Reading the genome: how do transcription factors achieve target specificity?

动物的发育和身体的正常生理反应依赖于对基因组内基因活动的精确控制，这一过程由称为转录因子的调节蛋白控制。对于生物学家来说，转录因子如何通过与细胞核中特定的DNA序列结合来识别它们控制的基因仍然是一个相当大的挑战。我们知道，决定基因何时何地开启的信息是在DNA序列中编码的；然而，我们还不理解这一调控密码。已知的一组控制多方面发育的调节蛋白是HOX家族，它包括一组在从苍蝇到人类的所有动物中发现的密切相关的因子。我们目前的理解是，特定的HOX蛋白负责控制沿着体轴产生的结构，例如手臂、腿或肋骨的发育部位。HOX基因的突变对动物的外表产生了戏剧性的影响，例如，苍蝇的超双胸基因的缺失可能导致四个翅膀的产生，而不是正常的两个翅膀的苍蝇，而人类的HOX基因突变可能导致肢体异常，如发育额外的手指。虽然从遗传学上很清楚，单个Hox蛋白对发育的影响非常不同，但矛盾的是，所有的Hox蛋白都非常相似，似乎识别几乎相同的DNA序列，这些DNA序列被认为决定了他们控制的基因集。这种特异性的普遍问题对大多数转录因子家族来说是共同的。一些特异性可能来自与辅因子DNA结合蛋白的特定相互作用，我们将对此进行探索。为了更多地了解这类重要的HOX蛋白如何能够控制特定的基因集合，我们使用果蝇作为一个模型系统。苍蝇的HOX蛋白与人类的HOX蛋白关系密切，但苍蝇的基因组要小20倍，这使得分析变得容易得多。我们可以通过在特定的苍蝇细胞培养系统中控制条件下表达HOX蛋白来测试HOX基因与DNA的相互作用以及结合的功能后果。了解HOX基因是如何识别特定的基因组序列和控制基因的，对于我们基本了解所有动物是如何发育的很重要，如果我们想要洞察进化是如何产生我们周围看到的巨大范围的身体计划，这也是很重要的。我们的实验将使用细胞培养系统来确定所有核心Hox蛋白在基因组中的结合位置，以及它们在有和没有关键DNA结合辅助因子的情况下对基因表达的影响。然后，我们将把这种结合与细胞核中DNA的一般特征联系起来，本质上是一段DNA序列可用于结合的程度，以进一步了解决定HOX蛋白结合位置的规则。这将有助于我们确定这些非常相似的蛋白质是如何导致不同的调控结果的。我们将使用这些数据来更好地理解HOX蛋白能够识别控制基因的DNA序列的规则，并发现每个HOX蛋白的差异。由于哺乳动物的HOX基因的组织和运作方式与果蝇的相同，我们的工作将有助于我们了解HOX基因是如何控制高等动物的发育的，包括人类。此外，最近发现，HOX基因与包括癌症在内的多种疾病有关，因此，更好地了解HOX基因是如何发挥作用的，在未来研究人类疾病的各个方面可能会很有用。由于大多数基因调节器家族显示出与Hox蛋白相似的特性，我们的研究也将有助于解决更一般的问题，即基因如何由一组类似的调节器具体控制。通过研究这种特异性的基础，我们将能够在理解基因组的调节密码方面取得进展。

Animal development and the normal physiological responses of the body rely upon the precise control of the activity of genes within the genome, a process controlled by regulatory proteins known as transcription factors. How transcription factors identify the genes they control by binding to specific DNA sequences in the nucleus remains a considerable challenge for biologists. We know that information dictating where and when a gene is switched on is encoded in the DNA sequence; however, we do not yet understand this regulatory code. One group of regulatory proteins known to control multiple aspects of development, the Hox family, includes a set of closely related factors found in all animals, from flies to humans. Our current understanding is that specific Hox proteins are responsible for controlling the structures that are produced along the body axis, such as where arms, legs or ribs develop. Mutations in Hox genes have dramatic consequences on the way animals look, for example the loss of the Ultrabithorax gene in the fly can result in the production of a four wing rather than a normal two wing fly and Hox mutations in humans can result in limb abnormalities such as the development of extra digits. While it is clear from genetics that individual Hox proteins have very different effects on development, paradoxically all Hox proteins are very similar and appear to recognise virtually identical DNA sequences that are believed to dictate the sets of genes they control. This general problem of specificity is common to most families of transcription factors. Some of the specificity may come from specific interactions with co-factor DNA binding proteins and we will explore this. To understand more about how the important class of Hox proteins are able to control specific sets of genes we use the fruit fly as a model system. The Hox proteins of the fly are closely related to those in humans but the fly genome is 20 times smaller, making analysis much easier. We can test Hox gene interactions with DNA, and the functional consequences of binding, by expressing Hox proteins under controlled conditions in a defined fly cell culture system. Understanding how Hox genes recognise specific genomic sequences and control genes is important for our basic understanding of how all animals develop and it is also important if we wish to gain insights into how evolution has produced the huge range of body plans we see around us. Our experiments will use a cell culture system to determine where all core Hox proteins bind in the genome and the effects they have on gene expression with and without their key DNA binding cofactors. We will then relate this binding to general features of the DNA in the nucleus, essentially how available a stretch of DNA sequence is for binding, to further understand the rules that determine where Hox proteins bind. This will help us to determine how these very similar proteins give rise to different regulatory outcomes. We will use these data to better understand the rules by which Hox proteins are able to recognise the DNA sequences that control genes, and discover differences for each of the Hox proteins. Since the Hox genes of mammals are organised and operate in the same way as those in flies, our work will help us understand how Hox genes control development of higher animals, including man. In addition, it has recently become clear that Hox genes are involved in several diseases, including cancers, thus a better understanding of how Hox genes work may, in the future, be useful when studying aspects of human disease. Since most families of gene regulators show similar properties to Hox proteins, our studies will also help address the more general issue of how genes are specifically controlled by sets of similar regulators. By studying the basis for this specificity, we will be able to make progress in understanding the regulatory code of the genome.