Epigenetic mechanisms in cell cycle regulation of neuronal stem rolls

表观遗传机制可能在神经干细胞产生哺乳动物新皮质的细胞周期调控中起关键作用。在这个项目中，我们通过分析1)组蛋白的乙酰化状态，2)表达来研究染色质的修饰机制。小鼠大脑皮层发育过程中神经前体细胞核内脱乙酰酶的水平。用抗乙酰化组蛋白113、H4抗体对胚胎第10天、胚胎12天、胚胎14天和胚胎16天小鼠脑壁神经干细胞的核提取液进行免疫印迹分析。用抗乙酰化组蛋白H3、H4抗体进行染色质免疫沉淀分析。用针对细胞周期调控基因5‘端启动子区域设计的聚合酶链式反应/冰冻引物对免疫沉淀DNA进行分析。用抗组蛋白脱乙酰酶抗体4对前面描述的鼻咽癌细胞核提取物进行蛋白质印迹分析。我们通过使用tet…产生了可能以npc特异性方式过表达sir2蛋白的转基因小鼠。更多的环素诱导系统和巢蛋白内含子II启动子。结果：1.在神经干细胞的细胞核中，我们检测到在神经发生过程中组蛋白H3、Lys 9的乙酰化水平升高；组蛋白H3和H4中的其他乙酰化赖氨酸残基没有变化。免疫沉淀组蛋白H3LYS9抗体对细胞周期蛋白依赖性激酶抑制物(CDKI)的5‘端启动子区域进行免疫沉淀。在神经发生过程中检测到NPC胞核中Sir2蛋白的表达水平降低；其他组蛋白脱乙酰酶未见降低。Sirt1是Sir2的小鼠同源基因，是NAD依赖的脱乙酰酶，对大脑皮层发育至关重要。据报道，SIRT1基因敲除小鼠在出生时就有脑外畸形。我们推测，NPC中Sir2蛋白的下调导致组蛋白H3Lys 9乙酰化水平降低，从而开放转录因子对CDKI启动子区域的访问。我们通过油炸产生转基因小鼠来研究上述假设。然而，在研究项目期间，我们未能获得过表达Sir2蛋白的转基因小鼠品系。较少

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