Functional Genomics Analysis of Gene Regulation and Cell State Dynamics

Abstract

Multiple modalities of genome function, including gene expression and gene regulation, integrate to drive cellular functions and orchestrate cell identity. Because cellular function changes across space and time, the underlying functional genome is correspondingly dynamic and associated with specific cell states. As a result, functional genomic technologies can indirectly capture cell states as represented by a modality of genome function. Furthermore, when functional genomic technologies are applied in a temporal manner or at single-cell resolution, they can be used as powerful tools to reconstruct dynamic biological processes and study cell state transitions. Regeneration is one of the most dynamic and tightly regulated biological processes, and in vertebrates, the skeletal muscle displays remarkable regenerative capacity. Skeletal muscle regeneration is mediated by a rare population of cells: the muscle stem cells (MuSCs), which undergo a series of cell state changes, including activation and differentiation, culminating in muscle repair. Following multiple rounds of injuries, the MuSC pool is replenished through a poorly understood self-renewal process that is crucial for long-term regenerative capacity. Thus, MuSC self-renewal, activation, and differentiation are elusive biological processes that are of high biomedical importance but remain largely uncharacterized due to their dynamism and complexity. They, therefore, represent an important application area for single-cell functional genomics. In this dissertation, I applied multiple existing single-cell technologies to reconstruct the epigenomic atlas of the regenerating muscle with unprecedented resolution. I focus especially on the cell state dynamism of MuSCs during muscle regeneration and characterize both the differentiation and self-renewal trajectories. Furthermore, I identified betaglycan as a novel marker and candidate regulator of MuSC self-renewal during muscle regeneration. In another domain but also inspired by the overarching goal of better understanding gene and cell fate regulation using functional genomics, I focus on technology development to capture a previously unexplored arm of gene regulation: the 3-dimensional (3D) cooperativity of gene regulatory elements in the genome. Gene regulatory elements represent an important aspect of genome function that is crucial for controlling gene transcription and cellular identity. While it is known that they form long-range 3D interactions, no current technology can capture their functional cooperativity at genome-wide scale. To fill this gap, I developed a novel technology, 3D-STARRseq, to capture 3D-genome interactions-mediated transcriptional cooperativity. Applying this technology to a cancer cell line, GM12878 cells, I identified for the first time, a relationship between long-range regulatory element cooperativity and gene expression specificity. I also identified for the first time unique patterns of transcriptional factor cooperativity associated with regulatory element 3D function, and uncovered a potential novel role of zinc finger repeat genomic regions in long-range gene regulation. These findings represent significant advancements both in the field of functional genomics and 3D-genome biology, and advance our understanding of cellular dynamics underlying muscle regeneration and muscle stem cell self-renewal.