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变化，神经祖细胞具有由其亚基定义的独特“npBAF”复合物。当神经祖细胞产生神经元时，含有 Baf45a 和 Baf53a 的 npBAF 复合物被替换。神经元特异性 ¿ nBAF??与同源亚基（Baf45b 和 Baf53b）的复合物，这种调节开关是有丝分裂退出、活性依赖性树突生长所必需的。 尽管多项证据表明该复合体在发育转换时基因组分布发生了巨大变化，但亚基组成到底如何驱动 BAF 复合体功能这一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械后果，以揭示复合物用于支持这两种重要表观遗传状态的机制。我们已经开发了一种综合方法，通过创建一个模型来检查发育转换的机械作用。老鼠表达与 BAF 复合物的中心 ATPase Brg 融合的光开关荧光蛋白 Dendra2 为了检查 npBAF/nBAF 发育开关对复合物稳定性和周转的作用，我们将使用该 Brg-Dendra2 小鼠品系来测量活细胞中复合物的周转。此外，先前的研究表明，分化伴随着染色质相关蛋白的物理活动性的变化。为了确定亚基切换对复合物动力学的影响，我们将在活细胞中使用光激活后荧光衰减 (FDAP) 来测量发育切换前后 BAF 复合物核迁移率的变化。最后，我们将使用超分辨率。光学显微镜技术 3D-PALM，用于检查 npBAF/nBAF 发育转换引起的结构变化 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，聚类和其他参数来描述复合物发育调节的结构效应在每个目标中，我们将确定负责复合物变化的 BAF 亚基。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。通过重要的表观遗传开关来调节神经发育的特定方面，揭示该复合物发育调节的生物物理基础将为了解多能性和分化的分子机制提供有价值的见解。 公共健康相关性：中枢神经系统的发育是一个受 DNA 获取调控的复杂过程。我们创造了一种小鼠品系，使我们能够直接研究控制整个神经发育过程中 DNA 获取的重要蛋白质复合物。控制这一复合体的物理原理将使我们能够了解支持神经发育的分子过程；这些原理可以深入了解神经发育障碍（例如自闭症谱系障碍），并导致神经系统损伤和组织退化的治疗。