Development of a novel method to chart genomic localization of protein complexes in vivo

绘制修饰的组蛋白的基因组结构和定义DNA结合蛋白的位置是 这对于深入理解控制细胞状态的调节机制至关重要。然而，主要 用于绘制DNA相关蛋白的方法-染色质免疫沉淀， 测序（ChIP-seq）-具有几个主要限制：（i）标准ChIP-seq不能分析超过 在一个样品中一次一个表位;（ii）免疫沉淀步骤是低效的，导致低信号， （iii）ChIP-seq信号实际上是来自大量细胞群体的平均读数， 输入材料要求。因此，我们的挑战是同时研究多个DNA的组织 从单一样品中提取结合蛋白。我们提出的项目将开发一种新的方法，称为“鞭- seq'被设计来克服这些限制，部分是通过完全避免免疫沉淀， 使用长的特异性寡核苷酸和DNA条形码。Whip-seq方法希望提供一个 简单，快速，低成本的手段，以解决许多迄今无法回答的问题，染色质生物学。在 在本提案中概述的实验中，我们将：在体外逐步开发Whip-seq方法 （目标1）;实施数据分析的计算框架（目标2）;确定 使用体内小鼠ES细胞的新测定（Aim 3）。Whip-seq有可能改变 染色质转化为一个高度敏感，廉价和强大的过程，可以在任何分子生物学进行 实验室Whip-seq将提供一种手段，以获得一个前所未有的看法，不同的组合 染色质状态的标记以及这些标记在疾病中如何失调，例如，癌的 高分辨率映射和易于实施可以使Whip-seq能够用作诊断工具。

Mapping the genomic organization of modified histones and defining locations of DNA binding proteins is critical for deep understanding of the regulatory mechanisms governing cellular states. However, the primary methodology used for mapping DNA associated proteins – chromatin immunoprecipitation followed by sequencing (ChIP-seq) – has several major limitations: (i) standard ChIP-seq is unable to profile more than one epitope at a time in one sample; (ii) the immunoprecipitation step is inefficient, resulting in low signal to noise ratios; and (iii) ChIP-seq signal is effectively an average readout from large populations of cells due to input material requirements. Thus, our challenge is to simultaneously study the organization of multiple DNA binding proteins from a single sample. Our proposed project will develop a novel methodology termed `Whip- seq' that is designed to overcome these limitations, in part by avoiding immunoprecipitation altogether through the use of long specialized oligonucleotides and DNA barcoding. The Whip-seq method aspires to provide a simple, rapid, low-cost means to address many to date unanswerable questions in chromatin biology. In the experiments outlined in this proposal, we will: Develop the Whip-seq method in a stepwise manner in vitro (Aim1); implement a computational framework for data analysis (Aim 2); and establish the performance of the new assay using in vivo mouse ES cells (Aim 3). Whip-seq has the potential to transform the mapping of chromatin into a highly sensitive, cheap and robust process that can be carried out in any molecular biology laboratory. Whip-seq will provide a means to obtain an unprecedented view of the different combinatorial signatures of chromatin states and how these signatures may be dysregulated in disease, e.g., cancer. The high-resolution mapping, and the ease of implementation, may enable Whip-seq to serve as a diagnostic tool.