Structural and Dynamic Changes of Chromatin Remodeling at a Developmental Switch

描述（由申请人提供）：最近的研究表明，哺乳动物神经系统的发育需要ATP依赖性染色质重塑BAF复合物家族中亚基组成的调节性变化。神经祖细胞具有由其亚基组成定义的独特的“npBAF”复合物，其对于自我更新是必需的。当神经祖细胞产生神经元时，含有Baf 45 a和Baf 53 a的npBAF复合物被具有同源亚基（Baf 45 b和Baf 53 b）的神经元特异性<$nBAF <$复合物取代。这种调节开关是有丝分裂退出，活性依赖性树突生长， 和其他有丝分裂后神经元特异性功能。虽然有几条证据表明，复杂的经历了巨大的变化，在基因组分布的发育开关，长期存在的问题，究竟是如何亚基组成驱动的BAF复合体的功能仍然没有答案。我们的研究将致力于了解亚基转换的生物物理和机械后果，揭示复合物支持这两种基本表观遗传状态的机制。我们已经开发了一种全面的方法来检查的机制作用的发展开关，通过创建一个小鼠表达的光开关荧光蛋白Dendra 2融合到BAF复杂的中央ATP酶Brg。为了研究npBAF/nBAF发育开关对复合物稳定性和周转率的作用，我们将使用这种Brg-Dendra 2小鼠品系来测量发育开关前神经祖细胞阶段活细胞中复合物的周转率，以及开关后分化的神经元中的周转率。此外，以前的研究表明，分化伴随着染色质相关蛋白的物理流动性的变化。为了确定亚基开关的复合物的动力学的影响，我们将使用活细胞中的光活化后的荧光衰减（FDAP）来测量BAF复合物核迁移率在发育开关之前和之后的变化。最后，我们将使用超分辨率光学显微镜技术，3D-PALM，检查结构变化所产生的npBAF/nBAF发育开关。使用3D-PALM对单个复合物进行高分辨率定位将使我们能够比较亚核结构、聚类和其他参数，以描述复合物发育调控的结构效应。在每一个目标中，我们将确定BAF亚单位负责复杂的变化。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，由一个重要的表观遗传开关调节神经发育的特定方面。揭示该复合物发育调节的生物物理基础将为多能性和分化的分子机制提供有价值的见解。 公共卫生相关性：中枢神经系统的发育是一个复杂的过程，受DNA的调节。我们已经创造了一种小鼠品系，这将使我们能够直接研究一种重要的蛋白质复合物，这种蛋白质复合物在整个神经发育过程中控制DNA的获取。揭示控制这种复合体的物理原理将使我们能够理解支持神经发育的分子过程;这些原理可以提供对神经发育障碍的洞察力（例如，自闭症谱系障碍），并导致神经系统损伤和组织变性的治疗。

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. PUBLIC HEALTH RELEVANCE: Development of the central nervous system is a complex process that is regulated by access to DNA. We have created a mouse strain that will allow us to directly study an important protein complex that controls access to DNA throughout neural development. Revealing the physical principles that control this complex will allow us to understand the molecular processes that support neural development; these principles could provide insight into neurodevelopment disorders (e.g., autism spectrum disorders), and lead to treatments for nervous system injuries and tissue degeneration.