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dc.contributor.authorHsia, Chieh-Ren
dc.description170 pages
dc.description.abstractDuring cancer metastasis, cancer cells migrate through confined interstitial spaces, requiring extensive deformation of the cell body and nucleus. Previous studies found that nuclear compression induces heterochromatin formation; furthermore, the cytoskeleton can transmit extracellular forces to the nucleus, eliciting chromatin rearrangement and changes in gene expression. However, it remains unclear whether physical stress on the nucleus during confined migration through three-dimensional (3D) environments can induce chromatin modification and accessibility changes, and subsequently gene expression changes. Using custom-made microfluidic migration devices that mimic interstitial spaces in vivo, we here show that confined cell migration of fibroblasts and cancer cells results in increased H3K9me3 and H3K27me3 heterochromatin marks compared to unconfined conditions, and that these marks persist for at least 4-5 days. The migration-induced heterochromatin formation is dependent on histone modifying enzymes. Moreover, nuclear envelope proteins lamin A/C and emerin, and stretch-sensitive ion channels, contribute to the migration-induced heterochromatin formation. Finally, pharmacological inhibition of heterochromatin formation results in impaired cell migration compared to vehicle treatment, suggesting that heterochromatin formation promotes confined migration. Taken together, our study uncovers the novel phenomenon of heterochromatin formation induced by confined cell migration, and provides insights on the molecular mechanisms and its biological significance. As histone modifications often accompanies chromatin accessibility changes, we performed Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) on cells migrating in 3D collagen matrices, and found that cells in confined collagen matrices exhibit changes towards less accessible chromatin, consistent with the increased H3K9me3 and H3K27me3 heterochromatin levels. Confined migration increased chromatin accessibility near gene promoters associated with diverse functions such as chromatin silencing, tumor invasion, and DNA damage response. On the other hand, confined migration primarily reduced intergenic chromatin accessibility, particularly near centromeres and telomeres. Furthermore, transcription was decreased after confined migration through the microfluidic devices. Taken together, we demonstrate that confined migration induces chromatin accessibility and transcription changes associated with heterochromatin formation, which provides insights on the long-term functional consequences of confined migration. Finally, to better study the long-term consequences of confined migration, I designed a novel nuclear envelope (NE) rupture reporter utilizing the Cre-loxP recombination system for permanent labeling, and a laser capture microdissection (LCM)-based single cell RNA-seq assay tailored for microfluidic migration devices. The NE rupture reporter produced more persistent labeling compared to the commonly used cGAS-mCherry reporter. However, the reporter exhibited high background labeling and, contrary to the design expectations, did not permanently labeled ruptured nuclei. The single cell RNA-seq assay demonstrates the feasibility of LCM for RNA collection from single cells in microfluidic devices. However, the high background noise hindered the interpretation of RNA-seq data. Nonetheless, the development and troubleshooting process of both tools provides useful insights for future optimization and new designs. This thesis, thus, provides the exciting discovery of migration-induced changes in chromatin states and accessibility, while presenting promising new tools that could enable future studies of long-term cell fates and gene expression changes resulting from confined migration.
dc.rightsAttribution 4.0 International
dc.subjectchromatin accessibility
dc.subjectconfined cell migration
dc.titleThe Impact of Confined Cell Migration on Chromatin States, Accessibility, and Gene Expression
dc.typedissertation or thesis, Molecular and Cell Biology University of Philosophy D., Biochemistry, Molecular and Cell Biology
dc.contributor.chairLammerding, Jan
dc.contributor.committeeMemberFromme, Chris
dc.contributor.committeeMemberSoloway, Paul

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Except where otherwise noted, this item's license is described as Attribution 4.0 International