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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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CRA-1 expression pattern is altered in xnd-1 mutants.(A) CRA-1::GFP expression in xnd-1 mutant gonads. Gonads dissected from CRA-1::GFP; xnd-1 hermaphrodites were co-stained with anti-GFP antibody (green) and DAPI (blue). Bar, 20 μm. (B) High-magnification images of representative germline nuclei show the expression of CRA-1::GFP (green) at the premeiotic tip and transition zone in wild type and xnd-1 mutants. Bar, 5 μm. (C) High-magnification images of nuclei at the indicated stages from wild type and xnd-1 mutant gonads stained with an anti-acetylated lysine antibody. Images show projections through 3D data stacks of whole nuclei. Bar, 5 μm. (D) Quantification of the number of acetylation foci observed per nucleus in (D). Bars represent the mean number of foci ± SEM. * P<0.0001, by the two-tailed Mann-Whitney test, 95% C.I. (E) Gonads dissected from the indicated genotypes were co-stained with an anti-H2AK5ac antibody (red) and DAPI (blue). Bar, 20 μm. Yellow dashed lines were utilized to facilitate visualization of the gonad’s outline when only the antibody signal is depicted. White dashed vertical lines indicate exit from mitosis and entrance into meiosis. (F) A new histone acetylation-regulating protein network and sentinel chromosome model. In this regulatory network, XND-1 negatively regulates CRA-1, which interacts with and antagonizes ACER-1, an acetyl-CoA hydrolase, thereby providing a mechanism for regulating histone acetylation via modulation of acetyl-CoA levels. We propose that regulation of histone acetylation is linked to regulation of meiotic DSB formation. Dashed line: XND-1 promotes efficient meiotic DSB formation independent of its role in the histone acetylation regulatory network (potentially through HIM-5). This additional mechanism of function remains to be characterized. How are regulation of histone acetylation and meiotic DSB formation linked? Highly heterochromatic regions (dark blue) are not the preferred environments for DSB formation. Chromatin with a minimal level of histone acetylation, either transcriptionally active chromatin (red) or regions with lower histone acetylation thresholds (light blue), are better environments for DSBs. An increase in global histone acetylation does not remarkably increase the DSB-preferred chromatin regions on the autosomes, where they are already more prevalent due to their euchromatic state, but can significantly increase this type of site on the X chromosomes, where they are rare due to the highly heterochromatic environment. Our data suggests that DSBs and/or early stages of DSB repair take place close to chromosome axes. This could be either via tethering of loop sequences to axes, with DSB formation/repair taking place at axes as indicated by the red asterisk, or via breaks forming directly at sequences located near the base of the loops and in close proximity to axes, without any tethering (not shown). While we cannot exclude the latter, we favor the former given the evidence of chromatin silencing following DSB formation ([38]; Fig. 5B).
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pgen.1005029.g007: CRA-1 expression pattern is altered in xnd-1 mutants.(A) CRA-1::GFP expression in xnd-1 mutant gonads. Gonads dissected from CRA-1::GFP; xnd-1 hermaphrodites were co-stained with anti-GFP antibody (green) and DAPI (blue). Bar, 20 μm. (B) High-magnification images of representative germline nuclei show the expression of CRA-1::GFP (green) at the premeiotic tip and transition zone in wild type and xnd-1 mutants. Bar, 5 μm. (C) High-magnification images of nuclei at the indicated stages from wild type and xnd-1 mutant gonads stained with an anti-acetylated lysine antibody. Images show projections through 3D data stacks of whole nuclei. Bar, 5 μm. (D) Quantification of the number of acetylation foci observed per nucleus in (D). Bars represent the mean number of foci ± SEM. * P<0.0001, by the two-tailed Mann-Whitney test, 95% C.I. (E) Gonads dissected from the indicated genotypes were co-stained with an anti-H2AK5ac antibody (red) and DAPI (blue). Bar, 20 μm. Yellow dashed lines were utilized to facilitate visualization of the gonad’s outline when only the antibody signal is depicted. White dashed vertical lines indicate exit from mitosis and entrance into meiosis. (F) A new histone acetylation-regulating protein network and sentinel chromosome model. In this regulatory network, XND-1 negatively regulates CRA-1, which interacts with and antagonizes ACER-1, an acetyl-CoA hydrolase, thereby providing a mechanism for regulating histone acetylation via modulation of acetyl-CoA levels. We propose that regulation of histone acetylation is linked to regulation of meiotic DSB formation. Dashed line: XND-1 promotes efficient meiotic DSB formation independent of its role in the histone acetylation regulatory network (potentially through HIM-5). This additional mechanism of function remains to be characterized. How are regulation of histone acetylation and meiotic DSB formation linked? Highly heterochromatic regions (dark blue) are not the preferred environments for DSB formation. Chromatin with a minimal level of histone acetylation, either transcriptionally active chromatin (red) or regions with lower histone acetylation thresholds (light blue), are better environments for DSBs. An increase in global histone acetylation does not remarkably increase the DSB-preferred chromatin regions on the autosomes, where they are already more prevalent due to their euchromatic state, but can significantly increase this type of site on the X chromosomes, where they are rare due to the highly heterochromatic environment. Our data suggests that DSBs and/or early stages of DSB repair take place close to chromosome axes. This could be either via tethering of loop sequences to axes, with DSB formation/repair taking place at axes as indicated by the red asterisk, or via breaks forming directly at sequences located near the base of the loops and in close proximity to axes, without any tethering (not shown). While we cannot exclude the latter, we favor the former given the evidence of chromatin silencing following DSB formation ([38]; Fig. 5B).

Mentions: To further understand the mechanics by which CRA-1, ACER-1 and XND-1 regulate histone acetylation, and potentially meiotic DSB formation, we took advantage of the power of genetic and cytological analysis that can be combined in C. elegans. First, immunostaining with an anti-XND-1 antibody revealed that XND-1 expression and localization are not affected in cra-1 mutants (S8 Fig.). However, the reciprocal experiment revealed that CRA-1::GFP expression was remarkably increased in both premeiotic tip and transition zone nuclei in xnd-1 mutants compared to wild type (Fig. 7A-B). Second, altered dynamics of histone acetylation were also observed correlating with the altered CRA-1::GFP expression pattern in xnd-1 mutants. While a reduction of acetylation levels was observed upon meiotic entry in wild type, no reduction was observed in xnd-1 mutants (Fig. 7C). Quantification of acetylation foci showed that histone acetylation is increased in the xnd-1 mutant both in premeiotic tip and transition zone nuclei compared to wild type (Fig. 7C-D) (P<0.0001), consistent with the altered pattern of expression observed for CRA-1 in xnd-1 mutants. These data suggest that XND-1 acts upstream of CRA-1 to regulate histone acetylation. This is further confirmed by the analysis of H2AK5ac, previously shown to be increased in xnd-1 mutants compared to wild type [29], but whose levels are reduced in both cra-1(RNAi) and xnd-1; cra-1(RNAi) worms (Fig. 7E). Therefore, XND-1 is responsible for the suppression of CRA-1 expression at the premeiotic tip and early stage of meiotic prophase, and loss of this suppression is accompanied by increased histone acetylation upon meiotic entry.


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)

CRA-1 expression pattern is altered in xnd-1 mutants.(A) CRA-1::GFP expression in xnd-1 mutant gonads. Gonads dissected from CRA-1::GFP; xnd-1 hermaphrodites were co-stained with anti-GFP antibody (green) and DAPI (blue). Bar, 20 μm. (B) High-magnification images of representative germline nuclei show the expression of CRA-1::GFP (green) at the premeiotic tip and transition zone in wild type and xnd-1 mutants. Bar, 5 μm. (C) High-magnification images of nuclei at the indicated stages from wild type and xnd-1 mutant gonads stained with an anti-acetylated lysine antibody. Images show projections through 3D data stacks of whole nuclei. Bar, 5 μm. (D) Quantification of the number of acetylation foci observed per nucleus in (D). Bars represent the mean number of foci ± SEM. * P<0.0001, by the two-tailed Mann-Whitney test, 95% C.I. (E) Gonads dissected from the indicated genotypes were co-stained with an anti-H2AK5ac antibody (red) and DAPI (blue). Bar, 20 μm. Yellow dashed lines were utilized to facilitate visualization of the gonad’s outline when only the antibody signal is depicted. White dashed vertical lines indicate exit from mitosis and entrance into meiosis. (F) A new histone acetylation-regulating protein network and sentinel chromosome model. In this regulatory network, XND-1 negatively regulates CRA-1, which interacts with and antagonizes ACER-1, an acetyl-CoA hydrolase, thereby providing a mechanism for regulating histone acetylation via modulation of acetyl-CoA levels. We propose that regulation of histone acetylation is linked to regulation of meiotic DSB formation. Dashed line: XND-1 promotes efficient meiotic DSB formation independent of its role in the histone acetylation regulatory network (potentially through HIM-5). This additional mechanism of function remains to be characterized. How are regulation of histone acetylation and meiotic DSB formation linked? Highly heterochromatic regions (dark blue) are not the preferred environments for DSB formation. Chromatin with a minimal level of histone acetylation, either transcriptionally active chromatin (red) or regions with lower histone acetylation thresholds (light blue), are better environments for DSBs. An increase in global histone acetylation does not remarkably increase the DSB-preferred chromatin regions on the autosomes, where they are already more prevalent due to their euchromatic state, but can significantly increase this type of site on the X chromosomes, where they are rare due to the highly heterochromatic environment. Our data suggests that DSBs and/or early stages of DSB repair take place close to chromosome axes. This could be either via tethering of loop sequences to axes, with DSB formation/repair taking place at axes as indicated by the red asterisk, or via breaks forming directly at sequences located near the base of the loops and in close proximity to axes, without any tethering (not shown). While we cannot exclude the latter, we favor the former given the evidence of chromatin silencing following DSB formation ([38]; Fig. 5B).
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pgen.1005029.g007: CRA-1 expression pattern is altered in xnd-1 mutants.(A) CRA-1::GFP expression in xnd-1 mutant gonads. Gonads dissected from CRA-1::GFP; xnd-1 hermaphrodites were co-stained with anti-GFP antibody (green) and DAPI (blue). Bar, 20 μm. (B) High-magnification images of representative germline nuclei show the expression of CRA-1::GFP (green) at the premeiotic tip and transition zone in wild type and xnd-1 mutants. Bar, 5 μm. (C) High-magnification images of nuclei at the indicated stages from wild type and xnd-1 mutant gonads stained with an anti-acetylated lysine antibody. Images show projections through 3D data stacks of whole nuclei. Bar, 5 μm. (D) Quantification of the number of acetylation foci observed per nucleus in (D). Bars represent the mean number of foci ± SEM. * P<0.0001, by the two-tailed Mann-Whitney test, 95% C.I. (E) Gonads dissected from the indicated genotypes were co-stained with an anti-H2AK5ac antibody (red) and DAPI (blue). Bar, 20 μm. Yellow dashed lines were utilized to facilitate visualization of the gonad’s outline when only the antibody signal is depicted. White dashed vertical lines indicate exit from mitosis and entrance into meiosis. (F) A new histone acetylation-regulating protein network and sentinel chromosome model. In this regulatory network, XND-1 negatively regulates CRA-1, which interacts with and antagonizes ACER-1, an acetyl-CoA hydrolase, thereby providing a mechanism for regulating histone acetylation via modulation of acetyl-CoA levels. We propose that regulation of histone acetylation is linked to regulation of meiotic DSB formation. Dashed line: XND-1 promotes efficient meiotic DSB formation independent of its role in the histone acetylation regulatory network (potentially through HIM-5). This additional mechanism of function remains to be characterized. How are regulation of histone acetylation and meiotic DSB formation linked? Highly heterochromatic regions (dark blue) are not the preferred environments for DSB formation. Chromatin with a minimal level of histone acetylation, either transcriptionally active chromatin (red) or regions with lower histone acetylation thresholds (light blue), are better environments for DSBs. An increase in global histone acetylation does not remarkably increase the DSB-preferred chromatin regions on the autosomes, where they are already more prevalent due to their euchromatic state, but can significantly increase this type of site on the X chromosomes, where they are rare due to the highly heterochromatic environment. Our data suggests that DSBs and/or early stages of DSB repair take place close to chromosome axes. This could be either via tethering of loop sequences to axes, with DSB formation/repair taking place at axes as indicated by the red asterisk, or via breaks forming directly at sequences located near the base of the loops and in close proximity to axes, without any tethering (not shown). While we cannot exclude the latter, we favor the former given the evidence of chromatin silencing following DSB formation ([38]; Fig. 5B).
Mentions: To further understand the mechanics by which CRA-1, ACER-1 and XND-1 regulate histone acetylation, and potentially meiotic DSB formation, we took advantage of the power of genetic and cytological analysis that can be combined in C. elegans. First, immunostaining with an anti-XND-1 antibody revealed that XND-1 expression and localization are not affected in cra-1 mutants (S8 Fig.). However, the reciprocal experiment revealed that CRA-1::GFP expression was remarkably increased in both premeiotic tip and transition zone nuclei in xnd-1 mutants compared to wild type (Fig. 7A-B). Second, altered dynamics of histone acetylation were also observed correlating with the altered CRA-1::GFP expression pattern in xnd-1 mutants. While a reduction of acetylation levels was observed upon meiotic entry in wild type, no reduction was observed in xnd-1 mutants (Fig. 7C). Quantification of acetylation foci showed that histone acetylation is increased in the xnd-1 mutant both in premeiotic tip and transition zone nuclei compared to wild type (Fig. 7C-D) (P<0.0001), consistent with the altered pattern of expression observed for CRA-1 in xnd-1 mutants. These data suggest that XND-1 acts upstream of CRA-1 to regulate histone acetylation. This is further confirmed by the analysis of H2AK5ac, previously shown to be increased in xnd-1 mutants compared to wild type [29], but whose levels are reduced in both cra-1(RNAi) and xnd-1; cra-1(RNAi) worms (Fig. 7E). Therefore, XND-1 is responsible for the suppression of CRA-1 expression at the premeiotic tip and early stage of meiotic prophase, and loss of this suppression is accompanied by increased histone acetylation upon meiotic entry.

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