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Meiosis-specific loading of the centromere-specific histone CENH3 in Arabidopsis thaliana.

Ravi M, Shibata F, Ramahi JS, Nagaki K, Chen C, Murata M, Chan SW - PLoS Genet. (2011)

Bottom Line: Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced.These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells.Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology, University of California Davis, Davis, California, United States of America.

ABSTRACT
Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced. Factors required for mono-orientation have been identified in yeast. However, comparatively little is known about how meiotic centromere behavior is specialized in animals and plants that typically have large tandem repeat centromeres. Kinetochores are nucleated by the centromere-specific histone CENH3. Unlike conventional histone H3s, CENH3 is rapidly evolving, particularly in its N-terminal tail domain. Here we describe chimeric variants of CENH3 with alterations in the N-terminal tail that are specifically defective in meiosis. Arabidopsis thaliana cenh3 mutants expressing a GFP-tagged chimeric protein containing the H3 N-terminal tail and the CENH3 C-terminus (termed GFP-tailswap) are sterile because of random meiotic chromosome segregation. These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells. Loss of the GFP-tailswap CENH3 variant in meiosis affects recruitment of the essential kinetochore protein MIS12. Our findings suggest that CENH3 loading dynamics might be regulated differently in mitosis and meiosis. As further support for our hypothesis, we show that GFP-tailswap protein is recruited back to centromeres in a subset of pollen grains in GFP-tailswap once they resume haploid mitosis. Meiotic recruitment of the GFP-tailswap CENH3 variant is not restored by removal of the meiosis-specific cohesin subunit REC8. Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail. Meiosis-specific CENH3 dynamics may play a role in modulating meiotic centromere behavior.

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Lack of centromere function in meiosis causes micronuclei to form in GFP-tailswap pollen.Immunolocalization of alpha-tubulin outlines the nuclear envelope in microspores of GFP-CENH3 and GFP-tailswap pollen (A–F). GFP-tailswap pollen contains multiple micronuclei. Centromere DNA FISH shows that micronuclei contain 1–2 chromosomes each, as opposed to 5 chromosomes in a normal A. thaliana haploid pollen genome (G–L). The pollen grain shown in J–L has three micronuclei. Two contain one chromosome each, while the third contains two chromosomes.
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pgen-1002121-g002: Lack of centromere function in meiosis causes micronuclei to form in GFP-tailswap pollen.Immunolocalization of alpha-tubulin outlines the nuclear envelope in microspores of GFP-CENH3 and GFP-tailswap pollen (A–F). GFP-tailswap pollen contains multiple micronuclei. Centromere DNA FISH shows that micronuclei contain 1–2 chromosomes each, as opposed to 5 chromosomes in a normal A. thaliana haploid pollen genome (G–L). The pollen grain shown in J–L has three micronuclei. Two contain one chromosome each, while the third contains two chromosomes.

Mentions: Analysis of microspores (pollen grains) from GFP-tailswap plants revealed that each contained 1–8 nuclei instead of the single nucleus that is always seen in wild type (Figure 2A–2F). These micronuclei varied in size, suggesting that they contained different numbers of chromosomes. Fluorescence in situ hybridization (FISH) using a probe that recognizes the 180 bp centromere tandem repeats confirmed that each micronucleus contained from 1–4 chromosomes (Figure 2J–2L). This observation suggests that randomly scattered chromosomes that lie in close proximity reassemble their own nuclear envelope at the end of telophase II, resulting in multiple micronuclei within each microspore. A similar defect has been reported in A. thaliana separase (esp) mutants defective in the enzyme that releases sister chromatid cohesion [19]. In mammalian somatic cells, micronuclei formation is triggered by the depletion of factors required for chromosome segregation [20]–[22]. Formation of micronuclei might be a general feature of perturbations that drastically affect chromosome movement in mitosis or meiosis.


Meiosis-specific loading of the centromere-specific histone CENH3 in Arabidopsis thaliana.

Ravi M, Shibata F, Ramahi JS, Nagaki K, Chen C, Murata M, Chan SW - PLoS Genet. (2011)

Lack of centromere function in meiosis causes micronuclei to form in GFP-tailswap pollen.Immunolocalization of alpha-tubulin outlines the nuclear envelope in microspores of GFP-CENH3 and GFP-tailswap pollen (A–F). GFP-tailswap pollen contains multiple micronuclei. Centromere DNA FISH shows that micronuclei contain 1–2 chromosomes each, as opposed to 5 chromosomes in a normal A. thaliana haploid pollen genome (G–L). The pollen grain shown in J–L has three micronuclei. Two contain one chromosome each, while the third contains two chromosomes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111537&req=5

pgen-1002121-g002: Lack of centromere function in meiosis causes micronuclei to form in GFP-tailswap pollen.Immunolocalization of alpha-tubulin outlines the nuclear envelope in microspores of GFP-CENH3 and GFP-tailswap pollen (A–F). GFP-tailswap pollen contains multiple micronuclei. Centromere DNA FISH shows that micronuclei contain 1–2 chromosomes each, as opposed to 5 chromosomes in a normal A. thaliana haploid pollen genome (G–L). The pollen grain shown in J–L has three micronuclei. Two contain one chromosome each, while the third contains two chromosomes.
Mentions: Analysis of microspores (pollen grains) from GFP-tailswap plants revealed that each contained 1–8 nuclei instead of the single nucleus that is always seen in wild type (Figure 2A–2F). These micronuclei varied in size, suggesting that they contained different numbers of chromosomes. Fluorescence in situ hybridization (FISH) using a probe that recognizes the 180 bp centromere tandem repeats confirmed that each micronucleus contained from 1–4 chromosomes (Figure 2J–2L). This observation suggests that randomly scattered chromosomes that lie in close proximity reassemble their own nuclear envelope at the end of telophase II, resulting in multiple micronuclei within each microspore. A similar defect has been reported in A. thaliana separase (esp) mutants defective in the enzyme that releases sister chromatid cohesion [19]. In mammalian somatic cells, micronuclei formation is triggered by the depletion of factors required for chromosome segregation [20]–[22]. Formation of micronuclei might be a general feature of perturbations that drastically affect chromosome movement in mitosis or meiosis.

Bottom Line: Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced.These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells.Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology, University of California Davis, Davis, California, United States of America.

ABSTRACT
Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced. Factors required for mono-orientation have been identified in yeast. However, comparatively little is known about how meiotic centromere behavior is specialized in animals and plants that typically have large tandem repeat centromeres. Kinetochores are nucleated by the centromere-specific histone CENH3. Unlike conventional histone H3s, CENH3 is rapidly evolving, particularly in its N-terminal tail domain. Here we describe chimeric variants of CENH3 with alterations in the N-terminal tail that are specifically defective in meiosis. Arabidopsis thaliana cenh3 mutants expressing a GFP-tagged chimeric protein containing the H3 N-terminal tail and the CENH3 C-terminus (termed GFP-tailswap) are sterile because of random meiotic chromosome segregation. These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells. Loss of the GFP-tailswap CENH3 variant in meiosis affects recruitment of the essential kinetochore protein MIS12. Our findings suggest that CENH3 loading dynamics might be regulated differently in mitosis and meiosis. As further support for our hypothesis, we show that GFP-tailswap protein is recruited back to centromeres in a subset of pollen grains in GFP-tailswap once they resume haploid mitosis. Meiotic recruitment of the GFP-tailswap CENH3 variant is not restored by removal of the meiosis-specific cohesin subunit REC8. Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail. Meiosis-specific CENH3 dynamics may play a role in modulating meiotic centromere behavior.

Show MeSH
Related in: MedlinePlus