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NatB domain-containing CRA-1 antagonizes hydrolase ACER-1 linking acetyl-CoA metabolism to the initiation of recombination during C. elegans meiosis.

Gao J, Kim HM, Elia AE, Elledge SJ, Colaiácovo MP - PLoS Genet. (2015)

Bottom Line: Moreover, perturbations to global histone acetylation levels are accompanied by changes in the frequency of DSB formation in C. elegans.CRA-1 is in turn negatively regulated by XND-1, an AT-hook containing protein.We propose that this newly defined protein network links acetyl-CoA metabolism to meiotic DSB formation via modulation of global histone acetylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
The formation of DNA double-strand breaks (DSBs) must take place during meiosis to ensure the formation of crossovers, which are required for accurate chromosome segregation, therefore avoiding aneuploidy. However, DSB formation must be tightly regulated to maintain genomic integrity. How this regulation operates in the context of different chromatin architectures and accessibility, and how it is linked to metabolic pathways, is not understood. We show here that global histone acetylation levels undergo changes throughout meiotic progression. Moreover, perturbations to global histone acetylation levels are accompanied by changes in the frequency of DSB formation in C. elegans. We provide evidence that the regulation of histone acetylation requires CRA-1, a NatB domain-containing protein homologous to human NAA25, which controls the levels of acetyl-Coenzyme A (acetyl-CoA) by antagonizing ACER-1, a previously unknown and conserved acetyl-CoA hydrolase. CRA-1 is in turn negatively regulated by XND-1, an AT-hook containing protein. We propose that this newly defined protein network links acetyl-CoA metabolism to meiotic DSB formation via modulation of global histone acetylation.

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Sites of early meiotic recombination events are spatially separated from transcriptionally active chromatin.(A) Gonads from rad-54 mutants were co-immunostained for HTP-3 (white), RAD-51 (red) and AcK (green, with a mouse anti-AcK antibody). DNA was stained with DAPI (blue). To facilitate visualization of individual chromosomes, images of pachytene nuclei correspond to 0.2 μm-thick projections from 3D data stacks consisting of 0.05 μm intervals. Bar, 4 μm. (B) Superimposition of at least 200 RAD-51 foci (red) captured from different nuclei from three different gonads at early pachytene in rad-54 mutants or γ-IR treated spo-11 mutants (20 min post-IR), showing that sites where DSB-bound RAD-51 foci are detected are devoid of AcK signal (green) (left panels). Average fluorescence intensity around the RAD-51 foci was measured by radial profile analysis (Image J) (right panels). Bar, 1 μm. (C) Co-immunostaining for CTD serine2-phosphorylated RNA polymerase II (pSer2) (red), AcK (green) and HTP-3 (white) in wild type worms. DNA was stained with DAPI (blue). To facilitate visualization of individual chromosomes, images of pachytene nuclei correspond to 0.2 μm-thick projections from 3D data stacks consisting of 0.05 μm intervals. Filled white arrowheads show examples where pSer2 and AcK are present at the same perichromosomal regions. Bar, 3 μm. (D) Fluoresence intensity was measured along the yellow line in (C) left panel, exemplifying how AcK and pSer2 are present at the tip of the chromatin loops. (E) Measuring the distances from RAD-51 foci to chromosome axes. Gonads were immunostained for RAD-51 (red) and HTP-3 (green). DNA was stained with DAPI (blue). Images were captured through whole nuclei at 0.05 μm intervals. Three-dimensional distances between the centers of RAD-51 foci and the centers of the axes (centers were defined by points of peak intensity) were measured with softWoRx Explorer (Applied Precision). Top image represents a stack of sections halfway through a whole nucleus. The horizontal and vertical distances from RAD-51 foci to the axes in the white box are shown at a higher resolution in the middle and bottom panels, respectively. Bar, 3 μm. (F) Left panel depicts the average fluorescence intensity of HTP-3 (gray line), DAPI (blue line) and pSer2 (red line) from the axis to chromosome periphery. Measurements were performed as in (D), and data represent average fluorescence intensity measurements for 41 germline nuclei from four different gonads. Yellow dashed line indicates the peak of pSer2 signal (tips of the chromatin loops). The HTP-3 signal corresponds to two parallel chromosome axes about 0.1 μm apart [30]. Thus, the axes are actually positioned about 0.05 μm from the center of the signal, depicted by a green dashed line (base of the chromatin loops). Right panel shows the distances between RAD-51 or RPA-1::YFP foci and chromosome axes. Gonads from wild type or rad-54 mutant worms were triple-stained as in (A) and gonads from brc-2; RPA-1::YFP worms were immunostained for YFP, HTP-3 and AcK. 3D distances from the centers of the RAD-51 or RPA-1::YFP foci to the center of the axes were measured in early meiotic prophase as performed in (E). Dashed lines depict the positions indicated in the left panel. Bars represent the mean distances ± SEM. (G) Measurement of distances between γ-irradiation induced RAD-51 foci and axes. A dose of 800 rads was used to induce DSBs in spo-11 mutants.—IR: analysis of early pachytene nuclei from rad-54 mutants. The hours indicate the time points post-irradiation in which the worms were dissected for immunostaining. Dashed lines depict the positions indicated in (F). Bars represent the mean distances ± SEM. (H) Distribution of RAD-51 foci between the autosomes and X chromosomes in spo-11 mutants exposed to 800 rads of γ-irradiation. Worms were dissected and fixed 20 min post-IR.
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pgen.1005029.g005: Sites of early meiotic recombination events are spatially separated from transcriptionally active chromatin.(A) Gonads from rad-54 mutants were co-immunostained for HTP-3 (white), RAD-51 (red) and AcK (green, with a mouse anti-AcK antibody). DNA was stained with DAPI (blue). To facilitate visualization of individual chromosomes, images of pachytene nuclei correspond to 0.2 μm-thick projections from 3D data stacks consisting of 0.05 μm intervals. Bar, 4 μm. (B) Superimposition of at least 200 RAD-51 foci (red) captured from different nuclei from three different gonads at early pachytene in rad-54 mutants or γ-IR treated spo-11 mutants (20 min post-IR), showing that sites where DSB-bound RAD-51 foci are detected are devoid of AcK signal (green) (left panels). Average fluorescence intensity around the RAD-51 foci was measured by radial profile analysis (Image J) (right panels). Bar, 1 μm. (C) Co-immunostaining for CTD serine2-phosphorylated RNA polymerase II (pSer2) (red), AcK (green) and HTP-3 (white) in wild type worms. DNA was stained with DAPI (blue). To facilitate visualization of individual chromosomes, images of pachytene nuclei correspond to 0.2 μm-thick projections from 3D data stacks consisting of 0.05 μm intervals. Filled white arrowheads show examples where pSer2 and AcK are present at the same perichromosomal regions. Bar, 3 μm. (D) Fluoresence intensity was measured along the yellow line in (C) left panel, exemplifying how AcK and pSer2 are present at the tip of the chromatin loops. (E) Measuring the distances from RAD-51 foci to chromosome axes. Gonads were immunostained for RAD-51 (red) and HTP-3 (green). DNA was stained with DAPI (blue). Images were captured through whole nuclei at 0.05 μm intervals. Three-dimensional distances between the centers of RAD-51 foci and the centers of the axes (centers were defined by points of peak intensity) were measured with softWoRx Explorer (Applied Precision). Top image represents a stack of sections halfway through a whole nucleus. The horizontal and vertical distances from RAD-51 foci to the axes in the white box are shown at a higher resolution in the middle and bottom panels, respectively. Bar, 3 μm. (F) Left panel depicts the average fluorescence intensity of HTP-3 (gray line), DAPI (blue line) and pSer2 (red line) from the axis to chromosome periphery. Measurements were performed as in (D), and data represent average fluorescence intensity measurements for 41 germline nuclei from four different gonads. Yellow dashed line indicates the peak of pSer2 signal (tips of the chromatin loops). The HTP-3 signal corresponds to two parallel chromosome axes about 0.1 μm apart [30]. Thus, the axes are actually positioned about 0.05 μm from the center of the signal, depicted by a green dashed line (base of the chromatin loops). Right panel shows the distances between RAD-51 or RPA-1::YFP foci and chromosome axes. Gonads from wild type or rad-54 mutant worms were triple-stained as in (A) and gonads from brc-2; RPA-1::YFP worms were immunostained for YFP, HTP-3 and AcK. 3D distances from the centers of the RAD-51 or RPA-1::YFP foci to the center of the axes were measured in early meiotic prophase as performed in (E). Dashed lines depict the positions indicated in the left panel. Bars represent the mean distances ± SEM. (G) Measurement of distances between γ-irradiation induced RAD-51 foci and axes. A dose of 800 rads was used to induce DSBs in spo-11 mutants.—IR: analysis of early pachytene nuclei from rad-54 mutants. The hours indicate the time points post-irradiation in which the worms were dissected for immunostaining. Dashed lines depict the positions indicated in (F). Bars represent the mean distances ± SEM. (H) Distribution of RAD-51 foci between the autosomes and X chromosomes in spo-11 mutants exposed to 800 rads of γ-irradiation. Worms were dissected and fixed 20 min post-IR.

Mentions: Our findings that meiotic DSBs are generated on the X chromosomes at lower levels (56%) compared to the autosomes, and that histone acetylation may promote DSB formation, are consistent with the idea that chromatin structure plays an important role in controlling DSB formation. Surprisingly, co-immunostaining of RAD-51 and acetylated lysine in rad-54 mutants shows that RAD-51 foci do not colocalize with the strong acetylation foci during meiosis (Fig. 5A). Superimposition of 200 RAD-51 foci captured from different nuclei at early pachytene shows a greatly reduced AcK staining on the RAD-51 sites (Fig. 5B). Interestingly, both acetylation signals and an active transcription marker, CTD ser2-phosphorylated RNA polymerase II (pSer2), exhibit perichromosomal enrichment, flanking the DAPI signal and away from chromosome axes marked by HTP-3, suggesting that transcriptionally active genes may be localized at the tips of the chromatin loops (Fig. 5C-D). Therefore, our observation that RAD-51 foci do not co-localize with AcK suggests that DSB formation and/or the early stages of meiotic DSB repair take place close to chromosome axes. This is further supported by measurements of the distances between RAD-51 foci and the axes on autosomes and X chromosomes during early meiotic prophase (transition zone and early pachytene stages) (Fig. 5E). RAD-51 foci were observed in close proximity to chromosome axes during early meiotic prophase in both wild type and rad-54 mutants. A similar result was obtained measuring distances of replication protein A (RPA), which binds to single-stranded DNA prior to RAD-51 following DSB end resection, in brc-2 mutants (Fig. 5F). Importantly, this proximity to axes was not observed for RAD-51 foci resulting from γ-irradiation (γ-IR) induced DSBs in spo-11 mutants (Fig. 5G), showing that the localization of RAD-51 foci close to chromosome axes is specific to endogenous DSB formation during early meiotic prophase. Furthermore, the distribution of DSBs generated by γ-IR on autosomes and X chromosomes, is also different from the distribution of endogenously produced DSBs. A X/A ratio of RAD-51 foci close to 1:5 is observed in irradiated-nuclei (P = 0.72) (Fig. 5H), indicating an even distribution of DSBs between the autosomes and X chromosomes.


NatB domain-containing CRA-1 antagonizes hydrolase ACER-1 linking acetyl-CoA metabolism to the initiation of recombination during C. elegans meiosis.

Gao J, Kim HM, Elia AE, Elledge SJ, Colaiácovo MP - PLoS Genet. (2015)

Sites of early meiotic recombination events are spatially separated from transcriptionally active chromatin.(A) Gonads from rad-54 mutants were co-immunostained for HTP-3 (white), RAD-51 (red) and AcK (green, with a mouse anti-AcK antibody). DNA was stained with DAPI (blue). To facilitate visualization of individual chromosomes, images of pachytene nuclei correspond to 0.2 μm-thick projections from 3D data stacks consisting of 0.05 μm intervals. Bar, 4 μm. (B) Superimposition of at least 200 RAD-51 foci (red) captured from different nuclei from three different gonads at early pachytene in rad-54 mutants or γ-IR treated spo-11 mutants (20 min post-IR), showing that sites where DSB-bound RAD-51 foci are detected are devoid of AcK signal (green) (left panels). Average fluorescence intensity around the RAD-51 foci was measured by radial profile analysis (Image J) (right panels). Bar, 1 μm. (C) Co-immunostaining for CTD serine2-phosphorylated RNA polymerase II (pSer2) (red), AcK (green) and HTP-3 (white) in wild type worms. DNA was stained with DAPI (blue). To facilitate visualization of individual chromosomes, images of pachytene nuclei correspond to 0.2 μm-thick projections from 3D data stacks consisting of 0.05 μm intervals. Filled white arrowheads show examples where pSer2 and AcK are present at the same perichromosomal regions. Bar, 3 μm. (D) Fluoresence intensity was measured along the yellow line in (C) left panel, exemplifying how AcK and pSer2 are present at the tip of the chromatin loops. (E) Measuring the distances from RAD-51 foci to chromosome axes. Gonads were immunostained for RAD-51 (red) and HTP-3 (green). DNA was stained with DAPI (blue). Images were captured through whole nuclei at 0.05 μm intervals. Three-dimensional distances between the centers of RAD-51 foci and the centers of the axes (centers were defined by points of peak intensity) were measured with softWoRx Explorer (Applied Precision). Top image represents a stack of sections halfway through a whole nucleus. The horizontal and vertical distances from RAD-51 foci to the axes in the white box are shown at a higher resolution in the middle and bottom panels, respectively. Bar, 3 μm. (F) Left panel depicts the average fluorescence intensity of HTP-3 (gray line), DAPI (blue line) and pSer2 (red line) from the axis to chromosome periphery. Measurements were performed as in (D), and data represent average fluorescence intensity measurements for 41 germline nuclei from four different gonads. Yellow dashed line indicates the peak of pSer2 signal (tips of the chromatin loops). The HTP-3 signal corresponds to two parallel chromosome axes about 0.1 μm apart [30]. Thus, the axes are actually positioned about 0.05 μm from the center of the signal, depicted by a green dashed line (base of the chromatin loops). Right panel shows the distances between RAD-51 or RPA-1::YFP foci and chromosome axes. Gonads from wild type or rad-54 mutant worms were triple-stained as in (A) and gonads from brc-2; RPA-1::YFP worms were immunostained for YFP, HTP-3 and AcK. 3D distances from the centers of the RAD-51 or RPA-1::YFP foci to the center of the axes were measured in early meiotic prophase as performed in (E). Dashed lines depict the positions indicated in the left panel. Bars represent the mean distances ± SEM. (G) Measurement of distances between γ-irradiation induced RAD-51 foci and axes. A dose of 800 rads was used to induce DSBs in spo-11 mutants.—IR: analysis of early pachytene nuclei from rad-54 mutants. The hours indicate the time points post-irradiation in which the worms were dissected for immunostaining. Dashed lines depict the positions indicated in (F). Bars represent the mean distances ± SEM. (H) Distribution of RAD-51 foci between the autosomes and X chromosomes in spo-11 mutants exposed to 800 rads of γ-irradiation. Worms were dissected and fixed 20 min post-IR.
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pgen.1005029.g005: Sites of early meiotic recombination events are spatially separated from transcriptionally active chromatin.(A) Gonads from rad-54 mutants were co-immunostained for HTP-3 (white), RAD-51 (red) and AcK (green, with a mouse anti-AcK antibody). DNA was stained with DAPI (blue). To facilitate visualization of individual chromosomes, images of pachytene nuclei correspond to 0.2 μm-thick projections from 3D data stacks consisting of 0.05 μm intervals. Bar, 4 μm. (B) Superimposition of at least 200 RAD-51 foci (red) captured from different nuclei from three different gonads at early pachytene in rad-54 mutants or γ-IR treated spo-11 mutants (20 min post-IR), showing that sites where DSB-bound RAD-51 foci are detected are devoid of AcK signal (green) (left panels). Average fluorescence intensity around the RAD-51 foci was measured by radial profile analysis (Image J) (right panels). Bar, 1 μm. (C) Co-immunostaining for CTD serine2-phosphorylated RNA polymerase II (pSer2) (red), AcK (green) and HTP-3 (white) in wild type worms. DNA was stained with DAPI (blue). To facilitate visualization of individual chromosomes, images of pachytene nuclei correspond to 0.2 μm-thick projections from 3D data stacks consisting of 0.05 μm intervals. Filled white arrowheads show examples where pSer2 and AcK are present at the same perichromosomal regions. Bar, 3 μm. (D) Fluoresence intensity was measured along the yellow line in (C) left panel, exemplifying how AcK and pSer2 are present at the tip of the chromatin loops. (E) Measuring the distances from RAD-51 foci to chromosome axes. Gonads were immunostained for RAD-51 (red) and HTP-3 (green). DNA was stained with DAPI (blue). Images were captured through whole nuclei at 0.05 μm intervals. Three-dimensional distances between the centers of RAD-51 foci and the centers of the axes (centers were defined by points of peak intensity) were measured with softWoRx Explorer (Applied Precision). Top image represents a stack of sections halfway through a whole nucleus. The horizontal and vertical distances from RAD-51 foci to the axes in the white box are shown at a higher resolution in the middle and bottom panels, respectively. Bar, 3 μm. (F) Left panel depicts the average fluorescence intensity of HTP-3 (gray line), DAPI (blue line) and pSer2 (red line) from the axis to chromosome periphery. Measurements were performed as in (D), and data represent average fluorescence intensity measurements for 41 germline nuclei from four different gonads. Yellow dashed line indicates the peak of pSer2 signal (tips of the chromatin loops). The HTP-3 signal corresponds to two parallel chromosome axes about 0.1 μm apart [30]. Thus, the axes are actually positioned about 0.05 μm from the center of the signal, depicted by a green dashed line (base of the chromatin loops). Right panel shows the distances between RAD-51 or RPA-1::YFP foci and chromosome axes. Gonads from wild type or rad-54 mutant worms were triple-stained as in (A) and gonads from brc-2; RPA-1::YFP worms were immunostained for YFP, HTP-3 and AcK. 3D distances from the centers of the RAD-51 or RPA-1::YFP foci to the center of the axes were measured in early meiotic prophase as performed in (E). Dashed lines depict the positions indicated in the left panel. Bars represent the mean distances ± SEM. (G) Measurement of distances between γ-irradiation induced RAD-51 foci and axes. A dose of 800 rads was used to induce DSBs in spo-11 mutants.—IR: analysis of early pachytene nuclei from rad-54 mutants. The hours indicate the time points post-irradiation in which the worms were dissected for immunostaining. Dashed lines depict the positions indicated in (F). Bars represent the mean distances ± SEM. (H) Distribution of RAD-51 foci between the autosomes and X chromosomes in spo-11 mutants exposed to 800 rads of γ-irradiation. Worms were dissected and fixed 20 min post-IR.
Mentions: Our findings that meiotic DSBs are generated on the X chromosomes at lower levels (56%) compared to the autosomes, and that histone acetylation may promote DSB formation, are consistent with the idea that chromatin structure plays an important role in controlling DSB formation. Surprisingly, co-immunostaining of RAD-51 and acetylated lysine in rad-54 mutants shows that RAD-51 foci do not colocalize with the strong acetylation foci during meiosis (Fig. 5A). Superimposition of 200 RAD-51 foci captured from different nuclei at early pachytene shows a greatly reduced AcK staining on the RAD-51 sites (Fig. 5B). Interestingly, both acetylation signals and an active transcription marker, CTD ser2-phosphorylated RNA polymerase II (pSer2), exhibit perichromosomal enrichment, flanking the DAPI signal and away from chromosome axes marked by HTP-3, suggesting that transcriptionally active genes may be localized at the tips of the chromatin loops (Fig. 5C-D). Therefore, our observation that RAD-51 foci do not co-localize with AcK suggests that DSB formation and/or the early stages of meiotic DSB repair take place close to chromosome axes. This is further supported by measurements of the distances between RAD-51 foci and the axes on autosomes and X chromosomes during early meiotic prophase (transition zone and early pachytene stages) (Fig. 5E). RAD-51 foci were observed in close proximity to chromosome axes during early meiotic prophase in both wild type and rad-54 mutants. A similar result was obtained measuring distances of replication protein A (RPA), which binds to single-stranded DNA prior to RAD-51 following DSB end resection, in brc-2 mutants (Fig. 5F). Importantly, this proximity to axes was not observed for RAD-51 foci resulting from γ-irradiation (γ-IR) induced DSBs in spo-11 mutants (Fig. 5G), showing that the localization of RAD-51 foci close to chromosome axes is specific to endogenous DSB formation during early meiotic prophase. Furthermore, the distribution of DSBs generated by γ-IR on autosomes and X chromosomes, is also different from the distribution of endogenously produced DSBs. A X/A ratio of RAD-51 foci close to 1:5 is observed in irradiated-nuclei (P = 0.72) (Fig. 5H), indicating an even distribution of DSBs between the autosomes and X chromosomes.

Bottom Line: Moreover, perturbations to global histone acetylation levels are accompanied by changes in the frequency of DSB formation in C. elegans.CRA-1 is in turn negatively regulated by XND-1, an AT-hook containing protein.We propose that this newly defined protein network links acetyl-CoA metabolism to meiotic DSB formation via modulation of global histone acetylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
The formation of DNA double-strand breaks (DSBs) must take place during meiosis to ensure the formation of crossovers, which are required for accurate chromosome segregation, therefore avoiding aneuploidy. However, DSB formation must be tightly regulated to maintain genomic integrity. How this regulation operates in the context of different chromatin architectures and accessibility, and how it is linked to metabolic pathways, is not understood. We show here that global histone acetylation levels undergo changes throughout meiotic progression. Moreover, perturbations to global histone acetylation levels are accompanied by changes in the frequency of DSB formation in C. elegans. We provide evidence that the regulation of histone acetylation requires CRA-1, a NatB domain-containing protein homologous to human NAA25, which controls the levels of acetyl-Coenzyme A (acetyl-CoA) by antagonizing ACER-1, a previously unknown and conserved acetyl-CoA hydrolase. CRA-1 is in turn negatively regulated by XND-1, an AT-hook containing protein. We propose that this newly defined protein network links acetyl-CoA metabolism to meiotic DSB formation via modulation of global histone acetylation.

Show MeSH
Related in: MedlinePlus