Integrative Analysis of DNA Replication Timing in Cancer

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Project Summary/Abstract: Genome instability is the most significant feature associated with poor prognosis in many cancers. But despite being a near-universal characteristic of human cancer, our knowledge of how genome instability initiates and contributes to tumor development is lacking. Because of its wide presence and close association with aggressive tumors, insight into the possible mechanisms and consequences of genome instability is crucial in the search for new and targeted cancer therapies and diagnostics. Here a new model is proposed, backed by significant preliminary findings, for the maintenance of genomic stability by cis-acting long non-coding RNA (lncRNA) elements that regulate multiple aspects of large-scale chromosome function. The main hypothesis of this project is that the recently discovered, cis-acting ASAR lncRNAs (ASynchronously Replicating Autosomal RNA), are expressed by normal cells on all autosomes to regulate fundamental chromosome behavior such as DNA replication timing, monoallelic gene expression, 3- dimensional chromosome localization, and mitotic chromosome condensation, but are frequently disrupted in cancer leading to genomic instability. Expression of the first two members of the ASAR family (ASAR6, ASAR15) is required for stability on chromosome 6 and 15 respectively, and disruption of either lncRNA leads to aberrant chromosomes through increased stalling of replication forks and mis-segregation of chromosomes. The long-term objective of this research is to identify all ASARs on human autosomes, validate their functional role in genome stability, and use ASARs as a tool to understand and ultimately identify and treat cancers with genomic instability. In Aim 1 a genome wide search for expression of putative ASARs will be performed utilizing a single-cell derived, haplotype-resolved human primary cell model system, followed by assessment of the functional qualities of potential ASARS with nucleotide sequencing assays that measure DNA replication timing, allele specific RNA expression, and 3D chromosome localization. To probe the potential effects of ASARs on human health, a search for significant disruption of ASARs in a dataset of ~10,000 human tumor samples will be performed. The research will be conducted as part of a comprehensive training plan involving advanced skill development in bioinformatics and biostatistics, professional development such as public speaking and networking, and will take place within an extensive intellectual community composed of cancer biologists at the Knight Cancer Institute, molecular and medical geneticists, and computational biologists at OHSU. The post-doctoral research direction will translate the knowledge of chromosome biology and skillsets in genomics developed during the pre-doctoral period to study the mechanisms of action of non protein-coding germline risk loci in human cancer. The successful results of the proposed research will reveal basic functions of chromosomes and provide new insight towards understanding genomic instability, a common abnormality in cancer.