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

基本信息

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
项目类别：
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

项目摘要

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个产生单倍体减数分裂产物需要多轮染色体分离。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意地使减数分裂重组聚集在调节其频率的“热点”。我们的模型系统——裂变酵母，导致了离散DNA的发现。序列基序及其结合蛋白将重组的起始位置定位在热点处。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。大约 4 亿年前分化的物种之间的依赖机制是保守的，并且值得注意的是，短的、随机的 DNA 筛选更广泛地涉及关联。由裂变酵母基因组中的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。 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，并且我们的长期 DNA 位点仅鉴定出结合/激活蛋白。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，以定义单次其次，我们将使用体内热点活性所需的离散 DNA 序列基序的核苷酸分辨率。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蛋白的 DNA 位点特异性结合。第三，我们将检验不同顺式作用调节模块的假设。每个都通过涉及染色质重塑的共同下游机制促进重组。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。