https://escholarship.org/uc/item/0fg13906
UC San Francisco Electronic Theses and Dissertations – Publication Date: 2024
Takagishi, Seesha
ABSTRACT
Although there are many environmental stimuli and possible cellular responses, there are a finite number of transcription factors (TFs) in the cell to coordinate these responses. One way the information carrying capacity of a single signal is multiplexed is through the combinatorial effect of TFs, where two or more inputs modulate the expression of a single gene. Functionally, it has been hypothesized that these combinatorial dynamics are utilized by cells to fine tune cellular responses, such as with the NFAT TFs and the immune response. NFAT (nuclear factor of activated t-cells) activity is regulated both physically and temporally. Physically, NFAT is known to bind several cofactors that modulate the subsequent transcriptional response. Temporally, NFAT isoforms are known to have divergent nuclear translocation responses to the same environmental stimulus. Additionally, a single environmental stimulus could activate several pathways simultaneously, such as the case with NFAT and NFkB, which are both known to behave dynamically. Here, we leverage an epistasis experimental strategy and optogenetics to dissect combinatorial gene regulation involving NFAT1. We replicate the sustained nuclear translocation of NFAT1 and pulsatile nuclear translocation of NFAT4 in response to IgE-mediated mast cell activation in the mast model cell line RBL-2H3. For the first time, we showed that IgE antigen stimulation results in many transcriptional changes, of which only a subset are NFAT1 or NFAT4 specific. We demonstrated that there are distinct and overlapping roles of NFAT1 and NFAT4 in the IgE antigen transcriptional response, and this response involves other factors. We used the optogenetic tool CLASP (Controllable Light Activated Shuttling and Plasma membrane sequestration) for NFAT1 and successfully controlled NFAT1 nuclear translocation with diverse blue light inputs. We found that blue light alone produced significant transcriptional activity in our cells of interest, but also found that NFAT1-CLASP produced specific NFAT1-like effects upon light-induced translocation. Overall, we identified the NFAT specific signalling downstream of IgE stimulation and demonstrated a system capable of dissecting the information encoded in NFAT1 nuclear translocation dynamics.
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