Multiple isoforms of promyelocytic leukaemia protein (PML) participate in various cellular activities including innate immune responses, gene transcription and cell apoptosis. Recent work in our laboratory demonstrated PML isoform II (PML-II) was required for type I interferon (IFN) and IFN-stimulated gene (ISG) transcription by regulating transcription factor recruitment at gene enhancers/promoters. This thesis examines the mechanistic role of PML-II in this process. Considering that the different C-terminal fragments of PML isoforms play distinct roles in PML function, the function of the PML-II C-terminal domain was investigated. A C-terminal region 615-758, defined by deletions Δ1 and Δ2, was essential for its function in the IFN response, while the N-terminal RBCC structure was dispensable. Removal of residues 615-758 greatly impaired PMLII binding with transcription factors NF-κB and STAT1, coactivator (CBP) and SWI/SNF chromatin remodeling complex component Brg-1. These binding sites were further refined using smaller deletions to show that residues 645-665 were critical for PML-II binding with these transcription-related factors. The effect of PML-II on chromatin remodeling and histone modification at target promoters was investigated. Both PML-II and Brg-1 regulated the recruitment of transcription factor (STAT1) and the enrichment/disposition of H3K9me3 histone marks at ISG promoters but had no effect on H3K4me3 at these promoters. Depletion of PML-II also impaired ISG promoter binding by SWI/SNF core subunits Brg-1 and BAF155. Recruitment of activators, coactivators, and other regulators/mediators to ISG promoters occurred sequentially and was interrupted by PML-II depletion. This suggested that PML-II is required for forming and stabilizing the whole transcriptional complex which contains various factors including STAT1, CBP, Brg-1 and BAF155. Finally, the role of PML-II in IFNα-induced cell apoptosis was studied. Knockdown of PML-II enhanced ERK and AKT signaling, suggesting activation of both pro-survival pathways. Moreover, depletion of PML-II greatly decreased expression of IFNα-induced pro-apoptotic proteins including ISG15/54, OAS1, PUMA and TRAIL. It also impaired IFNα-mediated inhibition of AKT signaling and consequently increased cell resistance to IFNα -induced cell apoptosis. Collectively, in this study we have shown the specific involvement of the PML-II C-terminal domain in IFN responses, and mapped the sequences within this domain that are necessary for its interaction with relevant transcription factors. I have also examined in detail the physical and functional interaction of PML-II with the SWI/SNF complex and shown the points in the transcription activation pathway at which PML-II acts. Finally, we have shown how impaired PML-II activity reduces the pro-apoptotic signaling that normally occurs downstream of an IFN response, suggesting a mechanism by which PML may provide its known tumour-suppressor function.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:742281 |
| Date | January 2018 |
| Creators | Meng, Xueqiong |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/102618/ |
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