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

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是如何调节的是必不可少的。这一目标的一个关键障碍是无法纯化和鉴定DNA上的蛋白质复合物，同时保留天然相互作用。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化背景下鉴定结合的调节因子来克服这一问题。ChIP-spec技术通过将由DNA结合TAL（转录激活因子样）亲和标签（TAL标签）组成的“DNA序列特异性分子手柄”引入感兴趣的细胞类型中来工作。 在交联以保持蛋白质-蛋白质和蛋白质-DNA相互作用后，剪切染色质化的基因组，并沿着与靶基因组区域结合的蛋白质一起纯化TAL-标签蛋白。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的蛋白质。我们建议开发和部署这种新的技术，以纯化蛋白质和蛋白质复合物，结合到基因组位点在其天然染色质化的情况下。 这种ChIP-spec技术将通过分离和鉴定推定的调控蛋白，为解剖发育和疾病中的基因组调控机制提供一种强有力的方法。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案例中，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-spec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-spec技术与定量蛋白质组学技术结合，以实现基因组调控蛋白的从头检测，以及3）部署ChIP-spec测定以绘制细胞分化期间的蛋白质复合物动力学。 人胚胎干细胞、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特化和发育提供新的见解。这些复合物也将为未来开发基因调控模型和基序发现算法的工作提供基础。在医学上，该技术可能促进新的治疗方法，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是非常宝贵的。

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.