Correlative light and electron microscopy of telomeric repeat-binding factor 1 in mammalian cells

• Main Author: von Otter, Eric
• Other Authors: Sara Sandin
• Format: Theses and Dissertations
• Language: English
• Published: 2019
• Subjects: Science::Biological sciences::Biophysics; Science::Chemistry::Crystallography::Electron microscopy
• Online Access: https://hdl.handle.net/10356/106933; http://hdl.handle.net/10220/50047
• Institution: Nanyang Technological University

Telomeres are specialized nucleoprotein structures at the ends of eukaryotic chromosomes. Telomeric chromatin consists of ‘TTAGGG’ DNA repeats sequences, nucleosomes and specific telomere-binding proteins called “shelterin”. Shelterin is a six-protein complex that is essential for genome stability. Due to the end-replication problem, telomeres shorten with each round of cell division. In stem cells and germ cells, telomere length is maintained by activation of the telomerase enzyme. In somatic cells, however, which have little or no telomerase activity, short telomeres are recognized as sites of DNA damage, resulting in an irreversible cell-cycle withdrawal known as “cellular senescence”. As a result, telomere length limits the cellular lifespan to a finite number of divisions. Importantly, one of the hallmarks of cancer is replicative immortality, which requires circumventing the proliferative boundary established by telomere shortening and cellular senescence. Further, the accumulation of senescent cells in human tissues with age has been implicated in age-related disease and human aging. Therefore, telomeric chromatin plays central roles in both cancer and age-related disease. Shelterin contains two telomere repeat-binding factors (TRF1 and TRF2) that binds specifically to the double stranded region of telomeric DNA. It has been proposed that the end protection is the result of a lariat-structure called the “t-loop” which sequesters the chromosome end, thus shielding it from the DNA damage response and DNA repair machinery. Evidence for the t-loop model is based on imaging of isolated telomeres or reconstitutions of telomeric DNA and purified shelterin components. However, the organization of telomeres inside cells remains largely unknown. To address this, I have used correlative light and electron microscopy (CLEM) to visualize and study telomeres in mammalian cells. In order to selectively visualize telomeric chromatin by CLEM, a TRF1 construct containing both eGFP and APEX2 tags was generated. EM analysis of cell sections shows that telomeres have a median diameter of 156 nm and 196 nm in HT1080 and MEFs, respectively. Following a low salt treatment of the cells, MEF telomeres de-compact and increase in diameter to 218 nm. Analysis of the ultrastructure of telomeres by electron tomography reveals a mesoporous meshwork of interconnected chromatin fibers and globular domains. Chromatin fibers range from 5 nm to 30 nm in diameter with a mean diameter of 18.5 nm which decreases to 13.1 nm in low salt conditions. In order to establish a comparative data-set of telomeric and non-telomeric chromatin, nucleosomes were stained for CLEM by eGFP_APEX2 labelling of H2B. The comparison shows that telomeric chromatin fibers more closely resemble heterochromatin regions, whereas telomeric chromatin in low salt conditions more closely resembles euchromatin chromatin in MEFs. In summary, this thesis uses correlative light and electron microscopy in combination with electron tomography to study telomeric chromatin in situ and provide new insights into the ultrastructure of telomeres in mammalian cells.