The role of BCL-3 feedback loops in regulating NF-kappaB signalling

Abstract

NF-kappaB signalling induces transcriptional upregulation of a wide array of genesin response to inflammatory signalling caused by, for example, TNFalpha cytokine.In addition to inducing the expression of factors which mediate an intracellularresponse, such stimuli also cause the expression of further signalling factors,including TNFalpha itself, to propagate and refine an initial stimulus. However, whilesuch positive feedback signalling can be seen to be beneficial in amplifyingpotentially small initial stimuli, excessive production can cause hyper-inflammatoryresponses; an occurrence linked to several autoimmune diseases. Therefore,correct regulation - in regards to both too little and too much TNFalpha signal production- is essential for a balanced immune response.In this thesis I have focussed on the effects of the IkappaB protein family member BCL-3 on TNFΑ transcription: demonstrating NF-kappaB dependent induction of both TNFΑand BCL3 genes and a subsequent negative role for BCL-3 in regulating TNFΑtranscription in the human fibrosarcoma HT1080 cell line - forming an IncoherentFeed Forward Loop (I-FFL) motif. Notably, I have shown a differential rate ofinduction of TNFΑ (rapid) and BCL3 (delayed) transcript levels; demonstrating thatwhile the TNFΑ gene has a pre-stimulus RNA polymerase II bound and poised fora rapid response, the BCL3 promoter requires histone modification and chromatinremodelling for binding of NF-kappaB and RNA polymerase II. Extensive characterisationof the temporal sequence of events constituting BCL3 promoter remodelling, mRNAplus protein levels and NF-kappaB nuclear localisation through live cell microscopyallowed the construction of a mathematical model which has been tested to ensure itcan accurately recreate biological behaviour.This model has been utilised to show that the delayed production of inhibitoryBCL-3 produces distinct TNFΑ transcript dynamics: (i.) initially allowing a highmagnitude response but coupled to later strong repression of TNFΑ expression and(ii.) producing a non-monotonic response to pulsed stimuli. This behaviour cannotbe quantitatively recreated with models in which BCL3 transcription is inducedsimultaneously with TNFΑ and proposed physiological benefits are outlined. Basedon this work, time delays in I-FFLs are proposed as a novel mechanism to producevaried output dynamics.Future research tools have also been developed in this work - including generationof an expression vector to visualise BCL-3 protein in live cells (utilising a BACrecombinant engineering approach) - plus further research questions and predictionsregarding TNFalpha signalling have been raised by additional modelling work.

Date of Award	31 Dec 2012
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Dean Jackson (Supervisor) & Michael White (Supervisor)