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.

Polymorphisms in the interferon regulatory factor 5 (IRF5) gene have been consistently replicated and shown to confer risk for or protection from the development of systemic lupus erythematosus (SLE). IRF5 expression is significantly upregulated in SLE patients and upregulation associates with IRF5-SLE risk haplotypes. IRF5 alternative splicing has also been shown to be elevated in SLE patients. Given that human IRF5 exists as multiple alternatively spliced transcripts with distinct function(s), it is important to determine whether the IRF5 transcript profile expressed in healthy donor immune cells is different from that expressed in SLE patients. Moreover, it is not currently known whether an IRF5-SLE risk haplotype defines the profile of IRF5 transcripts expressed. Using standard molecular cloning techniques, we identified and isolated 14 new differentially spliced IRF5 transcript variants from purified monocytes of healthy donors and SLE patients to generate an IRF5 variant transcriptome. Next-generation sequencing was then used to perform in-depth and quantitative analysis of full-length IRF5 transcript expression in primary immune cells of SLE patients and healthy donors by next-generation sequencing. Evidence for additional alternatively spliced transcripts was obtained from de novo junction discovery. Data from these studies support the overall complexity of IRF5 alternative splicing in SLE. Results from next-generation sequencing correlated with cloning and gave similar abundance rankings in SLE patients thus supporting the use of this new technology for in-depth single gene transcript profiling. Results from this study provide the first proof that 1) SLE patients express an IRF5 transcript signature that is distinct from healthy donors, 2) an IRF5-SLE risk haplotype defines the top four most abundant IRF5 transcripts expressed in SLE patients, and 3) an IRF5 transcript signature enables clustering of SLE patients with the H2 risk haplotype.

Thesis (M.Sc.). / Written for the Dept. of Microbiology and Immunology. Title from title page of PDF (viewed 2008/05/14). Includes bibliographical references.

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 3 (IRF3) plays an important role in activating the innate immune response in a variety of conditions, including viral infection. As well as regulating the immune response to viruses, IRF3 is involved in regulating cellular functions including apoptosis. Apoptosis and the inflammatory response to viral infection are very different; therefore, it is obvious that IRF3 plays dramatically different roles in the cell depending on the conditions. We previously identified a non-activating phosphorylation of IRF3 in response to adenovirus (Ad) in which Serine-173 is phosphorylated. In addition to Ad infection, IRF3- S173 is phosphorylated in response to genotoxic stresses including ultraviolet (UV) irradiation and etoposide. In this study, I show that this phosphorylation event is involved in a variety of processes including protein stability, cell survival and IRF3 regulation. Thus, phosphorylation of IRF3-S173 is a novel and important event in a complex regulatory pathway of an integral protein.

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.

Interferon regulatory factor 3 (IRF3) plays an important role in activating the innate immune response in a variety of conditions, including viral infection. As well as regulating the immune response to viruses, IRF3 is involved in regulating cellular functions including apoptosis. Apoptosis and the inflammatory response to viral infection are very different; therefore, it is obvious that IRF3 plays dramatically different roles in the cell depending on the conditions. We previously identified a non-activating phosphorylation of IRF3 in response to adenovirus (Ad) in which Serine-173 is phosphorylated. In addition to Ad infection, IRF3- S173 is phosphorylated in response to genotoxic stresses including ultraviolet (UV) irradiation and etoposide. In this study, I show that this phosphorylation event is involved in a variety of processes including protein stability, cell survival and IRF3 regulation. Thus, phosphorylation of IRF3-S173 is a novel and important event in a complex regulatory pathway of an integral protein.

No / 1 In this study we examined the signalling events that regulate lipopolysaccharide (LPS)-stimulated induction of interferon regulatory factor (IRF)-1 in human umbilical vein endothelial cells (HUVECS). 2 LPS stimulated a time- and concentration-dependent increase in IRF-1 protein expression, an effect that was mimicked by the cytokine, tumour necrosis factor (TNF)-¿. 3 LPS stimulated a rapid increase in nuclear factor kappa B (NFKB) DNA-binding activity. Preincubation with the NFKB pathway inhibitors, N-¿-tosyl-L-lysine chloromethyl ketone (TLCK) or pyrrolidine dithiocarbamate (PDTC), or infection with adenovirus encoding IKB¿, blocked both IRF-1 induction and NFKB DNA-binding activity. 4 LPS and TNF¿ also stimulated a rapid activation of gamma interferon activation site/gamma interferon activation factor (GAS/GAF) DNA-binding in HUVECs. Preincubation with the Janus kinase (JAK)-2 inhibitor, AG490 blocked LPS-stimulated IRF-I induction but did not affect GAS/ GAF DNA-binding. 5 Preincubation with TLCK, PDTC or infection with I¿Ba adenovirus abolished LPS-stimulated GAS/GAF DNA-binding. 6 Incubation of nuclear extracts with antibodies to RelA/p50 supershifted GAS/GAF DNA-binding demonstrating the involvement of NF¿B isoforms in the formation of the GAS/GAF complex. 7 These studies show that NF¿B plays an important role in the regulation of IRF-1 induction in HUVECs. This is in part due to the interaction of NF¿B isoforms with the GAS/GAF complex either directly or via an intermediate protein.

Interferon Regulatory Factor -1 (IRF-1) is a transcription factor that acts as a tumour suppressor in cancer cells. The inactivation or deletion of IRF-1 either in one or both allele has been frequently reported in leukaemia and myelodysplasia (MDS). On the other hand nucleophosmin (NPM), a nucleo-cytoplasmic shuttling phosphoprotein is also known to be aberrant in some form of leukaemia. NPM was first proposed as a binding partner of IRF-1 in 1997 and suggested to inactivate IRF-1 by inhibiting its DNA binding ability. No further researches on the interaction between IRF-1 and NPM1 was reported prior to the start of my PhD. In the research presented here the interaction and mechanism by which IRF-1 might be inactivated by NPM was studied. Under the context of both NPM and IRF-1 being frequently associated with leukaemia and MDS, the study was done to determine the role of NPM under its naïve condition and a most frequent mutated condition (NPMc+), where the C-terminal of NPM was frequently mutated to give rise to a cytoplasmic NPM in certain leukaemia. In this current research, the direct interaction between IRF1 and NPM was further confirmed both in vitro as well as within the cells. Following this, the effect of this interaction in respect to the leukaemic condition having NPMc+ mutation was done, by comparing the end results on AML2 (leukaemic cells with intact wild type NPM) and AML3 (leukaemic cells having a single NPM allele mutated to form NPMc+) cells. In this research, overexpression of wild type NPM (NPMwt) was found to increase IRF-1 transcriptional activity. On further analysis, the DNA binding activity of IRF-1 due to the presence of NPMwt or NPMc+ was not always inhibited, instead it shows a change in binding specificity, where NPMwt bound IRF-1, lacks DNA binding ability and DNA bound IRF-1 has a reduced binding towards NPM. This is being studied further in terms of NPM overexpression and increased IRF-1 transcriptional activity, as the order of addition (order of interaction in vivo) plays a major role in activating or deactivating IRF-1. This along with the increased transcriptional activity of IRF-1 suggests a novel function of NPM, where it could act in favour of IRF- 1 activity. Additionally, the NPM induced change in IRF-1 localisation was confirmed by the cytoplasmic localised IRF-1 in NPMc+ expressing cells and nucleolar sequestration in NPMwt overexpressing cells. This gives a novel mechanism by which NPM regulates IRF-1. Further, the NPMc+ specific colocalisation of IRF-1 urges to study the other proteins that may have been re-localised in AML cells due to the NPMc+ specific interaction. A mass spectrometric analysis on the cellular distribution of total proteins were analysed between AML2 (cells with NPMwt) and AML3 (cells containing NPMc+). Specific proteins related to cancer have been identified to be differentially distributed rather than being a random translocation. With this being said, a peptide phage display technology coupled with next generation sequencing was done to identify NPMwt binding peptides that can be used in drug discovery process or as small molecule inhibitors or activators. Three different peptides were selected at the end of the study that bind very effectively to NPMwt. These peptide can either aid or restrict NPM activity and need to be validated and studied in the future.

Cardiovascular disease, and myocardial infarction (MI) in particular, remains a major burden in the developed world today. In fact, the remodeling process, which follows the initial ischemic episode of MI, is a major determinant of heart failure. Although several key mechanistic pathways involving cell growth and death have been identified, there is limited knowledge surrounding the role of the innate immune response as a positive or negative regulator of cardiac remodeling. Recent data strongly support a role for key regulatory components within the toll-like receptor (TLR) family as potent modulators of cardiac remodeling post-MI. It has been demonstrated that targeted gene knockdown of TLR4, as well as downstream adaptor proteins and kinases, significantly improve cardiac function and overall survival. While the well-known NF-κB transcriptional factor that is downstream to TLR4 signaling has been linked to remodeling, there has been no evidence thus far describing a role of the parallel interferon regulatory factor-3 (IRF3) signaling cascade in any facet of this process. Several key findings suggest that IRFs contribute to both cell growth and apoptosis, thus providing an appealing, and novel target for interrogation. In this thesis I describe how IRF3 contributes to maladaptive remodeling post-MI. In my first set of experiments, I demonstrate that IRF3 is acutely upregulated within the cardiomyocyte following MI and that this response contributes to excessive apoptosis post-MI. A targeted deletion of the IRF3 gene enhances cardiac function, decreases infarct size, and improves survival following MI. In the second set of experiments I demonstrate that IRF3 attenuates angiogenesis at the ischemic border zone by upregulating the expression of thrombospondins. I have shown that IRF3 deficiency, which liberates endogenous anti-angiogenic signals, promotes angiogenesis following ischemic injury. These data suggest that IRF3 is a potent regulator of cardiac remodeling and may be an effective therapeutic target to ameliorate maladaptive cardiac repair post-MI.