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（R01 GM117446） 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、复制较晚、转录沉默的异染色质。 最近的研究发现在重复的情况下存在广泛的转录去抑制 致癌和衰老过程中的区域。去压抑加速了这些的复制 区域，从而损害基因丰富的转录活性区域的复制 染色质。尽管低水平之间的关联非常重要且普遍 转录和晚期复制的机制，但这种联系的机制基础仍不清楚。 核糖体 DNA (rDNA) 以及铜诱导的 CUP1 阵列 芽殖酵母是阐明这些机制的理想实验系统 有两个主要原因。首先，在每个位点，进行一次操作，Sir2 耗尽 rDNA 和 CUP1 上的库珀管理可激活转录和 复制，提供一个简单的工具来操纵这两个过程。二、每个rDNA CUP1 重复序列具有单一的、序列定义的复制起点，从而创建 复制起始因子和核小体的统一且可预测的定位 他们的附近。这一特性使得这些系统以及一般的酵母起源非常适合 深度测序方法在表观基因组分析中的应用 与哺乳动物起源相比，它具有巨大的优势，因为哺乳动物起源缺乏 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析核小体和前体的精确位置。 复制复合体（pre-RC）在重复区域和独特区域 基因组。使用这些方法，我们发现转录激活 rDNA 和 CUP1 起源导致邻近前核小体的占据减少 RC，尽管在两个位点通过不同的方式：在 CUP1，转录减少 核小体占据靠近预 RC 加载位点，而在 rDNA 处 预 RC 加载位点附近的核小体占据情况不会改变；相反，RNA Pol-II 将 pre-RC 推至核小体密度较低的相邻区域。使用这个 我们开发的实验系统和染色质分析工具，我们将 确定与 pre-RC 相邻的高核小体占用是否会抑制复制 核小体的启动以及是否染色质压缩介导的抑制 重塑者强制转录沉默染色质的晚期复制。