An Imaging Approach to Examine Telomere Dynamics and Regulation of Gene Expression with Aging

Telomeres are repetitive non-coding nucleotide sequences (TTAGGG)n capping the ends of chromosomes. Improved methods to measure the shortest (not just average) telomere lengths (TLs) are needed. Progressive telomere shortening with increasing age has been associated with shifts in gene expression through models such as the telomere position effect (TPE), which suggests reduced interference of the telomere with transcriptional activity of increasingly more distant genes. A modification of the TPE model, referred to as Telomere Position Effects over Long Distance (TPE-OLD), explains why some genes 1-10 MB from a telomere are still affected by TPE, but genes closer to the telomere are not. Therefore, demonstrating the regulatory roles of telomere length shortening on genes with accurate TL measurement will improve our understanding to the 3D genomic DNA landscape including telomeres. In this doctoral dissertation, I developed a user-friendly software for automatic electrophoresis gel quantification and contributed to developing the Telomere Shortest Length Assay (TeSLA), a technique that detects telomeres from all chromosome ends from <1 kb to 18 kb using small amounts of input DNA. Using cells with more TL information provided by TeSLA, I conducted an imaging approach to systematically examine the occurrence of TPE-OLD at the single cell level. Compared to existing methods, the pipeline allows rapid analysis of hundreds to thousands of cells, which is necessary to establish TPE-OLD as an acceptable mechanism of gene expression regulation. I examined two human genes, for which TPE-OLD has been described before, ISG15 (Interferon Stimulated Gene 15) and TERT (TElomerase Reverse Transcriptase). For both genes I found less interaction with the telomere on the same chromosome in old cells compared to young cells. Experimentally elongated telomeres in old cells rescued the level of telomere interaction for both genes. However, the dependency of the interactions on the age progression from young to old cells varied. One model for the differences between ISG15 and TERT may relate to the markedly distinct interstitial telomeric sequence arrangement in the two genes. Overall, this provides a strong rationale for the role of telomere length shortening in the regulation of gene expression.