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.

项目概要 减数分裂耦合一轮 DNA 复制、同源物之间的高频重组以及两 多轮染色体分离以产生单倍体减数分裂产物。减数分裂重组是必需的 减数分裂中同源物的正确分离，并产生减数分裂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其频率的“热点” 分布在整个基因组中。我们的模型系统裂殖酵母发现离散 DNA 序列基序及其结合蛋白将重组的起始位置定位在热点处。他们这样做是通过 诱导组蛋白 PTM 和核小体置换，从而促进局部重组的催化 - 通过基础重组机制（Spo11/Rec12 复合体）启动 dsDNA 断裂。一般的DNA位点- 大约 4 亿年前分化的物种之间的依赖机制是保守的，并且 关联意味着更广泛地保守。值得注意的是，短的、随机的 DNA 筛选 裂变酵母基因组中碱基对替换生成的序列，直接分析了 约 46,000 个独立克隆中的减数分裂重组——鉴定出 202 个不同的短 DNA 序列 激活重组热点的元素。这些惊人的发现表明最减数分裂重组， 与大多数转录一样，它是由离散的 DNA 位点及其结合蛋白定位和调节的。然而， 202 个热点激活 DNA 序列中只有 5 个在单核苷酸上进行了功能定义 分辨率，并且仅鉴定出其中三个 DNA 位点的结合/激活蛋白。我们的长期 目标是系统地定义减数分裂的离散 DNA 位点和结合/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“靶向正向遗传学”（TFG），它可以 在一次实验中生成超过 100,000 个独立的等位基因替换，以定义单次 核苷酸解析体内热点活性所需的离散DNA序列基序。其次，我们将使用 一种称为“DNA 亲和捕获质谱法”(DAC-MS) 的方法，与串联质量相结合 标记（TMT），三级质谱（MS3）策略，可以同时分析许多样品， 鉴定候选结合/激活蛋白。将验证候选者的 DNA 位点特异性结合 和体内热点激活。第三，我们将检验以下假设：不同的顺式作用调节模块 每个都通过涉及染色质重塑的共同下游机制促进重组。这 系统的、多方面的方法将为机制（和离散代码）提供新的见解 减数分裂位置重组，这对减数分裂非整倍体的病因学有影响（例如，唐氏 综合征），用于连锁图谱和基因组的进化动力学。