利用基因组定点分离技术研究可塑性蛋白转录调控机制及其在稳定长期记忆中的作用

Neuron plasticity is considered as the biological basis for learning and memory. The key molecules, which involved in the formation and regulation of neuronal plasticity, have been purified and studied. The transcriptional and translational regulation of these key molecules, as well as the post-translational modifications, in the neurons determined the capacity and maintenance of the plasticity changes upon stimuli. To systematically screen for the transcriptional regulation machines in the promoter of the plasticity genes, we developed a zinc finger protein based method to target on specific DNA sequences to isolate and purify the promoter-associated proteins upon various stimulation conditions. The system is based on the specific binding of DNA sequences by artificially designed zinc finger proteins. The system allowed us to purify the learning induced protein modifications as well as composition changes of transcriptional regulators on specific gene promoter. Therefore, when collecting samples at different learning stages, we were able to dissect the short-term and long-term regulation mechanisms for the plasticity gene regulation during memory formation and maintenance. Moreover, this system could also be used to purify the promoters to search for specific DNA modifications within the promoter region, as well as the specific histone modifications. As an unbiased screening system, this zinc-figure based method would enable us to identify novel mechanisms for the transcriptional regulation on the plasticity genes, which is specific for the nerve system and important for memory regulation. With the newly identified gene regulation mechanisms, we have found long-term regulation machines in some of the plasticity genes as potential markers for long-term memory. Further studies would definitely improve our understanding on the molecular basis for the learning and memory defects in various diseases, such as mental retardation and neurodegenerative diseases.

神经元的可塑性是记忆的生理基础。与调节神经元可塑性相关的蛋白质已经被成功分离与鉴定,这些可塑性关键基因的转录及其精密调控在长期记忆的形成与维持中起到了决定性的作用。然而，目前缺乏对其研究，仅有很少的调节机制被确立。我们利用锌指结合技术，发展了一种定点分离基因组及其附着蛋白的方法，用以系统地捕捉基因组关键位点的结合蛋白，并可以检测附着在基因组上的蛋白的翻译后修饰。本研究将用小鼠皮层与海马区脑组织作为研究材料，特异性地在可塑性相关蛋白基因启动子区域分离捕捉长期记忆形成与维持所必须的转录调控蛋白复合物,从而系统地得到调控神经元可塑性基因表达的分子机制，找到相应的新调节蛋白。这项研究，也将加深对长期记忆形成与维持的分子细胞学基础认识，并且能够找到长期记忆过程在染色体上的分子标记。获得的数据将有利于探索可塑性相关蛋白的调控表达机制的缺失所引发的各种神经系统疾病的分子机制。

神经元的突触可塑性被广泛认为是记忆存储的分子基础。学习后，外界信息通过改变特定神经元的突触连接强度和突触可塑性，从而形成神经网络存储记忆。但研究可塑性蛋白受神经活动调控的机制尚没有系统性的方法。同时，我们尚不清楚存储记忆的神经网络如何避免被新的刺激改变突触连接，从而使已经形成的长时程记忆不受新的记忆干扰。.Neurexin作为重要的突触前膜黏附分子，在突触可塑性调控过程中起着重要的作用。我们发现Nrxn1的α启动子的转录活性和可变剪接位点4的剪接均在短暂的神经活动后，出现长时程的变化。为了深入探究这一神经活动引起的长时程可塑性变化机制，本项目通过建立以噬菌体Gp8蛋白和LacZ为筛选标记的细菌单杂交系统，对特定基因位点进行特异性锌指蛋白高的通量筛选，从而为研究突触可塑性相关基因的调控机制提供了DNA-蛋白互作的检测平台。随后，我们分别针对Nrxn1α启动子和可变剪接位点4设计了与之特异结合的人工锌指蛋白。通过特异性锌指蛋白下拉实验，分离并鉴定出脑内分别与Nrxn1α启动子区和可变剪接位点4结合的调控蛋白，并进行了下游验证和机制研究。.结果表明，短暂的神经元活性刺激能够促使Ash1L在Nrxn1α启动子上富集，增加启动子区H3K36me2修饰，进而抑制Nrxn1α基因转录。此外，Ash1L+/-小鼠出现一定程度的模式识别能力缺陷和恐惧记忆泛化现象。同时，在特定海马记忆印记细胞中，学习能够引起Suv39h1介导的长时程Nrxn1可变剪接位点4包含率上升，从而抑制了该神经网络与其他神经元的再连接能力，因此保护了网络中记忆的完整性。在Suv39h1敲除小鼠中，神经元兴奋无法引起长时程可变剪接变化，小鼠已经形成的记忆稳定性也出现相应下降。.综上所述，学习引起的神经元兴奋会引起表观遗传调控因子主动参与可塑性分子的调控，从而影响记忆网络的连接和可塑性，保证了所储存记忆的稳定性。本研究不仅为为后续研究表观修饰对突触可塑性基因调控提供新示例，并且在分子层面解释了脑内记忆保护机制，为治疗阿尔茨海默病，创伤后应激综合症等疾病提供了新的思路。