Epigenetic mechanisms in cell cycle regulation of neuronal stem rolls

表观遗传机制可能在神经干细胞（NPCs）的细胞周期调控中发挥重要作用。在这个项目中，我们通过分析1）组蛋白的乙酰化状态，2）表达来研究染色质修饰机制。小鼠大脑皮质发育过程中NPC细胞核内脱乙酰酶水平的变化。方法：1.用抗乙酰化组蛋白113、H4抗体对胚胎（E）10、E12、E14和E16天小鼠脑壁NPC的核提取物进行western blot分析.用抗乙酰化组蛋白H3、H4抗体进行染色质免疫沉淀测定。免疫沉淀后的DNA经PCR /酶切鉴定，根据细胞周期调控基因5 '端启动子区设计引物.用抗组蛋白去乙酰化酶抗体对先前描述的NPC的核提取物进行蛋白质印迹分析4。我们利用泰特技术建立了转基因小鼠，这些小鼠可能以NPC特异的方式过表达Sir 2蛋白。 关于我们 racycline诱导系统和nestin内含子II启动子。在NPC细胞核中，在神经元发生过程中，我们检测到乙酰化组蛋白H3赖氨酸9的水平升高，而组蛋白H3和H4中的其他乙酰化赖氨酸残基不变.用抗-组蛋白H3 lys 9抗体免疫沉淀细胞周期蛋白依赖性激酶抑制剂（CDKIs）的5'启动子区.在神经元发生过程中，NPCs细胞核中Sir 2蛋白水平下降，其他组蛋白去乙酰化酶的表达没有下降.我们构建了5个转基因小鼠品系，但没有一个品系能够通过强力霉素诱导Sir 2蛋白的过表达。讨论Sirt 1是Sir 2的小鼠同源物，是NAD依赖的脱乙酰酶，在大脑皮层发育中起重要作用。据报道，Sirt 1基因敲除小鼠在出生时具有露脑畸形。我们推测，NPCs中Sir 2蛋白表达下调导致乙酰化组蛋白H3 lys 9水平降低，从而导致转录因子开放进入CDKIs的启动子区。然而，在研究项目期间，我们无法获得能够过表达Sir 2蛋白的转基因小鼠品系。少

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