@misc{oai:ir.soken.ac.jp:00001017,
 author = {三上, 剛和 and ミカミ, ヨシカズ and MIKAMI, Yoshikazu},
 month = {2016-02-17},
 note = {The centromere plays a fundamental role in accurate chromosome segregation during mitosis and meiosis in eukaryotes. Its functions include sister chromatid adhesion and separation, microtubule attachment, chromosome movement, formation of the heterochromatin structure, and mitotic checkpoint control. One essential function of the centromere is the formation of the kinetochore, which is the structure responsible for microtubule binding and chromosome movement. Although chromosome segregation errors cause genetic diseases including some cancers, the mechanism by which the kinetochore interacts with the microtubules of the spindle apparatus during cell division is not fully understood. <br />　Traditional electron microscopy of chromosomes revealed, that the kinetochore of vertebrate cells is a trilaminar button-like structure on the surface of the centromeric heterochromatin. The inner kinetochore plate plays an essential role in kinetochore assembly. The outer kinetochore plate serves both as a microtubule binding structure and as a mitotic checkpoint structure that includes the Bub and Mad complexes. The inner kinetochore contains the centromeric DNA as well as centromere proteins (CENPs) A and C. CENP-H is also one of the inner kinetochore proteins. CENP-H localizes to the centromere throughout the cell cycle, and is found only in active centromeres, including neocentromeres. In this thesis, I investigated the function of CENP-H. <br />　Firstly, I attempted to generate a conditional knockout of CENP-H in chicken DT40 cells. Analysis of CENP-H-deficient cells revealed that CENP-H is essential for cell growth and mitotic progression. <br />　Secondly, the functional region of CENP-H was identified by using a CENP-H conditional knockout cell line. The minimal region of CENP-H required for centromere localization and cell viability was determined by a series of GFP-tagged deletion derivatives of CENP-H. On the basis of the complementation assay and cellular localization data from the CENP-H deletion derivatives, the minimal region necessary for centromere targeting (aa72 to 225) was identified, which was also essential for cell viability. <br />　Thirdly, interaction of CENP-H with other proteins was investigated. A yeast two-hybrid screening was performed using CENP-H as a bait, and I picked up 24 proteins (104 positive clones) including Hec1. In this thesis, I. focused on the relationship between Hec1 and CENP-H. Hec1 was originally identified as a retinoblastoma protein-associated protein, and it was shown that microinjection of Hec1 antibodies into cultured cells disrupts mitotic progression. The two-hybrid system was used to map the region in chicken CENP-H that is responsible for the interaction with Hec1. This analysis showed that the Hec1 interaction region contains the minimal region for CENP-H function identified by the complementation assay. Because CENP-H interacts with Hec1 in the yeast two-hybrid system, cellular localization of both proteins was investigated. A DT40 cell line that co-expressed CENP-H-Flag and Hec1-GFP was created, and immunostaining with anti-Flag antibodies was performed at various cell cycle stages. Hec1 localized to the centrosome but not to the centromere during G1 and S phases. Hec1 moved into the nucleus and localized to centromeres in G2, and it remained associated with the centromere during mitosis. During G2 and mitosis, CENP-H-Flag and Hec1-GFP signals were colocalized at centromeres. To investigate interactions between CENP-H and Hec1 in DT40 cells further, a coimmunoprecipitation assay was performed. A cell line that expressed CENP-H-Flag was prepared to perform this assay. Hec1 was detected in the immunoprecipitates when the exstract was treated with anti-Flag antibodies. Hec1 is a member of the Nuf2 complex, which contain Nuf2, Spc24, and Spc25. Nuf2 was also coimmunoprecipitated with CENP-H. A cell line that co-expressed CENP-H-Flag and Spc24-GFP and another cell line that coexpressed CENP-H-Flag and Spc25-GFP were also created. Coimmunoprecipitation experiments with anti-Flag antibodies using these cells were performed and the immunoprecipitates were analyzed by Western blotting with anti-GFP antibodies. Both Spc24-GFP and Spc25-GFP signals were detected. These results indicate that CENP-H interacts with the Nuf2 complex at centromeres in DT40 cells. <br />　Lastly, photobleaching experiments were carried out on Hec1 and CENP-H. An iFRAP (inverse fluorescence recovery after photobleaching) analysis was performed to examine the stability of the Hec1-GFP association with centromeres. The fluorescence intensities of the unbleached area and of the whole cell were measured across time. During G2, the fluorescence intensity, of the unbleached region decreased gradually, while that of the bleached region was recovered. After approximately 150s, the signal intensities of the whole cell and of the unbleached region became equivalent. In contrast, little loss of fluorescence intensity of Hec1-GFP in the unbleached region was observed during mitosis, and the fluorescence intensity of the bleached region remained constant. These findings indicate that Hec1-GFP associates stably with the centromere during mitosis. The stability and mobility of CENP-H-GFP during both mitosis and interphase was then investigated. The fluorescence intensity remained unchanged in both bleached and unbleached regions for at least 30 min, suggesting that CENP-H is stable at centromeres throughout the cell cycle. These findings indicate that CENP-H and Hec1 form a stable association with the centromere during mitosis. <br />　Considering the results of these analyses, it is proposed that the Nuf2 complex, including Hecl1, is stably associated with the centromere through the interaction with CENP-H during mitosis and provides a site for assembly of the checkpoint proteins (outer kinetochore proteins) that regulate cell cycle progression. The Nuf2 complex may serve as a connector between the inner and outer kinetochores. <br />, 総研大乙第149号},
 title = {The finctional region of CENP-H interacts with the Nuf2 complex that localizes to centromere during mitosis.},
 year = {}
}