Genetically Encoded Small Illuminants for 4D nucleome imaging

用于 4D 核组成像的基因编码小光源

• 批准号: 9003351
• 负责人: R.Holland Cheng
• 金额: $ 33万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9003351
• 关键词: Acetylation; Acylation; Animals; Antibodies; Binding; Biological; Biological Process; Cell Nucleus; Cells; Chemicals; Chromatin; Complementary DNA; Coupled; Cryoelectron Microscopy; DNA Repair; Deposition; Development; Dyes; Electron Microscopy; Environment; Enzymes; Epigenetic Process; Event; Exhibits; Figs - dietary; Fluorescence; Fluorescence Resonance Energy Transfer; Gene Expression; Genetic; Histone H2A; Histones; Image; Implant; Label; Libraries; Life; Ligand Binding; Maps; Mediating; Metabolic; Methodology; Methods; Methylation; Molecular; N-terminal; Nuclear; Nucleosomes; Nude Mice; Peptide Library; Peptides; Phosphorylation; Physiological; Post-Translational Protein Processing; Precipitation; Property; Proteins; Regulation; Reporting; Series; Signal Transduction; Site; Staining method; Stains; Techniques; Technology; Testing; Time; Tissues; Transgenic Animals; Ubiquitination; Xenograft procedure; cellular imaging; combinatorial; dalton; design; functional status; histone modification; histone-binding proteins; human H2AX protein; imaging system; in vivo; interest; molecular imaging; neoplastic cell; new technology; novel; public health relevance; screening; small hairpin RNA; spatiotemporal

 DESCRIPTION (provided by applicant): We propose to develop a novel technology that allows one to image the spatiotemporal dynamics of the epigenetic functional status of histones within the chromatin in real time, thus enabling 4D-nucleome imaging in living cells at single cell level. The proposed molecular imaging system is developed by using a one-bead-one-compound (OBOC) combinatorial library screen to identify short peptide(s) that activate fluorescence of organic dyes or molecular rotors. Activation of fluorescence is coupled to alterations of their chemical environment including conformational change upon ligand binding and phosphorylation, acetylation, methylation, or ubiquitination of the peptide. These peptides can then be genetically fused to target proteins such as histones to enable functional cellular imaging in living cells in real time. We will adapt our newly developed technology to the epigenetics studies in this application. Hypothesis: The Genetically Encoded Small Illuminant (GESI) technology, comprised of OBOC combinatorial peptide library design and serial screening of huge arrays of immobilized bead (~1 million diversities) under pre-defined conditions, enables identification of short peptide- dye pairs that can be used as genetically encoded illuminants to probe post-translational modification of histones in nucleosomes, temporary and spatially in living cells in real time, thus enabling 4D nucleome imaging. Fluorescently activated GESI sites can be covalently marked for subsequent correlative fluorescent and electron microscopy, and chromatin precipitation via dye/GESI interaction. Impact: GESI peptides can specifically bind to and activate the fluorescence of selected organic dyes. Some GESI peptides will do so only after binding to cellular components such as Ca2+, conformational changes or post-translational modifications (PTMs). Therefore, when expressed in a living cell, they can illuminate the spatiotemporal regulation and modification of proteins of interest. The genetic illuminants are small (1200-1900 daltons), thus can be readily inserted along the sequence of the native proteins without interfering with their physiological functions. Multiplexing is possible and allows us to study cross talks of different histone PMTs and/or recruitments of histone binding proteins in real time. Specific aims of the proposed project are: Aim 1. To design and synthesize a series of organic dyes suitable for GESI reporting in the nucleus of living cells. Aim 2. To develop GESIs to track the spatiotemporal dynamics of subtype of histone H2A (H2AX and H2AZ). Aim 3. To develop GESIs to newly identify acylations status of N-terminus histone H2A/H3 inside living cells.

 描述(申请人提供)：我们建议开发一种新的技术，允许人们实时成像染色质中组蛋白的表观遗传功能状态的时空动态，从而能够在单个细胞的活细胞中进行4D-核基因组成像 水平。所提出的分子成像系统是通过使用一珠一化合物(OBOC)组合文库筛选来识别能够激活有机染料或分子转子的荧光的短肽(S)而开发的。荧光的激活与其化学环境的变化相耦合，包括配体结合时的构象变化和多肽的磷酸化、乙酰化、甲基化或泛素化。然后，这些多肽可以通过基因融合到目标蛋白质，如组蛋白，从而能够在活细胞中实时进行功能细胞成像。我们将把我们最新开发的技术应用于表观遗传学研究。假设：基因编码的小发光(GeSI)技术，包括OBOC组合肽库设计和预先定义的条件下对大量固定珠子(约100万个多样性)的连续筛选，能够识别短肽-染料对，这些短肽-染料对可以用作遗传编码光源，实时探测活细胞中核小体中临时和空间上的组蛋白翻译后修饰，从而实现4D核组成像。荧光激活的GeSI位点可以共价标记，用于随后的相关荧光和电子显微镜，以及通过染料/GeSI相互作用的染色质沉淀。影响：GeSI多肽可以与选定的有机染料特异结合并激活其荧光。一些GeSI多肽只有在与细胞成分如钙离子、构象变化或翻译后修饰(PTM)结合后才会这样做。因此，当它们在活细胞中表达时，可以阐明蛋白质的时空调节和修饰。 利息。遗传光源很小(1200-1900道尔顿)，因此可以很容易地沿着天然蛋白质的序列插入，而不会干扰它们的生理功能。多路复用是可能的，并允许我们实时研究不同组蛋白PMT和/或组蛋白结合蛋白的招募的串扰。本项目的具体目标是：目的1.设计和合成一系列适合活细胞内GeSI报告的有机染料。目的2.建立GEIS示踪组蛋白H_2A亚型(H_2AX和H_2AZ)的时空动力学模型。目的3.建立组蛋白N-端组蛋白H_2A/H_3酰化状态的基因定位系统。