The effect of local inter-nucleosomal interactions and chromatin remodeling on in vivo chromatin fiber folding

将真核细胞基因组组织成染色质使细胞能够调节所有依赖于DNA的 流程。目前的染色质结构模型认为，染色质存在于四个层次，与蛋白质结构相似。 染色质的二级结构是将染色质折叠成类似30纳米的结构 纤维，这是由同一DNA链上的核小体之间的局部相互作用所介导的。次要的 结构被认为是转录抑制的最强机制之一，在此期间 相邻核小体之间的相互作用阻止了转录因子结合和 聚合酶延长。然而，在这个水平上研究染色质结构一直是最困难的。近期 从细胞中提纯的染色质的研究未能观察到规则折叠的染色质纤维，这让人产生怀疑 关于30纳米纤维的存在。基因组学和显微技术，其特征是染色质 其细胞背景，一直无法达成研究二级结构所需决议， 导致染色质结构低于千碱基对水平常被称为“盲点”。 最近开发的一种名为Micro-C的基因组学技术打破了这一技术障碍 酿酒酵母。Micro-C使用微球菌核酸酶修改成熟的Hi-C方案 将交联染色质消化到核小体，将DNA连接在交联核小体之间，以及 然后鉴定连接的序列。尽管Hi-C方法最多只能达到1-4千基的分辨率，但Micro-C 提供了150碱基对单核小体分辨率下核小体间相互作用的图谱。Micro-C 在指数增长的培养实验中发现了无序的二级结构 同一基因中核小体之间的“皱缩”相互作用，但几乎没有发现折叠的证据 染色质纤维。然而，染色质折叠并不是活跃生长的酵母的普遍特征。 生命的一个阶段是静止期，在这个阶段中，次级结构有望发挥更重要的作用 (Q)，细胞进入长期的、非复制的和转录不活跃的程序的可逆阶段。 之前发表的初步数据表明，染色质折叠控制的全球增加 Q过程中的转录抑制，并暗示Isw2染色质重塑酶参与了这一过程 压抑的结构。在本提案中描述的工作中，我将使用Micro-C来测试这些假设 绘制全基因组LOG和Q细胞染色质结构图。一旦Q细胞被建立为 功能二级结构，我将能够揭示染色质折叠的机制并确定其 在转录抑制中的作用。我还将调查Isw2如何影响二级染色质结构，以及 检验Q期间染色质折叠增加指导Isw2靶向的模型。这些实验将填补 我们对染色质结构认识上的一个关键缺口，是第一个确定染色质结构和功能的机制 染色质在细胞内折叠，并建立染色质结构和重塑之间的关系。

The organization of the eukaryotic genome into chromatin allows the cell to regulate all DNA-dependent processes. Current models of chromatin structure hold that it exists in four levels, similar to protein structure. The secondary structure of chromatin is the folding of chromatin into structures such as the 30 nanometer fiber, which is mediated by local interactions between nucleosomes on the same DNA strand. Secondary structure is considered to be one of the strongest mechanisms of transcriptional repression, during which interactions between neighboring nucleosomes block events such as transcription factor binding and polymerase elongation. However, studying chromatin structure at this level has been the most difficult. Recent studies of chromatin purified from cells have failed to observe regularly folded chromatin fibers, casting doubt on the existence of 30 nanometer fibers. Genomics and microscopy methods, which characterize chromatin in its cellular context, have been unable to reach resolutions necessary for examining secondary structure, leading chromatin structure below the kilobase pair level to be frequently referred to as “a blind spot.” The recent development of a genomics technique called Micro-C has broken this technical barrier in Saccharomyces cerevisiae. Micro-C modifies the well-established Hi-C protocol by using Micrococcal nuclease to digest crosslinked chromatin down to nucleosomes, ligating DNA between crosslinked nucleosomes, and then identifying ligated sequences. Whereas Hi-C methods reach resolutions of 1-4 kilobases at best, Micro-C provides maps of inter-nucleosomal interactions at 150 base pair single-nucleosome resolution. Micro-C experiments in exponentially growing cultures discovered secondary structure in the form of disordered “crumpling” interactions between nucleosomes in the same gene, but found little evidence for a folded chromatin fiber. However, chromatin folding is not predicted to be a prevalent feature of actively growing yeast. A life stage during which secondary structure is expected to play a more significant role is quiescence (Q), a reversible phase in which cells enter a long-lived, non-replicative, and transcriptionally inactive program. Previously published and preliminary data suggest that a global increase in chromatin folding controls transcriptional repression during Q, and implicate the Isw2 chromatin remodeling enzyme in mediating this repressive structure. In the work described in this proposal, I will test these hypotheses by using Micro-C to map chromatin structure in log and Q cells genome-wide. Once Q cells are established as a model of functional secondary structure, I will be able to uncover the mechanisms of chromatin folding and determine its role in transcriptional repression. I will also investigate how Isw2 affects secondary chromatin structure, and test the model that an increase in chromatin folding during Q directs Isw2 targeting. These experiments will fill a critical gap in our knowledge of chromatin structure, be the first to determine the mechanisms and functions of chromatin folding within cells, and establish relationships between chromatin structure and remodeling.