Cancer cells differ from their normal cellular counterparts in many important characteristics. These 'hallmarks'of cancer are acquired during the multistep development of human cancer. Not surprisingly, genetic alterations are present in most, if not all cancers, and are thought to lie at the heart of these phenotypic alterations. In addition, genomic instability, defined as an increase in the rate at which spontaneous mutations occur, is considered to be one of the hallmarks of cancer and is thought to be required to generate the numerous genetic changes that are present in individual tumor cells. Previous studies from my lab indicate that certain tumor-derived chromosome rearrangements exhibit a chromosome wide delay in replication timing (DRT) and a subsequent delay in mitotic chromosome condensation (DMC). In addition, we found that DRT/DMC causes an ~50 fold increase in the rate at which secondary rearrangements occur on the affected chromosome, providing formal proof that DRT/DMC causes genomic instability. In our most recent studies, we have used chromosome-engineering strategies to identify an autosomal locus that when disrupted results in DRT/DMC. These studies led to the identification of a discrete cis-acting locus that controls replication timing, mitotic condensation and structural stability of human chromosome 6. Moreover, molecular characterization of this locus identified a large non-coding RNA gene, which we named Asynchronous replication and Autosomal RNA on chromosome 6 (ASAR6). As the name implies, ASAR6 displays asynchronous replication timing between alleles. In addition, ASAR6 displays random monoallelic expression, and disruption of the expressed allele results in DRT/DMC and structural instability of human chromosome 6. Importantly, recent work from the Y. Marahrens'indicated that deletion of the Xist gene, in adult somatic cells, results in delayed replication, abnormal chromatin structure and instability of the chromosome. Therefore, disruption of ASAR6 results in an apparent phenocopy of deletion of Xist. Because we have detected DRT/DMC on numerous human and mouse chromosomes, our observations have led to the novel hypothesis that 'inactivation/stability centers'are present on all mammalian chromosomes. We believe that these observations represent the discovery of a new fundamental property of all mammalian chromosomes. Thus, we are proposing that every mammalian chromosome contains four cis-acting elements that ensure proper replication, segregation and stability: 1) centromeres, 2) telomeres, 3) origins of replication, and 4) inactivation/stability centers. This proposal represents collaboration between the Thayer and Grompe labs, and is designed to characterize the ASAR6-syntenic locus in the mouse. In addition, this proposal is designed to determine if the structural instability of individual chromosomes caused by disruption of an autosomal inactivation/stability center results in an increase in tumor formation using conditional knockout strategies in the mouse.
The experiments described in this proposal are designed to develop a mouse model for the chromosome structure instability that has recently been observed in human cancers. These experiments are designed to determine if disruption of the Asar4/6 inactivation/stability center, located on the mouse chromosome 4, results in an increase in chromosome structure instability, loss of mono-allelic expression and increased tumorigenesis. The successful completion of the experiments described in this proposal will contribute to our basic understanding of the mechanisms functioning to maintain structural integrity of individual chromosomes and of the impact that this type of genomic instability has on tumor formation.