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（由申请人提供）：我们提出开发一种新技术，其允许人们在真实的时间内对染色质内组蛋白的表观遗传功能状态的时空动态进行成像，从而使得能够在单细胞水平上在活细胞中进行4D核组成像。 水平所提出的分子成像系统是通过使用一珠一化合物（OBOC）组合文库筛选来鉴定激活有机染料或分子转子的荧光的短肽而开发的。荧光的激活与其化学环境的改变相关联，包括配体结合和肽的磷酸化、乙酰化、甲基化或泛素化后的构象变化。然后，这些肽可以与靶蛋白如组蛋白进行基因融合，以实现活细胞中真实的功能性细胞成像。我们将调整我们新开发的技术，以表观遗传学研究在此应用。 假设：基因编码小光源（GESI）技术，包括OBOC组合肽库设计和固定化珠大阵列的系列筛选（~ 100万个二聚体），使得能够鉴定短肽-染料对，所述短肽-染料对可以用作遗传编码的发光体以在真实的时间内探测核小体中组蛋白的翻译后修饰，在活细胞中是暂时的和空间的，从而实现4D核组成像。可共价标记经活化的GESI位点，用于随后的相关荧光和电子显微镜检查，以及通过染料/GESI相互作用进行染色质沉淀。 影响：GESI肽可以特异性结合并激活选定有机染料的荧光。一些GESI肽仅在结合细胞组分如Ca 2+、构象变化或翻译后修饰（PTM）后才这样做。因此，当在活细胞中表达时，它们可以阐明蛋白质的时空调节和修饰， 兴趣遗传发光体很小（1200-1900道尔顿），因此可以容易地沿天然蛋白质的序列沿着插入而不干扰其生理功能。多路复用是可能的，使我们能够研究不同的组蛋白PMT和/或招募组蛋白结合蛋白在真实的时间的交叉会谈。 拟议项目的具体目标是：目标1。设计并合成一系列适用于活细胞核内GESI报告的有机染料。目标2.建立GESIs用于追踪组蛋白H2 A亚型（H2 AX和H2 AZ）的时空动态。目标3.建立GESIs，用于新的鉴定活细胞内组蛋白N端H2 A/H3的酰化状态。