冷泉港实验手册：验证蛋白-DNA相互作用的功能重要性 http://cshprotocols.cshlp.org/content/2012/7/pdb.top070060.full 日期：2012-07-20 来源：互联网 作者：keeii 点击：28次 蛋白技术 7月，最新一期的冷泉港实验手册《Cold Spring Harbor Protocols》发布。按照惯例，有两篇protocol是可以免费阅读的，其中一篇介绍了如何验证蛋白-DNA相互作用的功能重要性。这篇文章节选自《真核生物的转录调控：概念、策略与技术》一书，是由美国加州大学洛杉矶分校（UCLA）的研究人员撰写的。 鉴定与感兴趣的控制区域相互作用的DNA结合蛋白已变得相当简单。然而，特定的蛋白-DNA相互作用的功能相关性还难以建立。作者介绍了一些检验这种相关性的方法。但同时强调，没有一种实验本身是结论性的。作者介绍了每种方法所获得的信息，并解释了它为什么产生了有用但不确定的结果。就所需的劳动量和获得的信息而言，每种方法差别很大。 作者写道，鉴定与控制区域相互作用的DNA结合蛋白相对简单，但要明确特定转录因子通过与确定的控制元件结合而直接调控靶基因还是一个相当困难的任务。其中涉及到鉴定重要的DNA序列元件及与这些元件结合的蛋白的实验策略。 然而，这种鉴定并不能证明生物相关性。例如，在细胞核提取物种检测蛋白-DNA的相互作用反映了很多因素：(1)细胞中蛋白的丰度，(2)蛋白提取的效率，(3)活性蛋白的稳定性，(4)提取过程中翻译后修饰的维持，(5)体外DNA结合分析所使用的条件，(6)蛋白与控制元件的亲和力。 作者认为，体外检测蛋白-DNA相互作用不足以说明它的体内相关性。然而，证实哺乳动物细胞内蛋白-DNA相互作用的功能重要性的明确方法尚未开发。目前大家常采用两种来开展功能相关性研究，那就是ChIP和RNAi。 ChIP目前已成为鉴定蛋白与特定基因组区域之间关联的极为常用方法。一个严格对照的ChIP实验的阳性结果可以提供关键的信息，即一个候选蛋白确实非常靠近感兴趣的控制元件。不过，ChIP并不是一个功能分析，而仅仅显示了体内关联。因为一些研究表明，DNA结合蛋白在体内并非能激活所有DNA元件，还必须获得其他的功能相关性证据。 功能丧失（loss-of-function）研究也是目前很常用的。利用RNAi，可以快速knockdown编码几乎所有蛋白的基因的表达，只要能获得对RNAi敏感的细胞群体。不过，RNAi也无法轻松克服功能丧失研究所固有的限制，包括两个或多个因子之间冗余度所造成的差异以及很难区分直接和间接影响。 在这篇文章中，作者介绍了12种不同的方法，从ChIP和RNAi开始，它们可以检测一个特定的蛋白-DNA相互作用是否在功能上重要。他们介绍了每种方法的原理和操作，并解释了它们为什么产生了有用但不确定的结果。作者认为，如果主要目标是建立一种相互作用的功能重要性，那么应采取几种不同的方法。 这篇文章可免费阅读，地址为： http://cshprotocols.cshlp.org/content/2012/7/pdb.top070060.full Confirming the Functional Importance of a Protein–DNA Interaction 1. Michael F. Carey, 2. Craig L. Peterson and 3. Stephen T. Smale Adapted from Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques, 2nd edition, by Michael F. Carey, Craig L. Peterson, and Stephen T. Smale. CSHL Press, Cold Spring Harbor, NY, USA, 2009. Next Section Abstract Identifying DNA-binding proteins that interact with a control region of interest has become quite straightforward. However, the functional relevance of a given protein–DNA interaction is difficult to establish. The hypothesis that an interaction is relevant can be tested by several different experiments, 12 of which are outlined in this article. It must be remembered that none of these experiments by itself is conclusive. The information gained from each approach is described and explanations are given for why each yields useful but inconclusive results. The approaches vary widely with respect to the amount of effort required and the quality of information obtained. Previous SectionNext Section INTRODUCTION AND OVERVIEW In the modern era of molecular biology, identifying DNA-binding proteins that interact with a control region of interest is relatively straightforward, but establishing definitively that a specific transcription factor directly regulates a target gene by binding to a defined control element can be among the most difficult of tasks. There are experimental strategies to identify important DNA sequence elements, as well as proteins that bind those elements. In most instances, a transcription factor will have been implicated as a potential gene regulator by a TRANSFAC or JASPAR database search, by mass spectrometry analysis of proteins that bind the element in vitro, by a genome-wide chromatin immunoprecipitation (ChIP) analysis of a transcription factor’s binding sites, or perhaps by a yeast one-hybrid screen. The identification of candidate DNA-binding proteins provides a significant advance because it allows one to hypothesize that the protein is responsible for the function of the control element in regulating the nearby gene. By itself, however, identification does not prove biological relevance. For example, detecting a protein–DNA interaction in a nuclear extract reflects many factors: (1) the abundance of the protein in the cells from which the extract was prepared, (2) the efficiency with which the protein was extracted from the cells, (3) the stability of the active protein within the extract, (4) the maintenance of essential posttranslational modifications during extract preparation, (5) the conditions used for the in vitro DNA-binding assay, and (6) the affinity of the protein for the isolated control element (Table 1). View this table: • In this window • In a new window Table 1. Factors that influence the in vitro detection and in vivo relevance of protein–DNA interactions The criteria for detecting protein–DNA interactions in vitro are very different from those that determine which protein interacts functionally with the control element in vivo (i.e., which protein regulates the endogenous gene by binding to the control element) (Table 1). These criteria include (1) the abundance and stability of the protein in the cell nucleus, (2) the affinity of the protein for the site, (3) the ability of the protein to perform appropriate interactions with other proteins bound to adjacent sites and with non-DNA-binding cofactors, (4) appropriate posttranslational modifications that allow the protein to perform the necessary protein–DNA and protein–protein interactions, and (5) the appropriate subnuclear localization of the protein. When considering the above points, it is readily apparent that detecting a protein–DNA interaction in vitro provides only weak evidence of its relevance in vivo, even when the DNA sequence element to which the protein binds is known to be important. A similar level of uncertainty exists when a protein capable of recognizing a DNA sequence element is identified in a TRANSFAC or JASPAR database search, as the consensus recognition sequences used to create those databases were determined using in vitro protein–DNA interaction assays. Most DNA-binding proteins can recognize a broad spectrum of DNA sequences with a wide range of affinities. Furthermore, most DNA-binding proteins are members of multiprotein families, with each cell type containing several family members that recognize similar DNA sequences. Based on these considerations, it is likely that multiple proteins will be capable of binding a defined control element in vitro, including several members of a particular protein family, and perhaps members of another family that recognize a similar or overlapping sequence. The challenge is to determine which of these proteins is capable of performing the protein–protein and protein–DNA interactions that allow it to regulate the endogenous gene. If by chance only one predominant DNA-binding protein is detected in vitro, or if a database search identifies only one candidate protein, it still may not be the one responsible for the function of the control element in vivo; other proteins within the cells are almost certainly capable of binding the same element, even if they were not detected in initial database analyses or electrophoretic mobility-shift assay (EMSA) studies. A definitive approach for confirming the functional importance of a protein–DNA interaction in mammalian cells has not yet been developed. However, two powerful techniques have revolutionized efforts to evaluate the functional relevance of a DNA-binding protein that interacts with a DNA element of interest: ChIP and RNA interference (RNAi). Although both of these techniques were developed before the year 2000, they did not emerge as common methods for studying protein–DNA interactions in eukaryotic cells, in particular mammalian cells, until the 21st century. As described below, ChIP has now become an extremely common method for monitoring the association of proteins with specific genomic regions in living cells. A positive result in a well-controlled ChIP experiment provides crucial information not obtainable previously: namely, that a candidate protein does indeed associate in close proximity to a control element of interest. This knowledge adds considerable strength to the hypothesis that a candidate DNA-binding protein is responsible for the function of a DNA element. However, ChIP is not a functional assay and merely shows an in vivo association. Because several studies suggest that DNA-binding proteins are not functionally active at all DNA elements they bind in vivo (see, e.g., Walter et al. 1994; Li and Johnston 2001;Martone et al. 2003; Phuc Le et al. 2005; Beima et al. 2006; Hollenhorst et al. 2007; Dong et al. 2008), additional evidence of functional relevance must be obtained. Loss-of-function studies of a DNA-binding protein are critical for evaluating the functional importance of a protein–DNA interaction. However, until RNAi emerged as a common molecular biology tool, the methods available for performing such studies, particularly in mammalian cells, were either laborious (e.g., gene disruption by homologous recombination) or unpredictable, in that they had a relatively low probability of success (e.g., antisense RNA or dominant-negative methods). Using RNAi, it is now possible to rapidly knockdown the expression of the gene encoding almost any protein, as long as a cell population susceptible to RNAi is available. Unfortunately, despite its power, RNAi cannot easily overcome the limitations inherent in all loss-of-function approaches, including difficulties caused by redundancy between two or more factors and difficulties distinguishing direct from indirect effects. To reiterate, although ChIP and RNAi have become two of the most useful methods for evaluating the relevance of a protein–DNA interaction, neither yields definitive results, and both possess considerable limitations. In the absence of a definitive experiment (i.e., an experiment that allows one to visualize directly a protein binding to a control element within its natural chromosomal location and regulating transcription of the linked gene), the only viable approach is to hypothesize that a protein–DNA interaction is relevant and then to subject that hypothesis to as many rigorous tests as possible, with ChIP and RNAi experiments serving as two of the more common and crucial tests. The point at which a hypothesis that a protein–DNA interaction is functionally important for regulating a gene of interest has been confirmed beyond a reasonable doubt depends on subjective evaluation and is therefore best left to the judgment of the scientific community. This article presents 12 different approaches, beginning with ChIP and loss-of-function methods, that can be used to test whether a specific protein–DNA interaction is functionally important. We describe the information gained from each approach and explain why each yields useful but inconclusive results. The approaches vary widely with respect to the amount of effort required and the quality of information obtained. If a major goal is to establish the functional importance of an interaction, several different approaches should be pursued. It is important to emphasize that a rigorous examination of the relevance of a transcription factor–DNA interaction might not be an important objective of the investigator. The primary interest could instead be to determine whether a factor discovered during efforts to examine the regulation of a particular gene is important for a biological process. For example, even without strong evidence that the transcription factor regulates the gene of interest, it could itself have an interesting expression pattern that merits further exploration of its biological functions. In this instance, the next step is more obvious: The gene encoding the factor can be disrupted in a cell or animal, or gene expression can be reduced by RNAi. A comprehensive characterization of the knockout or knockdown phenotype can then be performed, including microarray experiments to identify genes that are misexpressed in the absence of the factor. If an interesting phenotype is observed, the investigator might begin a broadly based analysis of the factor, regardless of whether it interacts functionally with the control element used originally for its identification. This is a common and valid course of action. However, if the long-term goal is to link a protein to its relevant target genes, or to carefully dissect the mechanism by which a target gene is induced by a constellation of DNA-binding proteins, cofactors, and general transcription factors, the issues discussed in this article ultimately must be considered. 。 相关阅读 • 冷泉港实验手册专家讲述FISH和ELISPOT技巧 标签：冷泉港 • 上一篇：高手支招：miRNA功能分析的五大要点 • 下一篇：5款方便好玩的实验app推荐 分享到：0 精彩专题
对浙大院士课题组的论文事件已有众多评论，因为事不关己，只浏览了少数的评论。此前曾目睹过前老板被同事指控在投稿时随意删除或添加作者，因而得知审理这类案件国外机构会引用温哥华议定书（Vancouver Protocol, www.icmje.org）。 议定书明确了要被列为作者以下三条必须全部满足： 1. 对构思和设计，获取数据，或分析解释数据有显著贡献， 2.起草文章或修改文章重要的学术内容， 3.参与定稿。 （Authorship credit should be based on 1） substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; 2） drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published. Authors should meet conditions 1, 2, and 3.） 议定书并明确指出仅仅参与获取科研资金，或收集数据，或作常规的监管不足以被列为作者。 对照温哥华议定书，显然浙大院士和实验室主任的行为完全越轨了。浙大既然宣称要角逐世界一流，那么就必须要顺应国际惯例。如果浙大尚未有明确的科研守则，何不应尽快参照其他大学的守则写出草案，公示后成为正式文件向师生传达? 处理这起院士课题组的论文事件既棘手而又不讨好，那么为什么不防患于未然？与其给学生开爱国课，倒不如向师生介绍科研伦理，不出丑闻不给中国丢脸，不就是爱国吗？！ 再贴一段澳洲著名免疫学家Gustav Nossal 回忆诺贝尔奖得主Macfarlane Burnet时的讲话：One of the minor regrets, not really a big regret, is that Ive never published a paper with Mac Burnet. Ive published 500 papers, not a single one has Burnet as a co-author. He did not believe in putting his name on a paper if he hadnt done at least one third of the work himself. A sort of an honest unselfish approach, when it comes time to reap the glory you do it without having someone grabbing it instead of you. Macfarlane Burnet只有在自己对文章有三分之一以上贡献时才肯把自己列为作者 --- 这才是真正的大师！
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