Uncovering the hidden mechanisms governing the transcriptional regulation of inflammation

• Main Author: Fok, Ezio T
• Other Authors: Mhlanga, Musa
• Format: Doctoral Thesis
• Language: English
• Published: Faculty of Health Sciences 2021
• Subjects: Chemical and Systems Biology
• Online Access: http://hdl.handle.net/11427/32667

Inflammation provides broad immunological protection that is essential for our survival. This cellular response is characterised by a biphasic cycle consisting of an initial acute pro-inflammatory phase and a subsequent resolving anti-inflammatory phase. Underlying each of these phases are changes in the expression of hundreds of immune genes, which encode for inflammatory mediators called cytokines. Importantly, the biphasic nature of inflammation requires cytokine expression to be highly regulated and coordinated to different timescales during each phase of inflammation. For the initial proinflammatory response, cytokine expression needs to be rapid and robust to efficiently initiate host defence mechanisms and provide effective immunological protection. In contrast, the expression of anti-inflammatory cytokines is temporally delayed to ensure that anti-inflammation always follows pro-inflammation. In order to choreograph the expression of these cytokines during inflammation, numerous mechanisms within the cell serve to regulate and coordinate cytokine transcription. Within the eukaryotic nucleus, multiple modes of transcriptional regulation function cooperatively to provide the regulatory capacity that is required for complex transcription patterns to emerge. These include the organisation of the genome, which confine cognate chromosomal contacts that are causal to transcription, and long-non coding RNAs (lncRNAs) that function to discretely fine tune transcriptional activity. Although many of the mechanisms that regulate transcription have been well described, their role in cytokine expression during inflammation remains largely unknown. In particular, the mechanisms that facilitate rapid and robust cytokine expression during proinflammation and the regulatory networks that coordinate the biphasic regulation of inflammation are unresolved. In this work, two novel lncRNAs were discovered to transcriptionally regulate these key features of cytokine expression during inflammation. The first, UMLILO (Upstream Master LncRNA of the Inflammatory chemokine LOcus), was found to emanate from the ELR+ CXCL chemokine TAD and regulate the transcriptional activation of the pro-inflammatory ELR+ CXCL chemokines (IL-8, CXCL1, CXCL2 and CXCL3). By exploiting the pre-formed local 3D topology, UMLILO is able to epigenetically prime the chemokines for transcriptional activation. This involves the discrete deposition of H3K4me3 onto the promoters of the chemokines, which allows for the pre-loading of transcriptional machinery prior to their signal-dependent activation. This reveals a fundamental mechanism for the epigenetic priming and rapid activation of pro-inflammatory cytokine genes. The second lncRNA, called AMANZI (A MAster Non-coding RNA antagoniZing Inflammation), was found to coordinate the transcription of two functionally opposed cytokines: the master pro-inflammatory IL-1β and the broad antiinflammatory IL-37. AMANZI is encoded in the promoter of IL-1β, which results in its concomitant expression when IL-1β is transcriptionally active. Functionally, AMANZI mediates the formation of a dynamic chromosomal contact between IL-1β and IL-37. This leads to the delayed transcriptional activation of IL-37 ensuring that the pro-inflammatory function of IL-1β precedes IL-37 mediated anti-inflammation. This revealed a novel biphasic circuit that coordinated the expression of IL-1β and IL-37, through the activity of AMANZI, to regulate the two functionally opposed states of inflammation. Clinical observations in healthy individuals revealed that a polymorphism occurring in AMANZI (rs16944) was able to augment the state of this genetic circuit and shift the relative levels of IL-1β and IL-37 to influence an individual's inflammatory capacity. This affected the establishment of innate immunological memory, which is involved in the progression of many inflammatory conditions and the efficacy of certain vaccines. The work described here uncovers novel mechanisms that transcriptionally regulate key features of the inflammatory response. Importantly, this work implicates the role of two novel lncRNAs in inflammation, essentially contributing to the functional annotation to the genome and providing novel targets for the modulation of pathogenic inflammation.