Electronic Theses and Dissertations (Masters)

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Browsing Electronic Theses and Dissertations (Masters) by Keyword "Chromatin immunoprecipitation (ChIP)"

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    Investigating the regulation of PXDN expression by the early growth response 1(EGR1) transcription factor in the context of human fibrotic diseases
    (University of the Witwatersrand, Johannesburg, 2023) Makhanya, Thokozile
    Peroxidasin (PXDN) is a novel member of the peroxidase-cyclooxygenase family of haem containing proteins that catalyze oxidative reactions. It consolidates the extracellular matrix (ECM) by using hydrogen peroxide (H2O2) as a substrate to generate hypohalous acid intermediates, which help catalyze the formation of sulfilimine bonds between collagen IV protomers. The aberrant expression of PXDN has been linked to the development of various diseases where the architecture of the ECM is compromised such as cardiovascular diseases, ocular diseases, cancer, and fibrosis. Fibrosis develops due to repetitive tissue injury which is followed by aberrant wound healing that causes the excessive deposition of ECM proteins such as collagen into the injured tissue. In turn, ECM crosslinking enzymes such as PXDN are upregulated, and the matrix becomes thick and heavily crosslinked. This study aims to elucidate whether early growth response 1 (EGR1), a zinc-finger transcription factor which regulates cell proliferation, differentiation, apoptosis, and a key pro-fibrotic protein, can drive PXDN expression. To address the aim, HEK293 cells were treated with TGF-β1, a master activator of fibrotic genes, and western blot and immunofluorescence microscopy were performed to detect EGR1 and PXDN and their cellular localization, respectively. Chromatin immunoprecipitation (ChIP) was performed to determine if EGR1 binds to the PXDN promoter and the luciferase reporter assay was employed to determine if the interaction resulted in an alteration in gene expression. Our western blot findings showed that for EGR1, there was a statistically insignificant increase in protein expression in response to the TGF-β1 treatment. PXDN expression could not be quantified due to the high background on the blots. Further analysis by immunofluorescence microscopy showed that EGR1 expression was increased and was localised to the nuclei in response to the TGF-β1 treatment. We also observed that PXDN was predominantly expressed extracellularly and showed a significant increase in protein expression with treatment. The bioinformatics analysis has identified two putative EGR1 binding sites in the PXDN promoter and ChIP-PCR showed that binding occurred at one of these sites. This site was cloned into the pGL4.10 vector to determine whether EGR1 drives PXDN expression. Due to unsuccessful transfection optimization, the luciferase assay could not be performed and therefore for future work this assay needs to be performed to verify if EGR1 can drive PXDN expression. In conclusion, we showed that PXDN is a TGF-β1-responsive gene and may be regulated by EGR1. Studying the interaction of EGR1 and PXDN may establish roles for PXDN in fibrosis and further consolidate PXDN as a possible anti-fibrotic therapeutic target.