Interferon regulatory factor 1 (IRF-1), the founding member of IRF family, is a nuclear transcription factor first described as a transcription factor that binds to the upstream region of interferon induced genes following viral infection. In addition, IRF-1 has been reported to be involved in cell growth regulation, induction of apoptosis, immune responses, post-transcriptional modification, and cell transformation by oncogenes. Thus, IRF-1 shows accumulative evidence supporting the theory that IRF-1 functions as a tumour suppressor. However, we still lack the knowledge in the regulation and function behind IRF-1 and many other tumour suppressors due to the lack of synthetic tools that can aid in understanding the mechanism of cancer biology. Here we described the creation of synthetic tools that can be applied to study the role of a transcription factor(s) in cancer biology. Firstly, we described the creation, using recombineering technology, of universal bacterial artificial chromosome (BAC) targeting vector. This targeting vector, carry a cre-conditioned STOP cassette that can be targeted at a desired specific area. The resulted targeting vector can aid the generation of mice models with a conditioned knock-in subtle mutation(s). The resulted cre-conditioned mice models are an essential tool for any outstanding research project in cancer biology. Secondly, we described the development of Flp-In System™ from Invitrogen; the system can ease the generation of isogenic stable mammalian expression cell lines. Using this system, we created two isogenic stable cell lines expressing wild-type IRF-1 and a mutant that abolish IRF-1 DNA binding ability (W11R). Both cell lines were investigated using microarray analysis revealing new IRF-1 target genes. We reported the up-regulation of expected standard interferon regulatory genes such as, interleukin-24 (IL-24) and interferon regulatory factor-2 binding protein-2 (IRF2BP2) and the up-regulation of standard apoptotic genes such as, early growth response-1 (EGR-1) and prostate transmembrane protein, androgen induced-1 (PMEPA1) confirming the role of IRF-1 as a tumour suppressor. However, we also reported the up-regulation of secreted phosphoprotein-1 (SPP1) and SH3 and PX domains-2A (SH3PXD2A) which are matricellular protein produced by cancer cells playing a role in cellular adhesion, invasion, tumour growth progression and metastasis. Thus, we proposed a new biological role of IRF-1 in cellular movement. Thirdly, we described the development of a synthetic stable reporter cell line which can report IRF-1 transcriptional activity; such reporter cell line can be used once large scale screening is needed. The created stable reporter cell line was used to screen a kinase inhibitor library which has revealed C3 as an IRF-1 modifier. The newly identified IRF-1 modifier regulates IRF-1 transcriptional activity by inhibiting platelet-derived growth factor receptor (PDGFR) and/or vascular endothelial growth factor receptor (VEGFR) tyrosine kinase. Finally, we validated the synthetic Flp-In System™ by testing the system using a novel oncoprotein model. We have developed a stable cell line that overexpresses an oncoprotein named Anterior Gradient 2 (AGR-2). We have found that AGR-2 can attenuate IRF-1 protein levels dependent of p53. In addition, AGR-2 has been identified as a cellular survivor factor during unfolding protein response. In conclusion, this study descried the creation and the validation of synthetic tools: synthetic cassette for cre-conditioned mice creation, the Flp-In System™ for isogenic stable cell line creation, and IRF-1 reporter cell line for high throughput screening. All synthetic tools were validated and used to investigate IRF-1, a transcription factor that plays a role in cancer and immune system.

The transcription factor Interferon Regulatory Factor-1 (IRF-1) has been demonstrated to suppress tumour growth through the regulation of many anti-oncogenic genes. Pro- and anti-apoptotic factors, cell cycle control genes, DNA damage response genes and prometastatic factors are all under the control of IRF-1, which effects both transcriptional activation and repression. In addition to these cell autonomous tumour suppressor activities, IRF-1 is also a key regulator of the immune system and, as such, mediates immune surveillance of tumours. Numerous studies have confirmed that loss or mis-regulation of IRF-1 is a key factor in several different types of cancer. Despite strong evidence for the crucial role of IRF-1 in cancer, and frequent assertions that this protein warrants further investigation as a drug target, very little is known about its regulation. Furthermore, since recent studies have linked upregulation of IRF-1 to the development of autoimmune diseases, it is particularly important that drugs be able to decouple autoimmune and anti-cancer functions of IRF-1 to avoid harmful side effects. This thesis describes how phosphorylation of IRF-1 in its regulatory C-terminal Mf1 domain modulates transactivatory and tumour suppressor activity. Phosphospecific antibodies were developed as tools to study the C-terminal phosphorylation. Using these, it was shown that treatment of cells with Interferon-γ(IFN-γ) not only causes accumulation of IRF-1 protein, but also results in phosphorylation of IRF-1 at two sites in the C-terminal Mf1 domain. Phosphomimetic mutants demonstrated that these phosphorylations enhanced the transactivatory activity of IRF-1 at various promoters, but did not affect repressor activity. Gel shift assays revealed that dual phosphorylation of IRF-1 (IRF-1 D/D) promoted DNAbinding and suggested this was through increased interaction with the cofactor/histone acetylase p300 which induces a conformational change in IRF-1, favouring DNA-binding. Acetylation by p300 appears to be important although it is not yet clear whether this directly or indirectly affects IRF-1 activity. Since the tumour suppressor activity of IRF-1 is of particular interest, the effect of phosphorylation was examined in clonogenic and invasion assays. IRF-1 D/D more efficiently suppressed colony formation in both anchorage dependent and independent assays, and may improve inhibition of invasion in Transwell assays. Thus, cell treatment with the therapeutic agent IFN-γ nduces phosphorylation of IRF-1, resulting in enhanced DNA binding of IRF-1 through improved p300 binding. In cells the outcome is more effective tumour suppression and inhibition of metastasis.

Interferon regulatory factor-1 (IRF-1) is a transcription factor and tumour suppressor, involved in many diverse cellular processes including immune responses and growth regulation. An interesting feature of IRF-1 is that it can both activate and repress gene expression, possibly by acting with co-activator or co-repressor proteins. In a previous phage display assay, a homologous peptide to the known repressor protein, zinc finger 350 (ZNF350), was found to bind to the C-terminus of IRF-1. ZNF350, also known as ZBRK1 (Zinc finger and BRCA1-interacting protein with KRAB domain-1), is a member of the Krüppel-associated box (KRAB)-containing zinc finger (KZF) proteins, which is a group of the widely distributed transcriptional repression proteins in mammals. ZNF350 has previously been shown to repress the expression of a number of genes including ANG1 and GADD45A, often in complex with other proteins. This study confirms the direct interaction between IRF-1 and ZNF350 and identifies key residues, including the LXXLL repression motif within the C-terminus of IRF-1, necessary for the binding interface. The two proteins have additionally been shown to interact within a cellular environment, shown by using techniques including immunoprecipitation and a proximity ligation assay. In addition, the ZNF350/IRF-1 complex formation appears to occur in the basal state of the cell, as opposed to in response to cellular stress such as viral infection or DNA damage. On the basis of ZNF350 being a negative regulator of transcription, a novel technique was developed to identify putative targets of both ZNF350 and IRF-1. This involved an initial bioinformatics screen using candidate IRF-1 binding site data obtained from CENTIPEDE, an algorithm that combines genome sequence information, with cell-specific experimental data to map bound TF binding sites. This allowed for the identification of novel target genes that contained the ZNF350 consensus binding site, GGGxxCAGxxxTTT, within close proximity to an IRF-1 consensus site, such as the immune response gene IL-12A. Lastly, a peptide phage display screen was combined with high-throughput sequencing to identify other potential binding partners of ZNF350 and perhaps help to understand the mechanism by which transcriptional repression is controlled by complex formation.

The results of experiments aimed at determining whether cytotoxic T lymphocytes (CTLs) can kill keratinocytes (KCs) expressing endogenously loaded antigen indicated that antigen specific cytotoxic T lymphocytes could recognize and kill keratinocytes expressing ovalbumin (OVA) or SIINFEKL peptide. Exposure of the KCs to interferon-gamma (IFN-γ) enhanced this CTL-mediated KC lysis and increased CTL epitope presentation on the surface of target cells. Expression of HPV 16 E7 protein in KCs affected CTL-mediated lysis. Expression of HPV 16 E7 inhibited IFN-γ-mediated up-regulation of SIINFEKL/H-2Kb complexes on keratinocytes, and also inhibited IFN-γ-mediated up-regulation of IRF-1 expression, and consequent up-regulation of TAP1 transcription. Further, overexpression of IRF-1 partially corrected the HPV 16 E7-mediated inhibition of enhanced susceptibility of KC lysis induced by IFN-γ. Thus, the effects of HPV 16 E7 on CTL-mediated lysis of IFN-γ exposed KCs are likely mediated by inhibition of MHC class I antigen presentation by IFN-γ. These findings may help explain why HPV-infected epithelial cells can escape from immune surveillance mediated by CTLs in vivo and in vitro.