| Co-Investigator(Kenkyū-buntansha) |
TAKEDA Shunichi Kyoto University, Professor, 大学院・医学研究科, 教授 (60188191)
KIKUCHI Hidehiko Miyazaki Medical College, Assistant Professor, 医学部, 助手 (10301384)
TAKAMI Yasunari Miyazaki Medical College, Associate Professor, 医学部, 助教授 (80236356)
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| Budget Amount *help |
¥8,800,000 (Direct Cost: ¥8,800,000)
Fiscal Year 2000: ¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 1999: ¥4,700,000 (Direct Cost: ¥4,700,000)
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| Research Abstract |
In the past several years a number of HDAC family members, like those of HAT family members, have been identified in eukaryotes. We have cloned cDNAs and genomic DNAs encoding the three chicken HDACs, chHDAC-1, 2 and 3. chHDAC-3 was localized in both nuclei and the cytoplasm, although chHDAC-1 and 2 were preferentially localized in nuclei. Using the gene targeting technique for the DT40 chicken B cell line, we generated various DT40 mutants, respectively, devoid of chHDAC-1,2 and 3, and six H1 variants and one H2B variant. Systematic analyses of the resultant mutants led us to some noticeable conclusions, as follows.
1) The protein patterns are altered in the mutants, respectively, lacking particular H1 and H2B variants. The variable proteins are specific for the corresponding mutants, suggesting that the H1 and core variants should play individual roles in regulation of the expression of specific genes, probably through alterations in the chromatin structure localized in specific genom
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ic regions.
2) The participation of chHDAC-1 in protein pattern changes is slight, but chHDAC-2 predominantly participates in the accumulation of the IgM H-chain. chHDAC-2 controls the amount of the IgM H-chain at the steps of both transcription of its gene and the alternative processing of its pre-mRNA.
3) chHDAC-3 is essential for the viability of DT40 cells.
4) Both the 1-23 N-terminal amino acids and the 389-417 C-terminal amino acids of chHDAC-3 are essential for the cell viability. The proper NES of chHDAC-3 should be required for both the nuclear export and viability function. The deacetylation activity of chHDAC-3 is also necessary for its viability function. In addition, we have studied the interactions of p46 and p48 subunits of CAF-1 with HAT-1, or histones H2B and H4, and then obtained some noticeable conclusions, as follows.
5) Distinct regions of the p46 polypeptide are required for its in vitro interaction with histones H2B and H4 and chHAT-1.
6) Leucine zipper motif of chHAT-1 is essential for in vivo and in vitro interactions with the p48 subunit of chCAF-1. Less
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