Host cell factor-1 (HCF-1) is an evolutionarily conserved protein first discovered through its interaction with the herpes simplex viral transactivator VP16. Both the amino-terminal VP 16 interaction domain and the adjacent, less studied basic domain of HCF-1 are essential in cell cycle regulation. However, the mechanism(s) of this regulation is unknown. Our objective was to provide insight into the importance of the basic domain by studying proteins that interact with this region. Using the yeast two-hybrid system with the HCF-1 basic region as bait, we identified a novel protein named HCF-1 interacting zinc finger protein (HIZ). The purpose of this research was to characterize HIZ and determine if its interaction with HCF-1 could reveal a novel cellular target for HCF-1. A putative HIZ full-length sequence was determined and its primary structure was studied in detail. HIZ contains 16 C₂H₂ zinc fingers in its amino terminal. HIZ also contains a novel glutamine-rich motif adjacent to a potent autonomous transactivation domain. The presence of a DNA-bincing domain structure and a strong, functional transactivation domain indicate that HIZ is a novel transcriptional factor. Northern blot analysis showed tissue specific expression of HIZ with highest levels in the testis, skeletal muscle, liver and pancreas, suggesting possible roles in spermatogenesis, differentiation and metabolism. The HIZ-HCF-1 interaction was verified 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 using glutathione-s-transferase (GST) pull-down assays and the minimal HIZ domain for HCF-1 interaction was mapped to the same region critical for transactivation. Recent studies have identified the transcription factors Miz-1, GABP, LZIP, Zhangfei and PGC-1β as proteins that interact with the two domains of HCF-1 important in cell cycle control. Also, these studies have shown that interaction with HCF -1 is important in regulation of their transactivation potential. Thus, the effect of HCF-1 on HIZ activation was studied using transient transfection assays. Similar to its effect on the known cell cycle inhibitor, Miz-1, our studies showed that cotransfection with HCF-1 significantly inhibited HIZ transactivation. The expression pattern of HIZ in a matched tumour/normal tissue array showed that HIZ is expressed at a significantly lower level in tumours of the lung and uterus in comparison to normal tissues from the same individual. This finding suggests a correlation between HIZ expression and tumour formation, possibly in conjunction with the known cell cycle regulator HCF -1. / Thesis / Master of Science (MSc)

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.

Doctor en Farmacología / Autorizada por el autor, pero con restricción para ser publicada a texto completo en el Portal de Tesis Electrónicas, hasta diciembre de 2018 / El cáncer es la 2° causa de muerte en Chile. El desarrollo del cáncer se ha propuesto que es consecuencia de la pérdida de función de los genes supresores de tumores beneficiando la acción de los oncogenes (genes que promueven el crecimiento de tumores). La activación del Factor inducible por hipoxia 1α (HIF1α) en un ambiente reducido en oxígeno (hipoxia) o por óxido nítrico (NO), permiten la proliferación y adaptación metabólica de las células tumorales. Por otro lado, Caveolina-1 es una proteína de andamiaje, que ha sido descrita como un supresor de tumores y promotor de metástasis. Resultados de nuestro laboratorio han demostrado que E-Cadherina y Caveolina-1 actúan cooperativamente en supresión de tumores in vivo. Sin embargo, Caveolina-1 suprime el crecimiento de tumores incluso en células carentes de E-Cadherina, de un modo menos eficiente. La isoforma endotelial de la sintasa de óxido nítrico (NOS3), es uno de los blancos inhibidos por Caveolina-1 más ampliamente descritos en la literatura, que recientemente ha sido implicado en mantenimiento tumoral. Cómo la inhibición de NOS mediada por Caveolina-1 afecta la activación de HIF1α contribuyendo a la función supresora de tumores de Caveolina-1 en ausencia de E-Cadherina, aún no ha sido evaluado. En este trabajo, evaluamos la posibilidad que la inhibición de NOS por Caveolina-1 pueda reducir la transcripción dependiente de HIF1α y contribuir así a la función supresora de tumores de Caveolina-1. Líneas celulares transfectadas con un plasmidio que codifica para Caveolina-1 [HT29(US), B16F10 y HEK293T] o células en que la expresión endógena de Caveolina-1 fue disminuida utilizando un shRNA [MDA-MB231], fueron tratadas en hipoxia (1% O2, 4-24 h). La actividad transcripcional de HIF, la expresión de genes blanco de HIF1α, la distribución subcelular de HIF1α, Caveolina-1 y NOS3, fueron evaluados mediante ensayos de gen reportero, RT-PCR/qPCR, Western Blot y microscopía confocal, respectivamente. Además, se realizaron ensayos de formación de tumores en ratones inmunosuprimidos SCID-Beige y en ratones inmunocompetentes C57BL/6. Los principales hallazgos de este trabajo fueron, que Caveolina-1 reduce la actividad transcripcional de HIF1α y la expresión de VEGF en hipoxia en las líneas celulares analizadas. Además, la reducción del crecimiento tumoral de células de melanoma murino B16F10(Cav-1) fue coincidente con la reducción de la expresión del mRNA de vegf in vivo. El tratamiento de las células con el dador de NO (DETA/NO) o el inhibidor de arginasa BEC, previnieron la inhibición de la actividad transcripcional de HIF1α mediada por Caveolina-1. Además, la inhibición de NOS con L-NAME o con el inhibidor selectivo de NOS3, L-NIO, reducen el incremento en la actividad transcripcional de HIF en hipoxia. In vivo, la sobreexpresión de HIF1α en células HT29(US)(Cav-1) revierte la supresión de tumores mediada por Caveolina-1 en ratones SCID-Beige. Finalmente, el tratamiento sistémico de ratones C57BL/6 con L-NAME, reduce el volumen tumoral a niveles comparables con los observados en los tumores formados por células B16F10(Cav-1). En resumen, nuestros resultados sugieren que la inhibición de NOS3 mediada por Caveolina-1 reduce la actividad transcripcional de HIF1α y la expresión de sus genes blanco, in vitro e in vivo, contribuyendo a la función supresora de tumores de Caveolina-1 en ausencia de E-Cadherina / Cancer, the 2nd most important cause of death in Chile, is thought to develop due to loss of tumor suppressor and gain of oncogene function. Activation of Hypoxia-inducible factor 1α (HIF1α) in the low oxygen environment (hypoxia) present in tumors or by nitric oxide (NO), favors cancer cell proliferation and metabolic adaptation. Caveolin-1 is a scaffolding protein that reportedly functions both as a tumor suppressor and promoter of metastasis. Results from this laboratory have shown that E-cadherin and Caveolin-1 cooperate in tumor suppression in vivo. However, Caveolin-1 expression suppresses tumor growth even in cancer cells lacking E-cadherin, albeit less efficiently. The endothelial isoform of nitric oxide synthase (NOS3), one of the best-established targets for inhibition by Caveolin-1, has recently been implicated in maintence of tumor growth. Whether, Caveolin-1-mediated NOS inhibition may impact on HIF1α activation and account for tumor suppression by Caveolin-1 in the absence of E-cadherin has not been yet assessed. Here, we evaluated the possibility that NOS inhibition by Caveolin-1 may reduce HIF1α - dependent transcription and thereby contribute to the tumor suppressor function of Caveolin-1. Cell lines transfected with a Caveolin-1-encoding plasmid [HT29(US), B16F10 and HEK293T] or cells where endogenous Caveolin-1 protein levels [MDA-MB231] were reduced using shRNA-technology, were exposed to hypoxia (1% O2, 4-24 h). In these cells, HIF transcriptional activity, HIF1α target-gene expression, HIF1α, Caveolin-1 and NOS3 protein levels and subcellular localization were evaluated by gene reporter assays, RT-PCR/qPCR, Western Blot and confocal microscopy, respectively. Tumor forming capacity was evaluated in immunodeficient SCID-Beige and immunocompetent C57BL/6 mouse strains. The main findings of this study are that Caveolin-1 reduced HIF1α transcriptional activity and VEGF expression in hypoxia in vitro in all cell lines. Also, reduced tumor growth of B16F10(Cav-1) melanoma cells in C57BL/6 mice correlated with reduced vegf gene expression in vivo. Treatment of cells with the NO donor (DETA/NO) o arginase inhibition with BEC, prevented Caveolin-1-mediated inhibition of HIF1α transcriptional activity. Furthermore, NOS inhibition with L-NAME or selective NOS3 inhibition with L-NIO, reduced hypoxia-enhanced HIF transcriptional activity. In vivo HIF1α overexpression in HT29(US)(Cav-1) cells reversed tumor suppression due to Caveolin-1 in SCID-Beige mice. Finally, systemic treatment of C57BL/6 mice with L-NAME, reduced tumor volumes to levels comparable to those observed for tumors formed by B16F10(Cav-1) cells. In summary, our observations suggest that Caveolin-1-mediated inhibition of NOS3 activity reduces HIF1α transcriptional activity and target gene expression, both in vitro and in vivo, and that this ability contributes to tumor suppression by Caveolin-1 in the absence of E-cadherin / CONICYT, FONDECYT, FONDAP, MECESUP

acase@tulane.edu / The goal of the following experiments was to determine the cellular mechanisms through which estrogen receptor activity is maintained in hippocampal cells following termination of ovarian function. Aim 1 determined that kinase signaling contributes to the maintenance of estrogen receptor activity in the hippocampus of ovariectomized mice in addition to local synthesis of brain derived “neuroestrogens”. Inhibition of both the mitogen activated protein kinase (MAPK) and phosphoinositide-3 kinase (PI3K) cascades with intracerebroventricular infusion of specific kinase inhibitors reduced estrogen response element (ERE)-dependent gene expression in the hippocampus of ovariectomized mice. Aim 2 determined that neuroestrogen synthesis, MAPK signaling, and PI3K signaling interact to regulate the transcriptional output of estrogen receptors in response to insulin like growth factor-1 receptor (IGF-1R) activation in the Neuro-2A cell culture model. Rapid IGF-1R-dependent MAPK signaling promotes, while PI3K signaling inhibits, IGF-1R-dependent activation of endogenous estrogen receptors in Neuro-2A cells. Long-term IGF-1R stimulation reduces ERE-dependent gene expression in part through phosphorylation of estrogen receptor alpha (ERα). Rapid IGF-1R-dependent activation but not long-term repression of estrogen receptor activity in Neuro-2A cells requires neuroestrogen synthesis. Aim 3 determined that exposure to 40 days of continuous unopposed estradiol at the time of ovariectomy results in lasting enhancement of estrogen receptor activity in the hippocampus and lasting enhancement of hippocampus dependent memory in female mice beyond the period of short-term estradiol exposure. Together these three aims determine that neuroestrogen synthesis and kinase signaling interact to actively maintain estrogen receptor signaling in neuronal cells and these autonomous neuronal mechanisms of estrogen receptor activation have functional consequences on cognition long after cessation of ovarian function. / 1 / Kevin J Pollard

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2008. / Härtill 3 uppsatser.

Heterozygous mutations and deletions of the gene that encodes the transcription factor hepatocyte nuclear factor 1β (HNF1B) are the commonest known monogenic cause of developmental kidney disease. However, diagnosis remains challenging due to phenotypic variability and frequent absence of a family history. There is also no consensus as to when HNF1B genetic testing should be performed. This thesis includes work looking at the identification of HNF1B-associated disease. An HNF1B score was developed in 2014 to help select appropriate patients for genetic testing. The aim in chapter 2 was to test the clinical utility of this score in a large number of referrals for HNF1B genetic testing to the UK diagnostic testing service for the HNF1B gene. An HNF1B score was assigned for 686 referrals using clinical information available at the time of testing; performance of the score was evaluated by receiver-operating characteristic curve analysis. Although the HNF1B score discriminated between patients with and without a mutation/deletion reasonably well, the negative predictive value of 85% reduces its clinical utility. HNF1B-associated disease is due to an approximate 1.3 Mb deletion of chromosome 17q12 in about 50% of individuals. This deletion includes HNF1B plus 14 additional genes and has been linked to an increased risk of neurodevelopmental disorders, such as autism. The aim in chapter 3 was to compare the neurodevelopmental phenotype of patients with either an HNF1B intragenic mutation or 17q12 deletion to determine whether haploinsufficiency of the HNF1B gene is responsible for this aspect of the phenotype. Brief behavioural screening showed high levels of psychopathology and impact in children with a deletion. 8/20 (40%) patients with a deletion had a clinical diagnosis of a neurodevelopmental disorder compared to 0/18 with a mutation, P=0.004. 17q12 deletions were also associated with more autistic traits. Two independent clinical geneticists were able to predict the presence of a deletion with a sensitivity of 83% and specificity of 79% when assessing facial dysmorphic features as a whole. These results demonstrate that the 17q12 deletion but not HNF1B intragenic mutations are associated with neurodevelopmental disorders; we conclude that the HNF1B gene is not involved in the neurodevelopmental phenotype of these patients. Extra-renal phenotypes frequently occur in HNF1B-associated disease, including diabetes mellitus and pancreatic hypoplasia. Faecal elastase-1 levels have only been reported in a small number of individuals, the majority of which have diabetes. In chapter 4 we measured faecal elastase-1 in patients with an HNF1B mutation or deletion regardless of diabetes status and assessed the degree of symptoms associated with pancreatic exocrine deficiency. We found that faecal elastase-1 deficiency is a common feature of HNF1B-associated renal disease even when diabetes is not present and pancreatic exocrine deficiency may be more symptomatic than previously suggested. Faecal elastase-1 should be measured in all patients with a known HNF1B molecular abnormality complaining of chronic abdominal pain, loose stools or unintentional weight loss. Hypomagnesaemia is a common feature of HNF1B-associated disease and is due to renal magnesium wasting. The aim in chapter 5 was to measure both serum and urine magnesium and calcium levels in individuals with an HNF1B molecular defect and compare to a cohort of patients followed up in a general nephrology clinic in order to assess their potential as biomarkers for HNF1B-associated disease. The results of this pilot study show that using a cut-off for serum magnesium of ≤0.75 mmol/L was 100% sensitive and 87.5% specific for the presence of an HNF1B mutation/deletion. All individuals in the HNF1B cohort had hypermagnesuria with fractional excretion of magnesium >4%; a cut-off of ≥4.1% was 100% sensitive and 71% specific. This suggests serum magnesium levels and fractional excretion of magnesium are highly sensitive biomarkers for HNF1B-associated renal disease; if these results are confirmed in a larger study of patients with congenital anomalies of the kidneys or urinary tract they could be implemented as cheap screening tests for HNF1B genetic testing in routine clinical care.

Plants modulate their growth and development according to the prevailing light conditions. To detect light signals plants have an array of photoreceptors including the phytochromes which monitor the red and far-red light regions of the light spectrum. Phytochromes regulate gene expression in response to light in part by physically interacting with nuclear-localized bHLH transcription factors called PHYTOCHROME INTERACTING FACTORS (PIFs). PIFs are known to function as negative regulators of photomorphogenesis. Here we show that PIF1, the PIF family member with the highest affinity for phys, is degraded after pulses or continuous red, far-red or blue light in a phytochrome dependent manner. In etiolated seedlings, phyA plays a dominant role in regulating the degradation of PIF1 after a pulse of red, far-red or blue light; while phyB, phyD and other phys also influence PIF1 degradation after prolonged illumination. PIF1 interacted with phyA and phyB in a blue light-dependent manner, and the interactions with phys are necessary for the light-induced degradation of PIF1. In response to red, far-red or blue light treatments PIF1 is rapidly phosphorylated, poly-ubiquitinated and degraded via the ubiquitin/26S proteasomal pathway. In addition, we show that PIF1 negatively regulates photomorphogenesis at the seedling stage. The overexpression of a light-stable truncated form of PIF1 causes constitutively photomorphogenic phenotypes in the dark. pif1 seedlings displayed more open cotyledons and slightly reduced hypocotyl length compared to wild type under diurnal (12h light/12h dark) blue light conditions. Double mutant analyses demonstrated that pif1phyA, pif1phyB, pif1cry1 and pif1cry2 have enhanced cotyledon opening compared to the single photoreceptor mutants under diurnal blue light conditions. Taken together, these data suggest that PIF1 functions as a negative regulator of photomorphogenesis and that light-activated phys induce the degradation of PIF1 through the ubi/26S proteasomal pathway to promote photomorphogenesis. / text

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.

1 / Sarah Elizabeth Galvez

This study explored the possibility that upregulation of Hypoxia Inducible Factor-1 (Hif-1)-responsive genes in Human Umbilical Vein Endothelial Cells (HUVEC) would promote and stabilize HUVEC formation into inchoate vascular beds within artificial collagen gels. This experiment was designed to explore the above possibility by sub-cloning Hif-1[alpha], the related chimeric construct Hif-1[alpha]/VP16, and the marker gene dsRed into retroviral expression vectors, producing retroviral vectors containing these genes, and stably transducing HUVEC using these retroviruses. Transduced HUVEC were to be observed in cell culture as well as after implantation into artificial collagen gels that have previously supported vascular bed formation by HUVEC. Our results show, preliminarily, that HUVEC transduced with Hif-1[alpha]/VP16 go into cell-cycle arrest. Attempts to transduce HUVEC with Hif-1[alpha] failed to achieve high enough transduction efficiency to determine the cells angiogenic potential. This study concluded that more experiments need to be conducted to better characterize the effects of hypoxia-responsive gene upregulation in controlling HUVEC angiogenesis and cell-cycle signaling and that straightforward transduction of HUVEC by Hif-1[alpha]/VP16 is probably not sufficient, in itself, to induce in vitro vascular bed formation.