A novel ChIP-spec technology to isolate protein complexes at unique genomic loci

一种新颖的 ChIP-spec 技术，可在独特的基因组位点分离蛋白质复合物

基本信息

批准号：
8440468
负责人：
RICHARD YOUNG
金额：
$ 24.38万
依托单位：
WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-12-01 至 2014-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8440468
关键词：
Affinity Algorithms Binding Binding Proteins Biological Assay Biological Process Categories Cell Line Cells Chromatin Code Complex Coupled DNA DNA Binding DNA Sequence DNA-Protein Interaction Detection Development Disease Disease model Elements Fibroblasts Foundations Functional RNA Future Gene Targeting Genes Genetic Enhancer Element Genome Genomics Goals Hela Cells Human Human Genome Insulator Elements Knowledge Liquid Chromatography Literature Maps Mass Spectrum Analysis Measures Medicine Methods Modeling Molecular Peptides Protein Binding Proteins Proteomics Regenerative Medicine Regulation Regulator Genes Sampling Shotguns Signal Transduction Site Somatic Cell Specificity Structure System Techniques Technology Testing Therapeutic Time Transcription Coactivator Transcriptional Regulation Work Xanthomonas cell growth cell type crosslink genetic regulatory protein human disease human embryonic stem cell human stem cells insight interest liquid chromatography mass spectrometry new therapeutic target novel novel therapeutic intervention promoter protein complex protein purification public health relevance tandem mass spectrometry

项目摘要

DESCRIPTION (provided by applicant): The human genome contains protein coding genes, highly conserved non-coding elements, enhancer elements, insulator elements, and expansive transcriptionally silenced domains. Coordinated regulation of these genomic regions determines cellular identity. Determining the identity of proteins that interact with the genome is essential or understanding how the genome is regulated. A critical barrier to this goal has been the inability to purify and identify protein complexes on DNA while preserving native interactions. The ChIP-spec (Chromatin ImmunoPurification coupled to mass spectrometry) technology has the potential to overcome this problem by offering sequence specific purification of genomic loci and identification of bound regulatory factors in their native chromatinized context. The ChIP-spec technology works by introducing a "DNA sequence specific molecular handle" comprised of a DNA binding TAL (Transcription-Activator Like) affinity tag (TAL-tag) into a cell type of interest. After cross linking to preserve protein-protein and protein-DNA interactions, the chromatinized genome is sheared and the TAL-tag protein purified along with proteins bound to the targeted genomic region. Known and novel proteins are then identified by either standard immunodetection techniques or quantitative mass spectrometry. We propose to develop and deploy this novel technique to purify proteins and protein complexes that are bound to genomic loci in their native chromatinized context. This ChIP-spec technique will provide a powerful approach to dissecting genome regulatory mechanisms in development and disease by isolating and identifying putative regulatory proteins. We will develop our technology using well-established human model cell lines and deploy the technique in a test case to measure dynamic exchange of proteins during cellular differentiation. To reach these goals, we propose to 1) Develop the ChIP-spec technology to defined endpoints of affinity tag targeting, site specificity, and immunopurification using human model cell lines, 2) Couple the ChIP-spec technology to quantitative proteomic technologies to enable de novo detection of genomic regulatory proteins, and 3) Deploy the ChIP-spec assay to map protein complex dynamics during cellular differentiation. Purification of transcriptional regulatory complexes in human embryonic stem cells, somatic cells, and disease model cells are likely to provide new insights into how transcriptional regulation and the cell signaling contributes to cell specification and development. These complexes will also provide the foundation for future work developing gene regulatory models and motif finding algorithms. In medicine, this technique may facilitate new therapeutic approaches and provide new therapeutic targets for human disease. Finally, this work will determine the DNA binding specificities of TAL proteins in a human genomic context. Such information will be invaluable to human gene targeting and therapeutic genome editing efforts.

描述（由申请人提供）：人类基因组包含蛋白质编码基因、高度保守的非编码元件、增强子元件、绝缘子元件和广泛的转录沉默结构域。这些基因组区域的协调调节决定了细胞身份。确定与基因组相互作用的蛋白质的身份或了解基因组的调控方式至关重要。实现这一目标的一个关键障碍是无法在保留天然相互作用的同时纯化和鉴定 DNA 上的蛋白质复合物。 ChIP-spec（染色质免疫纯化与质谱联用）技术有可能通过提供基因组位点的序列特异性纯化和在其天然染色质环境中识别结合的调节因子来克服这个问题。 ChIP-spec 技术的工作原理是将由 DNA 结合 TAL（转录激活剂样）亲和标签 (TAL-tag) 组成的“DNA 序列特异性分子手柄”引入感兴趣的细胞类型。交联以保留蛋白质-蛋白质和蛋白质-DNA 相互作用后，染色质化基因组被剪切，TAL 标签蛋白质以及与目标基因组区域结合的蛋白质一起被纯化。然后通过标准免疫检测技术或定量质谱法鉴定已知的和新颖的蛋白质。我们建议开发和部署这种新技术来纯化与天然染色质环境中的基因组位点结合的蛋白质和蛋白质复合物。这种 ChIP-spec 技术将通过分离和鉴定假定的调节蛋白，提供一种强大的方法来剖析发育和疾病中的基因组调节机制。我们将使用成熟的人类模型细胞系开发我们的技术，并将该技术部署在测试用例中，以测量细胞分化过程中蛋白质的动态交换。为了实现这些目标，我们建议 1) 开发 ChIP 规格技术，以使用人类模型细胞系定义亲和标签靶向、位点特异性和免疫纯化的终点，2) 将 ChIP 规格技术与定量蛋白质组学技术相结合，以实现基因组调节蛋白的创新检测，以及 3) 部署 ChIP-spec 检测来绘制细胞分化过程中的蛋白质复合物动态图。人类胚胎干细胞、体细胞和疾病模型细胞中转录调控复合物的纯化可能为转录调控和细胞信号传导如何促进细胞规范和发育提供新的见解。这些复合物还将为未来开发基因调控模型和基序寻找算法的工作奠定基础。在医学上，这项技术可能会促进新的治疗方法，并为人类疾病提供新的治疗靶点。最后，这项工作将确定 TAL 蛋白在人类基因组背景下的 DNA 结合特异性。这些信息对于人类基因靶向和治疗性基因组编辑工作将具有无价的价值。