Towards a mechanistic understanding of telomere loop structures in S. cerevisiae

Abstract

Only with the evolution from circular to linear genomes, allowing easy exchange of genetic information by sexual reproduction, complex organisms with large genomes could evolve. But the linearity of a chromosome inherits a major problem, namely two chromosome ends that have to be protected. Telomeres at the very end of the chromosomes ensure cell survival. These nonprotein coding DNA repeats are essential features of chromosomes, as their loss leads to irreversible cellular senescence and chromosome loss. Paradoxically, telomeres resemble DNA double-strand breaks (DSBs), however, unlike DSBs, they are refractory to repair events. This socalled “end protection” function carried out by telomeres ensures that chromosomes do not fuse together in an end-to-end manner and avoids the DNA damage response machinery from being activated, leading to cell cycle arrest. Although end protection has largely been attributed to the major telomere binding complexes such as shelterin in mammals and the CST complex in yeast, it has also been proposed that a three-dimensional structure at the telomere may contribute to safeguarding telomeres. These so called telomere loops (t-loops) have been demonstrated via electron- and super-resolution-microscopy in mammalian cells, however, the short length and base composition of yeast telomeres prevent such approaches. By using a combination of Chromatin Immunoprecipitation (ChIP) and transcriptional readouts it has been demonstrated that yeast telomeres loop back onto their respective subtelomeres; however, both methods are indirect and unsatisfactory in terms of analyzing the dynamic regulation of loop structures. In this study we have established a new assay based on Chromosome Conformation Capturing (3C) to directly detect and quantify interactions between a telomere and its subtelomeric region in S. cerevisiae, as a measure of telomere looping. In this manner we could exploit the genetic advantages of the yeast system to understand the mechanistic details of telomere loop formation and maintenance. Since telomere shortening leads to an unprotected telomere, we wondered whether telomere length may have an impact on looping. We were able to show a significant looping defect in cells that lack telomerase as well as in other mutants that harbor short telomeres. On the contrary, elongated telomeres were able to maintain the looped structure. This suggests that a critical telomere length is essential to maintain the telomere loop and that telomeres in senescent cells are likely in an open conformation, rendering them susceptible to nucleolytic end resection and unscheduled DNA repair events. Gene looping is another kind of looped chromatin that brings promoter and terminator together. Gene loops depend on transcription, a functional transcription initiation complex and several components of the mRNA processing machinery. Indeed, we could detect a telomere looping defect upon loss of RNA polymerase II and in a mutant of the transcription preinitiation complex (sua7-1). It has been shown that certain chromatin loops that bring promoter and enhancer regions in close proximity depend on a non-coding RNA species that interacts with components of the promoter associated mediator complex. Depletion of total RNA levels or mutation of mediator resulted in a telomere looping defect, indicating a similar regulation at the telomere.