Systematic elucidation of DNA sequence codes that regulate meiotic recombination

项目摘要 减数分裂耦合一轮DNA复制，同源物之间的高频重组，和两轮DNA复制。 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 在减数分裂中同源物的适当分离，它产生了遗传多样性， 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”处 分布在整个基因组中。我们的模型系统，分裂酵母，导致了离散DNA的发现， 序列基序和它们的结合蛋白将重组的起始定位在热点处。他们这样做， 诱导组蛋白PTM和核小体置换，其促进重组的局部催化- 通过基础重组机制（Spo 11/Rec 12复合物）启动dsDNA断裂。一般的，DNA位点- 依赖机制在大约4亿年前分化的物种之间是保守的， 通过关联暗示甚至更广泛地保守。值得注意的是，一个短的，随机的DNA屏幕 在分裂酵母基因组中碱基对取代产生的序列-直接分析了 在大约46，000个独立的克隆中进行减数分裂重组，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大多数减数分裂重组， 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，在这方面， 在202个热点激活DNA序列中，只有5个在单核苷酸水平上被功能性地定义， 分辨率，和结合/激活蛋白只被确定为三个DNA位点。我们的长期 目的是系统地确定减数分裂的离散DNA位点和结合/激活蛋白编码， 重组首先，我们将使用一种新开发的称为“靶向正向遗传学”（TFG）的工具，该工具可以 在单个实验中产生超过100，000个独立的等位基因替换，以定义单个 核苷酸分辨率的离散DNA序列基序所需的热点活动在体内。第二，我们将使用 一种称为“DNA亲和捕获质谱法”（DAC-MS）的方法， 标记（TMT），三级质谱（MS 3）策略，可以同时分析许多样品， 以鉴定候选结合/激活蛋白。将验证候选物的DNA位点特异性结合 和体内热点激活。第三，我们将检验不同的顺式作用调节模块 每一种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、多方面的方法将提供对机制（和离散代码）的新见解， 定位减数分裂重组，这对减数分裂非整倍性的病因学有影响（例如，唐氏 综合征），连锁图谱，和基因组的进化动力学。

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.