Date of Award
2026
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Viviana I. Risca
Abstract
Linker histone H1 is a key regulator of chromatin fibre structure, thought to promote formation of more compact nucleosome clutches as demonstrated in vitro through nucleosome array experiments. In cells, H1 depletion has been shown to impair stem cell differentiation, facilitate malignant B-cell transformation, and derepress satellite DNA, which implicates H1 in the maintenance of key regulatory and genome integrity pathways. However, how H1 shapes chromatin fibre structure across epigenetic states in the intact cellular environment, and its subsequent contributions to transcriptional regulation, remains to be fully elucidated. To address these questions, I applied RICC-seq 2.0, an improved version of radiation-induced correlated cleavage with sequencing, that probes DNA-DNA contacts within a ~3 nm physical radius in intact unfixed cells. Through a cross-species comparison, I establish a dynamic range of compaction measurements correlating with increasing linker histone stoichiometry and nucleosome repeat length observed across organisms. Then, through a multi-Gaussian decomposition of fragment length distributions, I apply an unbiased systematic analysis for extracting fibre structure parameters that can be compared across conditions and used to contrast and inform simulation models of chromatin fibre geometry. Applying this method to a doxycycline inducible CRISPRi system for acute pan-H1 depletion in K562 cells, I show that H1 depletion leads to a significant loss of higher-order nucleosome stacking contacts consistent with decompaction, a genome-wide shortening of nucleosome repeat length, and a reduction in the contrast between heterochromatic and euchromatic fibre structure. These structural changes are reversible upon recovering H1 levels via doxycycline washout, and are most pronounced at the short-range mesoscale under 1kb; while large-scale compartments and domain organisation are largely preserved. Upon such decompaction, I note increasing transcriptional derepression with ~1500 upregulated genes; and therefore a decoupling of gene expression changes from large scale compartment changes in our system. Upregulated genes are enriched for Polycomb complex targets CBX8, CBX2, and SUZ12 while H3K27me3 is globally reduced but not preferentially lost at upregulated loci. Regions gaining accessibility are compacted at baseline with a chromatin fibre structure resembling that of Polycomb-marked heterochromatin. These findings support a model in which H1-mediated fibre compaction maintains a fibre geometry preferrable to both PRC2 and PRC1. Furthermore, to test the susceptibility of chromatin states to genotoxic stressors, I adapted END- seq and GLOE-seq to isolate and sequence genome-wide break density in heavy-ion irradiated cells, in collaboration with the NASA Human Research Program. Here, we find that epigenetic state influences radiation-induced DNA damage susceptibility where euchromatic regions have a greater propensity for clustered damage events by heavy ion irradiation while more compact heterochromatin regions confer more protection against this irradiation regime. This work aims to measure chromatin compaction and link it as a mediator of transcriptional regulation and genome integrity under heavy ion genotoxic stress.
License and Reuse Information
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Recommended Citation
Canaj, Hera, "Chromatin Compaction in Transcriptional Regulation and Genome Integrity" (2026). Student Theses and Dissertations. 856.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/856
Comments
A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy