A toolkit to reversibly disrupt nuclear bodies and move genes among compartments

 描述(由申请人提供)：该提案的目标是创建一种工具包，其中内源性核体(NBS)和染色质隔间可以被可逆破坏，内源性基因可以在隔间之间移动。这一工具包将使人们能够研究NBS和染色质之间的功能相互作用，并将通过4D核组联盟向科学界广泛提供。很明显，染色质在分化过程中会重组，但目前还不清楚NBS是如何与染色质相互作用和影响的 这次重组。例如，很难研究镇压的组织是如何 染色质隔室可能被播种或受到核仁等NBS的影响，因为目前的工具主要局限于对外源表达成分的研究和/或不能迅速逆转。这个工具包将在小鼠胚胎干细胞(MESCs)中开发，因为它们是可以由最终用户分化成多种细胞类型的原始细胞。在……里面 具体目标1，生长素诱导降解(AIDS)将被用来构建其中的NBS和染色质隔间被快速和可逆地破坏的系统。AID序列将稳定地整合到内源基因的等位基因中，这些基因编码A)对核仁的完整性至关重要的蛋白质(作为测试NB)和B)锚定外周异染色质的片层蛋白(PH，作为测试间隔)。与目前的RNAi敲除或基于cre/lox的敲除技术不同，这种方法将允许靶蛋白快速降解(几分钟/小时对几天)，不依赖于细胞周期，并且快速可逆。在特定目标2中，一种新的改进的Teto/TetR系统将被用来将感兴趣的特定蛋白质与单个基因捆绑在一起，并在间隔室之间移动基因。将特定的核仁和片层结构蛋白与活跃和不活跃的mESC基因捆绑在一起作为测试案例，并将确定最有效地在间隔室之间移动基因的蛋白质。这种系链系统将比目前的系统更有用，因为它不会影响靶区的染色质背景(如大的重复阵列)，也不会有靶外效应的风险(如在基于核酸酶缺陷的dCas9方法中)。与转录激活因子样效应器(TALE)方法相比，标记独特的基因也更简单、更快，在TALE方法中，必须合成针对多个序列的TALE。在具体目标3中，我们描述了分化细胞的原理实验的证明，以测试异染色室的冗余并表征PH内的微域。我们通过合作证明了其他研究人员对这些工具感兴趣。普通科学界成功地使用这些工具将允许详细研究NBS在染色质组织中所起的作用，并提供拴系工具来研究基因在发育和疾病期间如何在核间室之间移动。

 DESCRIPTION (provided by applicant): The proposal goal is to create a toolkit in which endogenous nuclear bodies (NBs) and chromatin compartments can be reversibly disrupted and endogenous genes can be moved among compartments. This toolkit will allow study of the functional interactions between NBs and chromatin and will be made widely available to the scientific community through the 4D Nucleome consortium. It is clear that chromatin reorganizes during differentiation, yet it is not well-understood how NBs interact with chromatin and influence this reorganization. For example, it has been difficult to study how organization of the repressive chromatin compartment might be seeded or affected by NBs such as the nucleolus because current tools are primarily limited to the study of exogenously expressed components and/or are not rapidly reversible. This toolkit will be developed in mouse embryonic stem cells (mESCs) because they are primary cells that can be differentiated into multiple cell types by end users. In Specific Aim 1, auxin inducible degrons (AIDs) will be employed to construct systems in which NBs and chromatin compartments are rapidly and reversibly disrupted. The AID sequence will be stably integrated into both alleles of endogenous genes coding for A) proteins essential for the integrity of the nucleolus (as a test NB) and B) lamina proteins that anchor peripheral heterochromatin (PH, as a test compartment). In contrast to current RNAi knockdown or cre/lox-based knockout techniques, this approach will allow rapid degradation of target proteins (minutes/hrs vs days), is not dependent on the cell cycle and is rapidly reversible. In Specific Aim 2, a novel modified TetO/TetR system will be used to tether specific proteins of interest to individual genes and move genes among compartments. Tethering of specific nucleolar and laminar structural proteins to both active and inactive mESC genes will be used as test cases and proteins that most effectively move genes among compartments will be identified. This tethering system will be more useful than current systems because it will not affect the chromatin context of the target region (as do large repetitive arrays) nor risk off-target effects (as in nuclease deficient dCas9-based approaches). Labeling unique genes is also simpler and quicker as compared to the transcription activator-like effector (TALE) approach, where TALEs targeting multiple sequences must be synthesized. In Specific Aim 3 we describe proof of principle experiments in differentiating cells to test the redundancy of the heterochromatic compartment and characterize microdomains within the PH. We demonstrate by collaboration that other researchers are interested in these tools. Successful implementation of these tools by the general scientific community would permit detailed study of the role that NBs play in chromatin organization and provide tethering tools to study how genes move between nuclear compartments during both development and disease.