Research Interests – The Challenges and Solutions at TELOMERES

The preservation and faithful transmission of genomes is fundamental to life. The partition of eukaryotic genomes into linear chromosomes posed two profound challenges to genome preservation and transmission. The first is for the cells to distinguish natural chromosome ends, or telomeres, from abnormal double strand breaks—in order to leave the natural ends intact, but repair the aberrant breaks. In addition, because of a fundamental quirk of DNA polymerase, a small amount of DNA is lost from telomeres with each round of genome replication, making it imperative for an organism to devise compensatory mechanisms. To overcome the two challenges at telomeres, eukaryotic organisms have managed to (1) coat chromosome ends with special proteins to stabilize the termini, and (2) invent elaborate molecular machines to add telomeric DNA (e.g., telomerase). When these telomere proteins and machines malfunction, there is heightened risk for genomic instability and related diseases such as cancer and aging. 

We are intrigued by many unanswered questions surrounding telomeres: How do the machines that synthesize telomeric DNA work and how are they regulated?  What happens to these machines in disease states, especially in cancers? Which protein factors at telomeres are valuable therapeutic targets? How do different organisms evolve distinct telomere structures and telomere synthesizing machines? 

We investigate these questions using both unicellular model organisms and cancer cells. Typically for an interesting issue, we first identify a model system that shares essential features of the human telomere machinery under investigation, make mechanistic discoveries using the model system, and then apply the insights to the analysis of human factors.

Projects

1) The mechanism and regulation of telomerase. 

The telomeric DNA consists of many copies of a short repeated sequence, and this sequence is rich in G nucleotide on the strand that carries the 3’-OH group (e.g., TTAGGG in humans). This “G-strand” is extended by telomerase, an RNA-protein complex; the RNA provides the template, while the catalytic protein (TERT) synthesizes telomeric DNA through reverse transcription. Telomerase is abnormally activated in about 80% of cancer cells, and is responsible for a major hallmark of these cells (i.e., replicative immortality). Improved understanding of telomerase may thus lead to new therapeutic strategies for cancer. Unique among reverse transcriptases, telomerase is able to copy iteratively the template region of the RNA, thus adding multiple telomere repeats onto the G-strand without dissociation. This property (known as repeat addition processivity) requires an “anchor site” in telomerase that interacts with the 5’ region of telomeric DNA during the “translocation” reaction. We and others have mapped anchor site to the conserved TERT essential N-terminal (TEN) domain and shown that this unique telomerase domain is regulated by a conserved OB fold protein (named TPP1 in humans and Est3 in budding yeast). A current focus is to understand how the interaction between Est3 and TEN domain triggers the activation of telomerase. Several of our proposals regarding telomerase regulation have received support from recent cryo-EM structures of telomerases complexes.

2) The mechanisms of primase-DNA Polymerase α complex (primase-Pol α) and its regulation by CST. 

Because telomerase mediates the extension of only the G-strand, another complex called primase-Pol α is required to “fill-in” the telomere C-strand. This C-strand synthesis reaction has received far less attention than telomerase-mediated G-strand extension, but is no less important. The key regulator of primase-Pol α in C-strand synthesis is a conserved telomere-binding complex named CST. Mutations in human CST subunits were recently revealed to be the underlying cause of a complex hereditary disease named Coats plus.

The investigation of C-strand synthesis has been thwarted by the inability to express and purify adequate quantities of CST for mechanistic studies. To overcome this difficulty, we screened multiple CST homologues for ease of expression and purification, and succeeded eventually in obtaining CST from C. glabrata (a fungal pathogen). We then proceeded to isolate the C. glabrata primase-Pol α and demonstrated its activation by CST. Further analysis pinpointed the STN1 subunit of CST as the key activator protein and uncovered a critical protein-protein interaction between STN1 and primase-Pol α. While these observations were made initially through the analysis of C. glabrata proteins, we have since obtained similar results on the human homologs, thus demonstrating a high degree of mechanistic conservation in regard to the regulation of telomere C-strand synthesis. Ongoing studies are directed toward understanding the roles of all three CST subunits in primase-Pol α regulation in telomere maintenance and during replication stress.

3) The roles of DNA repair factors in telomere maintenance and protection

DNA repair factors play multi-faceted roles at telomeres: they promote telomere maintenance by assisting the replication machinery to overcome replications barriers, but they also trigger abnormal DNA repair and genomic stability when telomere structures are perturbed. Thus the telomere nucleoprotein complex must be capable of optimizing the replication function of repair factors without triggering rampant repair. Especially interesting to us is how direct interactions between telomere proteins and DNA repair factors are utilized to achieve this intricate regulation at telomeres. We address this question using U. maydis, a yeast-like fungus that exhibits strong similarities to mammals with regard to the DNA repair machinery. In a recent study, we showed that the telomere protein Pot1 regulates the homology-directed DNA factors Rad51 and Brh2 (BRCA2 ortholog) in a dichotomous and context-dependent manner through direct Pot1-Rad51 interaction. In normal cells, Pot1 binds to Rad51 to promote the replication function of Rad51 (most likely by stabilizing stalled replication forks). However, when Pot1 is depleted, Rad51 and Brh2 play essential roles in triggering aberrant telomere repair. Accordingly, deleting rad51 or brh2 suppresses the growth defects and telomere aberrations triggered by Pot1 deficiency. These results underscore the crucial function of telomeres in suppressing abnormal repair and reveal how direct protein-protein interactions can be utilized to achieve dichotomous regulation of the DNA repair machinery at telomeres.

4) Targeting telomeres for neuroblastoma therapy 

Neuroblastoma is an aggressive childhood cancer with significant morbidity and mortality. Recent studies indicate that telomere maintenance mechanisms in this cancer have strong prognostic value and strong impacts on disease progression. In collaboration with Nai-Kong Cheung (Sloan Kettering Cancer Center), we profiled the telomere characteristics of a panel of neuroblastoma cell lines and surgical samples using a combination of assays that assess telomere length distribution, single strand overhangs, extrachromosomal telomere repeats, as well as telomere DNA damage. Our results point to pervasive “telomere trimming” and telomere DNA damage as signatures of high-risk neuroblastoma. The presence of telomere trimming, a recombination pathway that induces rapid telomere shortening, suggests that neuroblastoma is under strong selection pressure to maintain high levels of telomerase or ALT activity. Accordingly, inhibiting these telomere maintenance pathways may be an especially efficacious strategy against this pediatric cancer. More recently, we uncovered a tight mechanistic connection between telomere/telomerase proteins and tumor cell differentiation. The distinct differentiation states of neuroblastoma tumor cells (i.e., the ADRN and MES cell states) have been shown to affect disease progression and response to therapy. Our findings suggest that manipulating telomeres may impact not only on cell proliferation, but also malignancy and therapeutic response.