Development of enhanced gene specific technology for the isolation of proteins binding at a single locus in vivo.

识别结合并调节细胞内基因表达的蛋白质是一个重要的、持续的但耗时的过程。目前的方法，如染色质免疫沉淀(CHIP)，需要预先知道特定的蛋白质可能与细胞中特定的DNA区域结合。然后，这种蛋白质被用作一个‘把手’，分离出细胞中可能与该蛋白质结合的所有DNA。(这通常是通过使用针对特定蛋白质的抗体来实现的)。然后对该DNA进行评估，以确定相对于其他序列，原始DNA是否被浓缩。通过使用蛋白质‘手柄’作为分离可能与目标蛋白质相互作用的其他额外蛋白质的手段，该方法可以进行一定程度的扩展，从而可以费力地组装关于给定启动子的总蛋白质占有率的某种图像。然而，该方法存在几个缺陷：-1)需要确定特定蛋白质作为起点，2)需要获得针对目标蛋白质的高质量抗体，3)这些抗体在不同批次和不同蛋白质目标之间的亲和力/特异性不同，导致不同实验之间的结果可能存在很大差异，4)目标蛋白质在整个细胞内结合的所有DNA区域将被分离，以及在这些其他DNA位点与其相互作用的大多数其他蛋白质。因此，通过后续实验如质谱学来鉴定DNA区域特异性结合蛋白是不可能的。规避这些问题的一个聪明的方法是使用与感兴趣的DNA区域互补的小DNA分子(寡核苷酸)。它们有一个单一的化学手柄(去硫代生物素)，这样当这些寡核苷酸与它们的目标DNA序列结合时，它们可以从细胞中检索出来，以及那些仍然与目标DNA结合的蛋白质。到目前为止，这种方法只被用于检测与DNA结合的重复蛋白质，从而人为地提高了该方法的灵敏度。此外，这种方法的关键步骤之一是变性(融化)细胞的DNA，以允许寡核苷酸与其结合。这代表了一个可能的不利因素，因为我们希望捕捉的一些DNA：蛋白质相互作用可能会在这个过程中丢失。我们建议开发一种绕过这些问题的方法，提供一种试剂，这种试剂将适用于世界各地的科学家，一次过识别与特定的单个基因相关的所有蛋白质。我们的方法是使用锌指蛋白(或类似的作用蛋白)，这种蛋白可以设计成与几乎任何DNA序列结合，连接到多个在体内被修饰的小标签(生物素受体多肽)，这将为DNA分离提供一个令人难以置信的强大“把手”。我们认为，这种方法不需要任何DNA变性，可以有多个‘手柄’来提高灵敏度，将为分离整个基因组中的单个DNA区域和结合它们的蛋白质提供一种通用和高度敏感的途径。其他途径将包括通过增加它们在体内的修饰程度来增强这些酶在细胞内的“手感”效力。最终目标将是使科学家能够快速而自信地建立起细胞内发生的事件的图景，这些事件发生在DNA的特定调控区域，在细胞分裂和分化、干细胞更新以及疾病发生和发展等过程中。

The identification of proteins that bind to and regulate gene expression within the cell is an important, ongoing but time consuming process. Current methodologies, such as chromatin immunoprecipitation (ChIP) require foreknowledge that a particular protein may be binding to a particular region of DNA in the cell. This protein is then used as a 'handle' to isolate all the DNA in the cell to which that protein might be binding. (This is generally achieved by using antibodies specific for the particular protein). Then an assessment of this DNA is carried out to determine whether the original DNA of interest is enriched relative to other sequences. The method can be extended somewhat by using the protein 'handle' as a means to isolate other additional proteins that might be interacting with the target protein, thus some kind of picture can be laboriously assembled as to the general protein occupancy of a given promoter. The methodology however suffers from several flaws:- 1) one needs to identify a specific protein as a starting point, 2) one needs to have access to high quality antibodies specific for the target protein, 3) such antibodies differ in their affinity/specificity between individual batches and between protein targets, leading to potentially large variations in results between experiments, 4) ALL the DNA regions that are bound by the target protein throughout the cell will be isolated, along with a majority of the other proteins that it interacts with at these other DNA loci. Thus identifying DNA region specific binding proteins via follow on experiments such as mass spectrometry is impossible.One clever approach to circumvent these problems has been to use small DNA molecules (oligonucleotides) complementary to the DNA region of interest. These have a single chemical handle attached (des-thiobiotin), so that when these oligonucleotides bind to their target DNA sequence they can be retrieved from the cell, along with those proteins that have remained bound to the target DNA. So far this method has only been used to examine proteins bound to DNA that is repetitive, thus artificially boosting the sensitivity of the approach. Also, one of the key steps in this method is denaturing (melting) the cell's DNA to allow the oligonucleotide to bind it. This represents a possible downside, in that some of the DNA:protein interactions we were hoping to capture may be lost during the process. We propose to develop a method that circumvents these issues, delivering a reagent that will have applications for scientists throughout the world, in identifying in one go, all of the proteins that are associated with a specific, individual gene. Our approach is to use zinc finger proteins (or similar acting TALE proteins), that can be designed to bind virtually any DNA sequence, linked to multiple small tags (biotin acceptor peptides) that become modified in vivo, and that will provide an incredibly strong 'handle' for DNA isolation. We suggest that this approach, which does not require any DNA denaturation and can have multiple 'handles' to increase sensitivity, will provide a versatile and highly sensitive route for the isolation of individual DNA regions throughout the genome and the proteins that bind them. Additional routes will involve enhancing the 'handle' efficacy of these enzymes within the cell via increasing their degree of modification in vivo. The ultimate aim will be to enable scientists to rapidly and confidently build up a picture of events in the cell as they happen at specific regulatory regions of DNA, during processes such as cell division and differentiation, stem cell renewal and disease onset and progression.