Epigenetic mechanisms in cell cycle regulation of neuronal stem rolls

神经元干卷细胞周期调控的表观遗传机制

基本信息

批准号：
18390302
负责人：
TAKAHASHI Takao
金额：
$ 10.88万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18390302/
关键词：
epigenetics cerebral cortical histogenesis neuronal progenitor cells mice cell cycle acetylation

项目摘要

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 developmentMethods1. 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 antibodies4. 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.Results1. 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.DiscussionSirt1, 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

表观遗传机制可能在产生哺乳动物新皮层的神经元干细胞（NPC）中具有关键作用，我们通过分析冰淇淋素的修饰机制用抗乙酰化组蛋白113，H4抗体对胚胎日（E）10，E12和E16小鼠小脑L进行的NPC进行。2。免疫沉淀的DNA是为5'p romoter的循环调节基因的PCR /糖霜引物。3。 NPCS特异性的蛋白质使用TET ...诱导系统和Nestin Intron II启动子。在NPC的核中。通过-H3 LYS 9抗体。3。依赖性的脱乙酰基酶是关键的小脑发育。推测NPC中SIR2的下调导致乙酰化H3 Lys 9的水平降低，从而导致对CDKIS的启动子区域的开放式转录因子。为了获得可以在研究项目期间过表达SiR2蛋白的转基因小鼠系。