Structural and Dynamic Changes of Chromatin Remodeling at a Developmental Switch

发育开关时染色质重塑的结构和动态变化

• 批准号: 8312453
• 负责人: Hamilton Courtney Hodges
• 金额: $ 5.22万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8312453
• 关键词: ATP phosphohydrolase; Actins; Address; Affect; Cell Nucleus; Cells; Cellular Structures; Characteristics; Chromatin; Chromatin Structure; Chromosomes, Human, Pair 9; Complex; DNA; DNA Binding Domain; Dendrites; Development; Dimensions; Disease; Epigenetic Process; Exons; Family; Fibroblasts; Fluorescence; Fluorescence Microscopy; Fluorescent Probes; Genes; Genomics; Glutamates; Histones; Image; Individual; Inherited; Lead; Life; Measures; Methods; Microscopy; Mitotic; Molecular; Monitor; Mouse Strains; Mus; National Institute of Biomedical Imaging and Bioengineering; Nervous System Trauma; Nervous system structure; Neuraxis; Neurons; Nuclear; Nuclear Structure; Optics; Pattern; Play; Process; Proteins; Regulation; Resolution; Role; Site; Staging; Techniques; Tissues; autism spectrum disorder; base; brahma; chromatin remodeling; design; embryonic stem cell; homologous recombination; insight; nerve stem cell; neurodevelopment; photoactivation; pluripotency; protein complex; self-renewal; small hairpin RNA

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.

描述（由适用提供）：最近的研究表明，哺乳动物神经系统的发展需要调节ATP依赖性染色质重塑BAF复合物中亚基组成的调节变化。神经祖细胞具有由其亚基组成定义的独特的“ NPBAF”复合物，这对于自我更新至关重要。当神经祖细胞产生神经元时，含有BAF45A和BAF53A的NPBAF复合物被神经元特异性„ nbaf？与同源亚基（BAF45B和BAF53B）取代。这种受调节的开关是有丝分裂出口所需的，依赖活性的树突形出生的开关， 以及其他有丝分裂后的神经特异性功能。尽管有几条证据表明该复合物在发育开关处发生了基因组分布的巨大变化，但长期存在的问题是亚基组成如何驱动BAF复合物的功能仍然没有得到解答。我们的研究将旨在理解亚基转换的生物物理和机械后果，以揭示该复合物用来支持这两个基本表观遗传态的机制。我们开发了一种全面的方法来检查发育开关的机械作用，通过创建表达可拍照的荧光蛋白dendra2融合到BAF复合物的中央ATPase BRG中的小鼠。为了检查NPBAF/NBAF开发人员开关对复杂稳定性和营业额的作用，我们将使用此BRG-Dendra2小鼠应变来测量开发开关前神经祖细胞阶段的活细胞中的复合物的营业额，以及开关后分化的神经元中的复合物。此外，先前的研究表明，通过染色质相关蛋白的物理迁移率的变化来实现分化。为了确定亚基开关的效果，我们将在活细胞中使用光激活后（FDAP）后使用荧光衰减来测量发育开关前后BAF复合核迁移率的变化。最后，我们将使用超分辨率光学显微镜技术3D-PALM来检查由NPBAF/NBAF开发器开关引起的结构变化。使用3D-PALM对单个复合物进行的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，以描述该复合物的发育调节的结构效应。在这些目标中的每一个中，我们将确定负责该综合体变化的BAF亚基。在我们的研究结束时，我们将定义由基本表观遗传转换调节的生物物理相互作用和机制，以调节神经发育的特定方面。揭示复合物发育调节的生物物理基础将对多能和分化的分子机制产生有价值的见解。 公共卫生相关性：中枢神经系统的发展是一个复杂的过程，受到DNA访问的调节。我们创建了一种小鼠菌株，该菌株将使我们能够直接研究一个重要的蛋白质复合物，该蛋白质复合物在整个神经发育过程中控制访问DNA。揭示控制这一复合物的物理原理将使我们能够理解支持神经元发展的分子过程。这些原则可以提供有关神经发育障碍（例如自闭症谱系障碍）的见解，并导致治疗神经系统损伤和组织变性。