Spatiotemporal Control of the Epigenome via Photoactivatable Nuclear Localization

通过光激活核定位对表观基因组的时空控制

• 批准号: 8642354
• 负责人: Klaus M. Hahn
• 金额: $ 39.72万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8642354
• 关键词: Acetylation; Address; Animals; Avena sativa; Binding; Biological; Caenorhabditis elegans; Cell Culture Techniques; Cell Nucleus; Cells; Complementary DNA; Coupled; Cytoplasm; DNA Binding Domain; DNA Methylation; DNA Methyltransferase; DNA Modification Methylases; DNA Modification Process; DNA Repair; Deacetylase; Deacetylation; Development; Developmental Process; Docking; Endoderm; Engineering; Enzymes; Epigenetic Process; Epitopes; Event; Gene Activation; Gene Expression; Gene Silencing; Genes; Genetic; Genome; Histocompatibility Testing; Histone Acetylation; Histone Deacetylase; Histones; Homologous Gene; Knock-out; Life; Light; Malignant Neoplasms; Mammalian Cell; Mediating; Modification; Mutation; Nuclear; Nuclear Export; Nuclear Import; Nuclear Localization Signal; Organism; Peptides; Play; Promoter Regions; Proteins; Recruitment Activity; Relative (related person); Repression; Research Personnel; Role; Scaffolding Protein; Signal Transduction; Site; Specific qualifier value; System; Testing; Time; Yeasts; abstracting; cell type; design; directed evolution; epigenetic marker; epigenome; heterochromatin-specific nonhistone chromosomal protein HP-1; histone modification; knockout gene; mitochondrial membrane; public health relevance; research study; spatiotemporal; stem cell biology; tool; transcription factor

DESCRIPTION (provided by applicant): Abstract Gene activation and silencing via epigenetic modifications is important for determining cell fate and is critical for the proper development of multicellular organisms. Determining the relative importance of various epigenetic markers in specific developmental processes can be challenging because these modifications are often dynamic and vary between cell and tissue types. Common approaches such as a gene knockout of an enzyme required for epigenetic modifications can be used to assess the global importance of the modification, but do not provide a straightforward approach for testing the importance of the modification at specific loci, cell types and developmental time points. Here, we aim to create a general approach for the control of epigenetic modifications that is reversible and can be applied at specific times in development to a specified set of cells. In preliminary studies we have developed a light activatable protein that localizes to the cytoplasm in the dark, but enters the nucleus when the cell is illuminated with blue light. Our hypothesis is that this switch, called LANS for Light Activatable Nuclear Shuttle, can be used to control the activity of proteins that must be in the nucleus to be functional. As proof of concept, we have shown that we can use LANS to control the activity of a transcription factor in yeast with light. Here, we wil explore if light activated nuclear localization can be used to control enzymes and scaffolding proteins required for histone and DNA methylation and acetylation. To target predetermined loci, we will fuse LANS with naturally occurring and engineered DNA binding domains. We will test the LANS switch in mammalian cell culture, and we will explore whether the switch can be used to manipulate cell fate in C. elegans via light mediated control of a histone deacetylase.

描述（由申请人提供）：通过表观遗传修饰的抽象基因激活和沉默对于确定细胞命运至关重要，对于多细胞生物的正确发展至关重要。确定各种表观遗传标记在特定发育过程中的相对重要性可能具有挑战性，因为这些修饰通常是动态的，并且在细胞和组织类型之间有所不同。可以使用常见方法，例如表观遗传修饰所需的酶的基因敲除，可用于评估修饰的全球重要性，但不提供直接的方法来测试在特定基因座，细胞类型和发育时间点修饰的重要性。在这里，我们旨在为控制表观遗传修饰的控制创建一种通用方法，该方法可逆，可以在开发的特定时间应用于指定的一组单元。在初步研究中，我们开发了一种可激活的蛋白质，该蛋白在黑暗中定位在细胞质中，但是当细胞用蓝光照亮时，进入细胞核。我们的假设是，该开关（称为LANS用于光活化的核班车）可用于控制核中必须具有功能性的蛋白质的活性。作为概念证明，我们已经证明我们可以使用LAN来控制光线中酵母中转录因子的活性。在这里，我们将探索是否可以使用光激活的核定位来控制组蛋白和DNA甲基化和乙酰化所需的酶和脚手架蛋白。为了靶向预定的基因座，我们将与自然存在和工程的DNA结合域融合。我们将测试哺乳动物细胞培养的LAN开关，我们将探讨该开关是否可以通过组蛋白脱乙酰基酶的光介导的控制片中的秀丽隐杆线虫中的细胞命运。