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Systems biology of the type 1 interferon response
thesisposted on 31.01.2017, 00:53 by Forster, Samuel Charles
The innate immune response represents the first line of defence against detrimental conditions, controlling the response to infectious agents, abnormal cellular conditions or metabolic products. This system plays a crucial role in the early defence mechanisms, determining the immediate cellular response and recruiting and sculpting the adaptive immune cells. In the initial hours after challenge it is the responses initiated by the innate immune system that will play a pivotal role in determining the survival outcome. Fundamental to this process is protective Interferon (IFN) activation and signalling. This component of the response is essential for host survival, fundamental to vaccine responses, can be activated therapeutically and is often the target for pathogen evasion strategies. Uncontrolled or aberrant cytokine responses including IFNs can be toxic and induce autoimmunity. While there is considerable knowledge of transcriptional regulation and role of protein coding transcripts, equivalent knowledge of the non-coding RNA and their regulatory mechanism remain to be elucidated. Furthermore, a detailed understanding of microRNA regulation of the host biological response to IFN remains to be determined. Previous microarray based, large scale bioinformatics analysis have identified approximately 2000 genes regulated by IFN in a subtype, timing and a tissue specific manner. The work presented herein applies high throughput, whole transcriptome RNA-sequencing in combination with HITS-CLIP sequencing, experimental validation and computational analysis to further expand the understanding of this important system. Initial analysis identified key limitations in computational tools available for transcriptional analysis. To address this weakness and ensure maximum quality analysis of the generated transcriptional data, a comprehensive analysis framework, RNA-eXpress was developed. This framework provides a capacity to define and efficiently analyse next generation sequencing data regardless of instrument or mapping strategy. This method enables the discovery and annotation of the transcriptional landscape as required for thorough analysis of these large datasets. A full description of this framework is presented in Chapter 3. To generate a comprehensive overview of the IFN response extensive, whole transcriptome RNA-sequencing was performed examining the IFNβ response 0, 1 and 3 hours after stimulation. Utilizing the RNA-eXpress analysis approach, this work has identified differential gene expression in 2714 transcripts including 1149 which are down-regulated. This characterization included precursor microRNA and 84 putative long non-coding RNA. Gene set enrichment analysis on co-regulated clusters confirmed immune signatures and identified cell cycle and migration association pathways associated with down-regulated genes. An expanded version of the Interferome database was developed to facilitate this analysis and characterize the response relative to known datasets. Transcriptional regulatory mechanisms were correlated with transcriptional response applying a specifically developed enrichment analysis method. This work identified ISRE, STAT, IRF, NF-κB and Ets transcription factor signatures correlating with expression profile in induced genes. The uncharacterized Zinc Finger Protein 219 represented the only enriched transcription factor in down-regulated genes suggesting a post-transcriptional regulatory mechanism responsible for down-regulation. In addition approximately 25 percent of regulated genes exhibited modified 3’ UTR regions in response to IFNβ. These changes were validated in response to both IFNβ and Toll-like receptor ligands and were hypothesized to play a crucial role in the alteration of microRNA targeting. To characterize the nature of the IFNβ induced small RNA responses, including microRNA, high throughput small RNA sequencing was performed in parallel to the large RNA sequencing. This analysis identified 586 differentially regulated small RNA including 98 annotated known microRNAs, 86 predicted microRNAs and a diversity of other small RNA that were not investigated in this work. HITS-CLIP sequencing of the 3 hour time point confirmed interaction of 119 microRNA with the RISC complex, validating 55 of the predicted microRNA and identifying 3865 interactions with 3’ UTR regions. Network based methods were applied for computational analysis, detecting a key set of 11 anti-viral microRNAs that targeted genes, including Irf1. Key genes, highly targeted by microRNA including Cxcr4 and Rsad2. Application of a specifically developed fluorescent reporter construct validated these interactions and confirmed an additive impact of cumulative targeting by different microRNAs. Finally Stat1 and Tlr4, both of which demonstrated altered 3’UTR in response to IFN were shown to be selectively targeted in a 3’UTR dependent manner. This understanding of the structure and subtleties of the signalling pathways, made possible through novel computational methods, cutting-edge technology and extensive experimental analysis performed in this work provides a foundation for further studies in this area. Ultimately the development of this understanding provides an important basis of understanding with a potential to provide disease insights, facilitate the development of novel therapeutics and provide an increased knowledge of host-pathogen interactions.