Cytokine-induced changes in transcription factories

只有我们DNA中编码的部分遗传信息被复制到RNA中，然后‘翻译’成蛋白质；复制是有选择性的，错误复制会导致疾病(例如癌症)。我们希望分析关键的复制机器--被称为‘RNA聚合酶’--在它们的自然环境中。在过去的五十年里，人们已经开发出了从细胞中纯化这些复制机器的技术，现在我们已经有了它们的内容和结构的详细地图。我们还知道它们是执行额外功能的更大生产线的一部分；例如，主要的复制机-聚合酶II-与其他在制作RNA副本时进行工作的机器一起被发现。我们已经证明，这些生产线中的许多又被组织成更大的“工厂”。[我们将工厂定义为包含至少两个复制不同DNA片段的RNA聚合酶的场所，所有工厂的原因是相同的(无论是制造汽车、果酱还是RNA)-通过将相关机器和原材料集中在一个地方来提高产量。]不幸的是，这些工厂与细胞中的子结构紧密相连，到目前为止它们还不可能被分离出来(所以我们对它们的内容知之甚少)；幸运的是，我们刚刚开发出一种方法，允许我们将它们从子结构中释放出来(我们使用细胞自杀时自然使用的酶！)，这使我们能够继续部分净化工厂，而不是许多其他细胞成分。我们现在想利用这种方法，以及一种相关的方法来分析工厂中哪些基因正在被复制；我们想使用各种技术(包括测序来确定DNA和RNA含量，以及光学显微镜来绘制位置)来对它们的一些内容进行编目。我们尤其感兴趣的是比较工厂开始复制特定组基因之前和之后的情况。为此，我们将使用两种能够快速启动选定基因复制的天然制剂--肿瘤坏死因子α(TNFa)和转化生长因子β(TGFb)。[当我们的身体受到微生物感染时，TNFa会协调我们的炎症反应；它与细胞膜结合，并向细胞核发送信使-NFkappaB-以启动编码保护蛋白的基因的复制。如果感染是轻微的，该系统会产生能够对抗它的蛋白质。但如果情况严重，细胞可能会选择通过激活上述酶来自杀--从而将感染微生物一起送进坟墓。显然，系统必须做出正确的决定！TGFb向细胞核发送不同的信使，以启动一组不同的基因。由于这些基因控制着与细胞增殖和分化有关的许多细胞功能，TGFb在癌症、心脏病和糖尿病等疾病中发挥着重要作用。]由于这两种药物作用如此迅速，我们将把它们加入从正常脐静脉获得的细胞中，启动不同组的基因，10-480分钟后净化工厂，并将其内容与未经处理的细胞中的内容进行比较。我们还有一个更雄心勃勃的目标。人类染色体可以说是最大和最重要的生物分子，其结构的许多方面-以及结构如何影响功能-仍然不清楚。这有实际的后果：例如，我们还不能预测当一个基因被插入一个新的位置时会有多活跃，这显然限制了成功的基因疗法的发展。因此，我们这个时代的主要问题是：人类染色体的3D结构是什么，这种结构如何影响功能？我们认为，活跃基因进入工厂是主要的建筑主题之一；我们希望这一分析将有助于证明或反驳这一点。

Only some of the genetic information encoded in our DNA is copied into RNA and then 'translated' into protein; copying is selective, and mis-copying results in disease (e.g., cancer). We wish to analyze the critical copying machines - known as 'RNA polymerases' - in their natural surroundings. During the last fifty years, techniques have been developed to purify these copying machines from our cells, and we now have detailed maps both of their contents and structure. We also know they are part of a larger production line that carries out additional functions; for example, the major copying machine - polymerase II - is found with other machines that work on the RNA copies as they are made. We have shown that a number of these production lines are, in turn, organized into still-larger 'factories'. [We define a factory as a site containing at least two RNA polymerases copying different pieces of DNA, and the reason for having all factories is the same (whether they make cars, jam, or RNA) - to enhance production by concentrating relevant machines and raw materials in one place.] Unfortunately, these factories are so tightly bound to the substructure in the cell that hitherto they have been impossible to isolate (so we know little about their contents); fortunately, we have just developed a method that allows us to release them from the substructure (we use the enzymes the cell uses naturally when it commits suicide!), and this has enabled us to go on to partially purify the factories free of many other cellular components. We now want to exploit this method, and a related one for assaying which genes are being copied in the factories; we want to catalogue some of their contents using various techniques (including sequencing to determine DNA and RNA content, and light microscopy to map locations). We are especially interested in comparing factories before and after they start copying particular sets of genes. To this end, we will use two natural agents that rapidly switch on copying of selected genes - tumour necrosis factor alpha (TNFa) and transforming growth factor beta (TGFb). [TNFa orchestrates our inflammatory response when our body becomes infected with microbes; it binds to the cell membrane and sends a messenger - NFkappaB - into the nucleus to switch on the copying of the genes that encode protective proteins. If the infection is mild, the system produces proteins able to fight it. But if severe, the cell may choose to commit suicide by activating those enzymes described above - so taking the infecting microbes with it to the grave. Clearly, the system has to make the right decision! TGFb sends a different messenger into the nucleus to switch on a different set of genes. As these genes control many cell functions involved in cell proliferation and differentiation, TGFb plays important roles in diseases like cancer, heart disease, and diabetes.] As both agents act so rapidly, we will add them to cells obtained from normal umbilical veins, switch on different groups of genes, purify the factories after 10-480 min, and compare their contents with those from untreated cells.We also have a more ambitious aim. Human chromosomes are arguably the largest and most important biomolecules, and many aspects of their structure - and how structure affects function - remain obscure. This has practical consequences: for example, we cannot yet predict how active a gene will be when inserted into a new location, and this obviously limits the development of successful gene therapies. As a result, major questions of our age are: what is the 3D structure of the human chromosome, and how does that structure affect function? We suggest that the clustering of active genes into factories is one of the major architectural motifs; we hope this analysis will help prove or disprove whether this is so.