Systematic elucidation of DNA sequence codes that regulate meiotic recombination

项目摘要 减数分裂结合了一轮DNA复制，同源基因之间的高频重组，以及两轮 几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 在减数分裂中同系物的适当分离，并产生过程中所需的遗传多样性 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启动定位于热点。他们这样做是通过 诱导组蛋白PTM和核小体移位，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。将军，DNA站点- 相互依赖的机制在大约4亿年前分化的物种之间是保守的， 被关联所牵连的更广泛的保守性。值得注意的是，一组短小的随机DNA 由裂解酵母基因组中的碱基对替换产生的序列-它直接分析了 约46,000个独立克隆的减数分裂重组-鉴定了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了最减数分裂的重组， 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个激活热点的DNA序列中，只有5个在单核苷酸上进行了功能定义 分辨率，并且只在其中三个DNA位点鉴定了结合/激活蛋白。我们的长期合作 目的是系统地确定减数分裂的离散DNA位点和结合/激活蛋白密码 重组。首先，我们将使用一种新开发的名为“定向正向遗传学”(TFG)的工具，它可以 在一次实验中产生了超过10万个独立的等位基因替换，以定义在单个 核苷酸解析体内热点活动所需的离散DNA序列基序。第二，我们将使用 一种被称为“DNA亲和捕获-质谱仪”(DAC-MS)的方法，与串联质谱仪相结合 标记(TMT)、可同时分析多个样品的三级质谱学(MS3)策略、 鉴定候选结合/激活蛋白。候选人将接受DNA位点特异性结合的验证 活体内的热点激活。第三，我们将检验不同的顺式作用调节模块的假设 每一个都通过共同的下游机制促进重组，该下游机制涉及染色质重塑。这 系统的、多方面的方法将提供对以下机制(和离散代码)的新见解 位置减数分裂重组，这对减数分裂非整倍体(如Down‘s)的病因学有意义 综合症)，用于连锁作图，以及基因组进化动力学。

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.