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

在这个提议中，我们提出了一些基本问题，即在染色体的正常环境中激活基因表达的机制。哺乳动物基因组包含约1米长的DNA，其组装为染色质，并被压缩约10，000倍以将其全部作为浓缩的染色质装入细胞核。基因组被包装为称为核小体的结构的重复单元，其由被147个碱基对（bp）的DNA包围的组蛋白质束组成，所述DNA围绕核小体的外部旋转1.7圈。核小体本身通过约40 bp的短接头彼此分开，并组织为重复长度为约180 - 200 bp的高度规则阵列。为了激活基因表达，基因必须经历许多不同水平的染色质修饰和去凝聚。基因激活的整个过程由DNA上的控制模块严格控制，即mRNA合成开始的近端启动子元件和远端增强子元件。这些元件首先与称为转录因子的特定蛋白质相互作用，这些转录因子与DNA结合，然后招募修饰和重塑染色质的复合物，同时与转录装置接合，该转录装置读取基因产物合成所需的遗传信息。基因激活可以涉及从调控元件置换核小体和显著修饰包含基因及其增强子的整个核小体阵列。我们的实验室正积极致力于确定转录因子和染色质修饰复合物，相互作用，并激活人类GM-CSF基因的转录。这是一种在造血过程中和免疫系统内起作用的基因，以控制称为粒细胞和巨噬细胞的特定类别的白色血细胞的生长和功能。我们已经表明，该基因受到非常严格的调控，并且在刺激细胞（如T细胞和肥大细胞）中的促炎途径时诱导约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，并且必须仔细控制其表达。在我们的研究中，我们已经确定了一个可诱导的转录增强子，位于3000 bp的上游GM-CSF基因。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进行广泛的重塑。我们已经证明，诱导型转录因子如NFAT和AP-1或发育调节因子如加塔-2的募集导致核小体从增强子中被驱逐，并导致侧翼核小体的深刻重塑。我们发现至少4000 bp的DNA（~ 20个核小体）被重组，使得（i）规则阵列被破坏和随机化，（ii）平均核小体重复长度从正常值180-190 bp减少到150-160 bp的更短值。目前还不清楚这种相对较小的增强子和启动子元件如何在如此长的距离上改变染色质纤维的结构。我们有迹象表明，这可能是通过以下两种方式的组合来实现的：（i）不是从正常启动子元件而是从增强子启动的转录，以及（ii）由增强子募集然后在基因座上扩散的染色质重塑复合物。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其他遗传因素，阻止染色质重塑从活性基因扩散到邻近基因。

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.