Investigating the effects of IFNγ signalling on transcriptional, epigenetic and enhancer landscapes

Thesis

IFNγ signalling can have contradictory roles in a tumour microenvironment. Predominantly, IFNγ has an anti-tumour function, activating the immune system against the cancer. However, pro-tumour activities of IFNγ have also been observed. Its choice between the two opposing outcomes appears to be context dependent and the mechanism instructing this choice isn’t well understood. One prevailing determinant comes from the IFNγ-induced upregulation of PD-L1, an immunosuppressive ligand. Binding of PD-L1 to its receptor, PD-1, inhibits immune cells despite the presence of other activating signals, enabling cancer immune evasion. Increased knowledge of IFNγ-induced gene expression within cancer cells, including the regulation of PD-L1, would enable a more accurate prediction of IFNγ’s behaviour in the tumour microenvironment.

This thesis examines the effect of IFNγ signalling on the transcriptional, epigenetic and enhancer landscape in a hepatocellular carcinoma cell line, Hep3B. Using a variety of genome-wide sequencing techniques, I find that IFNγ orchestrates differential gene expression and enhancer activity dynamically in at least three distinct phases within 24 hrs of treatment. IFNγ treatments were found to regulate more enhancers than genes, with a large proportion of these enhancers becoming primed but not fully activated. Capture-C, a 3C based technique, revealed that IFNγ treatments significantly increase promoter:enhancer interactions, adding a further level of gene regulation.

IFNγ treatments induced upregulation of PD-L1 in Hep3B cells. Capture-C revealed that the PD-L1 promoter interacts with the closest neighbouring promoter and a CTCF site downstream of the gene. Neither of these interactions appeared to regulate IFNγ-induced PD-L1 expression. Instead, this thesis proposes that the proximal promoter may be sufficient for IFNγ-induced upregulation of PD-L1. Altogether, this study increases our understanding of IFNγ-induced gene regulation.

Authors: Lamstaes, A

• DOI: 10.5287/ora-kopq5m5ad
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: IFNγ; gene expression; regulatory elements; PD-L1; enhancers
• Subjects: Immunology; Genetics; Molecular biology