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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 regulates histone acetylation by antagonizing ACER-1.(A) Schematic representation of the ACER-1 protein. ACER-1 contains two acetyl-CoA hydrolase (ACH) domains. Arrowhead indicates position of out-of-frame deletion. Red bar shows peptide region used for ACER-1 antibody production. (B) Vectors expressing CRA-1-GFP were cotransfected with ACER-1-HA or empty vectors (control) in 293T cells. The interaction of CRA-1-GFP with ACER-1-HA was analyzed by immunoprecipitation (IP) of the cell lysate with anti-HA agarose beads and Western blotting of the precipitate with anti-GFP antibody. (C) Co-staining with anti-GFP (green) and DAPI (blue) of pachytene nuclei from control and acer-1(RNAi) CRA-1::GFP transgenic worms. White arrowheads indicate CRA-1::GFP aggregates. Bar, 5 μm. (D) Immunolocalization of ACER-1 (red) in DAPI-stained (blue) pachytene nuclei of wild type and acer-1 mutant worms. Images captured through the mid-section of nuclei were shown. Bar, 5 μm. (E) Measurement of ACER-1 immunostaining fluoresence intensity in the nucleus and cytoplasm. Fluoresence intensity was measured from images captured through the center of the nuclei in a rectangle area (1 μm x 6 μm) that covers both nucleus and cytoplasm for each cell with Image J. Data represent average signal measured from at least 20 nuclei from four different gonads for each genotype. (F) Measurement of acetyl-CoA in wild type and mutant worm lysates. * P<0.05 by the two-tailed t test, 95% C.I. (G) Western blot analysis of the global acetylation in wild type and mutant worm lysates detected with a pan acetylation antibody (AcK) and anti-H4ac antibody. The relative level of protein acetylation was determined by densitometric analysis of the western blot bands with ImageJ. Numbers represent mean ± SEM of data from at least three independent experiments. (H) Quantification of the number of acetylation foci observed per nucleus in germlines immunostained with anti-acetylated lysine antibody. Bars represent the mean number of foci ± SEM. * P<0.0001 by the two-tailed Mann-Whitney test, 95% C.I.
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pgen.1005029.g006: CRA-1 regulates histone acetylation by antagonizing ACER-1.(A) Schematic representation of the ACER-1 protein. ACER-1 contains two acetyl-CoA hydrolase (ACH) domains. Arrowhead indicates position of out-of-frame deletion. Red bar shows peptide region used for ACER-1 antibody production. (B) Vectors expressing CRA-1-GFP were cotransfected with ACER-1-HA or empty vectors (control) in 293T cells. The interaction of CRA-1-GFP with ACER-1-HA was analyzed by immunoprecipitation (IP) of the cell lysate with anti-HA agarose beads and Western blotting of the precipitate with anti-GFP antibody. (C) Co-staining with anti-GFP (green) and DAPI (blue) of pachytene nuclei from control and acer-1(RNAi) CRA-1::GFP transgenic worms. White arrowheads indicate CRA-1::GFP aggregates. Bar, 5 μm. (D) Immunolocalization of ACER-1 (red) in DAPI-stained (blue) pachytene nuclei of wild type and acer-1 mutant worms. Images captured through the mid-section of nuclei were shown. Bar, 5 μm. (E) Measurement of ACER-1 immunostaining fluoresence intensity in the nucleus and cytoplasm. Fluoresence intensity was measured from images captured through the center of the nuclei in a rectangle area (1 μm x 6 μm) that covers both nucleus and cytoplasm for each cell with Image J. Data represent average signal measured from at least 20 nuclei from four different gonads for each genotype. (F) Measurement of acetyl-CoA in wild type and mutant worm lysates. * P<0.05 by the two-tailed t test, 95% C.I. (G) Western blot analysis of the global acetylation in wild type and mutant worm lysates detected with a pan acetylation antibody (AcK) and anti-H4ac antibody. The relative level of protein acetylation was determined by densitometric analysis of the western blot bands with ImageJ. Numbers represent mean ± SEM of data from at least three independent experiments. (H) Quantification of the number of acetylation foci observed per nucleus in germlines immunostained with anti-acetylated lysine antibody. Bars represent the mean number of foci ± SEM. * P<0.0001 by the two-tailed Mann-Whitney test, 95% C.I.

Mentions: To understand the mechanism by which CRA-1 regulates global histone acetylation and meiotic DSB formation in C. elegans, we applied a proteomic approach to search for potential CRA-1 binding proteins. CRA-1::GFP was immunopurified from lysates of CRA-1::GFP transgenic worms and copurified proteins were indentified by mass spectrometry (see S1 Text). A list of identified proteins was generated following subtraction of proteins found in the control purification (S1 Table in S1 Text). While we did not identify a histone acetyltransferase by this approach, interestingly, we found ACER-1 (ORF C44B7.10) as a CRA-1 interacting protein. ACER-1 is a protein with homologs present from bacteria to humans (S5A Fig.) and it is a putative acetyl-CoA hydrolase/transferase (Fig. 6A). The interaction between CRA-1 and ACER-1 is further supported by co-expression in 293T cells, followed by immunoprecipitation and Western blot analysis (Fig. 6B). Moreover, depletion by RNAi of ACER-1 in CRA-1::GFP transgenic worms results in the aggregation of CRA-1::GFP in the nucleus (Figs. 6C, S5B), suggesting a direct relationship between CRA-1 and ACER-1 in C. elegans.


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 regulates histone acetylation by antagonizing ACER-1.(A) Schematic representation of the ACER-1 protein. ACER-1 contains two acetyl-CoA hydrolase (ACH) domains. Arrowhead indicates position of out-of-frame deletion. Red bar shows peptide region used for ACER-1 antibody production. (B) Vectors expressing CRA-1-GFP were cotransfected with ACER-1-HA or empty vectors (control) in 293T cells. The interaction of CRA-1-GFP with ACER-1-HA was analyzed by immunoprecipitation (IP) of the cell lysate with anti-HA agarose beads and Western blotting of the precipitate with anti-GFP antibody. (C) Co-staining with anti-GFP (green) and DAPI (blue) of pachytene nuclei from control and acer-1(RNAi) CRA-1::GFP transgenic worms. White arrowheads indicate CRA-1::GFP aggregates. Bar, 5 μm. (D) Immunolocalization of ACER-1 (red) in DAPI-stained (blue) pachytene nuclei of wild type and acer-1 mutant worms. Images captured through the mid-section of nuclei were shown. Bar, 5 μm. (E) Measurement of ACER-1 immunostaining fluoresence intensity in the nucleus and cytoplasm. Fluoresence intensity was measured from images captured through the center of the nuclei in a rectangle area (1 μm x 6 μm) that covers both nucleus and cytoplasm for each cell with Image J. Data represent average signal measured from at least 20 nuclei from four different gonads for each genotype. (F) Measurement of acetyl-CoA in wild type and mutant worm lysates. * P<0.05 by the two-tailed t test, 95% C.I. (G) Western blot analysis of the global acetylation in wild type and mutant worm lysates detected with a pan acetylation antibody (AcK) and anti-H4ac antibody. The relative level of protein acetylation was determined by densitometric analysis of the western blot bands with ImageJ. Numbers represent mean ± SEM of data from at least three independent experiments. (H) Quantification of the number of acetylation foci observed per nucleus in germlines immunostained with anti-acetylated lysine antibody. Bars represent the mean number of foci ± SEM. * P<0.0001 by the two-tailed Mann-Whitney test, 95% C.I.
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pgen.1005029.g006: CRA-1 regulates histone acetylation by antagonizing ACER-1.(A) Schematic representation of the ACER-1 protein. ACER-1 contains two acetyl-CoA hydrolase (ACH) domains. Arrowhead indicates position of out-of-frame deletion. Red bar shows peptide region used for ACER-1 antibody production. (B) Vectors expressing CRA-1-GFP were cotransfected with ACER-1-HA or empty vectors (control) in 293T cells. The interaction of CRA-1-GFP with ACER-1-HA was analyzed by immunoprecipitation (IP) of the cell lysate with anti-HA agarose beads and Western blotting of the precipitate with anti-GFP antibody. (C) Co-staining with anti-GFP (green) and DAPI (blue) of pachytene nuclei from control and acer-1(RNAi) CRA-1::GFP transgenic worms. White arrowheads indicate CRA-1::GFP aggregates. Bar, 5 μm. (D) Immunolocalization of ACER-1 (red) in DAPI-stained (blue) pachytene nuclei of wild type and acer-1 mutant worms. Images captured through the mid-section of nuclei were shown. Bar, 5 μm. (E) Measurement of ACER-1 immunostaining fluoresence intensity in the nucleus and cytoplasm. Fluoresence intensity was measured from images captured through the center of the nuclei in a rectangle area (1 μm x 6 μm) that covers both nucleus and cytoplasm for each cell with Image J. Data represent average signal measured from at least 20 nuclei from four different gonads for each genotype. (F) Measurement of acetyl-CoA in wild type and mutant worm lysates. * P<0.05 by the two-tailed t test, 95% C.I. (G) Western blot analysis of the global acetylation in wild type and mutant worm lysates detected with a pan acetylation antibody (AcK) and anti-H4ac antibody. The relative level of protein acetylation was determined by densitometric analysis of the western blot bands with ImageJ. Numbers represent mean ± SEM of data from at least three independent experiments. (H) Quantification of the number of acetylation foci observed per nucleus in germlines immunostained with anti-acetylated lysine antibody. Bars represent the mean number of foci ± SEM. * P<0.0001 by the two-tailed Mann-Whitney test, 95% C.I.
Mentions: To understand the mechanism by which CRA-1 regulates global histone acetylation and meiotic DSB formation in C. elegans, we applied a proteomic approach to search for potential CRA-1 binding proteins. CRA-1::GFP was immunopurified from lysates of CRA-1::GFP transgenic worms and copurified proteins were indentified by mass spectrometry (see S1 Text). A list of identified proteins was generated following subtraction of proteins found in the control purification (S1 Table in S1 Text). While we did not identify a histone acetyltransferase by this approach, interestingly, we found ACER-1 (ORF C44B7.10) as a CRA-1 interacting protein. ACER-1 is a protein with homologs present from bacteria to humans (S5A Fig.) and it is a putative acetyl-CoA hydrolase/transferase (Fig. 6A). The interaction between CRA-1 and ACER-1 is further supported by co-expression in 293T cells, followed by immunoprecipitation and Western blot analysis (Fig. 6B). Moreover, depletion by RNAi of ACER-1 in CRA-1::GFP transgenic worms results in the aggregation of CRA-1::GFP in the nucleus (Figs. 6C, S5B), suggesting a direct relationship between CRA-1 and ACER-1 in C. elegans.

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