The role of enhancers and transcription factors in the reorganisation of chromatin architecture

在这个建议中，我们提出了关于在染色体正常情况下激活基因表达的机制的基本问题。哺乳动物基因组包含约1米长的DNA，这些DNA被组装成染色质，并被压缩约10,000倍，以使其全部作为浓缩染色质进入细胞核。基因组被包装成称为核小体的重复结构单元，核小体由组蛋白束组成，周围是147个碱基对（bp）的DNA，在核小体的外部转1.7圈。核小体本身由一个约40 bp的短连接体彼此分开，并组织成一个高度规则的阵列，重复长度约为180 - 200 bp。为了激活基因表达，基因必须经历许多不同程度的染色质修饰和去致密化。基因激活的整个过程是由DNA上的控制模块严格控制的，即mRNA合成开始的近端启动子元件和远端增强子元件。这些元件的功能首先是与特定的蛋白质相互作用，这些蛋白质被称为转录因子，与DNA结合，然后招募修饰和重塑染色质的复合物，同时与转录装置结合，读取基因产物合成所需的遗传信息。基因激活可以涉及核小体从调控元件的位移和整个核小体阵列包含基因及其增强子的显著修饰。我们的实验室正在积极研究与人类GM-CSF基因相互作用并激活其转录的转录因子和染色质修饰复合物。这是一个在造血过程和免疫系统中起作用的基因，控制特定种类的白细胞的生长和功能，称为粒细胞和巨噬细胞。我们已经证明，这种基因受到非常严格的调控，并且在T细胞和肥大细胞等细胞的促炎途径刺激下被诱导~10,000倍，这些细胞也代表免疫系统的一部分。在这种情况下，GM-CSF的功能是在免疫系统的不同部分之间接收和发送信号，因此仔细控制其表达是至关重要的。在我们的研究中，我们已经确定了一个位于GM-CSF基因上游3000 bp的可诱导转录增强子。我们已经表明，这种增强子响应T细胞抗原受体的激活，并在染色质结构水平上经历广泛的重塑。我们已经证明，诱导转录因子如NFAT和AP-1的募集，或发育调节因子如GATA-2的募集，导致核小体从增强子中被驱逐，并对侧翼核小体进行深刻的重塑。我们发现至少4000 bp的DNA（约20个核小体）被重组，这样(i)常规阵列被打乱和随机化，（ii）核小体的平均重复长度从正常值180-190 bp减少到150-160 bp的短得多的值。目前尚不清楚这些相对较小的增强子和启动子是如何在如此远的距离上改变染色质纤维的结构的。我们有迹象表明，这可能是由以下两种组合完成的：(i)不是由正常启动子元件而是由增强子启动的转录，以及（ii）由增强子招募的染色质重塑复合物，然后扩散到整个位点。在这个提议中，我们希望测试这些机制是否确实在GM-CSF基因中起作用，我们希望找出重塑过程的精确细节。我们还旨在确定是否存在阻止染色质重塑从活性基因向邻近基因扩散的其他遗传元件。

In this proposal we are asking fundamental questions about the mechanisms employed to activate gene expression within the normal context of chromosomes. The mammalian genome encompasses about 1 metre of DNA which is assembled as chromatin and is compacted ~10,000 fold to fit it all into the cell nucleus as condensed chromatin. The genome is packaged as repeating units of structures termed nucleosomes which are made up of bundles of histone proteins surrounded by 147 base pairs (bp) of DNA which makes 1.7 turns around the outside of the nucleosome. Nucleosomes are themselves separated from each other by a short linker of about 40 bp, and are organised as a highly regular array with a repeat length of ~180 - 200 bp. In order to activate gene expression it is necessary for genes to undergo many distinct levels of chromatin modification and decondensation. The whole process of gene activation is tightly controlled by control modules on the DNA, namely proximal promoter elements where mRNA synthesis starts and distal enhancer elements. These elements function by first interacting with specific proteins named transcription factors that bind to DNA, which then recruit complexes that modify and remodel chromatin, and at the same time engage with the transcription apparatus that reads the genetic information required for the synthesis of gene products. Gene activation can involve the displacement of nucleosomes from regulatory elements and significant modification of the whole nucleosome array encompassing genes and their enhancers. Our laboratory is actively engaged in defining the transcription factors and chromatin modifying complexes that interact with and activate transcription of the human GM-CSF gene. This is a gene that functions during haemopoiesis and within the immune system to control the growth and function of specific classes of white blood cells termed granulocytes and macrophages. We have shown that this gene is very tightly regulated and is induced ~10,000 fold upon stimulation of pro-inflammatory pathways in cells such as T cells and mast cells, which also represent part of the immune system. In this context, GM-CSF functions to receive and send signals between different parts of the immune system, and it is essential that its expression is carefully controlled. In our studies we have identified an inducible transcriptional enhancer that is located 3000 bp upstream of the GM-CSF gene. We have shown that this enhancer responds to activation of the T cell antigen receptor, and undergoes extensive remodelling at the level of chromatin structure. We have shown that the recruitment of inducible transcription factors such as NFAT and AP-1, or developmentally regulated factors such as GATA-2, leads to the eviction of nucleosomes from the enhancer and profound remodelling of the flanking nucleosomes. We find that at least 4000 bp of DNA (~ 20 nucleosomes) is restructured such that (i) the regular array is disrupted and randomised, and (ii) the average nucleosome repeat length is reduced from the normal value of 180-190 bp to a much shorter value of 150-160 bp. It is not known how such relatively small enhancer and promoter elements can alter the structure of the chromatin fibre over such long distances. We have indications that this may be done by a combination of (i) transcription initiated not from the normal promoter element but from the enhancer, and (ii) chromatin remodelling complexes that are recruited by the enhancer and then spread across the locus. In this proposal we wish to test whether these mechanisms do indeed operate within the GM-CSF gene, and we want to work out the precise details of the remodelling process. We also aim to determine whether additional genetic elements exist that block the spread of chromatin remodelling from active genes into neighbouring genes.