Type of Thesis
Molecular, Cellular, & Developmental Biology
Dr. Thomas Cech
Dr. Jennifer Martin
Dr. Nancy Guild
Single-molecule live-cell imaging is a powerful tool for observing molecular dynamics in a living system. Here, this technique is employed to investigate the recruitment dynamics of telomerase and Polycomb Repressive Complex 2 (PRC2) to their targets in vivo. Telomerase is a ribonucleoprotein that maintains genome integrity by adding repetitive telomeric DNA sequences to chromosome ends in actively dividing cells. The model of human telomerase recruitment to telomeres is only partially understood. While it is known that telomerase forms both static and probing interactions with telomeres in S-phase, the nature of the telomeres that are statically visited is unknown as is the utility of TRF2 intensity as a measure of telomere length. By investigating the nature of the telomeres involved in static interactions over the course of S-phase, we were unable to confidently conclude that telomeres of a certain character were involved in static binding events.
PRC2 is a histone methyltransferase that deposits mono-, di-, and trimethylation marks onto chromatin to promote epigenetic silencing. In human cells, the mechanism by which PRC2 is recruited to specific sites is not completely understood. However, the importance of accessory proteins in dictating PRC2-chromatin binding has been demonstrated and in the present study, we seek to understand how PRC2 accessory proteins impact PRC2-chromatin binding in stem cells. Genome editing of a HaloTag into the endogenous SUZ12 loci of stem cells, coupled with single-molecule live-cell imaging is being used to examine the dynamics of PRC2 in stem cells. Preliminary data suggests that a higher fraction of the PRC2 present in stem cells is bound to chromatin as compared to osteosarcoma cells. Additionally, it appears that introducing mutations into both the AEBP2 and PCL homolog binding regions of SUZ12 decreases the fraction of chromatin-bound PRC2, perhaps reinforcing the idea that PRC2 has a decreased ability to bind chromatin upon disruption of the accessory protein binding domains.
Foster, Kyla, "Elucidating the Dynamics of Telomerase Recruitment to Telomeres and PRC2 Recruitment to Chromatin Using Single-Molecule, Live-Cell Imaging" (2019). Undergraduate Honors Theses. 1869.
Available for download on Sunday, April 11, 2021