Expression analyses of E12.5 embryonic brains from Nestin Cre+, Rest GTi/GTi, p53 fl/fl vs Rest GTi/GTi, p53 fl/fl littermates

We use mice containing a gene trap in the first intron of the Rest gene, which effectively eliminates transcription from all coding exons, to prematurely remove REST from neural progenitors. We find catastrophic DNA damage that occurs during S-phase of the cell cycle, with consequences including abnormal chromosome separation, apoptosis, and smaller brains. Further support for persistent effects is the latent appearance of proneural glioblastomas in adult mice also lacking the tumor suppressor, p53. A Rest deficient mouse line generated previously, using a conventional gene targeting approach, does not exhibit these phenotypes, likely due to a remaining C terminal peptide that still binds chromatin and recruits REST chromatin modifiers.Our results indicate that REST-mediated chromatin remodeling is required for proper S-phase dynamics, prior to its well-established role in relieving repression of neuronal genes at terminal differentiation. extract RNA from E12.5 brain in triplicates, compare gene expression profile between Gene Trap REST knockout mice and control littermates我们使用在Rest基因（Rest）第一内含子中携带基因陷阱（gene trap）的小鼠模型，该基因陷阱可有效阻断所有编码外显子（coding exons）的转录，以提前从神经前体细胞（neural progenitors）中清除REST蛋白。研究发现，细胞周期的S期（S-phase）会发生灾难性DNA损伤（catastrophic DNA damage），相关后果包括染色体分离异常、细胞凋亡（apoptosis）以及大脑体积缩小。关于效应持续性的进一步佐证来自：同时缺失抑癌基因p53（tumor suppressor p53）的成年小鼠体内，会迟发性出现神经源性胶质母细胞瘤（proneural glioblastomas）。此前通过传统基因靶向技术（conventional gene targeting approach）构建的Rest缺陷小鼠品系并未表现出上述表型，这大概率是因为其仍保留了一段可结合染色质并招募染色质修饰因子（chromatin modifiers）的C端肽（C terminal peptide）。本研究结果表明，在REST介导的染色质重塑（REST-mediated chromatin remodeling）发挥其在终末分化（terminal differentiation）阶段解除神经元基因（neuronal genes）抑制这一公认功能之前，其对于维持正常的S期动态是必需的。我们对胚胎第12.5天的脑组织（E12.5 brain）进行了三次重复的RNA提取，以此对比基因陷阱型Rest敲除小鼠与同窝对照小鼠（control littermates）的基因表达谱（gene expression profile）。