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.
The experiments described in this proposal will aid in our understanding of the mechanisms by which exposure to ionizing radiation leads to genetic instability. Because genetic instability is thought to be a driver of tumor cell evolution, the experiments that make up this proposal may aid in the development of therapies designed to prevent tumor progression. The experiments that make up this proposal will further our understanding of the role that long non-coding RNAs play during radiation induced carcinogenesis and may aid in the development of novel therapeutics designed to help maintain the integrity of the genome.