Role of STAT1 Cooperative DNA Binding in Interferon Signalling

这项提议旨在研究细胞如何对环境的突然变化做出快速反应。需要快速反应的一个例子是人体免疫系统的细胞，它们保护我们免受试图建立感染的微生物，即病毒和细菌的侵袭。一旦免疫细胞感觉到入侵微生物的存在，它们就会释放具有不同作用的信号分子。我们关注的是干扰病毒和细菌繁殖能力的信号分子，因此被称为干扰素(IFN)。自50年前发现以来，IFN已被视为信号分子的模型，不仅对微生物感染具有治疗效果，而且在治疗癌症和神经退行性变方面也具有疗效。然而，它们的效力也会导致自身免疫性疾病或严重的抑郁症。因此，干扰素及其活性仍受到密切研究也就不足为奇了，尤其是为了充分认识到它们的临床潜力。然而，关于干扰素是如何发挥作用的，人们已经了解了很多。一个重要的方面是，干扰素通过调节基因的活性对细胞行为产生影响。然而，干扰素不会进入细胞，而是附着在细胞膜上的受体上。在这里，他们激活一种名为STAT1的蛋白质，然后该蛋白质将信号传递给细胞核中的基因。STAT1是一种转录因子。转录因子与DNA--包含基因的物质--结合，以调节基因的表达。转录因子可以被认为是允许细胞外信号分子，如IFN，访问其目标基因的关键。STAT1，以及一般的转录因子如何找到数量很少的特定位点，并隐藏在大量过剩的非特定位点中，这是细胞信号传递中一个令人敬畏的、尚未完全理解的问题。当我们发现STAT1不仅可以单拷贝与DNA结合，而且它还可以聚合成更大的结构，这就提出了一个前进的方向。这些聚合物被认为然后访问干扰素目标基因库中的特定基因集。有人认为，聚合对转录因子控制单个细胞的行为和协调免疫等复杂现象具有广泛的重要性，但直接的实验证据很少。为了解决这一知识的缺乏，我们在PI的实验室中关于STAT1 DNA结合和聚合的一些关键发现上进行了扩展，并创造了一个具有正常STAT1功能的转基因小鼠品系，只是它在聚合方面存在具体缺陷。因此，它是研究聚合对基因调控和细胞行为的影响的一个很有前途的模型。在分子水平上，我们希望确定STAT1单分子和聚合物的靶基因，以及STAT1区分它们的“DNA密码”。因此，拟议的研究旨在解决一个基本的生物学问题。此外，老鼠是人类免疫系统功能的测试模型。因此，我们的突变STAT1小鼠使我们能够探索聚合在预防细菌和病毒感染方面的效果，这是免疫的核心原则。由于干扰素在不同的临床环境中是有效的，提高对它们如何实现靶基因特异性的理解对于根据临床需要更好地定制它们的使用至关重要。STAT1抑制剂可以限制过度炎症的可能性是另一个潜在的临床应用。目前的药物不分青红皂白地抑制STAT1。我们的试点工作表明，相反，靶向STAT1聚合可能具有更高特异性和更少副作用的新型STAT1抑制剂的前景。拟议中的实验将有助于确定这些有趣的可能性是否实际上是现实的。

This proposal aims to investigate how cells manage to respond quickly to sudden changes in their environment. An example of the need for rapid responsiveness is the cells of the body's immune system, which protect us against microbes, i.e. viruses and bacteria, that attempt to establish infections. Once the immune cells have sensed the presence of intruding microbes they release signalling molecules with diverse roles. We are concerned with signalling molecules that interfere with the ability of viruses and bacteria to multiply and therefore are called interferons (IFNs). Since their discovery 50 years ago, the IFNs have come to be regarded as the model signalling molecules with therapeutic efficacy not just against microbial infections, but moreover in the treatment of cancers and neurodegeneration. However, their very potency can also cause problems manifesting in autoimmune disorders or severe depression. It is thus not surprising that the interferons and their activities are still intensely studied not least to realize fully their clinical potential. A lot has been learned though about how the interferons function. One important aspect is that IFNs exert their influence on cellular behaviour by regulating the activity of genes. Yet interferons do not enter the cells but attach to receptors in the cell membrane. Here, they activate a protein called STAT1, which then carries the signal to the genes in the cell nucleus. STAT1 is a transcription factor. Transcription factors bind to DNA -the material containing the genes- in order to regulate the expression of genes. Transcription factors can be regarded as the keys that allow extracellular signalling molecules, like the IFNs, access to their target genes. How STAT1, and for that matter transcription factors in general, find specific sites that are few in number and hidden in a massive excess of unspecific sites, is a formidable, incompletely understood, problem in cell signalling. A way forward was suggested when we discovered that STAT1 can bind to DNA not only in single copies, but that it can also polymerise into larger structures. These polymers are believed to then access specific sets of genes within the IFN target gene repertoire. It was suggested that polymerization is widely important for transcription factors to steer the behaviour of individual cells and to orchestrate complex phenomena like immunity, but direct experimental evidence is scarce. To address this lack of knowledge we have expanded upon a number of crucial discoveries in the PI's lab regarding STAT1 DNA binding and polymerization, and have created a genetically modified mouse strain with normal STAT1 function, except that it is specifically deficient in polymerization. It is thus a promising model for studying the effects of polymerization on gene regulation and cellular behaviour. On the molecular level we hope to identify the target genes of STAT1 single molecules and polymers, and the "DNA code" by which STAT1 discriminates between them. As such the proposed research aims at solving a fundamental biological problem. Mice moreover are a tested model for the functioning of the human immune system. Our mutant STAT1 mice thus enable us to explore the effects of polymerization on protection against bacterial and viral infections, central tenets of immunity. As interferons are effective in diverse clinical settings, an improved understanding of how they achieve target gene specificity is critical to better tailor their use according to clinical need. The possibility that STAT1 inhibitors may limit excessive inflammation is another potential clinical application. Current pharmaceuticals inhibit STAT1 indiscriminately. Our pilot work indicates that targeting STAT1 polymerization, in contrast, might hold the prospect for novel STAT1 inhibitors with higher specificity and fewer side effects. The proposed experiments will help to decide whether these intriguing possibilities are in fact realistic.