Probing the Spatial Architecture and Function of Genome and Transcriptome

Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Genetics

First Advisor

Wang, Siyuan

Abstract

Spatial architecture is intimately linked with biological processes and functions. The genome is arranged in a three-dimensional pattern within the cell nucleus, playing a vital role in gene regulation and cellular activity. Similarly, the spatial distribution of RNA within cells and tissues is essential for proper function, and the organization of different cell types is critical for overall tissue performance. During my Ph.D., I applied and developed cutting-edge imaging-based methods to profile the spatial organization of genome and transcriptome across various cellular contexts, and further investigated the functional and regulatory mechanisms underlying these spatial arrangements.The emergence of chromatin tracing, a multiplexed fluorescent in-situ hybridization method, enables us to study the spatial arrangements of chromatin in single cells. In the first work, we performed chromatin tracing to visualize the fine-scale chromatin folding of inactive and active X chromosomes in female human cells. Our work revealed that highly variant single-cell domains exist on both inactive and active X chromosomes, and are resistant to the perturbations of major epigenetic components. While the role of three-dimensional (3D) chromatin folding in regulating gene expression and DNA replication has been extensively studied, the impact of genome architecture on the targeting of somatic hypermutation (SHM) in B cells remains unexplored. In our second study, we adapted the Multiplexed Imaging of Nucleome Architectures (MINA) method—which integrates RNA MERFISH, chromatin tracing, and protein staining—to human tonsil samples to examine how chromatin folding influences SHM susceptibility across the genome. Our study revealed that nuclear positions and looping interactions of genomic regions are linked to their susceptibility to SHM. Despite the critical role of chromatin folding, the factors governing chromatin topology remain incompletely understood, largely due to the lack of efficient technologies for screening new regulators of multiscale chromatin organization. In the third work, we developed an image-based high-content screening platform (Perturb-tracing) that combines pooled CRISPR screen, a new cellular barcode readout method (BARC-FISH), and chromatin tracing. With the application of this methods, we discovered tens of new regulators of chromatin folding at different length scales. Recent breakthroughs in spatial transcriptomics technologies have allowed researchers to study spatial RNA expression and diverse cellular identities, compositions, interactions, spatial organizations. Yet existing spatial transcriptomics tools are still limited in either transcriptomic coverage or spatial resolution. In the fourth work, we develop a new image-based spatial transcriptomics technology with whole-genome level coverage while retaining single-molecule spatial resolution in intact tissues. Our analyses reveal differential subcellular localizations of diverse transcripts, cell-type-specific and cell-type-invariant tissue zonation dependent transcriptome, and gene expression programs underlying preferential cell-cell interactions. Finally, we further develop our technology for direct spatial readout of gRNAs in an image-based high-content CRISPR screen.

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