PhD defence S.Y. (Calvin) Lo

Below is a brief summary of the dissertation:

Histone modifiers and chromatin remodelers contribute to DNA repair and replication as well as transcription, yet their roles in the replication stress response remain poorly understood, largely because suitable technologies have been lacking. This thesis develops such tools and uses them to investigate how chromatin landscapes, histone modifiers and chromatin remodelers shape the cellular response to replication stress.
To improve the reproducibility and throughput of quantitative imaging, this thesis first implements automated liquid handling and high-content imaging, and describes the workflow and challenges across liquid handling, microscopy, image analysis and data management. Quantitative mass spectrometry then identifies the chromatin remodeler SMARCAD1 as preferentially enriched at unperturbed replication forks. SMARCAD1 remodels 53BP1-associated nucleosomes to prevent untimely PCNA unloading, thereby stabilizing active forks in the presence of replication poisons. BRCA1-deficient tumors, whether chemotherapy-naïve or resistant, depend on this activity for fork progression and proliferation.
This thesis further shows that a checkpoint-regulated chromatin signaling cascade activates G9a, which cooperates with SUV39h1 to establish H3K9me1/me2/me3-marked heterochromatin at stressed forks, while excluding the demethylase KDM3A. Loss of this protection permits PRIMPOL access, ssDNA gap formation and chemosensitivity. These findings were enabled by ChromStretch, a single-molecule chromatin fiber method that resolves chromatin modifications at replication forks from minimal cell numbers.
Together, this work establishes chromatin remodeling and histone modification as key regulators of replication fork stability and genome integrity, with implications for therapeutic targeting in cancer.