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

局部核小体间相互作用和染色质重塑对体内染色质纤维折叠的影响

• 批准号: 9325353
• 负责人: Sarah Grace Swygert
• 金额: $ 5.67万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9325353
• 关键词: Affect; Base Pairing; Binding; Biochemical; Cell Cycle; Cells; ChIP-seq; Chromatin; Chromatin Fiber; Chromatin Modeling; Chromatin Structure; DNA; Data; Development; Disease; Drug resistance; Enzymes; Eukaryotic Cell; Event; Fiber; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Higher Order Chromatin Structure; Histone H2A; Histone H4; Knowledge; Life; Location; Maintenance; Maps; Measures; Mediating; Methods; Micrococcal Nuclease; Microscopy; Modeling; Mutate; Mutation; Nucleosomes; Phase; Play; Polymerase; Positioning Attribute; Process; Protocols documentation; Publishing; RNA Polymerase Inhibitor; Research; Resolution; Retinal blind spot; Role; Saccharomyces cerevisiae; Site; Structure; Surface; System; Tail; Techniques; Testing; Time; Transcriptional Regulation; Uncertainty; Work; Yeasts; base; cancer cell; cancer stem cell; cell type; chromatin remodeling; crosslink; density; experimental study; gene repression; genome-wide; in vivo; logarithm; mutant; nanometer; novel; programs; promoter; protein structure; transcription factor; transcriptome sequencing

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.

真核生物基因组组织成染色质允许细胞调节所有DNA依赖性 流程.目前的染色质结构模型认为，它存在于四个层次，类似于蛋白质结构。 染色质的二级结构是染色质折叠成诸如30纳米结构的结构。 纤维，这是由同一DNA链上的核小体之间的局部相互作用介导的。二次 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断了转录因子结合等事件， 聚合酶延伸然而，在这个水平上研究染色质结构是最困难的。最近 对从细胞中纯化的染色质的研究未能观察到规则折叠的染色质纤维， 30纳米纤维的存在。基因组学和显微镜方法，其特征染色质在 其细胞背景，一直不能达到检查二级结构所必需的分辨率， 导致染色质结构低于酶对水平，通常被称为“盲点”。 最近开发的一种称为Micro-C的基因组学技术打破了这一技术障碍， 酿酒酵母Micro-C通过使用微球菌核酸酶修改了完善的Hi-C方案 将交联的染色质消化成核小体，连接交联的核小体之间的DNA， 然后鉴定连接的序列。Hi-C方法最多可达到1-4个碱基的分辨率，而Micro-C方法最多可达到1-4个碱基的分辨率。 提供了150个碱基对单个核小体分辨率下的核小体间相互作用的图谱。微型C 在指数增长的培养物中的实验发现了无序形式的二级结构， 同一基因中核小体之间的“褶皱”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。 一个生命阶段，在此期间，二级结构预计将发挥更重要的作用是静止 (Q)这是一个可逆的阶段，细胞进入一个长寿命的、非复制的、转录不活跃的程序。 以前发表的和初步的数据表明，全球增加染色质折叠控制， 在Q过程中的转录抑制，并牵连Isw 2染色质重塑酶介导这一点， 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， 在全基因组范围内绘制log和Q细胞的染色质结构。一旦Q细胞被建立为 功能的二级结构，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研究Isw 2如何影响二级染色质结构， 测试Q期间染色质折叠的增加指导Isw 2靶向的模型。这些实验将填补 我们对染色质结构的认识中的一个关键空白，首先确定其机制和功能， 染色质折叠的细胞内，并建立染色质结构和重塑之间的关系。