Dissertations / Theses on the topic 'Kruppel-Like Transcription Factors'

Hepatocellular carcinoma (HCC) is a common malignancy of the liver that is one of the most frequent causes of cancer-related death in the world. Surgical resection and liver transplantation are the only curative options for HCC, and tumor invasion and metastasis render many patients ineligible for these treatments. Identification of the mechanisms that contribute to invasive and metastatic disease may enlighten therapeutic strategies for those not eligible for surgical treatments. In this dissertation, I describe two sets of experiments to elucidate mechanisms underlying HCC dissemination, involving the activities of Krüppel-like factor 6 and a particular p53 point mutation, R172H. Gene expression profiling of migratory HCC subpopulations demonstrated reduced expression of Krüppel-like factor 6 (KLF6) in invasive HCC cells. Knockdown of KLF6 in HCC cells increased cell transformation and migration. Single-copy deletion of Klf6 in a HCC mouse model results in increased tumor formation, increased metastasis to the lungs, and decreased survival, indicating that KLF6 suppresses both tumor formation and metastasis in HCC. To elucidate the mechanism of KLF6-mediated tumor and metastasis suppression, we performed gene expression profiling and ChIP-sequencing to identify direct transcriptional targets of KLF6 in HCC cells. This analysis revealed novel transcriptional targets of KLF6 in HCC including CDC42EP3 and VAV3, both of which are positive regulators of Rho family GTPases. Concordantly, KLF6 knockdown cells demonstrate increased activity of the Rho family GTPases RAC1 and CDC42, and RAC1 is required for migration induced following KLF6 knockdown. Moreover, VAV3 and CDC42EP3 are also required for enhanced cell migration in HCC cells with KLF6 knockdown. Together, this work describes a novel signaling axis through which KLF6-mediated repression of VAV3 and CDC42EP3 inhibits RAC1Gmediated HCC cell migration in culture, and potentially HCC metastasis in vivo. TP53 gene mutations are commonly found in HCC and are associated with poor prognosis. Prior studies have suggested that p53 mutants can display gain-of- function properties in other tumor types. Therefore, I sought to determine if a particular hotspot p53 mutation, p53R172H, provided enhanced, gain-of-function properties compared to p53 loss in HCC. In vitro, soft agar colony formation and cell migration is reduced upon knockdown of p53R172H, indicating that this mutation is required for transformation-associated phenotypes in these cells. However, p53R172H-expressing mice did not have enhanced tumor formation or metastasis compared to p53-null mice. These data suggest that p53R172H and p53 deletion are functionally equivalent in vivo, and that p53R172H is not a gain-of-function mutant in HCC. Inhibition of the related transcription factors p63 and p73 has been suggested as a potential mechanism by which mutant p53 exerts its gain-of-function effects. Analysis of p63 and p73 target genes demonstrated that they are similarly suppressed in p53-null and p53R172H-expressing HCC cell lines, suggesting a potential explanation for the phenotypes I observed in vivo and in vitro. Together, the studies described in this dissertation increase our understanding of the mechanisms underlying HCC progression and metastasis. Specifically, we find and characterize KLF6 as a novel suppressor of HCC metastasis, and determine the contribution of a common p53 point mutation in HCC. This work contributes to ongoing efforts to improve treatment options for HCC patients.

Hepatocellular carcinoma (HCC) is a common malignancy of the liver that is one of the most frequent causes of cancer-related death in the world. Surgical resection and liver transplantation are the only curative options for HCC, and tumor invasion and metastasis render many patients ineligible for these treatments. Identification of the mechanisms that contribute to invasive and metastatic disease may enlighten therapeutic strategies for those not eligible for surgical treatments. In this dissertation, I describe two sets of experiments to elucidate mechanisms underlying HCC dissemination, involving the activities of Krüppel-like factor 6 and a particular p53 point mutation, R172H. Gene expression profiling of migratory HCC subpopulations demonstrated reduced expression of Krüppel-like factor 6 (KLF6) in invasive HCC cells. Knockdown of KLF6 in HCC cells increased cell transformation and migration. Single-copy deletion of Klf6 in a HCC mouse model results in increased tumor formation, increased metastasis to the lungs, and decreased survival, indicating that KLF6 suppresses both tumor formation and metastasis in HCC. To elucidate the mechanism of KLF6-mediated tumor and metastasis suppression, we performed gene expression profiling and ChIP-sequencing to identify direct transcriptional targets of KLF6 in HCC cells. This analysis revealed novel transcriptional targets of KLF6 in HCC including CDC42EP3 and VAV3, both of which are positive regulators of Rho family GTPases. Concordantly, KLF6 knockdown cells demonstrate increased activity of the Rho family GTPases RAC1 and CDC42, and RAC1 is required for migration induced following KLF6 knockdown. Moreover, VAV3 and CDC42EP3 are also required for enhanced cell migration in HCC cells with KLF6 knockdown. Together, this work describes a novel signaling axis through which KLF6-mediated repression of VAV3 and CDC42EP3 inhibits RAC1Gmediated HCC cell migration in culture, and potentially HCC metastasis in vivo. TP53 gene mutations are commonly found in HCC and are associated with poor prognosis. Prior studies have suggested that p53 mutants can display gain-of- function properties in other tumor types. Therefore, I sought to determine if a particular hotspot p53 mutation, p53R172H, provided enhanced, gain-of-function properties compared to p53 loss in HCC. In vitro, soft agar colony formation and cell migration is reduced upon knockdown of p53R172H, indicating that this mutation is required for transformation-associated phenotypes in these cells. However, p53R172H-expressing mice did not have enhanced tumor formation or metastasis compared to p53-null mice. These data suggest that p53R172H and p53 deletion are functionally equivalent in vivo, and that p53R172H is not a gain-of-function mutant in HCC. Inhibition of the related transcription factors p63 and p73 has been suggested as a potential mechanism by which mutant p53 exerts its gain-of-function effects. Analysis of p63 and p73 target genes demonstrated that they are similarly suppressed in p53-null and p53R172H-expressing HCC cell lines, suggesting a potential explanation for the phenotypes I observed in vivo and in vitro. Together, the studies described in this dissertation increase our understanding of the mechanisms underlying HCC progression and metastasis. Specifically, we find and characterize KLF6 as a novel suppressor of HCC metastasis, and determine the contribution of a common p53 point mutation in HCC. This work contributes to ongoing efforts to improve treatment options for HCC patients.

Development of functional neural circuits involves a series of complicated steps, including neurogenesis and neuronal morphogenesis. To understand the molecular mechasnims of neurite complexity, especially neurite branching/arborization, the Drosophila brain, especially MBNs (mushroom body neurons) and PNs (projection neurons) in olfactory circuitry, was used in this dissertation work as the model system to study how two molecules, Dscam and Kr-h1 affect neurite complexity in the Drosophilabrain. For the Drosophila Dscam, through alternative splicing it could encode up to 152,064 distinct immunoglobulin/fibronectin type cell adhesion molecules. Each Dscam isoform is derived from one of the 19,008 ectodomain variants connected with one of the two alternative transmembrane segments and one of the four possible endodomain portions. Recent studies revealed that Dscam was widely required for neurite branching/arborizaiton. However, due to the technical difficulty, the functional roles of Dscam transmembrane variants and ectodomain variants remain unclear. In this thesis work, a microRNA based RNA interference was used to knock down distinct subsets of Dscam isoform. First, loss of Dscam[TM1] versus Dscam[TM2], two distinct Dscam transmembrane variants, disrupted the dendritic versus axonal morphogenesis, respectively. Furthermore, structural analysis suggested that the juxtamembrane portion of transmembrane segment was required for the Dscam protein targeting in dendrites/axons and this differential protein targeting might account for the functional distinction between Dscam[TM1] and Dscam[TM2]. Second, to further address the functional significance of having two Dscam transmembrane variants in axons versus dendrites, the possibility that there might be different usage of Dscam repertoire between axons and dendrites that lead to different levels of morphological complexity between axons and dendrites in the same neuron was examined. To this end, end-in targeting approaches were used to exchange Dscam populations between axons and dendrites. Though the genetic data suggested that Dscam populations were exchanged between axons and dendrites, the phenotypic analysis in various neuronal types revealed that depending on the neuronal types, exchange of Dscam populations between axons and dendrites might primarily affect either axonal or dendritic morphology, suggesting that different usage of Dscam population between axons and dendrites might regulate complex patterns of neurite morphology. Finally, the functions of Dscam exon 4 variants had been addressed in different model neurons in the Drosophilabrain. First, 12 Dscam exon 4 variants were divided into three groups based on their phylogenetic distance. Then, three miRNA constructs were engineered to knock down one group at a time. The genetic data suggested that different Dscam exon 4 variants are differentially required in different neurons to support their proper neuronal morphogenesis. In summary, this part of my thesis work identified and characterized previously unrecognized functions of all these distinct Dscam variants and provided novel insights into how diverse Dscam isoforms regulate the different aspects of neuronal morphogenesis. In the honey bee brain, Kr-h1 is upregulated during the behavioral shift from nursing to foraging when there is increased neurite branching in the brain. To directly examine the hypothesis that altered Kr-h1 expression might regulate morphological complexity of neurites, this research work involved the MARCM (mosaic analysis with a repressible cell marker) and TARGET (temporal and regional gene expression targeting) techniques to analyze the roles of Kr-h1 in Drosophila neuronal morphogenesis. Interestingly, increased expression of Kr-h1 blocked the axon branching and further disrupted the lobe formation in the mushroom body whereas the loss-of-Kr-h1 did not show any apparent neuronal morphogenetic defects. In addition, it was observed that Kr-h1 was expressed when MB (mushroom body) did not undergo active morphogenesis, suggesting its potential anti-morphogenetic activity. Indeed, loss of Kr-h1 (Kruppel homolog 1) enhanced the neuronal morphogenesis that was otherwise delayed due to the defective TGF-beta signaling. Furthermore, Kr-h1 expression was closely linked to ecdysone dependent signaling: Kr-h1 was first regulated by usp (ultraspiracle), which dimerized with various ecdysone receptors and then Kr-h1 expression was essential for proper ecdysone patterning in the larval CNS (central nervous system). Together, though Kr-h1could potentially regulate the neurite complexity, it seems primarily involved in the coordinating ecdysone signaling. In conclusion, the powerful genetic toolkit available in the Drosophila has allowed the investigation in the molecular mechanisms of neuronal morphogenesis and understanding of these mechanisms will enhance our understanding of how the complex nervous system is wired to perform the delicate behaviors.

Doctor of Philosophy (PhD)
Gene expression is known to be regulated at the level of transcription. Recently, however, there has been a growing realisation of the importance of gene regulation at the post-transcriptional level, namely at the level of pre-mRNA processing (5’ capping, splicing and polyadenylation), nuclear export, mRNA localisation and translation. Erythroid krüppel-like factor (Eklf) is the founding member of the Krüppel-like factor (Klf) family of transcription factors and plays an important role in erythropoiesis. In addition to its nuclear presence, Eklf was recently found to localise to the cytoplasm and this observation prompted us to examine whether this protein has a role as an RNA-binding protein, in addition to its well-characterised DNA-binding function. In this thesis we demonstrate that Eklf displays RNA-binding activity in an in vitro and in vivo context through the use of its classical zinc finger (ZF) domains. Furthermore, using two independent in vitro assays, we show that Eklf has a preference for A and U RNA homoribopolymers. These results represent the first description of RNA-binding by a member of the Klf family. We developed a dominant negative mutant of Eklf by expressing its ZF region in murine erythroleukaemia (MEL) cells. We used this to investigate the importance of this protein in haematopoietic lineage decisions by examining its effect on the multipotent K562 cell line. We provide evidence that Eklf appears to be critical not only for the promotion of erythropoiesis, but also for the inhibition of megakaryopoiesis.

To, Ka Yan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (p. 115-120).
Abstracts in English and Chinese.
Acknowledgement --- p.i
Abstract --- p.ii
中文摘要 --- p.vi
List of abbreviations --- p.ix
List of tables --- p.xii
List of figures --- p.xiii
Contents --- p.xvi
Chapter Chapter One: --- General introduction --- p.1
Chapter 1.1 --- Background of Hepatocellular carcinoma (HCC) --- p.2
Chapter 1.2.1 --- ZBP-89 structure and its expression in cancers --- p.3
Chapter 1.2.2 --- Transcriptional regulation of ZBP-89 --- p.5
Chapter 1.3.1 --- Apoptosis and necrosis --- p.6
Chapter 1.3.2 --- Mechanisms of Apoptosis --- p.7
Chapter 1.4 --- Bcl-2 family --- p.11
Chapter 1.5 --- Regulation of p53 cancer cells --- p.12
Chapter 1.6 --- The aim of the study --- p.13
Chapter Chapter Two: --- Up-regulation of Bak expression by Ad-ZBP-89 induce apoptosis in human liver cancer cells --- p.15
Chapter 2.1. --- Introduction --- p.16
Chapter 2.2. --- Materials and methods --- p.18
Chapter 2.2.1. --- Cell culture --- p.18
Chapter 2.2.2. --- RT-PCR --- p.19
Chapter 2.2.3. --- Western blotting --- p.21
Chapter 2.2.4. --- Adenovirus infection and Cell viability assay --- p.24
Chapter 2.2.5. --- Detection of apoptosis --- p.26
Chapter 2.2.6. --- RNA interference --- p.27
Chapter 2.2.7. --- Statistical analysis --- p.29
Chapter 2.3. --- Results --- p.30
Chapter 2.3.1. --- Endogenous expression of ZBP-89 and Bak of human liver cancer cells --- p.30
Chapter 2.3.2. --- Effects of Ad-ZBP-89 on proliferation in HCC cell lines --- p.31
Chapter 2.3.3. --- Effects of Ad-ZBP-89 on the expression of Bcl-2 family members --- p.34
Chapter 2.3.4. --- ZBP-89 induced Bak expression and release of cytochrome c --- p.39
Chapter 2.3.5. --- Effects of Ad-ZBP-89 on apoptosis rate in HCC cell lines --- p.41
Chapter 2.3.6. --- Effects of ZBP-89 siRNA on expression of Bcl-2 family members and proliferation in HCC cell lines --- p.43
Chapter 2.3.7. --- Effects of Bak siRNA and its combined effect with Ad-ZBP-89 on the expression of Bak and reduced apoptosis in HCC cell lines --- p.50
Chapter 2.4. --- Discussion --- p.55
Chapter Chapter Three: --- Identification of ZBP-89 protein as an apoptosis activator for a pro-apoptotic Bak gene promoter --- p.60
Chapter 3.1. --- Introduction --- p.61
Chapter 3.2. --- Materials and methods --- p.64
Chapter 3.2.1. --- Cell lines and tissues --- p.64
Chapter 3.2.2. --- Transient transfection and Luciferase activity assay --- p.64
Chapter 3.2.3. --- pGL3-Bak-promoter vector construction --- p.67
Chapter 3.2.4. --- "Preparation of mitochondrial, cytosolic and nuclear fractions" --- p.74
Chapter 3.2.5. --- Electrophoretic mobility shift assay --- p.75
Chapter 3.2.6. --- Overexpression of Bak --- p.76
Chapter 3.2.7. --- RT-PCR and Western blot analysis on HCC tissues samples --- p.80
Chapter 3.2.8. --- Statistical Analysis --- p.80
Chapter 3.3. --- Results --- p.81
Chapter 3.3.1. --- ZBP-89 activates Bak-luciferase promoter genes in HCC cells --- p.81
Chapter 3.3.2. --- ZBP-89 activates shortened Bak-luc-promoter in PLC/PRF/5 and SK-Hep-1 cells --- p.82
Chapter 3.3.3. --- ZBP-89 is a potential binding protein to the Bak promoter gene region -457/-407 --- p.85
Chapter 3.3.4. --- The combined effects of Bak overexpression and Ad-ZBP-89 induce apoptosis in HCC cells --- p.89
Chapter 3.3.5. --- The combined effects on Bak protein expression --- p.94
Chapter 3.3.6. --- Bak expression in HCC tissues --- p.98
Chapter 3.4. --- Discussion --- p.99
Chapter Chapter Four: --- Conclusion and Future Perspectives --- p.104
Chapter 4.1. --- Conclusion --- p.104
Chapter 4.2. --- Future Perspectives --- p.112
Reference --- p.113

By means of reverse-transcription polymerase chain reaction, overexpression of ZNF146 was detected in two human HCC cell lines HepG2 and Hep3B and a clear relationship between HCC and overexpression of ZNF146 has been established. Subcellular localization of ZNF146 protein in liver cells was studied by generation and expression of a green fluorescent protein (GFP) fusion protein. The nuclear localization and the reported DNA binding ability of ZNF146 protein provided a hint that ZNF146 may carry out its function in the cell system by interacting with specific genomic DNA sequences. Recombinant ZNF146 protein was expressed using bacterial and yeast system for the genomic DNA pull down assay in the identification of potential interacting genomic DNA sequences. Several potential genomic DNA sequences that interact with ZNF146 were identified and the gene MDM2 is the one of the candidates that is directly related to human carcinogenesis. MDM2 is a negative regulator of the tumor suppresser protein p53. Deregulation of MDM2 will impair the cell's ability in cell cycle arrest, DNA repair and apoptosis upon induced DNA damage.
Hepatocellular carcinoma (HCC) is a type of primary malignant liver tumor. And is one of the most frequent malignancies worldwide. The focus of this research project is the characterization of a Kruppel zinc finger protein, zinc Finger Protein 146 (ZNF146) using HCC as a disease model. The aim of this project is to understand the functional role ZNF146 and try to explore the mechanism of how ZNF146 might be involved in the carcinogenesis of HCC.
In order to have a better understanding with the protein ZNF146, SUMOylation properties of this protein has been studied. SUMO1 modification on ZNF146 has already been reported. And in our study, experimental result demonstrated that ZNF146 is also modified by SUMO2 and SUMO3 in liver cells. Other than the SUMOylation sites for SUMO1 protein which has been reported, modification sites for SUMO2 at the K247 and K275 positions were mapped, while K191R, K219R, K247R, K256R and K275R, five positions were mapped for SUMO3 modification. A more complete picture of the SUMOylation properties of ZNF146 has been revealed. Since we hypothesized that ZNF146 is related to the p53 tumor suppressor, cell cycle control and DNA repair pathway, a cell cycle study using flow cytometry was performed for the investigation of the effect on cell cycle regulation by ZNF146 overexpression. In our study, ZNF146 overexpression promoted the G1/S transition in the cell division cycle, which indicated that liver cells were more active for the progression of cell cycle.
On the other hand, using cDNA microarray technology expression profiles of ZNF146 overexpressing and non-overexpressing liver cell lines were compared and with real-time polymerase chain reaction, six candidate genes CRLF1, IFI44, ST6GAL1, LOC441601, IL18 and RAD17 were confirmed with their deregulation induced by the overexpression of ZNF146. Four of the candidates, IFI44, LOC441601, IL18 and RAD17 were found to be related to the p53 tumor suppressor activity or DNA damage, repair response and control. This observation, together with the result of genomic DNA pull down assay, gives us a hint that ZNF146 is possibly involved in liver carcinogenesis by affecting DNA repair and cell cycle control upon induced DNA damage.
The gene ZNF146 codes for a member of the Kruppel zinc finger proteins, however ZNF146 protein is different from most members of the Kruppel zinc finger proteins subfamily. It encodes a 33 kDa protein solely composed of 10 zinc finger motifs and is devoid of any non-zinc finger regulatory domain for interactions with other proteins. ZNF146 overexpression has been reported in a number of cancers including colon cancer and pancreatic carcinoma. However, the functional role of ZNF146 overexpression in tumorigenesis is yet to be solved and not much research on how ZNF146 might be invovled in the establishment of HCC was published.
To conclude, the experimental results of this study support the hypothesis that ZNF146 overexpression may deregulating the cell division cycle and some genes differentially regulated upon over-expression of ZNF146 are related to the regulations of DNA damage response. Future research on ZNF146 can be focused on the detail regulatory pathway of ZNF146 overexpression and its interaction between the p53 tumor suppressor, DNA damage response and cell cycle regulation, and a fuller picture of how ZNF146 overexpression might induce hepatocarcinogenesis can be revealed.
Yeung, Tsz Lun.
Adviser: Miu Yee (Mary) Waye.
Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3329.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2008.
Includes bibliographical references (leaves 287-304).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.

Les domaines de transactivation (TAD) acides sont présents dans plusieurs protéines oncogéniques, virales et dans des facteurs de différenciation de cellules souches. Ces domaines acides contrôlent la transcription à travers une myriade d’interactions avec divers partenaires ce qui provoque l’activation de la transcription ou leur propre élimination. Cependant, dans la dernière décennie, de plus en plus de recherches ont démontré que les TAD possédaient un sous-domaine activation/dégradation (DAD) responsable pour une fonction d'activation de la transcription dépendante de la dégradation de la protéine. Un tel phénomène peut être accompli par plusieurs moyens tels que des modifications post-traductionnelles, l’association à des cofacteurs ou la formation d’un réseau d’interaction complexe en chaînes. Or, aucune preuve concrète n’a pu clairement démontrer le fonctionnement de la dépendance paradoxale entre ces deux fonctions sur un activateur de transcription. Le DAD, a été observé dans plusieurs facteurs de transcription incluant la protéine suppresseur de tumeur p53 et le facteur de différenciation érythrocyte EKLF. Un aspect particulier des DAD est que la composition de leur séquence d’acide aminé est fortement similaire à celle des domaines de liaison à l’ubiquitine (UBD) qui jouent un rôle clé dans le contrôle de la transcription à travers leur interaction non-covalente avec l’ubiquitine. Ainsi, dans ce mémoire, nous avons étudié la possibilité que les TAD acides soient capables d’agir comme UBD pour réguler leur fonction paradoxale à travers des interactions non-covalentes avec l’ubiquitine. L’analyse est faite en utilisant la résonnance magnétique nucléaire (RMN) ainsi qu’avec des essais fonctionnels de dégradation. En somme, cette étude amène une plus grande compréhension des protéines impliquées dans le contrôle des TAD et caractérise le tout premier exemple de TAD capable d’interagir avec l’ubiquitine.
Acidic transactivating domains have been shown to be potential targets for a number of different therapies but their dynamic nature and their ability to bind many interacting partners has made it difficult to fully understand their functioning mechanisms. What we do know about these domains is that they readily control transcription through a myriad of interactions capable of either activating specific aspects of their function or simply, signal for their own demise. Within the acidic TADs lies an unusual degradation/activation domain (DAD) capable of activating transcription at the cost of its degradation. In other words, DAD transcriptional activation is dependent on the degradation of the protein. Such a phenomenon could be explained by a wide variety of hypotheses like the play of post-translational modifications, co-factors, or maybe just a really sophisticated time scaled network of interactions. However, no concrete explanation of how this dual dependent functioning domain works has yet to surface. The DAD has been observed within acidic TADs of several transcription factors including the tumor suppressor p53 and the red blood cell differentiation factor EKLF. Interestingly though, the amino acid sequence composition of DADs share a strong similarity with several types of sequences from domains that bind ubiquitin (UBDs). These domains have been shown in the past to, in addition to their role in degradation, play a key role in regulating transcription through non-covalent interaction with ubiquitin. Hence, in this project, we investigated weather acidic TADs had the ability to function as UBDs and form non-covalent interactions with ubiquitin and also to determine the functional significance of this interaction in regards to the dual function of acidic TADs.