSB maps and direct molecular assays, we find that DSB levels and chromatin structure within and near Tys vary widely between different elements and that local DSB suppression is not a universal feature of Ty presence.Given high strain-to-strain variability in Ty location and the high aggregate burden of Ty-proximal DSBs, we propose that meiotic recombination is an important component of host-Ty interactions and that Tys play critical roles in genome instability and evolution in both inbred and outcrossed sexual cycles.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.

ABSTRACT
Meiotic recombination is initiated by large numbers of developmentally programmed DNA double-strand breaks (DSBs), ranging from dozens to hundreds per cell depending on the organism. DSBs formed in single-copy sequences provoke recombination between allelic positions on homologous chromosomes, but DSBs can also form in and near repetitive elements such as retrotransposons. When they do, they create a risk for deleterious genome rearrangements in the germ line via recombination between non-allelic repeats. A prior study in budding yeast demonstrated that insertion of a Ty retrotransposon into a DSB hotspot can suppress meiotic break formation, but properties of Ty elements in their most common physiological contexts have not been addressed. Here we compile a comprehensive, high resolution map of all Ty elements in the rapidly and efficiently sporulating S. cerevisiae strain SK1 and examine DSB formation in and near these endogenous retrotransposable elements. SK1 has 30 Tys, all but one distinct from the 50 Tys in S288C, the source strain for the yeast reference genome. From whole-genome DSB maps and direct molecular assays, we find that DSB levels and chromatin structure within and near Tys vary widely between different elements and that local DSB suppression is not a universal feature of Ty presence. Surprisingly, deletion of two Ty elements weakened adjacent DSB hotspots, revealing that at least some Ty insertions promote rather than suppress nearby DSB formation. Given high strain-to-strain variability in Ty location and the high aggregate burden of Ty-proximal DSBs, we propose that meiotic recombination is an important component of host-Ty interactions and that Tys play critical roles in genome instability and evolution in both inbred and outcrossed sexual cycles.

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Meiotic DSBs in and around Ty elements.(A) Spo11 oligo densities around Ty elements. For each SK1 Ty, Spo11 oligo densities (hits per million mapped reads (hpM) per kb) were determined in the indicated window of adjacent sequence. Sites where Tys are present in S288C but absent in SK1 serve as controls. Bars are means and standard deviations; the dashed line is the genome average; p values are from Wilcoxon rank sum tests. For comparison, the internal Spo11 oligo density averaged across all Ty elements was 6.7 hpM/kb, approximately 30–40-fold lower than the mean for these flanking regions. (B) Spo11 oligo densities around Ty elements in different types of intergenic regions. (C–F) Physical detection of DSBs. (Left) Spo11 oligo distribution from Pan et al. (2011) and maps of ORFs (blue-filled polygons) and tRNA genes (horizontal bars). (Right) Southern blots of genomic DNA isolated from spo11-Y135F, sae2Δ and dmc1Δ strains at 6 hrs after entry into meiosis. Red numbers are DSB frequencies within the bracketed regions in each lane (% of total hybridization signal in the lane); quantification is provided separately for each lane, representing independent cultures. Red bars, probe positions; P, unbroken (parental) restriction fragments; asterisks, cross hybridizing bands. Flanking restriction sites plus internal sites used to generate genomic DNA markers (run on the same gels; not shown) are indicated: NcoI (N), BsaXI (XI), PpuMI (MI), Bsu36I (Bs), BglII (Bg), BspHI (HI), BamHI (B), ApaLI (Ap), SnaBI (Sn), NdeI (Nd). In (F), the inset shows a more exposed contrast of the phosphorimager signal for the region indicated by the dashed line. (G) Quantitative agreement between Spo11 oligo counts and DSB frequencies at hotspots near Ty elements in dmc1Δ mutants. DSB values are the means of the two independent cultures shown in panels C–F.
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pgen-1003732-g004: Meiotic DSBs in and around Ty elements.(A) Spo11 oligo densities around Ty elements. For each SK1 Ty, Spo11 oligo densities (hits per million mapped reads (hpM) per kb) were determined in the indicated window of adjacent sequence. Sites where Tys are present in S288C but absent in SK1 serve as controls. Bars are means and standard deviations; the dashed line is the genome average; p values are from Wilcoxon rank sum tests. For comparison, the internal Spo11 oligo density averaged across all Ty elements was 6.7 hpM/kb, approximately 30–40-fold lower than the mean for these flanking regions. (B) Spo11 oligo densities around Ty elements in different types of intergenic regions. (C–F) Physical detection of DSBs. (Left) Spo11 oligo distribution from Pan et al. (2011) and maps of ORFs (blue-filled polygons) and tRNA genes (horizontal bars). (Right) Southern blots of genomic DNA isolated from spo11-Y135F, sae2Δ and dmc1Δ strains at 6 hrs after entry into meiosis. Red numbers are DSB frequencies within the bracketed regions in each lane (% of total hybridization signal in the lane); quantification is provided separately for each lane, representing independent cultures. Red bars, probe positions; P, unbroken (parental) restriction fragments; asterisks, cross hybridizing bands. Flanking restriction sites plus internal sites used to generate genomic DNA markers (run on the same gels; not shown) are indicated: NcoI (N), BsaXI (XI), PpuMI (MI), Bsu36I (Bs), BglII (Bg), BspHI (HI), BamHI (B), ApaLI (Ap), SnaBI (Sn), NdeI (Nd). In (F), the inset shows a more exposed contrast of the phosphorimager signal for the region indicated by the dashed line. (G) Quantitative agreement between Spo11 oligo counts and DSB frequencies at hotspots near Ty elements in dmc1Δ mutants. DSB values are the means of the two independent cultures shown in panels C–F.

Mentions: We previously showed that Spo11 oligo counts covary linearly with DSB levels, so the frequency of mapped Spo11 oligos is a proxy for DSB frequency [19]. To assess global trends for DSB formation near Ty elements, we compiled densities of Spo11 oligos within 0.5, 1, and 2 kb windows on both sides of each SK1 Ty (Figure 4A and Table S1). These densities varied widely between different Ty insertion sites, covering 80 to 500-fold ranges, depending on window size. Many Ty-flanking regions differed substantially from genome average, both hotter and colder. There was no obvious distinction between Ty families, in that the five elements unambiguously identified as Ty2 showed 33-fold variation in local Spo11 oligo density, and overlapped extensively with densities for Ty1 elements (p = 0.25, Wilcoxon rank sum test) (Table S1).


Meiotic recombination initiation in and around retrotransposable elements in Saccharomyces cerevisiae.

Sasaki M, Tischfield SE, van Overbeek M, Keeney S - PLoS Genet. (2013)

Meiotic DSBs in and around Ty elements.(A) Spo11 oligo densities around Ty elements. For each SK1 Ty, Spo11 oligo densities (hits per million mapped reads (hpM) per kb) were determined in the indicated window of adjacent sequence. Sites where Tys are present in S288C but absent in SK1 serve as controls. Bars are means and standard deviations; the dashed line is the genome average; p values are from Wilcoxon rank sum tests. For comparison, the internal Spo11 oligo density averaged across all Ty elements was 6.7 hpM/kb, approximately 30–40-fold lower than the mean for these flanking regions. (B) Spo11 oligo densities around Ty elements in different types of intergenic regions. (C–F) Physical detection of DSBs. (Left) Spo11 oligo distribution from Pan et al. (2011) and maps of ORFs (blue-filled polygons) and tRNA genes (horizontal bars). (Right) Southern blots of genomic DNA isolated from spo11-Y135F, sae2Δ and dmc1Δ strains at 6 hrs after entry into meiosis. Red numbers are DSB frequencies within the bracketed regions in each lane (% of total hybridization signal in the lane); quantification is provided separately for each lane, representing independent cultures. Red bars, probe positions; P, unbroken (parental) restriction fragments; asterisks, cross hybridizing bands. Flanking restriction sites plus internal sites used to generate genomic DNA markers (run on the same gels; not shown) are indicated: NcoI (N), BsaXI (XI), PpuMI (MI), Bsu36I (Bs), BglII (Bg), BspHI (HI), BamHI (B), ApaLI (Ap), SnaBI (Sn), NdeI (Nd). In (F), the inset shows a more exposed contrast of the phosphorimager signal for the region indicated by the dashed line. (G) Quantitative agreement between Spo11 oligo counts and DSB frequencies at hotspots near Ty elements in dmc1Δ mutants. DSB values are the means of the two independent cultures shown in panels C–F.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3757047&req=5

pgen-1003732-g004: Meiotic DSBs in and around Ty elements.(A) Spo11 oligo densities around Ty elements. For each SK1 Ty, Spo11 oligo densities (hits per million mapped reads (hpM) per kb) were determined in the indicated window of adjacent sequence. Sites where Tys are present in S288C but absent in SK1 serve as controls. Bars are means and standard deviations; the dashed line is the genome average; p values are from Wilcoxon rank sum tests. For comparison, the internal Spo11 oligo density averaged across all Ty elements was 6.7 hpM/kb, approximately 30–40-fold lower than the mean for these flanking regions. (B) Spo11 oligo densities around Ty elements in different types of intergenic regions. (C–F) Physical detection of DSBs. (Left) Spo11 oligo distribution from Pan et al. (2011) and maps of ORFs (blue-filled polygons) and tRNA genes (horizontal bars). (Right) Southern blots of genomic DNA isolated from spo11-Y135F, sae2Δ and dmc1Δ strains at 6 hrs after entry into meiosis. Red numbers are DSB frequencies within the bracketed regions in each lane (% of total hybridization signal in the lane); quantification is provided separately for each lane, representing independent cultures. Red bars, probe positions; P, unbroken (parental) restriction fragments; asterisks, cross hybridizing bands. Flanking restriction sites plus internal sites used to generate genomic DNA markers (run on the same gels; not shown) are indicated: NcoI (N), BsaXI (XI), PpuMI (MI), Bsu36I (Bs), BglII (Bg), BspHI (HI), BamHI (B), ApaLI (Ap), SnaBI (Sn), NdeI (Nd). In (F), the inset shows a more exposed contrast of the phosphorimager signal for the region indicated by the dashed line. (G) Quantitative agreement between Spo11 oligo counts and DSB frequencies at hotspots near Ty elements in dmc1Δ mutants. DSB values are the means of the two independent cultures shown in panels C–F.
Mentions: We previously showed that Spo11 oligo counts covary linearly with DSB levels, so the frequency of mapped Spo11 oligos is a proxy for DSB frequency [19]. To assess global trends for DSB formation near Ty elements, we compiled densities of Spo11 oligos within 0.5, 1, and 2 kb windows on both sides of each SK1 Ty (Figure 4A and Table S1). These densities varied widely between different Ty insertion sites, covering 80 to 500-fold ranges, depending on window size. Many Ty-flanking regions differed substantially from genome average, both hotter and colder. There was no obvious distinction between Ty families, in that the five elements unambiguously identified as Ty2 showed 33-fold variation in local Spo11 oligo density, and overlapped extensively with densities for Ty1 elements (p = 0.25, Wilcoxon rank sum test) (Table S1).

Bottom Line: When they do, they create a risk for deleterious genome rearrangements in the germ line via recombination between non-allelic repeats.From whole-genome DSB maps and direct molecular assays, we find that DSB levels and chromatin structure within and near Tys vary widely between different elements and that local DSB suppression is not a universal feature of Ty presence.Given high strain-to-strain variability in Ty location and the high aggregate burden of Ty-proximal DSBs, we propose that meiotic recombination is an important component of host-Ty interactions and that Tys play critical roles in genome instability and evolution in both inbred and outcrossed sexual cycles.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.

ABSTRACT
Meiotic recombination is initiated by large numbers of developmentally programmed DNA double-strand breaks (DSBs), ranging from dozens to hundreds per cell depending on the organism. DSBs formed in single-copy sequences provoke recombination between allelic positions on homologous chromosomes, but DSBs can also form in and near repetitive elements such as retrotransposons. When they do, they create a risk for deleterious genome rearrangements in the germ line via recombination between non-allelic repeats. A prior study in budding yeast demonstrated that insertion of a Ty retrotransposon into a DSB hotspot can suppress meiotic break formation, but properties of Ty elements in their most common physiological contexts have not been addressed. Here we compile a comprehensive, high resolution map of all Ty elements in the rapidly and efficiently sporulating S. cerevisiae strain SK1 and examine DSB formation in and near these endogenous retrotransposable elements. SK1 has 30 Tys, all but one distinct from the 50 Tys in S288C, the source strain for the yeast reference genome. From whole-genome DSB maps and direct molecular assays, we find that DSB levels and chromatin structure within and near Tys vary widely between different elements and that local DSB suppression is not a universal feature of Ty presence. Surprisingly, deletion of two Ty elements weakened adjacent DSB hotspots, revealing that at least some Ty insertions promote rather than suppress nearby DSB formation. Given high strain-to-strain variability in Ty location and the high aggregate burden of Ty-proximal DSBs, we propose that meiotic recombination is an important component of host-Ty interactions and that Tys play critical roles in genome instability and evolution in both inbred and outcrossed sexual cycles.

Show MeSH
Related in: MedlinePlus