Date of Award

Spring 2022

Project Type

Thesis

Program or Major

Biological Sciences

Degree Name

Master of Science

First Advisor

Sherine F. Elsawa

Second Advisor

Don M Wojchowski

Third Advisor

Vicki Jeffers

Abstract

GLI3 is an important protein traditionally found in the Sonic Hedgehog pathway. It is involved in numerous events that take place during embryogenesis such as the differentiation and patterning of cells in the body. Monocytes are important innate immune cells which infiltrate the site of inflammation when a pathogen invades our body. Gli3 has also been shown to be upregulated in monocytes following pathogen stimulation, suggesting Gli3 could have an important role in mediating the release of inflammatory signals known as cytokines. The histone acetylase protein p300 traditionally acetylates histones to epigenetically modify them to allow DNA unwinding and subsequently gene expression. A physical interaction between p300 and GLI3 was shown in cancer cells and indicates that they could work together to modulate transcription of target genes, but no evidence of an interaction between these two proteins has been observed in other cell types. This thesis investigates how GLI3 and p300 work together in human monocytes to modulate inflammatory cytokine production. Quantitative PCR shows that GLI3 and p300 modulate pro-inflammatory cytokine expression, and both genes must be knocked down to decrease this cytokine expression. In addition, both GLI3 and p300 are required for CCL2 promoter activation in monocytes. Furthermore, GLI3 and p300 modulate CCL2 at the level of secretion. However, there was no synergistic effect of GLI3 and p300. Therefore, I attempted to clone GLI3 into a plasmid that expresses an engineered biotin ligase that promiscuously biotinylates nearby proteins known as TurboID. Using GLI3-TurboID, I can isolate biotinylated proteins and run mass spectrometry to identify novel proteins that GLI3 interacts with, furthering my potential research strategies. The data shown here is a step forward in understanding how the GLI proteins interact with the immune system during inflammatory conditions.

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