TFTag: A novel library of tagged transcription factors in Drosophila

每一个多细胞生物都由几十种甚至几百种不同的细胞类型组成，这些细胞类型由它们不同的蛋白质谱所定义。蛋白质的构建计划以基因的形式编码在DNA中，在哪里和何时制造它们的指令也是如此。这一过程被称为基因表达，是高度动态的，在发育过程中或对环境变化的反应中不断调整。每种细胞类型都有其特有的基因表达模式，而基因表达失调与包括癌症在内的多种疾病有关。因此，必须严格调节基因表达，以确保每个细胞都有正确的蛋白质含量。这在很大程度上是由一类特殊的蛋白质完成的，转录因子（tf），它们可以与DNA结合，并相互合作，影响哪些基因的转录量。这反过来又决定了哪些蛋白质存在于哪些细胞中以及其数量。因此，生命科学和医学领域的众多科学家都在研究tf。对tf的研究主要旨在揭示其表达水平、定位、基因组结合位点、相互作用伙伴以及功能丧失的影响等信息。这些分析大多严重依赖抗体的使用，而抗体的生产成本高、难度大，而且在伦理上存在问题。此外，涉及抗体的方法面向固定的生物材料，这使得动态过程的重述具有挑战性。一种常见的解决方案是利用遗传技巧来表达TF的一个版本，该版本与另一种蛋白质融合，反过来，它可以很容易地被检测到，因此被称为标签。然而，标签通常只适用于一种或几种方法，否则就太大，可能会干扰TF的功能。此外，标记TF的额外表达可以诱导伪影。此外，用于表达此类标记结构的工具仅适用于tf的一个子集。长期以来，果蝇（Drosophila melanogaster）一直是研究基本生物过程的主要模式生物之一。它得益于一个庞大的、联系良好的全球研究人员社区，以及大量遗传和分子资源的可用性。重要的是，它允许在体内进行研究，并且为产生新的工具而创建转基因系是直截了当的。在这里，我们建议建立一个生物信息学资源，包括一个转基因蝇系文库，其中所有果蝇TF基因都被标记在其原生基因组位点上。我们还将建立一个数据库，其中包含以前未研究过的tf子集的表达和DNA结合的基本数据。我们的资源将减轻以前标记蛋白库的许多缺点：1)我们的标记策略允许标签的轻松交换，使每个TF可用于各种下游应用；2)我们将创建单独的转基因系，其中TF的两端都被标记，这减少了标签干扰蛋白质功能的风险；3)对于用替代端表示的TF，我们将为每个端创建一条线，允许研究每个TF的不同版本。我们将免费向研究界的成员分发蝇线，同样，数据库也将免费使用。这些资源将为在任何可想象的生物学过程中使用选择方法研究任何果蝇TF提供可能性，并使科学家能够轻松地在许多未研究的TF中识别潜在的候选对象。我们期望我们的资源将极大地推动基因调控领域的研究。

Every multicellular organism consists of dozens or even hundreds of different cell types that are defined by their different protein repertoires. The building plans for the proteins are encoded in the form of genes in the DNA, as are the instructions of where and when to make them. This process, referred to as gene expression, is highly dynamic and is constantly adjusted during development or in response to, e.g., environmental changes. Each cell type has its characteristic pattern of gene expression, and mis-regulation is associated with various diseases, including cancer. Therefore, gene expression must be tightly regulated to ensure that each cell has the correct protein content. This is largely done by a specific class of proteins, the transcription factors (TFs), which can bind to DNA and cooperate with each other to influence which genes are transcribed in what quantities. This in turn determines which proteins are present in which cells and in which amount. TFs are, accordingly, studied by a wide range of scientists in the life and medical sciences.Research on TFs mainly seeks to uncover information about their expression level, localisation, binding sites in the genome, interaction partners, and effects of a loss of function. Most of these analyses rely heavily on the use of antibodies, the production of which is costly, difficult, and ethically questionable. Moreover, methods involving antibodies are geared towards fixed biological materials, which makes the recapitulation of dynamic processes challenging. A common solution is the use of genetic tricks to express a version of the TF that is fused to another protein, which can, in turn, be readily detected and is, accordingly, called a tag. However, tags are often suitable for only a single or few methods or otherwise are large and might interfere with the function of the TF. Moreover, the additional expression of the tagged TF can induce artefacts. Also, tools for the expression of such tagged constructs are only available for a subset of TFs.The fruit fly, Drosophila melanogaster, has long served as one of the prime model organisms to study basic biological processes. It benefits from a large, well-connected worldwide community of researchers and the availability of vast genetic and molecular resources. Importantly, it allows investigations in vivo, and the creation of transgenic lines for the generation of novel tools is straightforward.Here we propose to create a biological and bioinformatic resource consisting of a library of transgenic fly lines in which all Drosophila TF genes have been tagged at their native genomic locus. We will also establish a database with basic data on the expression and DNA binding of a subset of TFs that have not previously been studied. Our resources will alleviate many of the shortcomings of previous libraries of tagged proteins: 1) Our tagging strategy allows for the easy exchange of the tag, making each TF accessible for various downstream applications; 2) We will create individual transgenic lines in which either end of a TF has been tagged, which reduces the risk of an interference of the tag with protein function; 3) For TFs that are expressed with alternative ends, we will create a line for each end, allowing the study of different versions of each TF. We will distribute the fly lines free of charge to members of the research community, and the database will, likewise, be free to use. These resources will open the possibility to study any Drosophila TF with the methods of choice in any biological process imaginable and allow scientists to easily identify potential candidates among the many unstudied TFs. We expect that our resource will greatly advance research in the field of gene regulation.