期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 107, 期 23, 页码 10484-10489出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1002325107
关键词
CENP-A; centromere; super-resolution imaging; chromosome; boustrophedon
资金
- Wellcome Trust
- Engineering and Physical Sciences Research Council [EP/F042248/1]
- Darwin Trust of Edinburgh
- National Institutes of Health [R01GM06627]
- Ministry of Education, Culture, Sports, Science and Technology of Japan
- Grants-in-Aid for Scientific Research [22570008] Funding Source: KAKEN
- EPSRC [EP/F042248/1] Funding Source: UKRI
- Engineering and Physical Sciences Research Council [EP/F042248/1] Funding Source: researchfish
A longstanding question in centromere biology has been the organization of CENP-A-containing chromatin and its implications for kinetochore assembly. Here, we have combined genetic manipulations with deconvolution and super-resolution fluorescence microscopy for a detailed structural analysis of chicken kinetochores. Using fluorescence microscopy with subdiffraction spatial resolution and single molecule sensitivity to map protein localization in kinetochore chromatin unfolded by exposure to a low salt buffer, we observed robust amounts of H3K9me3, but only low levels of H3K4me2, between CENP-A subdomains in unfolded interphase prekinetochores. Constitutive centromere-associated network proteins CENP-C and CENP-H localize within CENP-A-rich subdomains (presumably on H3-containing nucleosomes) whereas CENP-T localizes in interspersed H3-rich blocks. Although interphase prekinetochores are relatively more resistant to unfolding than surrounding pericentromeric heterochromatin, mitotic kinetochores are significantly more stable, reflecting mitotic kinetochore maturation. Loss of CENP-H, CENP-N, or CENP-W had little or no effect on the unfolding of mitotic kinetochores. However, loss of CENP-C caused mitotic kinetochores to unfold to the same extent as their interphase counterparts. Based on our results we propose a new model for inner centromeric chromatin architecture in which chromatin is folded as a layered boustrophedon, with planar sinusoids containing interspersed CENP-A-rich and H3-rich subdomains oriented toward the outer kinetochore. In mitosis, a CENP-C-dependent mechanism crosslinks CENP-A blocks of different layers together, conferring extra stability to the kinetochore.
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