Genetically Encoded Small Illuminants for 4D nucleome imaging

用于 4D 核组成像的基因编码小光源

基本信息

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
项目类别：
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

项目摘要

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（由应用程序提供）：我们建议开发一种新技术，允许人们实时成像组蛋白内组蛋白表观遗传功能状态的空间时间动力学等级。提出的分子成像系统是通过使用单珠一个组合（OBOC）组合库筛选来开发的，以识别激活有机染料或分子转子的荧光的短肽。荧光的激活与其化学环境的改变，包括在配体结合和磷酸化，乙酰化，甲基化或泛素化肽时发生会议变化。然后可以将这些胡椒粉通常融合到靶向蛋白质（例如组蛋白）中，以实时在活细胞中实现功能性细胞成像。我们将在本应用程序中将新开发的技术调整为表观遗传学研究。 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 generically encoded illuminants to probe post-translational modification of histones in nucleosomes,实时在活细胞中进行临时和空间，从而实现4D核图像。可以将荧光活化的GESI位点标记为随后的矫正荧光和电子显微镜，以及通过染料/GESI相互作用的染色质沉淀。冲击：Gesi Pepperides可以特异性地结合并激活所选有机染料的荧光。只有在与细胞成分（例如Ca2+，构象变化或翻译后修饰（PTM））结合后，一些GESI Petides才能做到这一点。因此，当在活细胞中表达时，它们可以阐明空间时间调节和修饰兴趣。遗传照明剂很小（1200-1900 daltons），因此可以轻松地沿天然蛋白质的序列插入而不会干扰其物理功能。多路复用是可能的，并使我们能够实时研究不同组蛋白PMT和/或组蛋白结合蛋白的招募。拟议项目的具体目的是：目标1。设计和合成一系列适合活细胞核中GESI报告的有机染料。目标2。开发gesis以跟踪组蛋白H2A（H2AX和H2AZ）亚型的时空动力学。目的3。开发gesis以新识别活细胞内N端组蛋白H2A/H3的酰基化状态。