Single-telomere-specific (TTAGGG)n tract lengths and instabilities cannot be measured globally using current methods;the ability to do so would be a major, transformative addition to the arsenal of tools for analyzing critical telomere loss an telomere elongation events in cancer. The shortest telomere or a small subset of the shortest telomeres in a cell will determine the onset of senescence, apoptosis, or genome instability;single (TTAGGG)n tracts are crucial for the function of telomeres and the biological effects of telomere attrition and dysfunction. Telomere loss, breakage, fusion, and rejoining are highly elevated in cancer, but current methods for detecting and measuring these mutational events at the molecular level are limited and low-throughput. The technology we propose to develop here would permit quantitative, single- allele-resolution measurements of telomere length and instability, enabling unprecedented insights into the role(s) telomere loss, telomere breakage/re-joining, and telomerase or ALT dependent telomere elongation play in carcinogenesis, including mechanistic insights into molecular events mediating these processes and translational insights for the potential prognostic and tumor stratification applicability of the methods. In this method, input genomic DNA is labeled with fluorescent dyes specific for (TTAGGG)n sequences and for linked subtelomeric DNA. The labeled individual DNA fragments are linearized (stretched) and imaged in the nano-channels of Bionano Genomics system at very high throughput. The lengths of the telomere are measured accurately and the distances between probes in the subtelomere region are determined accurately to infer the identity of the telomere. Our goals for this R21 study are to establish feasibility for (1) high-throughput single-molecule detection and quantitation of (TTAGGG)n tracts in genomic DNA samples and (2) subtelomere probe development and efficient co-labeling of telomeres and subtelomeres in the context of total genomic DNA, including conversion of the labeled DNA to double- stranded DNA suitable for nano-channel analysis;and (3) proof-of-principle results for the technology and its applicability for cancer research using data generated and analyzed for a normal and a cancer cell line. There are significant technical challenges inherent in this early-stage technology development project, but the extraordinary payoff for cancer research will be a high-throughput ability to probe mechanisms of telomere length regulation and telomere mutation at single-telomere resolution, in small (ultimately single-cell) samples of both dividing and non-dividing cells.
Telomeres form the ends of chromosomes;their loss or dysfunction, and ultimately their long-term maintenance, are universal features of carcinogenesis. The methods we propose to develop here will allow high-throughput, high-resolution analysis of telomere mutation and elongation events, which could provide a unique tool for understanding the basic biology of telomeres in cancer, while also providing a new tool for possibly predicting the aggressiveness of cancers and the most appropriate therapies for treatment.
|McCaffrey, Jennifer; Young, Eleanor; Lassahn, Katy et al. (2017) High-throughput single-molecule telomere characterization. Genome Res 27:1904-1915|
|Young, Eleanor; Pastor, Steven; Rajagopalan, Ramakrishnan et al. (2017) High-throughput single-molecule mapping links subtelomeric variants and long-range haplotypes with specific telomeres. Nucleic Acids Res 45:e73|
|Nelson, David M; Jaber-Hijazi, Farah; Cole, John J et al. (2016) Mapping H4K20me3 onto the chromatin landscape of senescent cells indicates a function in control of cell senescence and tumor suppression through preservation of genetic and epigenetic stability. Genome Biol 17:158|
|Tutton, Stephen; Azzam, Greggory A; Stong, Nicholas et al. (2016) Subtelomeric p53 binding prevents accumulation of DNA damage at human telomeres. EMBO J 35:193-207|
|McCaffrey, Jennifer; Sibert, Justin; Zhang, Bin et al. (2016) CRISPR-CAS9 D10A nickase target-specific fluorescent labeling of double strand DNA for whole genome mapping and structural variation analysis. Nucleic Acids Res 44:e11|
|Sedic, Maja; Skibinski, Adam; Brown, Nelson et al. (2015) Haploinsufficiency for BRCA1 leads to cell-type-specific genomic instability and premature senescence. Nat Commun 6:7505|
|Porro, Antonio; Feuerhahn, Sascha; Delafontaine, Julien et al. (2014) Functional characterization of the TERRA transcriptome at damaged telomeres. Nat Commun 5:5379|