Mechanisms of Telomere Cohesion

Project Summary/Abstract Telomeres, the specialized structures at chromosome ends, are comprised of TTAGGG repeats, telomere repeat containing RNA (TERRA), and the shelterin protein complex. Sister chromatids are held together from the time of their replication in S phase until their separation in mitosis by cohesin rings. Ring-mediated cohesion is essential to ensure accurate distribution of chromosomes to daughter cells in mitosis. Cohesion between sisters is also important for recombination and repair, particularly at repetitive sequences like telomeres, where it keeps them aligned. For this, more intimate contacts (in addition to the cohesin ring) are needed and as such, there are telomere-specific requirements for cohesion. Establishment of cohesion at telomeres requires shelterin subunits and associated proteins. Resolution of cohesion between telomeres requires the PARP, tankyrase. Tankyrase localizes to telomeres by binding to the TTAGGG-repeat binding shelterin subunit TRF1, in late S/G2 to resolve cohesion. In tankyrase-depleted cells sister telomeres remain cohered in mitosis despite normal resolution of arms and centromeres. This persistent telomere cohesion is not just an aberrant state induced by depletion of tankyrase, it occurs naturally in certain human cell types that lack telomerase and have critically short telomeres: normal aged cells and cancer ALT cells. Unexpectedly, (and shown by our lab in the last few years) this persistent cohesion is beneficial to cells; it serves a protective role to prevent premature senescence in aged cells and growth arrest in ALT cancer cells. The goal of our research for the next five years is to elucidate the proteins and mechanisms required for establishment and resolution of cohesion. We will build on our previous work where we identified shelterin subunits and associated factors that are required for cohesion and we will focus on our most recent discovery indicating a role for RNA (TERRA and TERRA R-Loops) in telomere cohesion. We will determine how the telomeric components contribute to the establishment, maintenance, and resolution of telomere cohesion in normal human cells, aging, and cancer. For cohesion establishment, we will determine how and when the required proteins are deposited on telomeres and how far into the chromosome they extend. For cohesion resolution, we will determine how recruitment of tankyrase serves to resolve cohesion. We will characterize the proteins that tankyrase itself recruits to telomeres and investigate how tankyrase uses functional compartmentalization to orchestrate the resolution process. We will determine how TERRA RNA and TERRA R-loops contribute to telomere cohesion in normal cells and to persistent telomere cohesion in pathological conditions of aging and ALT cancer. And finally, we will elucidate the full proteome of the cohered telomeric state. Ultimately, a complete understanding of telomere cohesion will elucidate fundamental mechanisms of chromosome stability and genome integrity in normal human cells and provide insights into prevention of premature senescence in aging cells and into promotion of premature death in ALT cancer cells.