Regulation of Centromere Identity and Consequences for Genome Stability
Date of Award
Spring 2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Molecular Biophysics and Biochemistry
First Advisor
Kabeche, Lilian
Abstract
Cell division allows for the transmission of our genetic information from one generation to the next. This process is orchestrated by the centromere, a specialized region of the chromosome that mediates the attachment of spindle microtubules during mitotic chromosome segregation. As such, centromere dysregulation can lead to chromosome segregation errors and aneuploidy, a hallmark of genome instability and cancer that is characterized by an unbalanced number of chromosomes in a cell. Changes in gene dosage due to chromosome gain or loss can affect the expression of critical genes involved in cell cycle regulation, DNA repair, and mitotic fidelity, which can lead to uncontrolled proliferation, genome instability, and additional chromosome segregation errors. This highlights the centromere as a key genomic locus that must be tightly regulated to protect against aneuploidy as well as cancer development and progression. Therefore, my thesis work has centered on elucidating the molecular pathways underlying centromere regulation and understanding the consequences of their perturbation. Throughout my doctoral studies, I have discovered two novel pathways whereby the apical DNA damage response kinase ATR regulates centromere identity, integrity, and function. My first-author publication in Cell Reports describes a non-canonical role for ATR in protecting the occupancy of the histone variant CENP-A at centromeres. CENP-A epigenetically defines the centromere by recruiting the macromolecular protein complex known as the kinetochore, which is necessary for spindle microtubule attachment and faithful chromosome segregation. Using a suite of biochemistry and microscopy techniques, I found that ATR localizes to Promyelocytic Leukemia nuclear bodies (PML NBs) that house chromatin modifiers in the absence of DNA damage. ATR keeps the histone chaperone DAXX tethered to these bodies via phospho-regulation, which prevents aberrant changes to centromeric chromatin composition, including the loss of CENP-A- and gain of H3.3-containing nucleosomes. Disrupting this pathway with small-molecule ATR inhibitors increases mitotic chromosome segregation errors. This work highlights a novel, non-canonical role for ATR in protecting genome stability via centromere regulation. These findings led me to ask how centromeres are regulated in contexts of genotoxic stress. As ATR activity at PML NBs is necessary to prevent DAXX localization to centromeres, I hypothesized that ATR’s canonical response to DNA damage and translocation away from PML NBs would induce similar changes to centromeric chromatin composition. In conducting this research, I observed that prolonged treatment with replication inhibitors leads to CENP-A accumulation in nucleoli. My findings indicate that this phenotype is associated with CENP-A removal from centromeres, CENP-A degradation, and S-phase arrest. ATR activation following replication stress is required for nucleolar CENP-A accumulation, but the additional factors responsible for this translocation are unknown. Furthermore, the consequences of this pathway for mitotic fidelity and cell fate have yet to be determined. As a fragile locus with a paradoxical function in coordinating chromosome inheritance, the centromere is a double-edged sword that both protects and threatens genome stability. My research demonstrates two novel mechanisms for centromere regulation that hinge on DNA damage-independent and -dependent ATR activity, suggesting that centromere composition may be tuned to reinforce the cellular response to genotoxic stress. Furthermore, this work has the potential to inform targeted molecular therapies that exploit a cancer’s underlying instability. My work has elucidated novel roles for ATR in the regulation of centromere identity and integrity, thereby providing crucial insight into the clinical exploration of ATR inhibitors as a targeted cancer treatment. As centromere dysregulation can trigger the aneuploidy and instability that these therapies hope to exploit, my research may also uncover new diagnostic tools and targets related to centromere function.
Recommended Citation
Trier, Isabelle, "Regulation of Centromere Identity and Consequences for Genome Stability" (2024). Yale Graduate School of Arts and Sciences Dissertations. 1396.
https://elischolar.library.yale.edu/gsas_dissertations/1396
