Uncovering Mechanisms and Functions of Spatial Genome and Transcriptome
Date of Award
Spring 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Genetics
First Advisor
Wang, Siyuan
Abstract
Recent advances in spatial genomics and transcriptomics technologies have enabled the detection of various spatial biology phenotypes, such as chromatin organization and subcellular RNA localization. These phenotypes are important in many fundamental biological processes such as gene regulation, cell differentiation and cell migration, which are highly relevant to human health including developmental disorders, aging and cancer. Therefore, it is important to uncover the subsequent mechanisms and functions of these spatial phenotypes to advance our understanding in both basic biology and clinical questions. In this dissertation, I first introduce a strategy to study the mechanisms and functions of spatial biology via forward genetics. Together with colleagues from the Wang Lab, I developed an image-based CRISPR screen method termed barcode amplification by rolling circle and fluorescent in situ hybridization (BARC-FISH). Using this method, we screened 137 genes on their effects on multiscale genome organization, and identified a highly clinically important gene, CHD7, that regulates chromatin conformation in a CTCF-dependent manner. The phenotype landscape of image-based CRISPR screen can be further expanded by coupling with other spatial omics techniques, potentially enabling novel biological discoveries. Spatial omics techniques also enable unbiased marker discovery in a spatial context, which is another way for mechanistic and functional discoveries. In collaboration with the Nicoli Lab at Yale, I applied the spatial omics technique, multiplexed error-robust fluorescent in situ hybridization (MERFISH), to explore the role of mRNA localization in the organization and function of focal adhesions (FA). We identified key mRNA species that modulate FA function in a translation-independent manner, and demonstrated that untranslated mRNAs are tethered to FA by the RNA binding protein G3BP1. The untranslated mRNAs at FAs regulate the assembly and disassembly rate of FAs, and ultimately cell migration speed. This study challenges for the first time the canonical view that the function of mRNAs is solely to produce protein. Lastly, to facilitate the application of multiplexed fluorescent in situ hybridization (FISH) to the broader scientific community, I present a computational tool for convenient design of multiplexed FISH probes, including chromatin tracing (multiplexed DNA FISH), MERFISH and single-molecule FISH.
Recommended Citation
Hu, Mengwei, "Uncovering Mechanisms and Functions of Spatial Genome and Transcriptome" (2023). Yale Graduate School of Arts and Sciences Dissertations. 1055.
https://elischolar.library.yale.edu/gsas_dissertations/1055
