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基因组中基因活动的精确控制，这一过程由被称为转录因子的调节蛋白控制。转录因子如何通过结合细胞核中的特定DNA序列来识别它们所控制的基因，对生物学家来说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被编码在DNA序列中；然而，我们还不了解这一监管法规。Hox家族是一组已知控制发育多个方面的调节蛋白，包括一系列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蛋白负责控制沿着身体轴线产生的结构，比如手臂、腿或肋骨的发育。Hox基因的突变会对动物的外貌产生巨大的影响，例如，苍蝇的Ultrabithorax基因的缺失会导致产生四个翅膀而不是正常的两个翅膀的苍蝇，而人类的Hox基因突变会导致肢体异常，比如长出多余的手指。虽然从遗传学上可以清楚地看出，单个Hox蛋白对发育的影响非常不同，但矛盾的是，所有Hox蛋白都非常相似，似乎识别几乎相同的DNA序列，这些序列被认为决定了它们控制的基因集。大多数转录因子家族都存在这种特异性问题。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，我们将对此进行探讨。为了更好地了解重要的一类Hox蛋白如何能够控制特定的基因，我们使用果蝇作为模型系统。苍蝇的Hox蛋白与人类的Hox蛋白密切相关，但苍蝇的基因组要小20倍，这使得分析起来容易得多。我们可以测试Hox基因与DNA的相互作用，以及结合的功能后果，通过在一个确定的苍蝇细胞培养系统中在控制条件下表达Hox蛋白。了解Hox基因如何识别特定的基因组序列和控制基因，对于我们了解所有动物是如何发育的基本知识非常重要，如果我们希望深入了解进化是如何产生我们周围所见的各种体型的，这一点也很重要。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序列的可用性如何结合，以进一步了解决定Hox蛋白结合位置的规则。这将帮助我们确定这些非常相似的蛋白质如何产生不同的调控结果。我们将利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，并发现每种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苍蝇相同，我们的工作将帮助我们了解Hox基因是如何控制包括人类在内的高等动物的发育的。此外，最近已经清楚的是，Hox基因与包括癌症在内的几种疾病有关，因此，更好地了解Hox基因如何工作，在未来可能有助于研究人类疾病的各个方面。由于大多数基因调控家族显示出与Hox蛋白相似的特性，我们的研究也将有助于解决更普遍的问题，即基因是如何被一组类似的调控体特异性控制的。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控密码方面取得进展。

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.