Oncofetal HMGA2 as a key regulator of telomere stability through control of DNA topology

• Main Author: Dharshana, Ramani Priya
• Other Authors: Peter Droge
• Format: Theses and Dissertations
• Language: English
• Published: 2018
• Subjects: DRNTU::Science::Biological sciences
• Online Access: https://hdl.handle.net/10356/89230; http://hdl.handle.net/10220/46217
• Institution: Nanyang Technological University

Faithful replication of the whole genome is fundamental to maintain the genome integrity. During this process, the replication forks may encounter various endogenous and exogenous factors that could lead to replication fork stalling. Telomeres in particular experience numerous replication and topological fork barriers due to its intrinsic nature characterized by repetitive DNA sequences, heterochromatin, and formation of DNA secondary structures; a consequence of TERRA transcription. These impediments present telomeres as ‘difficult-to-replicate’ regions in the genome necessitating both temporal and spatial barrier resolution, which could otherwise lead to telomere deprotection and accumulation of DNA aberrations; a hallmark of cancer. To this end, tightly regulated mechanisms exist to ensure high fidelity telomeric replication. To study the various aspects of telomeres biology, I used the CRISPR/Cas9 tool to generate human embryonic stem cells and HT1080 fibrosarcoma cells stably expressing either GFP-FLAG or mCherry-miniSOG at the C-terminus of the shelterin protein TRF1. TRF1 interacts directly with telomeres and the visualization will help gain insights about telomere regulation in embryonic cells compared to malignant cells. The 3xFLAG tag will enable pull-down studies while the miniSOG will be used to induce telomere site-specific reactive oxygen species (ROS) to study the oxidative stress at telomeres. Recent studies identified the novel role of the oncofetal, non-histone chromatin protein, High Mobility Group AT-hook 2 (HMGA2) in providing first-line defense mechanism against endonucleolytic collapse of stalled forks in embryonic stem cells and cancer cells (Yu, Lim et al. 2014). However, the precise mechanism by which HMGA2 recognizes and interacts with the replication fork remains unclear. Herein, we demonstrate that the chaperoning effect of HMGA2 is conserved across a spectrum of cell lines in addition to ESCs and cancer cells. In the context of malignant cells, we discover differential chemosensitivity effect on the HMGA2 expressing cancer cells in response to chemotherapeutic drugs targeting Topoisomerase I, which is attributed to variable expression levels of HMGA2 in the tested cell lines. Furthermore, we affirm that chaperoning function is especially crucial for the stability of human telomeric and sub-telomeric regions during replication stress, thus reducing the catastrophic DSB occurrence at subtelomeres and telomeres. Our study, thus, reveals that HMGA2 protects the genome particularly the subtelomeric and telomeric stability by functioning as a global regulator of unconstrained supercoiled DNA. We henceforth propose that such a protective function of HMGA2, which initially evolved to protect ESC integrity, has been acquired by cancer cells to evade DNA-damaging chemotherapeutic drugs. Our findings, therefore, suggest that the interplay between HMGA2 and Topoisomerase might have far-reaching implications in personalized cancer therapy.