Induction of DNA complexes by type I and type II interferons

Cheng, Wai Kit (2016) Induction of DNA complexes by type I and type II interferons. MRes thesis, University of Nottingham.

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The Interferon (IFN) family is divided into two mainly classes which is type I IFN and type II IFN. Type I IFNs activate phosphorylation of STAT1 and STAT2 and induces their heterodimerization and association with IRF9 to form IFN-stimulated gene factor 3 (ISGF3) complex that binds to IFN-stimulated response elements (ISREs). Type II IFN induces STAT1:STAT1 homodimers (which is also known as GAF) that bind to IFN-gamma activated site (GAS) element. Besides, type I IFNs form GAF that bind to GAS element and activate STAT3 phosphorylation. Activated STAT3 is able to form either homodimers with STAT3 or heterodimers with activated STAT1 and bind to GAS element as well. Both ISGF3 and GAF complexes has distinct biological function but the balance between ISGF3 and GAF in type I IFN signalling pathway is not well understood. This project was aimed to investigate IFN-mediated induction of ISGF3 and GAF complexes of different cell lines. Electrophoresis mobility shift assay (EMSA) and western blot analysis were used to study the formation of ISGF3 and GAF complexes of each cell lines.

My experiments demonstrated serial dilution of protein extracts and increased final concentration of DTT in extraction buffers did not affect the formation of STAT3 homodimers, STAT3:STAT1 homodimers and GAF. Several optimization of experiment protocols were done to reveal these three DNA binding complexes. 4 out of 8 cell lines did not induce STAT3 homodimers although activated STAT3 was detected in these cells. 2 of these cells showed the formation of STAT3:STAT1 heterodimers but the concentration was lower than GAF.

On the other hand, 4 out of 8 cell lines induced ISGF3 complex under an hour of type I IFN-stimulation. Pre-treatment of low concentration of IFN-beta (priming) for 24 hours prior stimulation increased the DNA binding activity of ISGF3, GAF, STAT3 homodimers and STAT3:STAT1 heterodimers. However, priming affected differently on 8 cell lines.

In summary, there is cell type specific regarding the DNA binding complex such as ISGF3, STAT3 homodimers and STAT3:STAT1 heterodimers and IFN priming.

Item Type: Thesis (University of Nottingham only) (MRes)
Supervisors: Vinkemeier, Uwe
Subjects: Q Science > QR Microbiology > QR180 Immunology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 39050
Depositing User: Cheng, Wai
Date Deposited: 15 Feb 2017 13:59
Last Modified: 12 Oct 2017 21:55

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