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In the development of the central nervous system, thousands of neuronal subtypes must be generated, each with their own unique molecular properties. This process is governed by selector transcription factors, which specify cell-identities by binding to cell-type specific genomic regulatory elements. These regulatory elements, dispersed across extragenic regions of the genome, establish precise long-distance interactions with target gene promoters to regulate their expression. While prior studies have emphasized the roles of distally bound selector transcription factors in cell-type specification, the involvement of gene promoters in the regulation of gene expression remains underexplored. In this dissertation, I analyze the role of promoter elements in regulating neuronal gene expression programs using a comprehensive approach that combines high-throughput genomics and targeted experimental manipulations. In Chapter 2, I reveal a highly flexible regulatory system utilized in the nervous system: neuronal promoters are universal and can thus be activated by any enhancer found within their regulatory neighborhood. This model of promiscuous neuronal promoters raises two important questions: Is promoter promiscuity a universal phenomena? What are the promoter elements that facilitate universality? To address these questions in Chapter 3, I first find that promoters of genes associated with pluripotency exhibit incompatibility with neuronal enhancers. Then, to test what promoter elements encode for this incompatibility, and also which elements endow neuronal promoters with their promiscuity, I developed a novel promoter-screening strategy. Through this work, I discovered novel aspects of enhancer-promoter communication. First, core promoters are universal and can be induced by non-cell identity matched distal enhancers. Second, promoter-proximal regions serve to modulate expression from universal core promoters by either dampening or potentiating their responsiveness to distal enhancers. This work suggests that in addition to distal regulatory elements, promoter-proximal regions also play an active role in fine-tuning cell-type specific gene expression programs by either modulating induction or repressing ectopic expression. Finally, in Chapter 4, I explore another aspect of the regulation of cell-identity during development, shifting my focus away from selector transcription factors and instead on “secondary” transcription factors induced during differentiation. Here, I utilize a multi-omic approach to characterize the role of Mnx1 in motor neuron development. Analysis of its effects on gene expression, distal genomic binding patterns, and influence on the overall regulatory landscape reveals that Mnx1 plays a role in maintaining the motor neuron cell-identity by ensuring robust expression of motor neuron genes and preventing ectopic expression of genes normally restricted to alternate neuronal subtypes. This suggests that “secondary” transcription factors play a role in refining cellular identities established by selector transcription factors. Integrating these findings with prior research in central nervous system development underscores that while neuronal gene expression programs are primarily established through the actions of selector transcription factor-bound distal regulatory elements, promoters and secondary transcription factors contribute to the fine-tuning of transcription and, consequently, cell identity.

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E-cigarettes have recently become increasingly popular, especially amongst middle and high school students. Although they are marketed as safer alternatives to tobacco cigarettes, they produce toxic metals and carcinogenic nitrosamines. This thesis studies the effects of e-cigarette aerosol on the growth and proliferation of oral epithelial cells because the consequences of vaping, including a potential risk for aberrant growth leading to cancer, are not well understood. Cells were grown in matrigel, causing the formation of three-dimensional spheroids modeling the physiological architecture of the oral epithelium. Those spheroids were chronically exposed to vape with different treatment conditions to study the functional biological effects of the presence of nicotine, dosage, and different types of exposure. The diameter of the spheroids was measured throughout the process as an indicator of cell growth. It showed that the vape exposure, especially nicotine-rich aerosol, induces an increase in spheroid diameter in a dose-dependent manner. The increased cell growth is supported by enhanced metabolic activity as well as increased aldehyde dehydrogenase activity, a marker of stemness prominently upregulated in cancer stem cells. Protein was extracted at the end for protein expression analysis through Western blotting and the identification of the activation of survival signaling pathways and stem cell markers. Lastly, spheroids were co-cultured with Strep. mutans, a cariogenic bacterial resident in the oral cavity, and acutely exposed to vape. Co-culture with S. mutans did not significantly affect spheroid growth under the current experimental conditions or significantly change the expression patterns of proliferation and tumor initiation proteins. Future research will include tumorigenic assays and investigate how vape may induce carcinogenesis of the oral epithelium.

Nervous systems are made of a large diversity of neuron types, that are characterized by specific molecular markers, their unique morphology and connectivity patterns, or their electrophysiological profiles. Those terminal differentiation features are specified by combination of transcription factors, defining genetic programs that controls neuronal identity. Some transcription factors act as "master regulators" or "terminal selectors", binding directly and continuously to many effector genes of a specific neuron type. Neuronal identity is specified by the code that the combinations of those transcription factors define. It is hard to assess to which extent those mechanisms shape more complex nervous systems, while C. elegans has a small number of well- defined neuron classes that make it more experimentally tractable. Thanks to CRISPR tagged alleles, a novel multicolor C. elegans reporter transgene strain and recent scRNA-Seq datasets, it has been possible to determine the full expression pattern of the gene family that is most likely to control neuronal identity, the highly conserved homeodomain transcription factors. From this, it is now possible to study their functional effects on neuronal identity on the full complement of C. elegans neuron types. I show that previously understudied homeodomains can indeed act as terminal selectors. I find that multiple types of neurons that had no previous known master regulators can be attributed a homeodomain regulator. I focus on the SIX homeodomain subfamily and identify a new type of subordinate transcription factor, "subtype terminal selectors". I find that such a "subtype terminal selector" is sufficient and necessary to specify a subset of features separating two very closely related neuron types, under the control of a shared terminal selector that control both shared and subtype specific features in those classes. Homeodomains remain front and center in neuronal identity control and could plausibly contribute to the specification of every single neuron class in C. elegans. Moreover, they have very conserved roles starting even in primitive nervous systems and likely played a major contribution to the evolution of cellular diversity in the nervous system. With my subtype terminal selector examples, I show possible mechanisms through which an ancestrally shared neuron type could progressively diverge towards two distinct neuron types.

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