NF-κB signalling induces transcriptional upregulation of a wide array of genes in response to inflammatory signalling caused by, for example, TNFα cytokine. In addition to inducing the expression of factors which mediate an intracellular response, such stimuli also cause the expression of further signalling factors, including TNFα itself, to propagate and refine an initial stimulus. However, while such positive feedback signalling can be seen to be beneficial in amplifying potentially small initial stimuli, excessive production can cause hyper-inflammatory responses; an occurrence linked to several autoimmune diseases. Therefore, correct regulation – in regards to both too little and too much TNFα signal production – is essential for a balanced immune response. In this thesis I have focussed on the effects of the IκB protein family member BCL-3 on TNFΑ transcription: demonstrating NF-κB dependent induction of both TNFΑ and BCL3 genes and a subsequent negative role for BCL-3 in regulating TNFΑ transcription in the human fibrosarcoma HT1080 cell line – forming an Incoherent Feed Forward Loop (I-FFL) motif. Notably, I have shown a differential rate of induction of TNFΑ (rapid) and BCL3 (delayed) transcript levels; demonstrating that while the TNFΑ gene has a pre-stimulus RNA polymerase II bound and poised for a rapid response, the BCL3 promoter requires histone modification and chromatin remodelling for binding of NF-κB and RNA polymerase II. Extensive characterisation of the temporal sequence of events constituting BCL3 promoter remodelling, mRNA plus protein levels and NF-κB nuclear localisation through live cell microscopy allowed the construction of a mathematical model which has been tested to ensure it can accurately recreate biological behaviour. This model has been utilised to show that the delayed production of inhibitory BCL-3 produces distinct TNFΑ transcript dynamics: (i.) initially allowing a high magnitude response but coupled to later strong repression of TNFΑ expression and (ii.) producing a non-monotonic response to pulsed stimuli. This behaviour cannot be quantitatively recreated with models in which BCL3 transcription is induced simultaneously with TNFΑ and proposed physiological benefits are outlined. Based on this work, time delays in I-FFLs are proposed as a novel mechanism to produce varied output dynamics. Future research tools have also been developed in this work - including generation of an expression vector to visualise BCL-3 protein in live cells (utilising a BAC recombinant engineering approach) - plus further research questions and predictions regarding TNFα signalling have been raised by additional modelling work.

The oncogenic protein BCL-3, a member of the IκB family, was originally identified in a subset of human B-cell chronic lymphocytic leukemias that carry a translocation t(14,19), which results in BCL-3 overexpression. BCL-3 is also overexpressed in many solid tumors, such as in breast cancers and in cylindromas. This IκB protein activates or represses gene transcription through binding with the NF-κB proteins p50 and p52. Furthermore, BCL-3 is K63-linked polyubiquitinated, which leads to its translocation into the nucleus and to its target genes expression. BCL-3 is also K48-linked polyubiquitinated after GSK3 phosphorylation, which leads to its subsequent proteasomal degradation. However, the mechanisms underlying both its polyubiquitination and its ability to repress gene transcription remain poorly understood. In order to gain more insight into these BCL-3 functions, parallel screenings involving both yeast-two-hybrid experiments and biochemical purifications led to the identification of BCL-3-interacting partners. Those screenings identified CtBP as a molecule required for the ability of BCL-3 to repress gene transcription. CtBP is also required for the stability, for the oncogenic potential and for the ability of BCL-3 to inhibit UV-mediated cell apoptosis in keratinocytes. We also defined the E3 ligase TBLR1 as a key element involved in BCL-3 polyubiquitination and degradation through a GSK3-independent pathway and the proteasome subunit PSMB1 as a protein required for the GSK3-dependent and -independent proteasomal degradation of polyubiquitinated BCL-3. Importantly, all interactions require unique motifs within the amino-terminal domain of BCL-3. In conclusion, our data define multiple BCL-3-associated proteins that differentially and specifically regulate its function and stability and indicate that a better understanding of the mechanisms underlying the oncogenic properties of this IκB protein could be achieved through similar interactomic studies.

The transcription factor NF-κB interacts with many other co-regulator proteins that modulate its binding and transcriptional activity. One of these co-regulators, Pirin, is an iron-dependent metalloprotein that has been shown to enhance the DNA binding of NF-κB homodimers. Here, we characterize the interactions between Pirin and its known NF-κB binding partners and examined the role of Bcl-3, a protein that is required for Pirin’s interaction with p50. In addition, we use site-directed mutagenesis to alter conserved residues within Pirin’s metal binding environment and observed how it affected the DNA binding and conformation of the Pirin-NF-κB complex. These studies show that, while a similar enhancing effect on DNA binding is observed, the interactions of Pirin with different NF-κB members are distinct from each other and could possibly have different physiological purposes.