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

可逆地破坏核体并在区室之间移动基因的工具包

• 批准号: 9134116
• 负责人: MARK T GROUDINE
• 金额: $ 44万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9134116
• 关键词: Affect; Algorithms; Alleles; Auxins; Binding; Binding Proteins; Biological; Cell Cycle; Cell Nucleolus; Cell division; Cells; Chimeric Proteins; Chromatin; Chromosome Territory; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Collaborations; Communities; Development; Disease; Engineering; Epigenetic Process; Exhibits; Gene Expression; Gene Targeting; Gene-Modified; Genes; Genomics; Goals; Health; Heterochromatin; Hour; Individual; Kinetics; Knock-out; Label; Location; Measures; Methods; Molecular; Monitor; Movement; Mus; Nuclear; Nuclear RNA; Pattern Recognition; Peripheral; Play; Proteins; Quality Control; RNA Interference; Reporting; Research Personnel; Risk; Role; Running; Seeds; Site; Structural Genes; System; Techniques; Technology; Testing; Tetracyclines; Transcription Coactivator; Undifferentiated; Validation; base; blastomere structure; cell type; embryonic stem cell; homologous recombination; interest; knock-down; member; novel; nuclease; research study; tool; tool development

 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.

 描述（由申请人提供）：该提案的目标是创建一个工具包，其中内源性核体（NB）和染色质区室可以可逆地破坏，内源性基因可以在区室之间移动。该工具包将允许研究NB和染色质之间的功能相互作用，并将通过4D Nucleome联盟广泛提供给科学界。很明显，染色质在分化过程中重组，但还没有很好地理解NB如何与染色质相互作用并影响细胞的分化。 这次重组。例如，很难研究镇压性的组织是如何形成的。 染色质区室的基因可能被NB（如核仁）接种或影响，因为目前的工具主要限于研究外源表达的组分和/或不能快速逆转。该工具包将在小鼠胚胎干细胞（mESC）中开发，因为它们是可以由最终用户分化为多种细胞类型的原代细胞。在 具体目标1，生长素诱导降解决定子（AID）将用于构建系统，其中NB和染色质区室被快速和可逆地破坏。AID序列将被稳定地整合到编码A）核仁完整性所必需的蛋白质（作为测试NB）和B）锚外周异染色质（PH，作为测试区室）的纤层蛋白的内源基因的两个等位基因中。与目前的RNAi敲除或基于cre/lox的敲除技术相比，这种方法将允许靶蛋白的快速降解（分钟/小时vs天），不依赖于细胞周期并且是快速可逆的。在特定目标2中，一种新的改良TetO/TetR系统将用于将感兴趣的特定蛋白质拴系到单个基因上，并在隔室之间移动基因。将特定的核仁和层状结构蛋白与活性和非活性mESC基因的连接用作测试案例，并将鉴定最有效地在区室之间移动基因的蛋白质。这种拴系系统将比当前的系统更有用，因为它不会影响靶区域的染色质背景（如大的重复阵列），也不会有脱靶效应的风险（如在基于核酸酶缺陷的dCas 9的方法中）。与转录激活因子样效应物（TALE）方法相比，标记独特基因也更简单和更快，其中必须合成靶向多个序列的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，以测试异染色质区室的冗余度并表征PH内的微区。我们通过合作证明了其他研究人员对这些工具感兴趣。一般科学界成功实施这些工具将允许详细研究NB在染色质组织中发挥的作用，并提供拴系工具来研究基因在发育和疾病期间如何在核隔室之间移动。