| Project/Area Number |
18390302
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Pediatrics
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| Research Institution | Keio University |
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Principal Investigator |
TAKAHASHI Takao Keio University, School of Medicine, Professor (80171495)
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| Co-Investigator(Kenkyū-buntansha) |
KOSAKI Kenjiro Keio University, School of Medicine, Associate professor (30234743)
MITSUHASHI Takayuki Keio University, School of Medicine, Assistant professor (80338110)
YONEMOTO Junzo National Institute for Environmental Studies, 環境リスク研究センター, Leader (30072664)
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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 |
¥17,000,000 (Direct Cost: ¥14,900,000、Indirect Cost: ¥2,100,000)
Fiscal Year 2007: ¥9,100,000 (Direct Cost: ¥7,000,000、Indirect Cost: ¥2,100,000)
Fiscal Year 2006: ¥7,900,000 (Direct Cost: ¥7,900,000)
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| Keywords | epigenetics / cerebral cortical histogenesis / neuronal progenitor cells / mice / cell cycle / acetylation |
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| Research Abstract |
Epigenetic mechanism may have a critical role in cell cycle regulations of neuronal stem cells (NPCs) that generate mammalian neocortex. In this project, we examined chromatin modification mechanisms by analyzing 1) acetylated status of histone proteins, 2) expression. Levels of deacetylases in nuclei of NPCs during murine cerebral cortical development
Methods
1. Nuclear extracts of NPCs from embryonic day(E)10, E12, E14, and E16 mouse cerebral wall were subjected to western blot analyses with anti-acetylated histone 113, H4 antibodies.
2. Chromatin immunoprecipitation assays were performed with anti-acetylated histone H3, H4 antibodies. Immunoprecipitated DNA was then analyzed by PCR /icing primers designed for 5'promoter region of cell cycle regulatory genes.
3. Nuclear extracts of NPCs described previously were subjected to western blot analyses with anti-histone deacetylase antibodies
4. We generated transgenic mice that may overexpress Sir2 proteins in NPCs-specific manner by using tet
… More
racycline inducible system and nestin intron II promoter.
Results
1. In nuclei of NPCs, we detected increased level of acetylated histone H3 lys 9 during the course of neuronogenesis; other acetylated lysine residues in histone H3 and H4 were unchanged.
2. 5' promoter regions of cyclin dependent kinase inhibitors (CDKIs) were immunoprecipitated by and - histone H3 lys 9 antibody.
3. We detected decreased level of Sir2 protein in nuclei of NPCs during the course of neuronogenesis; other histone deacetylase were not decreased.
4. We generated five transgenic mice lines, however, neither line were able to overexpress Sir2 protein by doxycycline administration.
Discussion
Sirt1, mouse homolog of Sir2, is NAD dependent deacetylase that is critical for cerebral cortical development. Sirt1 knockout mice were reported to have exencephaly at birth. We speculate that down regulation of Sir2 protein in NPCs resulted in decreased levels of acetylated histone H3 lys 9, that lead to open access to transcription factors to promoter region of CDKIs.
We fried to generate transgenic mice to investigate above hypothesis in vivo. However, we were unable to obtain transgenic mice line that could overexpress Sir2 proteins during research project period. Less
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