Structural and Dynamic Changes of Chromatin Remodeling at a Developmental Switch

描述(申请人提供)：最近的研究表明，哺乳动物神经系统的发育需要ATP依赖的染色质重塑BAF复合体家族中亚单位组成的调节变化。神经前体细胞有一个独特的‘npBAF’复合体，由其亚单位组成定义，这是自我更新所必需的。当神经前体细胞分化为神经元时，含有Baf45a和Baf53a的npBAF复合体被具有同源亚基的神经元特异性复合体(Baf45b和Baf53b)所取代。这种调节开关是有丝分裂退出、依赖活性的树突生长所必需的， 以及其他有丝分裂后神经元特有的功能。尽管有几条证据表明，在发育转换期，BAF复合体的基因组分布发生了戏剧性的变化，但亚基组成如何驱动BAF复合体的功能这个长期存在的问题仍然没有答案。我们的研究将旨在了解亚基转换的生物物理和机制后果，以揭示复合体用于支持这两种基本表观遗传状态的机制。我们开发了一种全面的方法来研究发育开关的机制作用，方法是创建一只表达光切换荧光蛋白Dendra2的小鼠，该蛋白与BAF复合体的中心ATPase Brg融合。为了研究npBAF/nBAF发育开关对复合体稳定性和周转的作用，我们将使用这个Brg-Dendra2小鼠品系来测量发育开关前神经前体阶段活细胞和发育开关后分化神经元中复合体的周转。此外，以前的研究表明，分化伴随着染色质相关蛋白的物理迁移率的变化。为了确定亚单位切换对复合体动力学的影响，我们将使用活细胞中的光活化后荧光衰减(FDAP)来测量发育切换前后BAF复合体核迁移率的变化。最后，我们将使用超分辨率光学显微镜技术3D-Palm来研究npBAF/nBAF发育开关引起的结构变化。使用3D-Palm对单个复合体进行高分辨率定位将使我们能够比较亚核结构、集群和其他参数，以描述复合体发育调节的结构效应。在每个目标中，我们都将确定导致建筑群变化的BAF亚单位。在我们的研究结束时，我们将定义生物物理相互作用和机制，由一个必要的表观遗传开关来调节神经发育的特定方面。揭示该复合体发育调控的生物物理基础将有助于深入了解其多能性和分化的分子机制。

DESCRIPTION (provided by applicant): Recent studies have shown that development of the mammalian nervous system requires regulated changes of subunit composition in the family of ATP-dependent chromatin remodeling BAF complexes. Neural progenitor cells have a distinct 'npBAF' complex defined by its subunit composition, which is essential for self- renewal. When neural progenitors give rise to neurons, npBAF complexes containing Baf45a and Baf53a are replaced by neuron-specific ¿nBAF¿ complexes with homologous subunits (Baf45b and Baf53b). This regulated switch is required for mitotic exit, activity-dependent dendrite outgrowth, and other post-mitotic, neuron-specific functions. Although several lines of evidence indicate the complex undergoes a dramatic change in genomic distribution at the developmental switch, the long-standing question of exactly how subunit composition drives the function of the BAF complex remains unanswered. Our studies will be directed at understanding the biophysical and mechanistic consequences of subunit switching, to reveal the mechanisms used by the complex to support these two essential epigenetic states. We have developed a comprehensive approach to examine the mechanistic role of the developmental switch by creating a mouse expressing the photoswitchable fluorescent protein Dendra2 fused to the BAF complex's central ATPase Brg. To examine the role of the npBAF/nBAF developmental switch on complex stability and turnover, we will use this Brg-Dendra2 mouse strain to measure the complex's turnover in live cells at the neural progenitor stage before the developmental switch, and in differentiated neurons after the switch. Additionally, previous studies suggest that differentiation is accompanied by changes in the physical mobility of chromatin-related proteins. To identify the effect of the subunit switch the dynamics of the complex, we will use fluorescence decay after photoactivation (FDAP) in live cells to measure changes in BAF complex nuclear mobility before and after the developmental switch. Finally, we will use a super-resolution optical microscopy technique, 3D-PALM, to examine structural changes arising from the npBAF/nBAF developmental switch. High-resolution localization of individual complexes using 3D-PALM will allow us to compare sub-nuclear structure, clustering, and other parameters to describe the structural effects of the complex's developmental regulation. In each of these aims, we will identify the BAF subunits responsible for the complex's change. At the conclusion of our studies, we will have defined the biophysical interactions and mechanisms modulated by an essential epigenetic switch to regulate specific aspects of neural development. Revealing the biophysical basis for developmental regulation of the complex will yield valuable insight into the molecular mechanisms of pluripotency and differentiation.