Mechanisms of telomere regulation: characterizing TRF1-mediated phase separation of nucleosomes

• Main Author: Ng, Brendan Wei-Jie
• Other Authors: Lars Nordenskiöld
• Format: Final Year Project
• Language: English
• Published: Nanyang Technological University 2023
• Subjects: Science::Biological sciences
• Online Access: https://hdl.handle.net/10356/166549
• Institution: Nanyang Technological University

Telomeres are functionally conserved regions at the ends of chromosomes that confer protection. The dysregulation of telomeres has been implicated in diseases of aging and cancer, but not all underlying mechanisms of telomere regulation have been fully elucidated. Recently, liquid-liquid phase separation (LLPS) has been identified as an important regulator of telomere dynamics. In particular, shelterin proteins were shown to mediate phase separation events. In this study, we characterize the phase separation of human nucleosome arrays mediated by the shelterin component TRF1. Full-length TRF1 and 10x 157bp telomeric DNA repeats (t10-DNA) were expressed and purified from E. coli. Nucleosome arrays were reconstituted from t10-DNA and fluorescent-tagged histone octamers by salt dialysis. TRF1 was shown to bind to nucleosome arrays by electrophoretic mobility shift assay and Mg2+ pulldown assay. TRF1-mediated phase separation was characterized by confocal fluorescent microscopy at physiological salt conditions in single-colour and dual-colour mixing assays. Our results show a trend (n=4) of nucleosome compaction as a function of increasing TRF1 concentration, where phase separation occurs when the nucleosomal TRF1-binding sites are undersaturated, and transition to aggregate formation upon saturation. These findings can help to model TRF1’s role in telomere dynamics and contribute to understanding of multiple disease mechanisms.