Origin firing at repetitive sequences and genome replication - Admin Supplement

重复序列和基因组复制的起源 - 管理补充

• 批准号: 10626663
• 负责人: Antonio Bedalov
• 金额: $ 0.8万
• 依托单位: FRED HUTCHINSON CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10626663
• 关键词: Affect; Aging; Biological Phenomena; Cell Aging; Chromatin; Chromosome Structures; Copper; DNA Polymerase II; DNA Sequence; Genes; Genetic Transcription; Genome; Heterochromatin; Human Genome; Link; Location; Mediating; Methods; Modeling; Modernization; Mutation; Nucleosomes; Nucleotides; Peptide Initiation Factors; Positioning Attribute; Pre-Replication Complex; Prevalence; Process; RNA; Repetitive Sequence; Replication Initiation; Replication Origin; Repression; Resources; Rest; Ribosomal DNA; Saccharomycetales; Site; Specificity; System; Testing; Transcriptional Activation; Yeasts; base; carcinogenesis; deep sequencing; density; environmental change; epigenome; parent grant; parent project; tool

Abstract of the Parent Grant (R01 GM117446) Over half of the human genome is comprised of repetitive DNA sequences organized as gene-poor, late replicating, transcriptionally silent heterochromatin. Recent studies have discerned widespread transcriptional de-repression at repeated regions during carcinogenesis and aging. De-repression accelerates replication of these regions and thus compromises replication in the gene-rich transcriptionally active chromatin. Despite the importance and prevalence of the association between low levels of transcription and late replication, the mechanistic basis for this link remains unclear. The ribosomal DNA (rDNA) as well as copper-inducible CUP1 arrays in budding yeast are ideal experimental systems in which to elucidate these mechanisms for two main reasons. First, at each locus, a single manipulation, Sir2 depletion at the rDNA, and cooper administration at the CUP1, activates both transcription and replication, providing a simple tool to manipulate both processes. Second, each rDNA and CUP1 repeat features a single, sequence-defined origin of replication, which creates uniform and predictable positioning of replication initiation factors and nucleosomes in their vicinity. This feature makes these systems, and yeast origins in general, ideal for the application of deep sequencing methods for epigenome profiling we have developed, offering a tremendous advantage over mammalian origins whose lack of sequence-specificity confounds modern deep sequencing-based approaches. Our methods resolves at nucleotide level the precise location of nucleosomes and pre- replicative complexes (pre-RC) at the repetitive as well as in unique regions of the genome. Using these methods, we discovered that transcriptional activation at both rDNA and CUP1 origins leads to reduced occupancy of nucleosomes adjacent to pre- RC, though by different means at the two loci: At CUP1, transcription reduces nucleosome occupancy next to the pre-RC loading site, whereas at the rDNA the nucleosome occupancy next to the pre-RC loading site does not change; instead, RNA Pol-II pushes pre-RC to an adjacent region with low nucleosome density. Using this experimental system and the tools for chromatin profiling we have developed, we will determine whether high nucleosome occupancy adjacent to pre-RC inhibits replication initiation and whether chromatin compaction-mediated inhibition of nucleosome remodelers enforce late replication in transcriptionally silent chromatin.

家长补助金摘要（R 01 GM 117446） 超过一半的人类基因组由重复的DNA序列组成 组织为基因贫乏、复制延迟、转录沉默的异染色质。 最近的研究已经发现，在重复的转录去抑制， 在癌症发生和衰老过程中。去阻遏加速了这些 区域，从而损害在基因丰富的转录活性的复制， 染色质尽管低水平之间的关联的重要性和普遍性， 转录和后期复制的机制，这种联系的机制基础仍然不清楚。 核糖体DNA（rDNA）以及铜诱导的CUP 1阵列在 芽殖酵母是阐明这些机制的理想实验系统 这主要有两个原因。首先，在每个基因座处，单一操作，Sir 2缺失， rDNA和库珀在CUP 1的管理，激活转录和 复制，提供了一个简单的工具来操纵这两个过程。第二，每个rDNA CUP 1重复序列具有单一的、序列定义的复制起点， 复制起始因子和核小体的均匀和可预测的定位， 他们的附近。这一特点使得这些系统，以及一般的酵母来源， 深度测序方法在表观基因组分析中的应用 发达，提供了巨大的优势，超过哺乳动物的起源，缺乏 序列特异性混淆了现代基于深度测序的方法。 我们的方法在核苷酸水平上解决了核小体的精确位置， 复制复合物（前RC）的重复以及在独特的区域， 基因组使用这些方法，我们发现，转录激活在两个 rDNA和CUP 1起源导致邻近前- RC，尽管在两个基因座上的方式不同：在CUP 1，转录减少 核小体占据紧邻预RC加载位点，而在rDNA处， 前RC装载位点旁边的核小体占据率没有改变;相反，RNA Pol-II将前RC推向具有低核小体密度的相邻区域。使用此 实验系统和工具的染色质分析，我们已经开发，我们将 确定与前RC相邻的高核小体占据是否抑制复制 启动以及染色质致密化是否介导核小体的抑制 重塑者在转录沉默的染色质中加强晚期复制。