Cis-Acting Chromosomal Elements and Radiation-Induced Instability

描述（由申请人提供）：电离辐射（IR）被称为“通用致癌物”，因为它会在所有年龄段的大多数组织（包括胎儿）中诱发癌症。暴露于IR，无论是由于意外暴露还是作为癌症的治疗，都代表着世界范围内的重大健康问题。IR的致癌作用被认为不仅是由于直接的致突变作用，而且是由于对群体中许多细胞产生的“延迟”不稳定过程知之甚少，从而提高了癌症发展所需的多种突变的发生率。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，但负责的分子机制仍然未知。该建议旨在检验IR诱导的延迟不稳定性是由负责个体人类染色体的适当复制时机的离散顺式作用位点的破坏引起的这一假设。我实验室以前的研究表明，某些肿瘤来源的染色体重排表现出复制时间延迟（DRT），有丝分裂染色体浓缩延迟（DMC）和单个染色体的结构不稳定性。随后，我们发现，约5%的随机易位暴露于IR诱导显示这种DRT/DMC表型。最近，使用一种新的染色体工程策略，我们确定了一个离散的顺式作用位点，当破坏时，导致DRT/DMC的人类6号染色体上。此外，该基因座的特征鉴定了一个长的、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（ASAR 6）。ASAR 6的破坏导致DRT/DMC，其他顺式单等位基因的先前沉默等位基因的激活，以及人类6号染色体的结构不稳定性。此外，从Y. Marahrens的实验室表明，在成年体细胞中，小鼠Xist基因的缺失会导致复制延迟、染色质结构异常和X染色体的不稳定。因此，ASAR 6的缺失导致Xist缺失的表型。考虑到ASAR 6和Xist之间的结构和功能相似性，并且因为我们已经检测到影响许多人类和小鼠染色体的易位上的DRT/DMC，我们提出每个哺乳动物染色体都含有负责适当复制时机、单等位基因表达和结构稳定性的“失活/稳定中心”。与这一假设相一致，我们最近确定了第二个常染色体“失活/稳定中心”位于人类15号染色体上，我们将其命名为ASAR 15。该提案旨在使用IR在ASAR 6和ASAR 15处产生染色体突变，并确定DRT/DMC和IR诱导的不稳定性是否同时发生。此外，该提议旨在使用大规模平行测序来表征由DRT/DMC和IR诱导的不稳定性引起的二级重排的“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.