Cis-Acting Chromosomal Elements and Radiation-Induced Instability

说明(申请人提供)：电离辐射(IR)被称为“普遍致癌物质”，因为它会在所有年龄段的大多数组织中诱发癌症，包括胎儿。暴露于红外线，无论是由于意外暴露，还是作为癌症的一种治疗方法，在世界范围内都是一个重大的健康问题。IR的致癌作用被认为不仅是由于直接的诱变作用，而且是因为在人群中的许多细胞中产生了一个鲜为人知的“延迟”不稳定过程，从而提高了癌症发展所需的多种突变可能出现的速度。虽然已经提出了许多表观遗传和/或顺式作用模型来解释IR诱导的不稳定性，但相关的分子机制仍不清楚。这一建议旨在检验这样一种假设，即IR引起的迟发性不稳定性是由负责单个人类染色体正确复制时机的离散顺式作用基因座的中断引起的。我实验室以前的研究表明，某些肿瘤衍生的染色体重排表现出复制时间延迟(DRT)、有丝分裂染色体凝聚(DMC)延迟以及单个染色体的结构不稳定。随后，我们发现，在暴露于IR引起的随机易位中，~5%表现出这种DRT/DMC表型。最近，利用一种新的染色体工程策略，我们在人类6号染色体上发现了一个离散的顺式作用基因座，当该基因被干扰时，会导致人类6号染色体上的DRT/DMC。此外，对该基因的鉴定发现了一个长的、单等位基因表达的非编码RNA基因，我们将其命名为6号染色体上的异步复制和常染色体RNA(ASAR6)。ASAR6基因的中断会导致DRT/DMC，顺式基因中其他单等位基因的先前沉默的等位基因被激活，以及人类6号染色体的结构不稳定。此外，Y.Marahrens的实验室的工作表明，在成年体细胞中，小鼠Xist基因的缺失会导致X染色体的复制延迟、染色质结构异常和不稳定。因此，ASAR6的缺失导致Xist缺失的表型复制。鉴于ASAR6和XIST在结构和功能上的相似性，以及我们在影响大量人类和小鼠染色体的易位上检测到DRT/DMC，我们提出每个哺乳动物染色体都包含一个“失活/稳定中心”，负责正确的复制时机、单等位基因表达和结构稳定性。与这一假设一致，我们最近发现了位于人类15号染色体上的第二个常染色体“失活/稳定中心”，我们将其命名为ASAR15。这项建议旨在利用IR产生ASAR6和ASAR15的染色体突变，并确定DRT/DMC和IR诱导的不稳定性是否同时发生。此外，该方案旨在使用大规模并行测序来表征由DRT/DMC和IR诱导的不稳定性引起的二次重排的分子特征。

DESCRIPTION (provided by applicant): Ionizing radiation (IR) has been called a "universal carcinogen", because it will induce cancer in most tissues at all ages, including the fetus. Exposure to IR, either due to accidental exposure or as a treatment for cancer, represents a significant health problem world-wide. The carcinogenic effects of IR are thought to arise not only due to direct mutagenic affects but also to a poorly understood process of 'delayed' instability generated in many cells in the population, thereby enhancing the rate at which the multiple mutations necessary for the development of cancer may arise. While numerous epigenetic and/or cis-acting models have been proposed to explain IR-induced instability, the molecular mechanisms responsible remain unknown. This proposal is designed to test the hypothesis that IR induced delayed instability is caused by the disruption of discrete cis-acting loci responsible for proper replication timing of individual human chromosomes. Previous studies from my lab indicate that certain tumor-derived chromosome rearrangements exhibit a delay in replication timing (DRT), a delay in mitotic chromosome condensation (DMC), and structural instability of individual chromosomes. Subsequently, we found that ~5% of random translocations induced by exposure to IR display this DRT/DMC phenotype. Recently, using a novel chromosome-engineering strategy we identified a discrete cis-acting locus that when disrupted causes DRT/DMC on human chromosome 6. Moreover, characterization of this locus identified a long, monoallelically expressed, non-coding RNA gene, which we named Asynchronous replication and Autosomal RNA on chromosome 6 (ASAR6). Disruption of ASAR6 results in DRT/DMC, activation of previously silent alleles of other monoallelic genes in cis, and structural instability of human chromosome 6. In addition, work from the Y. Marahrens' lab indicates that deletion of the mouse Xist gene, in adult somatic cells, results in delayed replication, abnormal chromatin structure and instability of the X chromosome. Therefore, deletion of ASAR6 results in a phenocopy of deletion of Xist. Given the structural and functional similarities between ASAR6 and Xist, and because we have detected DRT/DMC on translocations affecting numerous human and mouse chromosomes, we are proposing that every mammalian chromosome contains an "inactivation/stability center" responsible for proper replication timing, monoallelic gene expression, and structural stability. Consistent with this hypothesis we recently identified a second autosomal "inactivation/stability center" located on human chromosomes 15, which we have named ASAR15. This proposal is designed to use IR to generate chromosomal mutations at ASAR6 and ASAR15, and to determine if DRT/DMC and IR-induced instability occur simultaneously. In addition, this proposal is designed to use massively parallel sequencing to characterize the 'molecular signature' of secondary rearrangements caused by DRT/DMC and IR induced instability.