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 fascinated 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 a project, 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.
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 major current focus is to understand how the interaction between Est3 and TEN domain triggers the activation of telomerase.
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 virtually identical 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 the other two CST subunits, CTC1 and TEN1. In addition, we are investigating the functions of CST and primase-Pol α during replication stress.
3) The mechanisms of ALT (alternative lengthening of telomeres)
Approximately 15-20% of cancer cells lack telomerase activity and utilize instead a RECOMBINATION-based mechanism called ALT for telomere elongation and for achieving replicative immortality. ALT is found frequently in sarcomas associated with poor prognosis; targeted treatments against ALT have the potential to provide strong therapeutic benefits. In collaboration with the lab of Bill Holloman (Microbiology and Immunology Department at Weill Cornell Medicine) and José Pérez Martín (IBFG, Spain), we developed a highly accurate model of the ALT pathway using a mutant of Ustilago maydis, a plant fungus. Also known as corn smut, this unusual model organism shares many similarities with human cells in regard to telomere structure and recombination proteins. As a result, we were able to reproduce in the U. maydis model all the characteristic telomere features of ALT cancer cells, including the accumulation extra-chromosomal telomeric DNA. Using this model, we further identified several recombination proteins that are essential for ALT, and hence potential therapeutic targets. We are continuing to dissect the mechanisms of ALT in the U. maydis model, and have begun to validate the role of putative treatment targets in ALT cancer cell lines.