Dynamic Chromatin Conformation - a Direct Imaging Approach to Structure-Function Relationships

MCB9728932摘要现在有压倒性的证据表明，组成真核基因组的线性核小体阵列的折叠和紧凑在转录调控中起着至关重要的作用。例如，从核小体在启动子和增强子控制序列上的精确定位到大规模压缩从而转录关闭基因块。为了确定染色质折叠的规律和机制，了解核小体的动态三维构象阵列是很重要的。发展了一种基于冷冻电子显微镜的技术，可以观察到单个寡核小体的3D水合构象。初步结果表明，核小体以3D之字形结构排列，根据连接物DNA的长度可以进行进一步的压缩。进一步的研究将确定(1)DNA连接物序列及其长度是否影响折叠模式，(2)盐浓度如何影响折叠，(3)组蛋白H1的作用是什么，它相对于核小体是如何定位的，(4)称为异染色质缩合的新的染色质结合蛋白是如何参与的，以及(5)转录RNA聚合酶如何与含有带有转录元件和停滞序列的DNA片段的核小体相互作用。这些研究意义重大，因为人们对核小体堆积和高阶染色质结构知之甚少，尽管这些特征被认为在染色质功能的调节中非常重要。

MCB9728932 Abstract There is now overwhelming evidence that the folding and compaction of the linear array of nucleosomes that comprises the eukaryotic genome plays a crucial role in transcriptional regulation. Examples range from the precise positioning of nucleosomes on promoter and enhancer control sequences to the large-scale compaction and hence transcriptional shutdown of blocks of genes. In order to define the rules and mechanisms of chromatin folding, it is important to understand the dynamic three dimensional (3D) conformation arrays of nucleosomes. A technique based on cryo-electron microscopy has been developed that allows the 3D hydrated conformation of individual oligonucleosomes to be observed. Preliminary results have demonstrated that nucleosomes array in a 3D zigzag structure that can undergo further compaction depending on the length of linker DNA. Further studies will determine (1) whether the DNA linker sequence as well as its length effects the folding pattern, (2) how salt concentration influences folding, (3) what the effect of histone H1 is and how it is positioned relative to the nucleosomes, (4) how a novel chromatin binding protein termed MENT is involved in heterochromatin condensation, and (5) how transcribing RNA polymerase interacts with nucleosomes that contain DNA segments with transcriptional elements and arrest sequences. The studies are highly significant since very little is known about nucleosome packing and higher orders of chromatin structure even though these features are believed to be extremely important in regulation of chromatin function.