Effect of Spatial Proximity on Chromosomal Translocations

描述(申请人提供)：空间邻近对染色体易位的影响染色体易位是一种已知与癌症发生和发展有关的基因组重排，它们涉及两个非同源染色体之间的遗传物质交换。在某些癌症中，特别是白血病、淋巴瘤和肉瘤，特定的易位是反复发生的，这意味着不同的患者出现的易位涉及相同两条染色体上的相同两个位点。这种特定易位的反复出现导致了一种假设，即基因组的某些区域更容易发生重排，可能是因为核空间中的空间接近。尽管相关证据表明，利用荧光原位杂交(FISH)等技术，基因组的常见易位区域在空间上非常接近，但空间邻近有助于形成染色体易位的想法尚未得到直接或彻底的检验。我们的实验室开发了一种新的遗传系统，利用它我们可以在酿酒酵母模式生物中检测非同源末端连接(NHEJ)介导的相互染色体易位。该系统允许同时诱导不同染色体上的DNA双链断裂，并实时检测相互易位的染色体。此外，酵母基因组最近被绘制在三维空间中，揭示了细胞核中非随机染色体区域的模式，着丝粒聚集在纺锤体极体附近，染色体臂伸展，端粒与核膜相连。这项提议的重点是直接测试空间邻近通过两种方式有助于形成经常性染色体易位的观点。首先，以三维基因组图为指导，两个DNA断裂点将被放置在基因组中的不同位置，彼此之间的空间距离不同。染色体易位形成的频率将与空间距离相关，通过染色体构象捕获(3C)分析来衡量。下一步，在这些指定的位置，染色质结构将通过改变细胞周期时相和删除端粒与核外周连接的因素来修改。空间邻近度和染色体易位频率将分别使用3C分析和易位基因株进行评估。这项研究将阐明导致染色体易位的分子机制，并为描述人类的同等过程奠定基础。

DESCRIPTION (provided by applicant): Effect of Spatial Proximity on Chromosomal Translocations Chromosomal translocations are a type of genomic rearrangement known to be involved in cancer development and progression, and they involve the exchange of genetic material between two non- homologous chromosomes. In certain cancers, especially leukemias, lymphomas, and sarcomas, specific translocations are recurrent, meaning different patients present with translocations involving the same two loci on the same two chromosomes. This recurrence of certain translocations leads to the hypothesis that certain regions of the genome are more susceptible to rearrangement, likely through a close spatial proximity in the nuclear space. Despite correlative evidence demonstrating close spatial proximity of commonly translocated regions of the genome using, for instance, Fluorescent In-Situ Hybridization (FISH) techniques, the idea that spatial proximity contributes to the formation of chromosomal translocations has not be directly or thoroughly tested. Our laboratory developed a novel genetic system with which we can detect Non-Homologous End- Joining (NHEJ)-mediated reciprocal chromosomal translocations in the S. cerevisiae model organism. This system allows for the simultaneous induction of DNA duplex breaks on separate chromosomes, and the real time detection of reciprocal chromosomal translocations. Additionally, the yeast genome has recently been mapped in three-dimensional space, revealing the patterns of non-random chromosomal territories in the nucleus, with the centromeres clustered near the spindle pole body, the chromosome arms outstretched, and the telomeres tethered to the nuclear membrane. The focus of this proposal is to directly test the idea that spatial proximity contributes to the formation of recurrent chromosomal translocations in two ways. First, using the three-dimensional genome map as a guide, two DNA break-sites will be placed at different locations in the genome, at different spatial distances from each other. The frequency of chromosomal translocation formation will be correlated to the spatial distance, as measure by the Chromosome Conformation Capture (3C) assay. Next, at these specified locations, the chromatin architecture will be modified by changing the cell cycle phase and by deleting factors involved in the telomere-tethering to the nuclear periphery. Spatial proximity and chromosomal translocation frequency will be assessed using the 3C assay and the translocation genetic strain, respectively. This study will shed light on the molecular mechanism leading to chromosomal translocations and set the stage for characterizing the equivalent processes in humans.