Systematic elucidation of DNA sequence codes that regulate meiotic recombination

系统阐明调节减数分裂重组的 DNA 序列代码

• 批准号: 10418872
• 负责人: Wayne P Wahls
• 金额: $ 42.32万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-10 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10418872
• 关键词: Affinity; Alleles; Aneuploidy; Animal Model; Base Pairing; Binding; Binding Proteins; Biological; Biological Assay; Biological Models; Catalysis; Chromosome Mapping; Chromosome Segregation; Code; Complex; Congenital Abnormality; Coupled; Couples; DNA; DNA Sequence; DNA biosynthesis; DNA sequencing; Defect; Down Syndrome; Elements; Etiology; Fission Yeast; Frequencies; Genetic Recombination; Genetic Transcription; Genetic Variation; Genome; Goals; Haploidy; Histones; Insecta; Length; Mass Spectrum Analysis; Measures; Meiosis; Meiotic Recombination; Molecular; Natural Selections; Nucleosomes; Nucleotides; Organism; Parents; Pathway interactions; Population; Positioning Attribute; Process; Proteins; Randomized; Regulation; Regulatory Element; Research; Resolution; SPO11 gene; Sampling; Sequence-Specific DNA Binding Protein; Site; Specificity; Spontaneous abortion; Technology; Testing; Vertebrates; Work; Yeast Model System; chromatin remodeling; diagnostic value; ds-DNA; experimental study; forward genetics; fungus; improved; in vivo; innovation; insight; next generation; precise genome editing; segregation; tool; yeast genome

Project Summary Meiosis couples one round of DNA replication, high-frequency recombination between homologs, and two rounds of chromosome segregation to produce haploid meiotic products. Meiotic recombination is required for the proper segregation of homologs in meiosis, and it generates genetic diversity required for the process of natural selection. Interestingly, meiotic recombination is clustered at “hotspots” that regulate its frequency distribution throughout the genome. Our model system, fission yeast, led to the discovery that discrete DNA sequence motifs and their binding proteins position the initiation of recombination at hotspots. They do so by inducing histone PTMs and nucleosome displacement, which promote locally the catalysis of recombination- initiating dsDNA breaks by the basal recombination machinery (Spo11/Rec12 complex). The general, DNA site- dependent mechanisms are conserved between species that diverged about 400 million years ago and are implicated by association to be even more broadly conserved. Remarkably, a screen of short, randomized DNA sequences generated by base-pair substitutions in the fission yeast genome—which directly analyzed rates of meiotic recombination in about 46,000 independent clones—identified 202 distinct, short DNA sequence elements that activate recombination hotspots. These striking findings suggest the most meiotic recombination, like most transcription, is positioned and regulated by discrete DNA sites and their binding proteins. However, only five of the 202 hotspot-activating DNA sequences have been defined functionally at single-nucleotide resolution, and the binding/activator proteins have only been identified for three of the DNA sites. Our long-term goal is to define systematically the discrete DNA sites and binding/activator protein codes of meiotic recombination. First, we will use a newly developed tool called “targeted forward genetics” (TFG), which can generate more than 100,000 independent allele replacements in a single experiment, to define at single- nucleotide resolution the discrete DNA sequence motifs required for hotspot activity in vivo. Second, we will use an approach called “DNA affinity capture with mass spectrometry” (DAC-MS), coupled with a tandem mass tagging (TMT), triple-stage mass spectrometry (MS3) strategy that can analyze many samples simultaneously, to identify the candidate binding/activator proteins. Candidates will be validated for DNA site-specific binding and hotspot activation in vivo. Third, we shall test the hypothesis that the different cis-acting regulatory modules each promote recombination via a common downstream mechanism that involves chromatin remodeling. This systematic, multifaceted approach will provide new insight into the mechanisms (and discrete codes) that position meiotic recombination, which has implications for the etiology of meiotic aneuploidies (e.g., Down's syndrome), for linkage mapping, and for the evolutionary dynamics of genomes.

项目摘要 减数分裂夫妇一轮DNA复制，同源物之间的高频重组 染色体隔离的一轮，产生单倍体减数分裂产物。需要减数分裂重组 减数分裂中同源物的适当分离，它产生了该过程所需的遗传多样性 自然选择。有趣的是，减数分裂重组聚集在调节频率的“热点”上 整个基因组的分布。我们的模型系统裂变酵母导致发现离散DNA的发现 序列基序及其结合蛋白将重组在热点处的启动。他们这样做 诱导的组蛋白PTM和核小体位移，从而促进重组的局部催化 启动DSDNA通过基本重组机制（SPO11/REC12复合物）破裂。一般的DNA位点 - 依赖机制是在大约4亿年前差异的物种之间保存的，并且 由协会实施，以更加广泛地保存。值得注意的是，简短的随机DNA屏幕 裂变酵母基因组中碱基对产生的序列，该序列直接分析了 约46,000个独立克隆中的减数分裂重组 - 已确定的202个不同的短DNA序列 激活重组热点的元素。这些引人注目的发现暗示了最重要的重组， 像大多数转录一样，由离散的DNA位点及其结合蛋白定位和调节。然而， 在202个热点激活的DNA序列中，只有五个在单核苷酸上定义了 分辨率和结合/激活蛋白仅在三个DNA位点被鉴定出来。我们的长期 目标是系统地定义离散的DNA位点和减数分裂的结合/激活蛋白代码 重组。首先，我们将使用一种新开发的工具，称为“靶向前遗传学”（TFG），可以 在单个实验中产生超过100,000个独立的等位基因替代品，以定义单个实验 核苷酸分辨率在体内热点活性所需的离散DNA序列基序。第二，我们将使用 一种称为“与质谱捕获DNA亲和力捕获”（DAC-MS）的方法，与串联质量结合 标记（TMT），三个阶段质谱法（MS3）策略，可以简单地分析许多样本， 确定候选结合/激活蛋白。候选者将通过DNA特定结合进行验证 在体内激活热点。第三，我们将检验以下假设：不同的顺式作用调节模块 每种都通过涉及染色质重塑的常见下游机制促进重组。这 系统的，多方面的方法将提供对机制（和离散代码）的新见解 位置减数分裂重组，对减数分裂性非整倍性的病因有影响（例如，Down's 综合征），用于链接映射和用于基因组的进化动力学。