Investigations into the mechanisms of telomere structure and function

Abstract

Telomere dysfunction is most commonly defined as critical shortening; i.e., loss of telomere sequence due to a variety of causes, usually the end-replication problem. However telomeres and their arsenal of associated proteins also provide essential end-capping structure that protects the ends of linear eukaryotic chromosomes. The overall goal of the studies presented here was to provide new insight into underlying mechanisms of telomeric structure and function. We examined the role of telomere function in Acute Myeloid Leukemia. We observed genomic instability in association with radiation-induced AML, and this association was observed following AML induction with both gamma (γ)- ray and 1 Gev 56Fe ion exposure. Furthermore, we observed a clonal fusion event involving telomeres in a human AML cell line. Taken together, our AML studies underline the importance of genome stability and its link to carcinogenesis. We previously reported a role for the DNA damage repair protein DNA-PKcs in mammalian telomere end-capping function, where inappropriate telomere fusions, as well as telomere fusions to other broken DNA ends, were observed in DNA-PKcs deficient backgrounds. DNA-PKcs has many proposed phosphorylation substrates, one of the most intriguing and relevant being the recently identified heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), an RNA binding protein that associates with telomeres. It is also now well accepted that telomeres are transcribed into telomeric repeat-containing RNAs, or TERRA, which are thought to contribute to telomeric chromatin structure. Taken together, we hypothesized that DNA-PKcs mediated phosphorylation of hnRNA1 plays an important role in tethering TERRA to telomere ends, thereby possibly contributing to telomere chromatin structure and function. Our data suggests that TERRA localization at telomeres is independent of hnRNP A1 and DNA-PKcs kinase activity. Rather, we observed decreased TERRA levels following DNA-PKcs kinase inhibition, suggesting DNA-PKcs indirectly regulates TERRA levels. Depletion of hnRNP A1 did not influence TERRA levels, but resulted in elevated frequencies of what have previously been termed "fragile" telomeres and telomere sister chromatid exchange (T-SCE), both indicative of a role for hnRNP A1 in facilitating telomere replication.