Limits...
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.

Show MeSH

Related in: MedlinePlus

Analysis of DSB distribution, gene expression and histone acetylation.(A) Gonads from the indicated genotypes were co-immunostained for HTP-3 (green), RAD-51 (red) and AcK (magenta). DNA was stained with DAPI (blue). Images are projections halfway through 3D data stacks of whole nuclei. The X chromosomes show low levels of AcK staining during early meiotic prophase (indicated by open arrowheads). Bar, 5 μm. (B) Graph depicts the distribution of RAD-51 foci on the X chromosomes and the autosomes during early meiotic prophase (from transition zone to mid pachytene combined). The average ratio of RAD-51 foci on the X versus autosomes is indicated. Dashed lines indicate a X/A ratio of 1:5. (C) Graph depicts the distribution of RAD-51 foci on the X and the autosomes during early meiotic prophase (from zones 2 to 4, early through mid-pachytene). The average ratios of RAD-51 foci on the X versus autosomes are indicated. The dashed line indicates a X/A ratio of 1:5. (D) The average ratios of RAD-51 foci on the X versus autosomes in indicated genotypes or treatments in the rad-54 mutant background. Data were analyzed as in (B). The numbers of nuclei scored for each genotype are indicated. Dashed red line depicts the ratio observed in rad-54 single mutants. * P<0.0001 by the extra sum-of-squares F test, compared to rad-54 single mutants. (E) Levels of spo-11 and DSB repair gene expression in dissected gonads from wild type and cra-1 mutants. Quantitative RT-PCR analysis revealed that the germline-specific gene expression for the depicted genes was not altered when comparing cra-1 to wild type and normalizing to gpd-1. Experiments were performed as in [26], utilizing the primers described therein. Error bars represent SEM for three biological replicates, each performed in duplicate. (F) Levels of spo-11 gene expression in indicated genotypes and treatment conditions. Quantitative RT-PCR was performed as in (E), except that whole worms were analyzed instead of dissected gonads. (G) Gonads from the indicated genotypes were co-immunostained for HTP-3 (green) and H4ac (red). DNA was stained with DAPI (blue). The white arrowheads indicate the H4ac-stained X chromosome tip. Bar, 3 μm. (H) Percentage of nuclei showing H4ac staining at the tip of the X chromosome in the indicated genotypes. The numbers of nuclei scored are indicated on the top. At least four gonads were scored for each genotype. (I) Average fluorescent intensity of H4ac along the X chromosomes and on the background was measured with Photoshop (Adobe systems Inc.) for each nucleus. The background fluorescent intensity was subtracted from the fluorescent intensity detected on the X chromosomes and this value was then normalized to the background fluorescent intensity. The numbers of nuclei measured are indicated on the top. At least four gonads were scored for each genotype. Bars represent the mean ± SEM. * P = 0.0006, ** P<0.0001, two-tailed Mann-Whitney test, 95% C.I.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4359108&req=5

pgen.1005029.g003: Analysis of DSB distribution, gene expression and histone acetylation.(A) Gonads from the indicated genotypes were co-immunostained for HTP-3 (green), RAD-51 (red) and AcK (magenta). DNA was stained with DAPI (blue). Images are projections halfway through 3D data stacks of whole nuclei. The X chromosomes show low levels of AcK staining during early meiotic prophase (indicated by open arrowheads). Bar, 5 μm. (B) Graph depicts the distribution of RAD-51 foci on the X chromosomes and the autosomes during early meiotic prophase (from transition zone to mid pachytene combined). The average ratio of RAD-51 foci on the X versus autosomes is indicated. Dashed lines indicate a X/A ratio of 1:5. (C) Graph depicts the distribution of RAD-51 foci on the X and the autosomes during early meiotic prophase (from zones 2 to 4, early through mid-pachytene). The average ratios of RAD-51 foci on the X versus autosomes are indicated. The dashed line indicates a X/A ratio of 1:5. (D) The average ratios of RAD-51 foci on the X versus autosomes in indicated genotypes or treatments in the rad-54 mutant background. Data were analyzed as in (B). The numbers of nuclei scored for each genotype are indicated. Dashed red line depicts the ratio observed in rad-54 single mutants. * P<0.0001 by the extra sum-of-squares F test, compared to rad-54 single mutants. (E) Levels of spo-11 and DSB repair gene expression in dissected gonads from wild type and cra-1 mutants. Quantitative RT-PCR analysis revealed that the germline-specific gene expression for the depicted genes was not altered when comparing cra-1 to wild type and normalizing to gpd-1. Experiments were performed as in [26], utilizing the primers described therein. Error bars represent SEM for three biological replicates, each performed in duplicate. (F) Levels of spo-11 gene expression in indicated genotypes and treatment conditions. Quantitative RT-PCR was performed as in (E), except that whole worms were analyzed instead of dissected gonads. (G) Gonads from the indicated genotypes were co-immunostained for HTP-3 (green) and H4ac (red). DNA was stained with DAPI (blue). The white arrowheads indicate the H4ac-stained X chromosome tip. Bar, 3 μm. (H) Percentage of nuclei showing H4ac staining at the tip of the X chromosome in the indicated genotypes. The numbers of nuclei scored are indicated on the top. At least four gonads were scored for each genotype. (I) Average fluorescent intensity of H4ac along the X chromosomes and on the background was measured with Photoshop (Adobe systems Inc.) for each nucleus. The background fluorescent intensity was subtracted from the fluorescent intensity detected on the X chromosomes and this value was then normalized to the background fluorescent intensity. The numbers of nuclei measured are indicated on the top. At least four gonads were scored for each genotype. Bars represent the mean ± SEM. * P = 0.0006, ** P<0.0001, two-tailed Mann-Whitney test, 95% C.I.

Mentions: To test for a biological significance of regulating global histone acetylation in the germline, we examined the distribution of DSBs between the X chromosomes and autosomes given their different chromatin states. To do this, we analyzed RAD-51 foci in rad-54 mutants, where RAD-51, a protein required for strand invasion/exchange during DSB repair, associates with DSB repair sites, but DSB repair is blocked and DSB-bound RAD-51 are “trapped” and can be easily scored [24,25,35]. Therefore, RAD-51 foci are used herein as a surrogate for DSBs, with the caveat that this may not represent the total number of DSBs since we cannot discard the possibility that not all DSBs, particularly in mutant backgrounds, may be processed to load RAD-51. This analysis was coupled to co-immunostaining with the pan acetylation antibody, which allows for ease of identification of the X chromosomes, as they exhibit greatly decreased histone acetylation compared to the autosomes during early meiotic prophase (Fig. 1G; [32]), and an antibody against the HORMA domain-containing protein HTP-3 to trace chromosome axes and distinguish between DSBs on different chromosomes (Fig. 3A).


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)

Analysis of DSB distribution, gene expression and histone acetylation.(A) Gonads from the indicated genotypes were co-immunostained for HTP-3 (green), RAD-51 (red) and AcK (magenta). DNA was stained with DAPI (blue). Images are projections halfway through 3D data stacks of whole nuclei. The X chromosomes show low levels of AcK staining during early meiotic prophase (indicated by open arrowheads). Bar, 5 μm. (B) Graph depicts the distribution of RAD-51 foci on the X chromosomes and the autosomes during early meiotic prophase (from transition zone to mid pachytene combined). The average ratio of RAD-51 foci on the X versus autosomes is indicated. Dashed lines indicate a X/A ratio of 1:5. (C) Graph depicts the distribution of RAD-51 foci on the X and the autosomes during early meiotic prophase (from zones 2 to 4, early through mid-pachytene). The average ratios of RAD-51 foci on the X versus autosomes are indicated. The dashed line indicates a X/A ratio of 1:5. (D) The average ratios of RAD-51 foci on the X versus autosomes in indicated genotypes or treatments in the rad-54 mutant background. Data were analyzed as in (B). The numbers of nuclei scored for each genotype are indicated. Dashed red line depicts the ratio observed in rad-54 single mutants. * P<0.0001 by the extra sum-of-squares F test, compared to rad-54 single mutants. (E) Levels of spo-11 and DSB repair gene expression in dissected gonads from wild type and cra-1 mutants. Quantitative RT-PCR analysis revealed that the germline-specific gene expression for the depicted genes was not altered when comparing cra-1 to wild type and normalizing to gpd-1. Experiments were performed as in [26], utilizing the primers described therein. Error bars represent SEM for three biological replicates, each performed in duplicate. (F) Levels of spo-11 gene expression in indicated genotypes and treatment conditions. Quantitative RT-PCR was performed as in (E), except that whole worms were analyzed instead of dissected gonads. (G) Gonads from the indicated genotypes were co-immunostained for HTP-3 (green) and H4ac (red). DNA was stained with DAPI (blue). The white arrowheads indicate the H4ac-stained X chromosome tip. Bar, 3 μm. (H) Percentage of nuclei showing H4ac staining at the tip of the X chromosome in the indicated genotypes. The numbers of nuclei scored are indicated on the top. At least four gonads were scored for each genotype. (I) Average fluorescent intensity of H4ac along the X chromosomes and on the background was measured with Photoshop (Adobe systems Inc.) for each nucleus. The background fluorescent intensity was subtracted from the fluorescent intensity detected on the X chromosomes and this value was then normalized to the background fluorescent intensity. The numbers of nuclei measured are indicated on the top. At least four gonads were scored for each genotype. Bars represent the mean ± SEM. * P = 0.0006, ** P<0.0001, two-tailed Mann-Whitney test, 95% C.I.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359108&req=5

pgen.1005029.g003: Analysis of DSB distribution, gene expression and histone acetylation.(A) Gonads from the indicated genotypes were co-immunostained for HTP-3 (green), RAD-51 (red) and AcK (magenta). DNA was stained with DAPI (blue). Images are projections halfway through 3D data stacks of whole nuclei. The X chromosomes show low levels of AcK staining during early meiotic prophase (indicated by open arrowheads). Bar, 5 μm. (B) Graph depicts the distribution of RAD-51 foci on the X chromosomes and the autosomes during early meiotic prophase (from transition zone to mid pachytene combined). The average ratio of RAD-51 foci on the X versus autosomes is indicated. Dashed lines indicate a X/A ratio of 1:5. (C) Graph depicts the distribution of RAD-51 foci on the X and the autosomes during early meiotic prophase (from zones 2 to 4, early through mid-pachytene). The average ratios of RAD-51 foci on the X versus autosomes are indicated. The dashed line indicates a X/A ratio of 1:5. (D) The average ratios of RAD-51 foci on the X versus autosomes in indicated genotypes or treatments in the rad-54 mutant background. Data were analyzed as in (B). The numbers of nuclei scored for each genotype are indicated. Dashed red line depicts the ratio observed in rad-54 single mutants. * P<0.0001 by the extra sum-of-squares F test, compared to rad-54 single mutants. (E) Levels of spo-11 and DSB repair gene expression in dissected gonads from wild type and cra-1 mutants. Quantitative RT-PCR analysis revealed that the germline-specific gene expression for the depicted genes was not altered when comparing cra-1 to wild type and normalizing to gpd-1. Experiments were performed as in [26], utilizing the primers described therein. Error bars represent SEM for three biological replicates, each performed in duplicate. (F) Levels of spo-11 gene expression in indicated genotypes and treatment conditions. Quantitative RT-PCR was performed as in (E), except that whole worms were analyzed instead of dissected gonads. (G) Gonads from the indicated genotypes were co-immunostained for HTP-3 (green) and H4ac (red). DNA was stained with DAPI (blue). The white arrowheads indicate the H4ac-stained X chromosome tip. Bar, 3 μm. (H) Percentage of nuclei showing H4ac staining at the tip of the X chromosome in the indicated genotypes. The numbers of nuclei scored are indicated on the top. At least four gonads were scored for each genotype. (I) Average fluorescent intensity of H4ac along the X chromosomes and on the background was measured with Photoshop (Adobe systems Inc.) for each nucleus. The background fluorescent intensity was subtracted from the fluorescent intensity detected on the X chromosomes and this value was then normalized to the background fluorescent intensity. The numbers of nuclei measured are indicated on the top. At least four gonads were scored for each genotype. Bars represent the mean ± SEM. * P = 0.0006, ** P<0.0001, two-tailed Mann-Whitney test, 95% C.I.
Mentions: To test for a biological significance of regulating global histone acetylation in the germline, we examined the distribution of DSBs between the X chromosomes and autosomes given their different chromatin states. To do this, we analyzed RAD-51 foci in rad-54 mutants, where RAD-51, a protein required for strand invasion/exchange during DSB repair, associates with DSB repair sites, but DSB repair is blocked and DSB-bound RAD-51 are “trapped” and can be easily scored [24,25,35]. Therefore, RAD-51 foci are used herein as a surrogate for DSBs, with the caveat that this may not represent the total number of DSBs since we cannot discard the possibility that not all DSBs, particularly in mutant backgrounds, may be processed to load RAD-51. This analysis was coupled to co-immunostaining with the pan acetylation antibody, which allows for ease of identification of the X chromosomes, as they exhibit greatly decreased histone acetylation compared to the autosomes during early meiotic prophase (Fig. 1G; [32]), and an antibody against the HORMA domain-containing protein HTP-3 to trace chromosome axes and distinguish between DSBs on different chromosomes (Fig. 3A).

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