Analysis of regulatory mechanisms of chromatin phosphorylation by kinases
| Project/Area Number |
18570164
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Molecular biology
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| Research Institution | University of Tsukuba |
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Principal Investigator |
HISATAKE Koji University of Tsukuba, Graduate School of Comprehensive Human Sciences, Professor (70271236)
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| Co-Investigator(Kenkyū-buntansha) |
SHIMADA Miho Saitama Medical University, Faculty of Medicine, Assistant Professor (50383287)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,890,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥390,000)
Fiscal Year 2007: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2006: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | chromatin / phosphorylation / transcription / expression regulation / 遺伝子 / キナーゼ |
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| Research Abstract |
We reconstituted chromatin in vitro that were composed entirely of recombinant histories via use of chromatin assembly factors, NAP-1, ACF and Topoisomerase I. The reconstituted chromatin was found to contain about 19 nucleosomes in a regular array by micrococcal digestion and supercoiling assays. Using this chromatin, we found that Aurora kinase phosphorylated histone H3 at serine 10 and 28 efficiently; while MSK1 failed to do so, indicating that the phosphorylation of histone H3 by MSK1 is inhibited dramatically by incorporation into the chromatin. Upon further addition of CREB, ART1, SRF and Elk-1, MSK1 phosphorylated histone H3 within the chromatin. In contrast, such augmentation of phosphorylation was not seen for Aurora kinase even when the same set of transcription factors were added to the chromatin. GST pull-down assays revealed that the all the four histones did interact with Aurora Abut failed to interact with MSK1, and MSK1 interacted with all the four activators, consistent with the requirement for the activators to phosphorylate chromatin-embedded histone H3.
We also found that HMGN1 facilitates the MSK1-mediated phosphorylation of histone H3 and during this process HMGN1 became phosphorylated at multiple sites by MSK1. Interestingly, this phosphorylation was not dependent upon the presence of activators. Analysis of the deletion mutants of MSK1 showed that the MSK1 possessed two interacting domains for CREB, both of which is presumably repressed by the intramolecular interactions. Indeed, removal of N-terminal 50 amino acids restored the CREB-mediated histone H3 phosphorylation, indicating that this particular region is the predominant repression domain within MSK1. The results suggested that the intra-molecular repression is de-repressed by the phosphorylation by p38 and by the interaction with CREB, revealing a complex regulatory network for this meted histone H3 phosphorylation.
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Report
(3 results)
Research Products
(11 results)
