Non-disruptive 3D Profiling of Combinatorial Epigenetic Marks in Single Cells: Development and Applications of the Epi-PHR Method

Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Genetics

First Advisor

Wang, Siyuan

Abstract

AbstractNon-disruptive 3D Profiling of Combinatorial Epigenetic Marks in Single Cells: Development and Applications of the Epi-PHR Method Yanbo Chen 2025 In eukaryotic organisms, gene expression is intricately regulated by epigenetic modifications and chromatin's three-dimensional (3D) organization. Epigenetic modifications, such as DNA methylation and histone modifications, control chromatin accessibility and transcriptional activity, thereby regulating gene expression without altering the underlying DNA sequence. Simultaneously, the 3D folding of chromatin creates spatially distinct compartments within the nucleus, adding a layer of regulatory control over gene expression. Currently, sequencing-based methods are mainstream for epigenetic mark profiling and 3D chromatin folding measurements, like chromatin immunoprecipitation sequencing (ChIP-seq) and Hi-C, which provide high-resolution data but lack spatial information and typically require bulk cell populations. Conversely, imaging-based techniques retain spatial context but are limited in genomic specificity and multiplexing capabilities. To overcome these limitations, this thesis introduces Epigenetic Proximity Hybridization Reaction (Epi-PHR), a novel, non-disruptive, image-based method that enables high-resolution spatial epigenetic profiling in single cells. Epi-PHR employs a proximity-dependent hybridization reaction that allows direct visualization of combinatorial histone modifications at numerous genomic loci while preserving the native 3D genome architecture. This technique combines fluorescence in situ hybridization (FISH) targeting specific genomic regions with antibody-based detection of histone modifications. Engineered DNA hairpins facilitate a controlled hybridization chain reaction upon spatial proximity. Incorporating proteinase digestion and optimized oligonucleotide hybridization strategies, Epi-PHR achieves high signal specificity with minimal background fluorescence, making it a robust tool for in situ epigenetic analysis. A significant advancement of Epi-PHR is its multiplexing capability, enabling simultaneous profiling of multiple histone modifications at hundreds of genomic loci within individual cells. A 200-gene probe library design allows for extensive epigenetic mapping at specific genomic loci. Additionally, Epi-PHR has been extended to dual-marker profiling, permitting concurrent visualization of two distinct epigenetic marks. Further enhancing its applications, Epi-PHR integrates with chromatin tracing, a high-throughput spatial genomics technique that maps chromatin folding and 3D genome structure in single cells. This integration enables simultaneous visualization of locus-specific histone modifications and chromatin architecture, uncovering spatial correlations between epigenetic states and nuclear compartmentalization. Epi-PHR has been applied to study allele-specific epigenetic regulation at the Dlk1-Meg3 imprinted locus in hybrid mouse trophoblast stem cells. Combining Epi-PHR with single-nucleotide polymorphism (SNP) FISH distinguishes maternal and paternal alleles, demonstrating differential enrichment of the H3K9me3 mark and distinct chromatin folding patterns and providing new insights into epigenetic imprinting mechanisms. The results presented in this thesis establish Epi-PHR as a versatile platform for in situ epigenetic profiling, offering a new approach to profile epigenome and measure the spatial organization of the chromatin at the same time. This new method has broad applications for studying gene regulation, cellular heterogeneity, and disease-associated epigenetic alterations, particularly in cancer and developmental disorders. Future directions include expanding the method to incorporate spatial transcriptomics, allowing for simultaneous mapping of gene expression and chromatin states within the same single cells, and applying Epi-PHR to clinical samples to identify novel epigenetic biomarkers for disease diagnosis and therapy.

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