Gene regulatory networks (GRNs) are central to every biological process from development to disease. GRNs are mediated through the activities of transcription factors (TFs), which interact in a sequence-specific manner with their target DNA elements to drive gene expression. In this thesis, two main aspects of GRNs are studied: (1) rewiring of GRNs by alternative TF isoforms, and (2) immune GRNs and strategies to modulate gene expression in immune diseases.
TF isoforms resulting from alternative splicing, alternative transcription start sites, or alternative transcription termination sites, are prevalent and can have profound changes in GRNs. However, the extent to which differences in TF isoforms affect global GRNs and how such regulatory network rewiring leads to altered gene expression programs remain unclear. In this thesis, a large clone collection of ~800 human TF isoforms was generated, and then used in high-throughput systematic experimental strategies to investigate the extent to which TF isoforms differ at the level of molecular protein-DNA interactions (PDIs) and transcriptional regulatory activities. The findings show that at least half of alternative TF isoforms exhibit functional differences and tend to behave like distinct proteins with different molecular capabilities. In the context of global GRNs, these findings reveal a widespread expansion of PDI and transcriptional regulatory capabilities through alternative TF isoforms. Altogether, this work constitutes an important step towards the long-term goal of contextualizing and functionalizing large numbers of TF isoforms in rewiring GRNs.
GRNs provide a wealth of information that can be leveraged in myriad ways including therapeutics. In particular, immune GRNs provide a framework for modulating cytokine gene expression, which are dysregulated in many human diseases. Proper cytokine gene expression is essential in development, homeostasis and immune responses. However, studies on the transcriptional control of cytokine genes over the last three decades have mostly focused on highly researched TFs and cytokines, resulting in an incomplete portrait of cytokine gene regulation. In this thesis, high-throughput assays were used to derive a comprehensive network that greatly expands the known repertoire of TF–cytokine gene PDIs and the set of TFs known to regulate cytokine genes. An enrichment of nuclear receptors was found and their role in cytokine regulation in primary macrophages was confirmed. Additionally, the network was used as a framework to identify TFs and synergistic TF pairs that can be targeted with FDA-approved drugs to modulate cytokine production. Finally, the PDI data was integrated with single cell RNA-seq datasets to identify druggable TF targets in cytokine-associated immune diseases (i.e., inflammatory bowel disease and COVID-19). Overall, this comprehensive cytokine GRN provides a rich resource to interrogate cytokine regulation in a variety of physiological and disease contexts.
Altogether, the work in this thesis accomplishes the following: (1) identifies alternative TF isoforms as a major driver of GRN rewiring, (2) delineates a comprehensive cytokine GRN that greatly expands three decades of research, and (3) leverages the cytokine GRN to identify candidate therapeutic TF targets in diseases associated with dysregulated cytokine gene expression. These findings contribute a significant step in the effort to understand mechanisms of GRN rewiring and to generate comprehensive GRNs that provide a framework for modulating gene expression, particularly in diseases. / 2023-11-01T00:00:00Z
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/43253 |
| Date | 01 November 2021 |
| Creators | Santoso, Clarissa S. |
| Contributors | Fuxman Bass, Juan I., Henderson, Andrew |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution-NonCommercial-NoDerivatives 4.0 International, http://creativecommons.org/licenses/by-nc-nd/4.0/ |
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