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.

真核基因组组织成染色质的组织使细胞可以调节所有DNA依赖性 过程。当前的染色质结构模型认为它以四个级别存在，类似于蛋白质结构。 染色质的二级结构是将染色质折叠为30纳米的结构 纤维是由同一DNA链上核小体之间的局部相互作用介导的。次要 结构被认为是转录表达的强大机制之一，在此期间 相邻核小体之间的相互作用阻止事件，例如转录因子结合和 聚合酶伸长。但是，在此水平上研究染色质结构是最困难的。最近的 从细胞中纯化的染色质的研究未能观察到定期折叠的染色质纤维，引起了疑问 关于30纳米纤维的存在。基因组学和显微镜方法，该方法表征了染色质 它的细胞环境无法达到检查二级结构所需的解决方案， 在千个酶对以下的领先染色质结构经常被称为“盲点”。 最新的基因组技术的发展称为Micro-C，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良好的HI-C方案 为了消化交联的染色质至核小体，将DNA连接到交联的核小体之间，然后 然后识别连接的序列。而HI-C方法最多可以达到1-4千碱基的分辨率，Micro-C 在150个基对单核小体分辨率下提供核间相互作用的地图。 Micro-C 成倍增长的培养物的实验发现了以无序形式的二级结构 同一基因中的核组组之间的“碎屑”相互作用，但几乎没有证据表明折叠 染色质纤维。但是，预测染色质折叠不是积极生长酵母的普遍特征。 预计二级结构将发挥更重要作用的生命阶段是静止 （q），一个可逆阶段，其中细胞进入了长期寿命，非复制和转录无效程序。 先前发表的和初步数据表明，染色质折叠控制的全球增加 Q期间的转录反射，并暗示ISW2染色质重塑酶在介导 压制结构。在本提案中描述的工作中，我将使用Micro-C进行测试这些假设 绘制对数和Q细胞全基因组中的染色质结构。一旦建立Q单元作为模型 功能二级结构，我将能够发现染色质折叠的机制并确定其 在转录表示中的作用。我还将研究ISW2如何影响二级染色质结构，并且 测试模型，即在Q指导ISW2靶向过程中增加染色质折叠的模型。这些实验将填充 我们对染色质结构的了解的关键差距，是第一个确定机制和功能的差距 细胞内的染色质折叠，并在染色质结构和重塑之间建立关系。