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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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Altering the CENH3 N-terminal tail domain leads to defects in meiotic chromosome segregation.a) CENH3 transgenes tested for fertility in a cenh3-1 homozygous mutant background. The male fertility was examined by Alexander staining. Viable pollen stains pink/red. Female fertility was judged by differential intereference contrast (DIC) microscopy of embryo sacs from at least 100 cleared mature ovules per genotype (Figure S1A). Single cell arrested ovules and ovules without an embryo sac (Figure S1B) were counted as non-viable, and ovules with 7–8 celled embryo sacs (Figure S1B) were counted as viable. Viable ovules may be haploid or aneuploid. b) Male meiotic chromosome spreads from wild type and GFP-tailswap plants. Metaphase I bivalents in the mutant are oval/round in shape, lacking the rhombus shape that indicates tension in wild type (compare A and F). Some metaphase I cells showed chromosomes that failed to congress to the spindle midzone (arrowed in K). Chromosome segregation at anaphase I is random in GFP-tailswap (G to I, L to N). Asynchronous homolog separation was seen at anaphase I (arrowed in G), and premature sister chromatid separation was also seen in meiosis I (arrowed in N). Decondensation at interkinesis was frequently delayed, especially for lagging chromosomes near the spindle midzone (arrowed in J, O). Metaphase II cells in the mutant show random chromosome alignment (U, Z). U shows one univalent (arrowed) and four bivalents plus the remaining univalent on the other side of the cell. Anaphase II chromosome segregation is random (V–X, AA–AC). Tetrad equivalent stages in GFP-tailswap (Y, AD) show several small nuclei instead of the expected four uniform nuclei seen in wild type. Scale bars −1 µm.
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pgen-1002121-g001: Altering the CENH3 N-terminal tail domain leads to defects in meiotic chromosome segregation.a) CENH3 transgenes tested for fertility in a cenh3-1 homozygous mutant background. The male fertility was examined by Alexander staining. Viable pollen stains pink/red. Female fertility was judged by differential intereference contrast (DIC) microscopy of embryo sacs from at least 100 cleared mature ovules per genotype (Figure S1A). Single cell arrested ovules and ovules without an embryo sac (Figure S1B) were counted as non-viable, and ovules with 7–8 celled embryo sacs (Figure S1B) were counted as viable. Viable ovules may be haploid or aneuploid. b) Male meiotic chromosome spreads from wild type and GFP-tailswap plants. Metaphase I bivalents in the mutant are oval/round in shape, lacking the rhombus shape that indicates tension in wild type (compare A and F). Some metaphase I cells showed chromosomes that failed to congress to the spindle midzone (arrowed in K). Chromosome segregation at anaphase I is random in GFP-tailswap (G to I, L to N). Asynchronous homolog separation was seen at anaphase I (arrowed in G), and premature sister chromatid separation was also seen in meiosis I (arrowed in N). Decondensation at interkinesis was frequently delayed, especially for lagging chromosomes near the spindle midzone (arrowed in J, O). Metaphase II cells in the mutant show random chromosome alignment (U, Z). U shows one univalent (arrowed) and four bivalents plus the remaining univalent on the other side of the cell. Anaphase II chromosome segregation is random (V–X, AA–AC). Tetrad equivalent stages in GFP-tailswap (Y, AD) show several small nuclei instead of the expected four uniform nuclei seen in wild type. Scale bars −1 µm.

Mentions: We previously observed sterility in GFP-tailswap plants but not in plants expressing GFP-CENH3, suggesting that the N-terminal tail of CENH3 might have a specific role in plant reproduction [14]. The sterile phenotype of GFP-tailswap could result from the absence of the CENH3 tail, or from the presence of the H3.3 tail. To differentiate between these possibilities, we created a chimera in which the A. thaliana CENH3 tail was replaced with an unrelated CENH3 tail domain from maize (Zea mays), and transformed it into cenh3-1 heterozygotes (Figure 1A). This GFP-maizetailswap protein was targeted to kinetochores and rescued the embryo-lethal phenotype of cenh3-1. In contrast, full-length maize CENH3 protein was targeted to A. thaliana kinetochores but failed to rescue cenh3-1 embryo lethality [13]. Complemented GFP-maizetailswap plants showed the vegetative phenotype previously seen in GFP-tailswap plants but were even more sterile than GFP-tailswap plants (although one partially fertile GFP-maizetailswap plant was recovered) (Figure 1A) [13].


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)

Altering the CENH3 N-terminal tail domain leads to defects in meiotic chromosome segregation.a) CENH3 transgenes tested for fertility in a cenh3-1 homozygous mutant background. The male fertility was examined by Alexander staining. Viable pollen stains pink/red. Female fertility was judged by differential intereference contrast (DIC) microscopy of embryo sacs from at least 100 cleared mature ovules per genotype (Figure S1A). Single cell arrested ovules and ovules without an embryo sac (Figure S1B) were counted as non-viable, and ovules with 7–8 celled embryo sacs (Figure S1B) were counted as viable. Viable ovules may be haploid or aneuploid. b) Male meiotic chromosome spreads from wild type and GFP-tailswap plants. Metaphase I bivalents in the mutant are oval/round in shape, lacking the rhombus shape that indicates tension in wild type (compare A and F). Some metaphase I cells showed chromosomes that failed to congress to the spindle midzone (arrowed in K). Chromosome segregation at anaphase I is random in GFP-tailswap (G to I, L to N). Asynchronous homolog separation was seen at anaphase I (arrowed in G), and premature sister chromatid separation was also seen in meiosis I (arrowed in N). Decondensation at interkinesis was frequently delayed, especially for lagging chromosomes near the spindle midzone (arrowed in J, O). Metaphase II cells in the mutant show random chromosome alignment (U, Z). U shows one univalent (arrowed) and four bivalents plus the remaining univalent on the other side of the cell. Anaphase II chromosome segregation is random (V–X, AA–AC). Tetrad equivalent stages in GFP-tailswap (Y, AD) show several small nuclei instead of the expected four uniform nuclei seen in wild type. Scale bars −1 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002121-g001: Altering the CENH3 N-terminal tail domain leads to defects in meiotic chromosome segregation.a) CENH3 transgenes tested for fertility in a cenh3-1 homozygous mutant background. The male fertility was examined by Alexander staining. Viable pollen stains pink/red. Female fertility was judged by differential intereference contrast (DIC) microscopy of embryo sacs from at least 100 cleared mature ovules per genotype (Figure S1A). Single cell arrested ovules and ovules without an embryo sac (Figure S1B) were counted as non-viable, and ovules with 7–8 celled embryo sacs (Figure S1B) were counted as viable. Viable ovules may be haploid or aneuploid. b) Male meiotic chromosome spreads from wild type and GFP-tailswap plants. Metaphase I bivalents in the mutant are oval/round in shape, lacking the rhombus shape that indicates tension in wild type (compare A and F). Some metaphase I cells showed chromosomes that failed to congress to the spindle midzone (arrowed in K). Chromosome segregation at anaphase I is random in GFP-tailswap (G to I, L to N). Asynchronous homolog separation was seen at anaphase I (arrowed in G), and premature sister chromatid separation was also seen in meiosis I (arrowed in N). Decondensation at interkinesis was frequently delayed, especially for lagging chromosomes near the spindle midzone (arrowed in J, O). Metaphase II cells in the mutant show random chromosome alignment (U, Z). U shows one univalent (arrowed) and four bivalents plus the remaining univalent on the other side of the cell. Anaphase II chromosome segregation is random (V–X, AA–AC). Tetrad equivalent stages in GFP-tailswap (Y, AD) show several small nuclei instead of the expected four uniform nuclei seen in wild type. Scale bars −1 µm.
Mentions: We previously observed sterility in GFP-tailswap plants but not in plants expressing GFP-CENH3, suggesting that the N-terminal tail of CENH3 might have a specific role in plant reproduction [14]. The sterile phenotype of GFP-tailswap could result from the absence of the CENH3 tail, or from the presence of the H3.3 tail. To differentiate between these possibilities, we created a chimera in which the A. thaliana CENH3 tail was replaced with an unrelated CENH3 tail domain from maize (Zea mays), and transformed it into cenh3-1 heterozygotes (Figure 1A). This GFP-maizetailswap protein was targeted to kinetochores and rescued the embryo-lethal phenotype of cenh3-1. In contrast, full-length maize CENH3 protein was targeted to A. thaliana kinetochores but failed to rescue cenh3-1 embryo lethality [13]. Complemented GFP-maizetailswap plants showed the vegetative phenotype previously seen in GFP-tailswap plants but were even more sterile than GFP-tailswap plants (although one partially fertile GFP-maizetailswap plant was recovered) (Figure 1A) [13].

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