3D organization of synthetic and scrambled chromosomes.
3D organization of synthetic and scrambled chromosomes.
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DOI：
10.1126/science.aaf4597
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发表时间：
2017-03-10
期刊：
影响因子：
--
通讯作者：
Koszul R
中科院分区：
文献类型：
--
作者：
Mercy G;Mozziconacci J;Scolari VF;Yang K;Zhao G;Thierry A;Luo Y;Mitchell LA;Shen M;Shen Y;Walker R;Zhang W;Wu Y;Xie ZX;Luo Z;Cai Y;Dai J;Yang H;Yuan YJ;Boeke JD;Bader JS;Muller H;Koszul R
发芽酵母染色体的整体组织由四个因素驱动和调节：(I)着丝粒在纺锤体极体上的捆绑和聚集；(Ii)在核膜上端粒的松散捆绑，在那里它们形成小的动态簇；(Iii)单个核仁，其中核糖体DNA(RDNA)簇与其他染色体隔离；以及(Iv)染色体的臂长。HI-C是染色体构象捕捉方法的一种基因组衍生品，它量化了存在于细胞群体细胞核中的所有DNA片段的接近程度，揭示了染色体在核空间的平均多尺度组织。我们利用Hi-C研究了人工合成染色体在酿酒酵母核内的轨迹，并将它们与天然染色体进行了比较。Sc2.0基因组设计规定了相对于天然染色体序列的基因内容和排列的高度保守性。然而，人工合成的染色体包含了数千种设计变化，特别是移除了转移RNA基因和重复序列，如转座子和亚端粒重复，以增强稳定性。它们还携带loxJunm位点，允许诱导基因组扰乱(通过loxP介导的进化来合成染色体重排和修改)，旨在加速基因组的可塑性。这些变化是否会影响染色体组织、DNA新陈代谢和适合性是完成Sc2.0项目的关键问题。为了解决这些问题，我们使用Hi-C来表征合成染色体的组织。将人工合成的染色体与天然染色体进行比较，没有发现实质性的变化，表明重新设计的序列，特别是删除重复序列，对平均染色体轨迹几乎没有影响。SC2.0合成染色体具有Hi-C接触图谱，其接触模式比天然染色体要平滑得多，特别是在亚端粒区域。这种改善的“可映射性”直接归因于沿着合成染色体长度的重复元件的移除。这些观察结果突显了自下而上的染色体合成带来的概念上的进步，它允许对实验系统进行改进，使复杂的问题更容易解决。尽管总体上有相似之处，但在两个实例中观察到了差异。首先，第三号染色体上HML和HMR沉默交配型盒式磁带的缺失导致了它们的特异性相互作用的丧失。其次，将大量的rDNA重复序列重新定位到离着丝粒簇更近的地方，迫使整个基因组发生实质性的构象变化--例如，将该阵列插入第三号染色体的小右臂中间，将该臂分裂为两个不相互作用的区域。然后，核仁结构被困在小染色体和大染色体臂之间的中间，在它们之间施加了物理屏障。除了描述Sc2.0的染色体组织外，我们还使用Hi-C来鉴定由打乱实验引起的染色体重排。引入Sc2.0染色体的数百个loxPeym位点之间的可诱导重组使基因组结构的组合重组成为可能。携带synIII和synIXR染色体的两个扰乱株的HI-C接触图显示了各种顺式事件，包括简单的缺失、倒置和复制，以及易位，后者代表了一类以前没有观察到的反式扰乱重排。这一庞大的数据集是一种资源，将在未来探索加扰系统的能力的研究中加以利用。通过研究Sc2.0染色体在核空间的轨迹，这项工作为未来研究全基因组工程方法对生命系统基本特征的影响铺平了道路。人工合成的染色体组织。(A)SynII和本地(野生型，WT)染色体II的Hi-C接触图。红色箭头指向仅存在于本地染色体图中的经过过滤的条带(白色载体)。Kb，千个基数。(B)在SynXII或天然染色体III上携带rDNA的菌株的Hi-C图谱的三维(3D)表示。(C)在(左)和(右)之后的synIXR(黄色)和SynIXR(粉色)的接触图和3D表示。转位断点由绿色和蓝色箭头表示。
The overall organization of budding yeast chromosomes is driven and regulated by four factors: (i) the tethering and clustering of centromeres at the spindle pole body; (ii) the loose tethering of telomeres at the nuclear envelope, where they form small, dynamic clusters; (iii) a single nucleolus in which the ribosomal DNA (rDNA) cluster is sequestered from other chromosomes; and (iv) chromosomal arm lengths. Hi-C, a genomic derivative of the chromosome conformation capture approach, quantifies the proximity of all DNA segments present in the nuclei of a cell population, unveiling the average multiscale organization of chromosomes in the nuclear space. We exploited Hi-C to investigate the trajectories of synthetic chromosomes within the Saccharomyces cerevisiae nucleus and compare them with their native counterparts. The Sc2.0 genome design specifies strong conservation of gene content and arrangement with respect to the native chromosomal sequence. However, synthetic chromosomes incorporate thousands of designer changes, notably the removal of transfer RNA genes and repeated sequences such as transposons and subtelomeric repeats to enhance stability. They also carry loxPsym sites, allowing for inducible genome SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution) aimed at accelerating genomic plasticity. Whether these changes affect chromosome organization, DNA metabolism, and fitness is a critical question for completion of the Sc2.0 project. To address these questions, we used Hi-C to characterize the organization of synthetic chromosomes. Comparison of synthetic chromosomes with native counterparts revealed no substantial changes, showing that the redesigned sequences, and especially the removal of repeated sequences, had little or no effect on average chromosome trajectories. Sc2.0 synthetic chromosomes have Hi-C contact maps with much smoother contact patterns than those of native chromosomes, especially in subtelomeric regions. This improved “mappability” results directly from the removal of repeated elements all along the length of the synthetic chromosomes. These observations highlight a conceptual advance enabled by bottom-up chromosome synthesis, which allows refinement of experimental systems to make complex questions easier to address. Despite the overall similarity, differences were observed in two instances. First, deletion of the HML and HMR silent mating-type cassettes on chromosome III led to a loss of their specific interaction. Second, repositioning the large array of rDNA repeats nearer to the centromere cluster forced substantial genome-wide conformational changes—for instance, inserting the array in the middle of the small right arm of chromosome III split the arm into two noninteracting regions. The nucleolus structure was then trapped in the middle between small and large chromosome arms, imposing a physical barrier between them. In addition to describing the Sc2.0 chromosome organization, we also used Hi-C to identify chromosomal rearrangements resulting from SCRaMbLE experiments. Inducible recombination between the hundreds of loxPsym sites introduced into Sc2.0 chromosomes enables combinatorial rearrangements of the genome structure. Hi-C contact maps of two SCRaMbLE strains carrying synIII and synIXR chromosomes revealed a variety of cis events, including simple deletions, inversions, and duplications, as well as translocations, the latter event representing a class of trans SCRaMbLE rearrangements not previously observed. This large data set is a resource that will be exploited in future studies exploring the power of the SCRaMbLE system. By investigating the trajectories of Sc2.0 chromosomes in the nuclear space, this work paves the way for future studies addressing the influence of genome-wide engineering approaches on essential features of living systems. Synthetic chromosome organization. (A) Hi-C contact maps of synII and native (wild-type, WT) chromosome II. Red arrowheads point to filtered bins (white vectors) that are only present in the native chromosome map. kb, kilobases. (B) Three-dimensional (3D) representations of Hi-C maps of strains carrying rDNA either on synXII or native chromosome III. (C) Contact maps and 3D representations of synIXR (yellow) and synIII (pink) before (left) and after (right) SCRaMbLE. Translocation breakpoints are indicated by green and blue arrowheads.
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