The molecular regulation of telomeres and telomerase

Telomeres are nucleoprotein caps present at each chromosomal end that play a key role in maintaining genomic stability. Telomeres shorten with each cell division, eventually reaching a critical length at which cellular senescence or death pathways are activated. The enzyme telomerase overcomes this shortening through de novo synthesis of telomeric DNA, and telomerase activity is present at high levels in cancer and stem cells. Telomerase is highly regulated by extracellular and intracellular signals, with this regulation having important consequences for telomere homeostasis. This thesis primarily focuses on the novel role of the glycolytic enzyme glyceraldehyde-3- phosphate dehydrogenase (GAPDH) in the regulation of telomeres and telomerase. Chapter 3 demonstrates an interaction between single-stranded 3’ C-rich telomeric overhangs and the N-terminal Rossman fold-containing NAD+ binding region of GAPDH. GAPDH is further revealed to inhibit telomerase activity in vitro and in cultured cells. This inhibition has been found to be dependent upon the C-terminal catalytic region of GAPDH. Furthermore, this chapter also demonstrates that nitric oxide modification of GAPDH impairs telomerase inhibition. Chapter 4 examines the relationship between the telomeric DNA binding activity of GAPDH and its telomerase inhibitory function. Several residues critical for mediating telomeric DNA binding were identified by site-directed mutagenesis and gel-shift assays. Expression of these GAPDH mutants in MCF7 breast cancer cells revealed that they retained the ability to inhibit telomerase, suggesting that telomeric DNA binding plays a role in positioning GAPDH on telomeres rather than inhibiting telomerase. However, the mutation K259N – located in a known protein-protein interaction region – abolishes telomerase inhibition and telomere shortening, demonstrating a critical role in telomerase inhibition for this region.This chapter also demonstrates for the first time an interaction between GAPDH and the telomerase RNA component hTERC, suggesting a switch between GAPDH binding of telomeric DNA and telomerase RNA. GAPDH specifically binds hTERC using identical components to those needed for the interaction with telomeric DNA. Furthermore, increased exogenous hTERC eliminates GAPDH-mediated telomerase inhibition. Recent studies from our laboratory have demonstrated that exogenous provision of several TGFβ superfamily cytokines can inhibit hTERT expression and telomerase activity. Chapter 5 focuses on the role in telomerase regulation played by the TGFβ superfamily type II receptors by inhibiting their action with siRNA or expression of dominant-negative (DN) proteins. Up-regulation of hTERT and telomerase activity resulted from receptor knockdown, confirming the telomerase inhibitory role for these receptors. However, longterm disruption of receptor signalling by stable expression of DN receptors resulted in telomerase inhibition in three of the four receptors examined. This data clearly demonstrates a role for TGFβ superfamily receptor signalling in telomerase regulation, though this regulation is likely complex in nature. In summary, this thesis investigates a new mechanismof telomere and telomerase regulation in GAPDH, while also furthering the understanding of the influence on telomerase activity by the TGFβ superfamily. The control of telomerase is important in the context of stem cell biology, cancer, and aging research and the findings from this thesis therefore have implications for all these fields.