Differential expression of the GMCSF gene in the immune system regulated by epigenetic factors
Creators: name: Sprod, OR
Publisher: name: University Of Tasmania
Year: 2023

description: Granulocyte-macrophage colony stimulating factor (GM-CSF) is a cytokine that stimulates the production of leukocytes as part of an immune response. The role that GM-CSF plays in the immune system is reliant on its tightly controlled expression, both temporally and spatially. The overall aim of this thesis was to investigate the factors that contribute to the correct temporal and spatial expression of GM-CSF in immune cells. It was found that while GM-CSF expression can be stimulated in murine T cells but not B cells, key transcription factors involved in GM-CSF gene expression were present in both cell types, leading to the hypothesis that epigenetic mechanisms underlie this differential response. In support of this, differences in DNA methylation, histone modifications and the presence of chromatin remodelling proteins were detected at the GM-CSF promoter between the two cell types. DNA methylation levels were higher at a CpG dinucleotide in the GM-CSF promoter in T compared to B cell lines, and DNA methylation of the GM-CSF promoter blocked expression from a reporter plasmid. Demethylation of the promoter was not sufficient to enable GM-CSF gene expression in B cells, although it increased its expression in T cells. The effect of removing the CpG dinucleotide, which is contained in an Sp1 transcription factor binding site, was also examined. In a transiently transfected reporter model, removal of the Sp1 site resulted in loss of promoter activity. However, in a stably integrated transgene model, the Sp1 mutant promoter exhibited an increased response to stimulation. The differential response of the promoter mutant between the transient and stably transfected models suggests that the chromatin environment of the gene plays an important role in transcriptional regulation. To further examine the importance of chromatin in GM-CSF gene regulation, histone modifications were examined at the GM-CSF promoter in T and B cell lines. Several key differences were observed. In T cells, acetylation of histone H3 was increased at the GM-CSF promoter relative to B cells. Increasing promoter acetylation levels by treatment with the histone deacetylase inhibitor Trichostatin A (TSA) facilitated expression of GM-CSF in the B cell lines in response to stimulation. Furthermore, TSA treatment in combination with DNA demethylation had a synergistic effect on GM-CSF expression in both T and B cells. In contrast to histone acetylation, histone H3 lysine 27 trimethylation was lower at the GM-CSF promoter in T cells relative to B cells. Finally, the chromatin remodelling protein Brg1, which is known to interact with acetylated histones, was present at the GM-CSF promoter in T cells at higher levels than in B cells. These data suggest that enrichment of histone H3 acetylation and Brg1 and decreased H3K27Me3 contribute to the establishment of a 'permissive' chromatin environment at the GM-CSF promoter in T cells, which is not present at the promoter in B cells. A 'permissive' chromatin environment can be established at the GM-CSF promoter in B cells following treatment with TSA, which increases histone acetylation. This allows remodelling of the promoter chromatin and subsequent gene expression in response to immune signals. However, this induced 'permissive' state is not maintained. Following removal of the inducing stimulus in A20 B cells, the chromatin is reset to its original 'repressive' state and the GM-CSF gene becomes unresponsive to subsequent stimulation.