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组织图并确定DNA结合蛋白的位置 对于深入理解控制细胞状态的调节机制至关重要。然而，主要的 DNA相关蛋白图谱的方法学--染色质免疫沉淀 排序(CHIP-SEQ)-有几个主要限制：(I)标准CHIP-SEQ无法分析超过 在一个样本中一次一个表位；(Ii)免疫沉淀步骤效率低下，导致信号低 噪声比；以及(Iii)码片序列信号实际上是从大量单元中的平均读出，这是由于 输入材料需求。因此，我们的挑战是同时研究多个DNA的组织 结合单个样本中的蛋白质。我们提议的项目将开发一种新的方法，名为‘鞭子-- 它旨在克服这些限制，部分原因是通过以下方式完全避免了免疫沉淀 使用长的专门化寡核苷酸和DNA条码。Wip-seq方法希望提供一个 简单、快速、低成本的方法可以解决染色质生物学中许多迄今无法回答的问题。在 在本提案中概述的实验中，我们将：在体外分步发展惠普-SEQ方法 (目标1)；实施数据分析的计算框架(目标2)；并确定 利用小鼠体内ES细胞进行新的检测(目标3)。Whip-seq有可能改变 染色质转化为一种高度敏感、廉价和强大的过程，可以在任何分子生物学中进行 实验室。WHIP-SEQ将提供一种手段来获得对不同组合的前所未有的看法 染色质状态的特征，以及这些特征在疾病中可能如何失调，例如癌症。这个 高分辨率测绘和易于实施，可能使WHIP-SEQ成为一种诊断工具。

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.