Chen, Yixiang (2014) The role of PML-II in type I interferon response and gene transcription regulation. PhD thesis, University of Warwick.

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Abstract

The promyelocytic leukaemia (PML) gene encodes seven principle protein isoforms due to mRNA alternative splicing; different isoforms have different functions. This study examines the role of PML isoform II (PML-II), in interferon (IFN) and innate immune responses. It was found that siRNA knockdown of PML-II not only inhibited the expression of IFNβ and a large number of IFN-stimulated genes (ISGs) such as ISG15, ISG20, ISG54, ISG56, IP-10 and IRF7, but also affected the induction of many proinflammatory cytokines and chemokines such as IL-6, IL-8, TNFα and RANTES. Signalling to activate these two pathways works principally through regulating interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB), respectively. Knockdown of PML-II indeed affected IRF3 and NF-κB activities. However, it had no significant effect on IRF3 and NF-κB nucleus translocation and IRF3 phosphorylation, two features of activation of these factors. These results suggested that PML-II does not affect these signalling pathways, but works downstream of IRF3/NF-κB activation. It was hypothesised therefore that PML-II played a positive role in the assembly of transcription factors at enhancers/promoters.

Using co-immunoprecipitation and chromatin immunoprecipitation experiments, it was found that PML-II could form protein complexes with co-activator CREB-binding protein (CBP) together with IRF3 or NF-κB. Depletion of PML-II reduced IRF3 and NF-κB binding to chromatin at promoters/enhancers of genes such as IFNβ, and also reduced the recruitment of co-activator CBP. PML-II depletion also impaired activation of IFNresponsive genes by signalling from the type I IFN receptor via the JAK-STAT signalling pathway in a similar fashion.

Type III IFNs (IFNλ) are induced and function similarly to IFNα/β while type II IFNγ induces downstream signalling through activating the same transcription factor STAT1 as utilized in the type I IFN response. Extending this study to these IFNs, depletion of PML-II greatly reduced type III IFN expression. It also reduced IFNγ-mediated signalling thus reducing the expression of IFNγ-stimulated genes. PML-II was also identified as a key positive regulatory component of TNFα-mediated NF-κB signalling to stimulate the production of the proinflammatory cytokines such as IL-6, IL-8, RANTES and IP-10 by affecting TFs assembly and CBP recruitment at promoters.

From these data, a model is proposed in which PML-II contributes to the transcription of multiple genes via its association with CBP-transcription factor complexes, which promotes the stable assembly of these complexes at promoters/enhancers of target genes. Through this mechanism, PML-II plays a significant positive role in the development of IFN responses, and hence in the response to virus infection.

Item Type:	Thesis (PhD)
Subjects:	R Medicine > RC Internal medicine
Library of Congress Subject Headings (LCSH):	Immune response -- Molecular aspects
Official Date:	April 2014
Dates:	Date Event April 2014 Submitted
Institution:	University of Warwick
Theses Department:	School of Life Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Leppard, Keith
Extent:	215 leaves :|billustrations (some colour), charts.
Language:	eng

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