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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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Reduced inter-kinetochore distance and meiotic spindle defects suggest lack of kinetochore function in GFP-tailswap.a) Centromere DNA FISH from metaphase I in wild type and in GFP-tailswap. Blue = DNA (DAPI), green = centromere DNA FISH (FITC). GFP-tailswap bivalents lack the centromere stretch exerted by the spindle in wild type, and have reduced inter-kinetochore distance. Representative bivalents are magnified in D and H. Scale bars −1 µm. b) Centromere DNA FISH shows random orientation of bivalent chromosomes in GFP-tailswap meiosis I. Blue = DNA (DAPI), green = centromere DNA FISH (FITC). Metaphase I chromosomes are frequently aligned at unusual angles in the mutant (B). Anaphase I chromosomes show random alignment and premature sister chromatid separation (D). Arrows in A and B show presumed orientation of sister centromeres. Arrows in D indicate separated univalents, while arrowheads show intact bivalents. Scale bars −1 µm.
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pgen-1002121-g003: Reduced inter-kinetochore distance and meiotic spindle defects suggest lack of kinetochore function in GFP-tailswap.a) Centromere DNA FISH from metaphase I in wild type and in GFP-tailswap. Blue = DNA (DAPI), green = centromere DNA FISH (FITC). GFP-tailswap bivalents lack the centromere stretch exerted by the spindle in wild type, and have reduced inter-kinetochore distance. Representative bivalents are magnified in D and H. Scale bars −1 µm. b) Centromere DNA FISH shows random orientation of bivalent chromosomes in GFP-tailswap meiosis I. Blue = DNA (DAPI), green = centromere DNA FISH (FITC). Metaphase I chromosomes are frequently aligned at unusual angles in the mutant (B). Anaphase I chromosomes show random alignment and premature sister chromatid separation (D). Arrows in A and B show presumed orientation of sister centromeres. Arrows in D indicate separated univalents, while arrowheads show intact bivalents. Scale bars −1 µm.

Mentions: A defect in kinetochore attachment to spindle microtubules in GFP-tailswap is suggested by the lack of apparent tension in metaphase I chromosomes and by random chromosome segregation (in the absence of pairing or recombination defects). The distance between opposing kinetochores (interkinetochore distance) during metaphase is a more precise measure of tension generated by the spindle during mitosis. To investigate interkinetochore distance in GFP-tailswap meiosis I, we used FISH with a centromere tandem repeat probe (Figure 3A). In meiotic chromosome spreads, the outer limit of centromere DNA staining indicates the likely position of the kinetochore. Wild type cells at the metaphase I to anaphase I transition showed centromere DNA foci whose outer edges were separated by a 405±68 nm distance (n = 15 bivalent chromosomes). Centromere DNA was clearly stretched out on either side of the non-hybridizing DNA representing chromosome arms (Figure 3A, panel D). In GFP-tailswap chromosomes at an equivalent stage, centromere DNA extremities were much closer to each other at 234±30 nm (n = 15 bivalent chromosomes) (Figure 3A, panel H). Furthermore, the centromere DNA stretch characteristic of wild-type chromosomes under tension was not seen in the mutant. We conclude that GFP-tailswap kinetochores may not be efficiently pulled by spindle microtubules.


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)

Reduced inter-kinetochore distance and meiotic spindle defects suggest lack of kinetochore function in GFP-tailswap.a) Centromere DNA FISH from metaphase I in wild type and in GFP-tailswap. Blue = DNA (DAPI), green = centromere DNA FISH (FITC). GFP-tailswap bivalents lack the centromere stretch exerted by the spindle in wild type, and have reduced inter-kinetochore distance. Representative bivalents are magnified in D and H. Scale bars −1 µm. b) Centromere DNA FISH shows random orientation of bivalent chromosomes in GFP-tailswap meiosis I. Blue = DNA (DAPI), green = centromere DNA FISH (FITC). Metaphase I chromosomes are frequently aligned at unusual angles in the mutant (B). Anaphase I chromosomes show random alignment and premature sister chromatid separation (D). Arrows in A and B show presumed orientation of sister centromeres. Arrows in D indicate separated univalents, while arrowheads show intact bivalents. Scale bars −1 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111537&req=5

pgen-1002121-g003: Reduced inter-kinetochore distance and meiotic spindle defects suggest lack of kinetochore function in GFP-tailswap.a) Centromere DNA FISH from metaphase I in wild type and in GFP-tailswap. Blue = DNA (DAPI), green = centromere DNA FISH (FITC). GFP-tailswap bivalents lack the centromere stretch exerted by the spindle in wild type, and have reduced inter-kinetochore distance. Representative bivalents are magnified in D and H. Scale bars −1 µm. b) Centromere DNA FISH shows random orientation of bivalent chromosomes in GFP-tailswap meiosis I. Blue = DNA (DAPI), green = centromere DNA FISH (FITC). Metaphase I chromosomes are frequently aligned at unusual angles in the mutant (B). Anaphase I chromosomes show random alignment and premature sister chromatid separation (D). Arrows in A and B show presumed orientation of sister centromeres. Arrows in D indicate separated univalents, while arrowheads show intact bivalents. Scale bars −1 µm.
Mentions: A defect in kinetochore attachment to spindle microtubules in GFP-tailswap is suggested by the lack of apparent tension in metaphase I chromosomes and by random chromosome segregation (in the absence of pairing or recombination defects). The distance between opposing kinetochores (interkinetochore distance) during metaphase is a more precise measure of tension generated by the spindle during mitosis. To investigate interkinetochore distance in GFP-tailswap meiosis I, we used FISH with a centromere tandem repeat probe (Figure 3A). In meiotic chromosome spreads, the outer limit of centromere DNA staining indicates the likely position of the kinetochore. Wild type cells at the metaphase I to anaphase I transition showed centromere DNA foci whose outer edges were separated by a 405±68 nm distance (n = 15 bivalent chromosomes). Centromere DNA was clearly stretched out on either side of the non-hybridizing DNA representing chromosome arms (Figure 3A, panel D). In GFP-tailswap chromosomes at an equivalent stage, centromere DNA extremities were much closer to each other at 234±30 nm (n = 15 bivalent chromosomes) (Figure 3A, panel H). Furthermore, the centromere DNA stretch characteristic of wild-type chromosomes under tension was not seen in the mutant. We conclude that GFP-tailswap kinetochores may not be efficiently pulled by spindle microtubules.

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