SMITH LAB | Molecular Mechanisms of Telomere Function

The Telomeric Complex

The focus of our research is to understand the molecular basis of telomere function in human cells. Telomeres, the ends of chromosomes, consist of long tandem arrays of G-rich repeats bound to specific proteins.

Telomeres act as protective caps and serve as templates for replication by the enzyme telomerase. Telomere integrity is essential for chromosome stability and maintenance, and telomeres play a key role in replicative senescence and cancer. To elucidate the molecular mechanisms that control telomere length and chromosome end protection we are investigating the protein components of the telomeric complex. Telomeres are coated by sequence specific DNA binding proteins TRF1 and TRF2 and their associated proteins, TIN2, TPP1, POT1, Tankyrase 1, and Rap1. We would like to understand how these proteins regulate access of telomeres to telomerase and how they protect chromosome ends from cellular DNA repair systems.

Tankyrase 1 is a member of the poly(ADP-ribose) polymerase (PARP) family of enzymes. Tankyrase1 ADP-ribosylates TRF1 inhibiting its ability to bind to telomeric DNA.

We have shown that tankyrase 1 is a positive regulator of telomere length; overexpression of tankyrase 1 in human tumor cells releases TRF1 from telomeres allowing access to telomerase and telomere elongation. We found that after TRF1 is released from telomeric DNA it becomes accessible to the ubiquitination machinery and is degraded by ubiquitin–mediated proteolysis, thereby preventing its rapid reassociation with telomeres. Our findings suggest a novel mechanism of sequential post-translational modification of TRF1 (ADP-ribosylation and ubiquitination) for regulating access of telomerase to telomeres.

Tankyrase 2, a closely related tankyrase 1 human homolog, shares a number of properties with tankyrase 1, including its ability to ADP-ribosylate TRF1 and induce telomere elongation.Tankyrase 1 and tankyrase 2 heterodimerize suggesting overlapping functions. We have generated tankyrase 2 knockout mice and will use these mice and tankyrase 2 knockout cell lines to elucidate the function of tankyrase 2 and to distinguish the roles of these related proteins at telomeres.

Unexpectedly, tankyrase 1 siRNA knockdown cells arrest in mitosis due to innapropriate sister telomere associations. The PARP actvity of Tankyrase 1 is required for normal mitotic progression.

Inhibiting tankyrase 1 expression, by siRNA, in human tumor cells lead to a mitotic arrest. We found that cells deficient for tankyrase 1 enter mitosis normally; chromosomes align on the metaphase plate, but are unable to proceed to anaphase. Fluorescent in situ hybridization using chromosome specific probes revealed that while sister chromatids are separated at their centromeres and along their arms, they remain associated at their telomeres, likely though proteinacious bridges. Only a catlytically active tankyrase 1, but not a PARP dead mutant, is able to rescue the mitotic phenotype.

. We propose that telomeres require a distinct tankyrase 1-dependent mechanism for sister chromatid resolution prior to anaphase. We are currently investigating if other telomeric proteins are involved in resolution or establishment of sister telomere associations, and screening for other interacting proteins which may also be involved.

We are also investigating other proteins in the TRF1 complex that influence telomere length regulation, including the TRF1/TRF2 interacting protein TIN2, and the recently identified telomere protein TPP1.

Recently we have identified a new telomere protein, TPP1 (TINT1, PIP1, PTOP1). We have shown that TRF1 and TRF2 are linked via TIN2, a previously identified TRF1-interacting protein, and its novel binding partner TPP1. TPP1 localizes to telomeres via TIN2, where it functions as a negative regulator of telomerase-mediated telomere elongation. TIN2 associates with TPP1, and TRF1 or TRF2 throughout the cell cycle, revealing a partially redundant unit in telomeric chromatin that may provide flexibility in telomere length control. Our findings suggest a dynamic cross talk between TRF1 and TRF2 and provide a molecular mechanism for telomere length homeostasis by TRF2 in the absence of TRF1. We are further exploring the function of TPP1 by inhibiting its expression using siRNA, as well as screening for both TIN2 and TPP1 interacting factors.