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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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Global histone acetylation is impaired in cra-1 mutants.(A) Wild type and cra-1 mutant germlines co-stained with a rabbit pan acetylation antibody (AcK) (white) and DAPI (blue). White arrowheads indicate entrance into meiosis (beginning of the transition zone). Bar, 30 μm. (B) High-magnification images of wild type and cra-1 mutant germline nuclei immunostained with AcK. Images represent 3D data stacks of whole nuclei. Bar, 5 μm. (C) Quantification of the number of acetylation foci observed per nucleus in (B). PMT, premeiotic tip; TZ, transition zone (leptotene/zygotene); LP, late pachytene. Bars represent the mean number of foci ± SEM. * P = 0.0029, ** P<0.0001, two-tailed Mann-Whitney test, 95% C.I. (D) Western blot analysis of protein lysine acetylation in wild type and cra-1 mutant whole worm lysates using the AcK, histone H3ac (pan-acetyl), histone H4ac (pan-acetyl) and H3K56ac antibodies. Arrow indicates histone H3 bands. Levels of protein acetylation are measured with ImageJ (National Institutes of Health, USA). Numbers represent mean ± SEM for data from at least four independent experiments. (E) Pachytene nuclei of wild type and cra-1 mutant gonads were co-stained with anti-H3K56ac antibody (red) and DAPI (blue). Bar, 5 μm. (F) Pachytene nuclei of wild type and cra-1 mutant gonads were co-stained with anti-H2AK5ac antibody (red) and DAPI (blue). Bar, 5 μm. (G) Pachytene nuclei of wild type gonads co-stained with AcK (red), anti-HIM-8 antibody (X chromosome marker, green) and DAPI (blue). Open arrowheads indicate X chromosomes. Bar, 5 μm.
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pgen.1005029.g001: Global histone acetylation is impaired in cra-1 mutants.(A) Wild type and cra-1 mutant germlines co-stained with a rabbit pan acetylation antibody (AcK) (white) and DAPI (blue). White arrowheads indicate entrance into meiosis (beginning of the transition zone). Bar, 30 μm. (B) High-magnification images of wild type and cra-1 mutant germline nuclei immunostained with AcK. Images represent 3D data stacks of whole nuclei. Bar, 5 μm. (C) Quantification of the number of acetylation foci observed per nucleus in (B). PMT, premeiotic tip; TZ, transition zone (leptotene/zygotene); LP, late pachytene. Bars represent the mean number of foci ± SEM. * P = 0.0029, ** P<0.0001, two-tailed Mann-Whitney test, 95% C.I. (D) Western blot analysis of protein lysine acetylation in wild type and cra-1 mutant whole worm lysates using the AcK, histone H3ac (pan-acetyl), histone H4ac (pan-acetyl) and H3K56ac antibodies. Arrow indicates histone H3 bands. Levels of protein acetylation are measured with ImageJ (National Institutes of Health, USA). Numbers represent mean ± SEM for data from at least four independent experiments. (E) Pachytene nuclei of wild type and cra-1 mutant gonads were co-stained with anti-H3K56ac antibody (red) and DAPI (blue). Bar, 5 μm. (F) Pachytene nuclei of wild type and cra-1 mutant gonads were co-stained with anti-H2AK5ac antibody (red) and DAPI (blue). Bar, 5 μm. (G) Pachytene nuclei of wild type gonads co-stained with AcK (red), anti-HIM-8 antibody (X chromosome marker, green) and DAPI (blue). Open arrowheads indicate X chromosomes. Bar, 5 μm.

Mentions: The presence of a NatB domain in CRA-1 prompted us to examine whether CRA-1 might regulate protein acetylation in the germline. Immunostaining of dissected gonads and Western blot analysis of whole worm lysates with a pan acetylation antibody revealed a decrease in protein lysine acetylation in cra-1 mutant germlines compared to wild type (Fig. 1A-D). Given that histones comprise a large portion of the proteins that undergo acetylation in the cells, we proceeded to examine whether this decrease might also reflect changes in histone acetylation. Western blot analysis of whole worm lysates showed that acetylation of histones, assessed with a histone H3 pan-acetyl antibody, a histone H4 pan-acetyl antibody, and a H3K56ac specific antibody, is decreased by 25%-62% in cra-1 mutants compared to wild type (Fig. 1D). This was further supported by the reduction in H3K56ac and H2AK5ac observed in whole mounted germlines of cra-1 mutants compared to wild type (Fig. 1E-F). These observations suggest, first, that the pan acetylation antibody can reveal alterations in global histone acetylation, possibly because histones are a major component of the broader pool of lysine acetylated proteins identified by this reagent; and second, that CRA-1 has a role in modulating histone acetylation that is not restricted to a single lysine residue on histones, and instead affects global levels of histone acetylation.


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)

Global histone acetylation is impaired in cra-1 mutants.(A) Wild type and cra-1 mutant germlines co-stained with a rabbit pan acetylation antibody (AcK) (white) and DAPI (blue). White arrowheads indicate entrance into meiosis (beginning of the transition zone). Bar, 30 μm. (B) High-magnification images of wild type and cra-1 mutant germline nuclei immunostained with AcK. Images represent 3D data stacks of whole nuclei. Bar, 5 μm. (C) Quantification of the number of acetylation foci observed per nucleus in (B). PMT, premeiotic tip; TZ, transition zone (leptotene/zygotene); LP, late pachytene. Bars represent the mean number of foci ± SEM. * P = 0.0029, ** P<0.0001, two-tailed Mann-Whitney test, 95% C.I. (D) Western blot analysis of protein lysine acetylation in wild type and cra-1 mutant whole worm lysates using the AcK, histone H3ac (pan-acetyl), histone H4ac (pan-acetyl) and H3K56ac antibodies. Arrow indicates histone H3 bands. Levels of protein acetylation are measured with ImageJ (National Institutes of Health, USA). Numbers represent mean ± SEM for data from at least four independent experiments. (E) Pachytene nuclei of wild type and cra-1 mutant gonads were co-stained with anti-H3K56ac antibody (red) and DAPI (blue). Bar, 5 μm. (F) Pachytene nuclei of wild type and cra-1 mutant gonads were co-stained with anti-H2AK5ac antibody (red) and DAPI (blue). Bar, 5 μm. (G) Pachytene nuclei of wild type gonads co-stained with AcK (red), anti-HIM-8 antibody (X chromosome marker, green) and DAPI (blue). Open arrowheads indicate X chromosomes. Bar, 5 μm.
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pgen.1005029.g001: Global histone acetylation is impaired in cra-1 mutants.(A) Wild type and cra-1 mutant germlines co-stained with a rabbit pan acetylation antibody (AcK) (white) and DAPI (blue). White arrowheads indicate entrance into meiosis (beginning of the transition zone). Bar, 30 μm. (B) High-magnification images of wild type and cra-1 mutant germline nuclei immunostained with AcK. Images represent 3D data stacks of whole nuclei. Bar, 5 μm. (C) Quantification of the number of acetylation foci observed per nucleus in (B). PMT, premeiotic tip; TZ, transition zone (leptotene/zygotene); LP, late pachytene. Bars represent the mean number of foci ± SEM. * P = 0.0029, ** P<0.0001, two-tailed Mann-Whitney test, 95% C.I. (D) Western blot analysis of protein lysine acetylation in wild type and cra-1 mutant whole worm lysates using the AcK, histone H3ac (pan-acetyl), histone H4ac (pan-acetyl) and H3K56ac antibodies. Arrow indicates histone H3 bands. Levels of protein acetylation are measured with ImageJ (National Institutes of Health, USA). Numbers represent mean ± SEM for data from at least four independent experiments. (E) Pachytene nuclei of wild type and cra-1 mutant gonads were co-stained with anti-H3K56ac antibody (red) and DAPI (blue). Bar, 5 μm. (F) Pachytene nuclei of wild type and cra-1 mutant gonads were co-stained with anti-H2AK5ac antibody (red) and DAPI (blue). Bar, 5 μm. (G) Pachytene nuclei of wild type gonads co-stained with AcK (red), anti-HIM-8 antibody (X chromosome marker, green) and DAPI (blue). Open arrowheads indicate X chromosomes. Bar, 5 μm.
Mentions: The presence of a NatB domain in CRA-1 prompted us to examine whether CRA-1 might regulate protein acetylation in the germline. Immunostaining of dissected gonads and Western blot analysis of whole worm lysates with a pan acetylation antibody revealed a decrease in protein lysine acetylation in cra-1 mutant germlines compared to wild type (Fig. 1A-D). Given that histones comprise a large portion of the proteins that undergo acetylation in the cells, we proceeded to examine whether this decrease might also reflect changes in histone acetylation. Western blot analysis of whole worm lysates showed that acetylation of histones, assessed with a histone H3 pan-acetyl antibody, a histone H4 pan-acetyl antibody, and a H3K56ac specific antibody, is decreased by 25%-62% in cra-1 mutants compared to wild type (Fig. 1D). This was further supported by the reduction in H3K56ac and H2AK5ac observed in whole mounted germlines of cra-1 mutants compared to wild type (Fig. 1E-F). These observations suggest, first, that the pan acetylation antibody can reveal alterations in global histone acetylation, possibly because histones are a major component of the broader pool of lysine acetylated proteins identified by this reagent; and second, that CRA-1 has a role in modulating histone acetylation that is not restricted to a single lysine residue on histones, and instead affects global levels of histone acetylation.

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