Dissertations / Theses on the topic 'Zinc finger'
To see the other types of publications on this topic, follow the link: Zinc finger.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'Zinc finger.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Looman, Camilla. "The ABC of KRAB zinc finger proteins." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3515.
Full text
2
Lanfear, Jeremy. "The molecular evolution of zinc-finger genes." Thesis, University of Oxford, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.291274.
Full text
3
Crawford, Catherine. "Characterisation of endogenous KRAB zinc finger proteins." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4225.
Full textAbstract:
The Krüppel-associated box (KRAB) zinc finger protein (ZFP) genes comprise one of the largest gene families in the mammalian genome, encoding transcription factors with an N-terminal KRAB domain and C-terminal zinc fingers. The KRAB domain interacts with a co-repressor protein, KAP-1, which can recruit various factors causing transcriptional repression of genes to which KRAB ZFPs bind. Little is currently known about the gene targets of the ~400 human and mouse KRAB ZFPs. Many KRAB ZFPs interact with factors other than KAP-1. To identify proteins that may interact with one particular KRAB ZFP, Zfp647, I previously carried out a yeast two-hybrid screen using the full-length Zfp647 sequence and a mouse embryonic cDNA library. I have now tested the interactions from this screen for their specificity for Zfp647. I show that Zfp647 can interact with itself and at least 20 other KRAB ZFPs through their zinc finger domains, and have confirmed the Zfp647 self-interaction by in vitro co-immunoprecipitation. In my yeast two-hybrid screen, Zfp647 bound to KAP-1 as well as another related protein, ARD1/Trim23. Zfp647 also interacts with proteins that function in ubiquitylation. I have found evidence to suggest that Zfp647 may also interact with proteins encoding jumonji domains both by yeast two-hybrid assay and by co-immunoprecipitation from NIH/3T3 cell extracts. We have previously found that Zfp647 localises to non-heterochromatic nuclear foci in differentiated ES cells, which also contain KAP-1 and HP1, and which lie adjacent to PML nuclear bodies in a high proportion of cells. I have found that these foci are also visible in pMEFs, but not NIH/3T3 tissue culture cells. Immunofluorescence studies with antibodies against proteins from the yeast twohybrid screen have not shown any significant co-localisation with Zfp647. KAP-1 is sumoylated ex vivo, as are two human KRAB ZFPs. Because Zfp647 lies adjacent to PML nuclear bodies and can associate with proteins involved in posttranslational modification, I tested whether Zfp647 is also modified. I characterised a sheep _-Zfp647 antibody previously created in the lab and have shown that it detects Zfp647 by western blot, but not by immunofluorescence. I show that treatment of NIH/3T3 cells with NEM, which prevents the removal of protein modifications, leads to the appearance of higher molecular weight forms of Zfp647. Modification of Zfp647 is not dependent on KAP-1, which is known to function as a SUMO E3 ligase. Attempts to classify the modification as either ubiquitin, SUMO or NEDD8 have suggested that Zfp647 may be mono-ubquitylated. The larger modified forms of Zfp647 are present in both NIH/3T3 and ES cells. Interestingly, I found that the modification profile of the protein changes over the course of ES cell differentiation, during which time Zfp647 relocalises to punctate nuclear foci; thus Zfp647 modification may be involved in this process.
4
Rebar, Edward John. "Selection studies of zinc finger-DNA recognition." Thesis, Massachusetts Institute of Technology, 1997. http://hdl.handle.net/1721.1/10383.
Full text
5
Simpson, Raina Jui Yu. "The multiple roles of zinc finger domains." Thesis, The University of Sydney, 2004. http://hdl.handle.net/2123/655.
Full textAbstract:
Zinc finger (ZnF) domains are prevalent in eukaryotes and play crucial roles in mediating protein-DNA and protein-protein interactions. This Thesis focuses on the molecular details underlying interactions mediated by two ZnF domains. The GATA-1 protein is vital for the development of erythrocytes and megakaryocytes. Pertinent to the protein function is the N-terminal ZnF. In particular, this domain mediates interaction with DNA containing GATC motifs and the coactivator protein FOG. The importance of these interactions was illustrated by the findings in Chapter 3 that naturally occurring mutations identified in patients suffering from blood disorders affect the interaction of the N-terminal ZnF with either DNA (R216Q mutation) or FOG (V205M and G208S mutations). In addition to the interaction FOG makes with GATA-1, it also interacts with the centrosomal protein TACC3. In Chapter 4, this interaction is characterised in detail. The solution structure of the region of FOG responsible for the interaction is determined using NMR spectroscopy, revealing that it is a true classical zinc finger, and characterisation of the interaction domain of TACC3 showed that the region is a dimeric coiled-coil. The FOG:TACC3 interaction appears to be mediated by a-helices from the two proteins. The data presented here represent some of the first described molecular details of how a classical ZnF can contact a protein partner. Interestingly, the a-helix used by the FOG finger to bind TACC3 is the same region utilised by DNA-binding classical zinc fingers to contact DNA. In addition to the multiple roles played by ZnFs, this domain is also known for its robustness and versatility. In Chapter 5, incomplete ZnF sequences were assessed for its ability to form functional zinc-binding domains. Remarkably, CCHX sequences (in the context of BKLF finger 3) were able to form discrete zinc-binding domains and also, mediate both protein-DNA and protein-protein interactions. This result not only illustrates the robust nature of ZnFs, it highlights the need for expanding ZnF sequence criteria when searching for functional zinc-binding modules. Together, the data presented here help further our understanding of zinc finger domains. Similar to the use of DNA-binding ZnFs in designer proteins, these data may start us on the path of designing novel protein-binding ZnFs.
6
Simpson, Raina Jui Yu. "The multiple roles of zinc finger domains." University of Sydney. Molecular and Microbial Biosciences, 2004. http://hdl.handle.net/2123/655.
Full textAbstract:
Zinc finger (ZnF) domains are prevalent in eukaryotes and play crucial roles in mediating protein-DNA and protein-protein interactions. This Thesis focuses on the molecular details underlying interactions mediated by two ZnF domains. The GATA-1 protein is vital for the development of erythrocytes and megakaryocytes. Pertinent to the protein function is the N-terminal ZnF. In particular, this domain mediates interaction with DNA containing GATC motifs and the coactivator protein FOG. The importance of these interactions was illustrated by the findings in Chapter 3 that naturally occurring mutations identified in patients suffering from blood disorders affect the interaction of the N-terminal ZnF with either DNA (R216Q mutation) or FOG (V205M and G208S mutations). In addition to the interaction FOG makes with GATA-1, it also interacts with the centrosomal protein TACC3. In Chapter 4, this interaction is characterised in detail. The solution structure of the region of FOG responsible for the interaction is determined using NMR spectroscopy, revealing that it is a true classical zinc finger, and characterisation of the interaction domain of TACC3 showed that the region is a dimeric coiled-coil. The FOG:TACC3 interaction appears to be mediated by a-helices from the two proteins. The data presented here represent some of the first described molecular details of how a classical ZnF can contact a protein partner. Interestingly, the a-helix used by the FOG finger to bind TACC3 is the same region utilised by DNA-binding classical zinc fingers to contact DNA. In addition to the multiple roles played by ZnFs, this domain is also known for its robustness and versatility. In Chapter 5, incomplete ZnF sequences were assessed for its ability to form functional zinc-binding domains. Remarkably, CCHX sequences (in the context of BKLF finger 3) were able to form discrete zinc-binding domains and also, mediate both protein-DNA and protein-protein interactions. This result not only illustrates the robust nature of ZnFs, it highlights the need for expanding ZnF sequence criteria when searching for functional zinc-binding modules. Together, the data presented here help further our understanding of zinc finger domains. Similar to the use of DNA-binding ZnFs in designer proteins, these data may start us on the path of designing novel protein-binding ZnFs.
7
Wang, Zhonghua Laity John H. "Characterization of novel structure-regulatory relationships within interacting two-finger Cys₂His₂ zinc finger protein motifs." Diss., UMK access, 2008.
Find full textAbstract:
Thesis (Ph. D.)--School of Biological Sciences. University of Missouri--Kansas City, 2008."A dissertation in cell biology and biophysics and molecular biology and biochemistry." Advisor: John H. Laity. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Sept.12, 2008. Includes bibliographical references (leaves 148-166). Online version of the print edition.
8
Fairall, Louise. "The interaction of zinc-finger proteins with DNA." Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.314849.
Full text
9
Knight, Robert D. "C2H2 zinc finger gene evolution in the Metazoa." Thesis, University of Reading, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312566.
Full text
10
Younce, Craig. "Zinc-Finger Protein MCPIP in Cell Death and Differentiation." Doctoral diss., University of Central Florida, 2009. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2279.
Full textAbstract:
Monocyte chemotactic protein-1 (MCP-1) plays a critical role in the development of cardiovascular diseases. How MCP-1 contributes to the development of heart disease is not understood. We present evidence that MCP-1 causes death in cardiac myoblasts, H9c2 by inducing oxidative stress, ER stress and autophagy via a novel Zn-finger protein, MCP-1 induced protein (MCPIP). MCPIP expression caused cell death and knockdown of MCPIP, attenuated MCP-1 induced cell death. Expression of MCPIP resulted in induction of iNOS and production of reactive oxygen (ROS). It caused induction of NADPH oxidase subunit phox47 and its translocation to the cytoplasmic membrane. Oxidative stress led to the induction of ER stress markers HSP40, PDI, GRP78 and IRE1α. ER stress lead to autophagy as indicated by beclin-1 induction, cleavage of LC3 to LCII and autophagolysosome formation. Here, MCPIP-induced processes lead to apoptosis as indicated by caspase 3 activation and TUNEL assay. This cell death involved caspase 2 and caspase 12 as specific inhibitors of these caspases prevented MCPIP-induced cell death. Inhibitors of oxidative stress inhibited ER stress, and cell death. Specific inhibitors of ER stress inhibited autophagy and cell death. Inhibition of autophagy inhibited cell death. Microarray analysis showed that MCPIP expression caused induction of a variety of genes known to be involved in cell death. MCPIP caused activation of JNK and p38 and induction of p53 and PUMA. These results collectively suggest that MCPIP induces ROS/RNS production that causes ER stress which leads to autophagy and apoptosis through caspase 2/12 and IRE1α –JNK/p38-p53-PUMA pathway. These results provide the first molecular insights into the mechanism by which elevated MCP-1 levels associated with chronic inflammation may contribute to the development of heart failure.
A role for inflammation and MCP-1 in obesity and diabetes has been implicated. Adipogenesis is a key process involved in obesity and associated diseases such as type 2 diabetes. This process involves temporally regulated genes controlled by a set of transcription factors, C/EBPβ, C/EBPδ, C/EBPα, and PPARγ. Currently PPARγ is considered the master regulator of adipogenesis as no known factor can induce adipogenesis without PPARγ. We present evidence that a novel Zn-finger protein, MCPIP, can induce adipogenesis without PPARγ. Classical adipogenesis-inducing medium induces MCP-1 production and MCPIP expression in 3T3-L1 cells before the induction of the C/EBP family of transcription factors and PPARγ. Knockdown of MCPIP prevents their expression and adipogenesis. Treatment of 3T3-L1 cells with MCP-1 or forced expression of MCPIP induces expression of C/EBPβ, C/EBPδ, C/EBPα, PPARγ and adipogenesis without any other inducer. Forced expression of MCPIP induces adipogenesis in PPARγ-/- fibroblasts. Thus, MCPIP is a newly identified master controller that can induce adipogenesis without PPARγ.
Heart failure is a major cause of death in diabetic patients. Hyperglycemia is a major factor associated with diabetes that causes cardiomyocyte apoptosis that leads to diabetic cardiomyopathy. Cardiomyoycte apoptosis is a key event involved in the pathophysiological progression of diabetic cardiomyopathy. We have recently found that in ischemic hearts, MCP-1 can induce the zinc-finger protein, MCP-1 induced protein (MCPIP) that causes cardiomyocyte apoptosis. Although there is evidence that inflammation may play a role in diabetic cardiomyopathy, the underlying mechanisms are poorly understood. In this study, we show that treatment of H9c2 cardiomyoblasts and Neonatal Rat Ventricular Myocytes (NRVM) with 28mmol/L glucose concentration results in the induction of both transcript and protein levels of MCP-1 and MCPIP. Inhibition of MCP-1 interaction with CCR2 via specific antibody or with the G-coupled receptor inhibitors propagermanium and pertussis toxin attenuated glucose-induced cell death. Knockdown of MCPIP with specific siRNA yielded similar results. Treatment of cells with 28mmol/L glucose resulted in increased ROS production and phox47 activation. Knockdown of MCPIP attenuated these effects. The increased ROS production observed in H9c2 cardiomyoblasts and NRVM’s resulted in increased ER stress proteins GRP78 and PDI. Knockdown of MCPIP attenuated expression of both GRP78 and PDI. Inhibition of ER stress with TUDC and 4’PBA prevented high glucose-induced cell death death. Treatment of cells with 28mmol/l glucose resulted in autophagy as determined by an increase in expression of beclin-1 and through increased cleavage of LC3I to LC3II. Knockdown of MCPIP attenuated expression of beclin-1 and prevented cleavage of LC3. Addition of the autophagy inhibitors 3’methyladenine and LY294002 attenuated high glucose-induced H9c2 cardiomyoblast death. We conclude that high glucose-induced H9c2 cardiomyoblast death is mediated via MCP-1 induction of MCPIP that results in ROS that leads to ER stress that causes autophagy and eventual apoptosis.Ph.D.
Department of Biomolecular Science
Burnett College of Biomedical Sciences
Biomedical Sciences PhD
11
Harris, Brett Stuart. "Zinc-finger transcription factors in the Schwann cell lineage." Thesis, University College London (University of London), 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.395480.
Full text
12
Elrod-Erickson, Monica (Monica Ann) 1969. "Structural and biochemical studies of zinc finger-DNA complexes." Thesis, Massachusetts Institute of Technology, 1998. http://hdl.handle.net/1721.1/49650.
Full text
13
Allen, Carl E. "Functional analysis of the large zinc finger protein, KRC /." The Ohio State University, 2000. http://rave.ohiolink.edu/etdc/view?acc_num=osu14881967817341.
Full text
14
Schaufler, Lawrence E. "NMR studies of the ADR1 zinc finger transcription factor /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/5079.
Full text
15
Jantz, Derek Neil. "The DNA-binding properties of semisynthetic zinc finger proteins." Available to US Hopkins community, 2003. http://wwwlib.umi.com/dissertations/dlnow/3080688.
Full text
16
Brayer, Kathryn Jo. "The Protein Binding Potential of C2H2 Zinc Finger Domains." Diss., The University of Arizona, 2008. http://hdl.handle.net/10150/195146.
Full textAbstract:
Cys2-His2 (C2H2) zinc finger domains were originally identified as DNA binding domains, and uncharacterized domains are typically assumed to bind DNA. However, a growing body of evidence suggests an important and widespread role for these domains in protein binding. Over 100 C2H2 zinc finger-protein interactions have been described. This study uses common bioinformatics tools to identify sequence features that predict a DNA- or protein-binding function. Several issues, including uncertainties about the full functional capabilities of the zinc fingers, complicated these efforts. Therefore, an unbiased approach which directly examined the potential for zinc fingers to facilitate DNA or protein interactions was used to determine the full functional capabilities of the C2H2 domains in two model proteins, human OLF-1/EBF associated zinc finger (OAZ) protein and Zif268. OAZ contains 30 zinc fingers in six clusters, some of which have been previously indicated in DNA or protein interactions. Zif268 is a well-known DNA binding protein with three C2H2 domains. DNA binding was assessed using a target site selection (CAST) assay, and protein binding was assessed using a yeast two-hybrid assay. Results indicate that clusters known to bind DNA could facilitate specific protein interactions, but clusters known to bind protein did not facilitate specific DNA interactions, indicating that DNA binding is a more restricted function of zinc fingers than has previously been recognized. These results also suggest that the role of C2H2 zinc finger domains in protein interactions has probably been underestimated. The implication of these findings for the prediction of zinc finger function is discussed.
17
Bhakta, Mital Subhash. "Highly Active Zinc Finger Nucleases by Extended Modular Assembly." Diss., The University of Arizona, 2012. http://hdl.handle.net/10150/265392.
Full textAbstract:
C2H2-zinc fingers (ZFs) are commonly found in transcription factors that code for nearly 3% of gene products in the human genome. ZF proteins are commonly involved in gene regulation during development, cell differentiation, and tumor suppression. Each "finger" is a domain composed of approximately 30 amino acids. Since the discovery of these domains over 25 years ago, several groups have contributed to the structural and biochemical knowledge to understand their DNA-binding properties. Taking advantage of the simplicity of manipulating the DNA-binding potential of a ZF, the technology has now evolved to make sequence-specific Zinc Finger Nucleases (ZFNs), Artificial Transcription Factors (ATFs), Zinc Finger Recombinases, and DNA detection tools. ZFPs have been used for various applications, ranging from regulating genes by ZF-ATFs to manipulating genomes in diverse organisms. ZFNs have remarkably revolutionized the field of genome engineering. ZFN-modified T-cells have now advanced into human clinical trials for cell-based therapies as a treatment against HIV. Despite the advances in the ZFN technology, one of the challenges in the field is obtaining effective ZFNs using publicly available tools. The traditional method of synthesizing custom ZF arrays was using modular assembly (MA). In this method, preselected ZFs from publicly available one-finger archives can be assembled modularly to make long arrays. MA of ZFNs provides a rapid method to create proteins that can recognize a broad spectrum of DNA sequences. However, three- and four-finger arrays often fail to produce active nucleases. The low success rate of MA ZF arrays was attributed to the fact that they suffer from finger-finger incompatibility referred to as context-dependent effects. However, we hypothesized that the low affinity of MA arrays was the limiting factor. The work presented in this dissertation describes our efforts at addressing these fundamental methodological challenges. We developed the Extended Modular Assembly method that overcomes the limitations of both the previous Modular Assembly. We performed a systematic investigation of number and composition of modules on ZFN activity and analyzed ZFN specificity both in vitro and in vivo. Our current experiments apply the ZFNs produced by our method to study the role of genetic variation in human disease.
18
El-Husseini, Alaa El-Din A. S. "Molecular cloning and characterization of a novel zinc finger protein, brain expressed RING finger protein (BERP)." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq25043.pdf.
Full text
19
Ullah, Mukta. "Characterization of the tetrameric monocytic leukemia zinc finger protein complex." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=99211.
Full textAbstract:
In human cells, histones bind DNA and form chromatin, a nucleoprotein complex important for various cellular processes. Abnormal histone modification is known to play causal roles in the development of cancer. Monocytic leukemia zinc finger protein (MOZ) is a MYST-family histone acetyltransferase whose gene is rearranged in chromosomal translocations giving rise to acute myeloid leukemia. This acetyltransferase functions as a potent transcriptional coactivator of Runx1 and Runx2, two homologous transcription factors that are important for definitive haematopoiesis and osteoblast maturation, respectively. Mouse knockouts have demonstrated that MOZ is required for the maintenance of hematopoetic stem cells, and is important for erythroid and myeloid cell differentiation.Chapter I reviews the importance of chromatin regulation, MYST histone acetyltransferases and ties them together to explain how gene regulation is achieved. Chapter II addresses the characterization of the tetrameric MOZ complex, and suggests that its activity can be modulated by the presence of associated subunits such as BRPF1, and ING5. We show by histone acetyltransferase assays that MOZ and MORF activity is indeed enhanced by associated proteins. We have mapped the interaction domains of BRPF1 required for binding by MOZ, ING5, and Eaf6 in an effort to understand the mechanism of activation. Given that MOZ and ING proteins contribute to oncogenesis by chromosomal translocations and loss of function respectively, the characterization of the multisubunit complex provides novel mechanistic insights into its function in normal human cells and under conditions of leukemia or other cancers.
20
Ooi, Aik Teong. "Sequence-Specific DNA Detection Utilizing Custom-Designed Zinc Finger Proteins." Diss., The University of Arizona, 2007. http://hdl.handle.net/10150/194236.
Full textAbstract:
DNA diagnostics are important technologies in molecular and cellular biology. By allowing identification of specific sequences, DNA-based diagnostics potentially provide more accurate and rapid results than protein- or antigen-based diagnostics, primarily because phenotypic changes come much later than changes in genotype. Despite this advantage, there are fewer diagnostic or imaging systems that target DNA than those targeting proteins, antibodies, or antigens.Each type of DNA-based diagnostic has its own, unique set of limitations; however, most can be attributed to issues related to sequence restriction, signal detection, specificity, or some combination thereof. For example, while PCR-based methods allow amplification and assessment of specific DNA sequences, they lack the ability to report information of specific cells, or cell types, within the heterogeneous pool of cells typically found in a tumor biopsy. In addition, none of the currently available DNA detection methods has the potential to be utilized in living cells, a disadvantage which limits the potential applications.The work presented here describes the design and development of a new methodology for the detection of specific double-stranded DNA sequences. This detection method is based on the concept that two inactive fragments of a reporter protein, each coupled to engineered zinc finger DNA-binding motifs, are able to reassemble and form an active complex in the presence of a predefined DNA sequence. This system, designated sequence-enabled reassembly (SEER), can achieve single base-pair specificity, and has the potential to be utilized in living cells.In this dissertation, we discuss the efforts from constructing to refining the system, as well as the future applications of SEER in diagnostics and therapeutics. Chapter I will provide an introduction to DNA detection methods, on which the principles of the SEER system are based. Chapter II describes the design and construction of an enzymatic SEER system, SEER-LAC, using beta-lactamase as the enzyme. In Chapter III, we outline the in vitro characterization of the SEER-LAC system, followed by its optimization in Chapter IV. Chapter V illustrates the efforts to develop SEER system for mammalian cell culture applications. In the final chapter, the future developments and applications of SEER are discussed.
21
Cradick, Thomas James. "Designing zinc finger nucleases that specifically cleave Hepatitis B viral DNA." Diss., University of Iowa, 2009. https://ir.uiowa.edu/etd/790.
Full textAbstract:
Hepatitis B virus chronically infects 350-400 million people worldwide. It often leads to hepatocellular carcinoma, which causes >1 million deaths yearly. Current therapies prevent new viral genome formation but do not target pre-existing viral genomic DNA, thus curing only ~1/2 of patients. We targeted hepatitis B virus DNA for cleavage using zinc finger nucleases, which cleave as dimers. Co-transfection of our zinc finger nuclease pair with a target plasmid containing the hepatitis B virus genome resulted in specific cleavage. After three days in culture, 26% of the target remained linear, while ~10% was cleaved and mis-joined tail-to-tail.
A portion of cleaved plasmids are repaired in cells, often with deletions and insertions. To track misrepair, we introduced an XbaI restriction site in the spacer between the zinc finger nuclease sites. Targeted cleavage and misrepair destroys the XbaI site. After three days in culture, ~6% of plasmids were XbaI resistant. 13 of 16 clones sequenced contained frameshift mutations that would lead to dramatic truncations of the viral core protein. These results demonstrate for the first time the feasibility of targeting episomal viral DNA genomes in cells using zinc finger nucleases. This strategy is broadly applicable toward inactivating other DNA viruses within cells.
A major concern for the therapeutic use of zinc finger nucleases is off-target cleavage. To measure specificity, we employed in vitro assays and developed a bioinformatics method to find off-target cleavage sites in cultured cells. These sites can then be PCR amplified and tested using a mutation detection assay that we developed.
22
Fu, Fengli. "Computational approaches to understand interactions between zinc finger proteins and DNA." [Ames, Iowa : Iowa State University], 2010. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3403797.
Full text
23
Nomura, Akiko. "Redesign of the metal coordination sites in the zinc finger peptides." 京都大学 (Kyoto University), 2003. http://hdl.handle.net/2433/149185.
Full text
24
Edwards, Jessica K. "Investigating the roles of zinc finger homeobox 3 in circadian rhythms." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:1444375c-b7de-425a-a2ed-53b715833737.
Full text
25
Méndez, Vidal Cristina. "Molecular studies of WIG-1, A P53-induced zinc finger protein /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-732-0.
Full text
26
Heath, Helen Elizabeth. "CTCF: comprehending.the complex functions of an 11-zinc-finger transcription factor." [S.l.] : Rotterdam : [The Author] ; Erasmus University [Host], 2007. http://hdl.handle.net/1765/10861.
Full text
27
Nobelen, Suzanne van de. "Touched by CTCF analysis of a multi-functional zinc finger protein /." [S.l.] : Rotterdam : [The Author] ; Erasmus University [Host], 2008. http://hdl.handle.net/1765/12282.
Full text
28
Dutnall, Robert Nicholas. "Structural and functional studies of a zinc finger DNA-binding domain." Thesis, University of Cambridge, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360030.
Full text
29
Greisman, Harvey Allan. "A strategy for selecting zinc finger proteins for desired DNA sequences." Thesis, Massachusetts Institute of Technology, 1997. http://hdl.handle.net/1721.1/10773.
Full text
30
Delahaye, Celia. "Characterisation of the non-canonical zinc finger protein ZFP263 in mouse." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/278697.
Full textAbstract:
Multicellular organisms are composed of a number of different specialised cells that all carry the same genetic material but are highly divergent in their functions and characteristics. This diversity is only allowed because sets of specific genes are expressed in one type of cells and silent in others. A precise control mechanism is required to fine-tune gene regulation and relies on chromatin structure and regulatory proteins. One of the largest families of DNA-binding factors that influence this in human and mouse is the KRAB zinc finger protein (KZFP) family. KZFPs are thought to have rapidly evolved alongside transposable elements and be mediators of transcriptional repression. The few KZFPs that have been characterised so far have been shown to be involved in a wide range of regulatory and biological processes; hence it is hard to make functional generalisations. During my PhD, I studied one member of the KZFP family in mouse, ZFP263, with the aim of understanding its mechanism of action in mouse embryonic stem cells (mESCs) and its role in mice. My work has shown that ZFP263 is an ancient protein highly conserved in mammals and under purifying selection. One of its two functional domains however is divergent from the consensus sequence found in most KZFPs and suggests that ZFP263 might have lost the ability to recruit repressive chromatin states. My research identified the targets of ZFP263 binding in mESCs and showed that it does not bind and silence transposable elements. Indeed it targets unique regions of the genome, mostly within transcribed regions of genes. These genes show a wide range of expression levels and are involved in several key biological processes. Surprisingly, binding sites are not associated with the canonical KZFP co-factor but mostly co-localize with active histone marks. My findings lead me to hypothesise that ZFP263 has evolved to target active epigenetic states to unique regions that are positive regulators of transcription, in contrast to the more canonical model of KZFP function. To test this hypothesis, I have generated targeted mutations at Zfp263 in mice using CRISPR-Cas9 and my preliminary results suggest that Zfp263 mutants have growth defects indicating a role for this protein in mouse development. My findings indicate that ZFP263 is a unique KZFP with non-canonical properties and provide novel insights into the evolution and functions of KZFPs in mammals.
31
Tsotsoros, Samantha. "Platinum Complexes and Zinc Finger Proteins: From Target Recognition to Fixation." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/610.
Full textAbstract:
Bioinorganic chemistry strives to understand the roles of metals in biological systems, whether in the form of naturally occurring or addition of non-essential metals to natural systems. Metal ions play vital roles in many cellular functions such as gene expression/regulation and DNA transcription and repair. The study of metal-protein-DNA/RNA interactions has been relatively unexplored. It is important to understand the role of metalloprotein interactions with DNA/RNA as this enhanced knowledge may lead to better understanding of diseases and therefore more effective treatments. A major milestone in the development of this field was the discovery of the cytotoxic properties of cisplatin in 1965 and its FDA approval in 1978. Since then, two other chemotherapeutic drugs containing platinum, carboplatin and oxaliplatin, have been used in the clinic. These three compounds are all bifunctional with the ligands surrounding platinum In the cis conformation and rearrangement of the ligands to the trans orientation results in a loss of cytotoxic properties due to rapid deactivation through binding to S-containing proteins. This enhanced reactivity yields new opportunities to study the reactions between proteins and DNA. One of the first crosslinking experiments used transplatin to crosslink NCp7 to viral RNA in order to understand how/where the protein bound to RNA. We have studied the interaction between cis and trans dinuclear platinum complexes and the C-terminal zinc finger (ZF). The trans complex reacts at a faster rate than the cis isomer and causes N- terminal specific cleavage of the ZF. The dinuclear structure plays a critical role in the peptide cleavage as studies with transplatin (the mononuclear derivative) does not result in cleavage. Monofunctional trans platinum-nucleobase complexes (MPNs) serve as a model for the binding of transplatin to DNA. This provides an interesting opportunity to study their reactions with S-containing proteins, such as HIV1 NCp7. MPNs have been shown to bind to the C-terminal ZF of HIV1 NCp7, resulting in zinc ejection. This occurs through a two-step process where the nucleobase π-stacks with Trp37 on the ZF, followed by covalent binding at the labile Cl site to Cys. MPNs have also shown antiviral activity in vitro. The labile Cl on MPNs reduces specificity of these compounds, as it leaves an available coordination site on the platinum center for binding to other S-proteins or DNA. Therefore, we have moved to an inert PtN4 coordination sphere, [Pt(dien)L]2+ (dien= diethylenetri- amine). Due to the strong bond between platinum and nitrogen, covalent reactions are highly unlikely to occur at rapid rates. The strength of the pi-stacking interaction between nucleobases (free and platinated) and the aromatic amino acid, tryptophan (Trp), showed an enhanced binding constant for platinated nucleobases. This was confirmed by density functional theory (DFT) calculations as the difference in energy between the HOMO of Trp and the LUMO of the nucleobase was smaller for the platinum complex. The studies were extended to the Trp-containing C-terminal ZF of HIV1 NCp7 and an increase in association constant was seen compared to free Trp. Reaction of PtN4 nucleobases compounds with a short amino acid sequence con- taining either Ala (no pi-stacking capabilities) or Trp (pi-stacking interactions) revealed an enhanced rate of reactivity for the Trp-containing peptide. This result supports the theory of a two-step reaction mechanism where the platinum-nucleobase complex recognizes the pep- tide through a pi-stacking interaction with Trp followed by covalent binding to the platinum center. The [Pt(dien)L]2+ motif allows for systematic modification of the structural elements surrounding platinum in a search for the most effective compound. Methylation of the dien ligand should, in theory, increase lipophilicity of the compounds, however, due to 2+ charge of the compounds, this simple association does not hold true. Analysis of the cellular accumulation profiles showed little change in the uptake with the addition of methyl groups to the dien ligand, in agreement with the non-linear change in lipophilicity. Modification of L using different nucleobases allows for the tuning of the strength of the π-stacking interaction between Trp and the platinum complex. The addition of inosine (which lacks a H-bonding donor/acceptor at the C2 position) resulted in a lower association constant with both N-AcTrp and the C-terminal zinc finger of HIV1 NCp7. Interestingly, the addition of xanthosine resulted in an ehanced pi-stacking interaction with the C-terminal zinc finger of HIV1 NCp7; likely as a results of the addition of a H-bonding donor (double-bonded O) at the C2 position. The ability of PtN4 nucleobase complexes to inhibit formation of the NCp7 complexation with viral RNA was studied by mass spectrometry and gel electrophoresis. Dissociation of the NCp7-RNA complex was seen upon addition of PtN4 compounds. These compounds were also able to retard formation of the NCp7-RNA complex when pre-incubated with the protein. These results have important implications as inhibition of complex formation between NCp7 and viral RNA has negative implications for viral replication. Despite the success of platinum-nucleobase compounds, it is important to evaluate all potential pi-stacking ligands. A series of pyridine- and thiazole-based compounds were evaluated for the strength of the pi-stacking interaction with N-AcTrp and the C-terminal ZF of HIV1 NCp7. There was notable increase in association constant for the platinum- DMAP (4-dimethylaminopyridine) complex compared to other ligands studied. This result highlights the importance of exploring multiple avenues for the design of specifically targeted inhibitors and further confirms the viability of the medicinal chemistry dual approach of target recognition (non-covalent) followed by target fixation (covalent).
32
Yang, Sile. "Functional Characterisation and Therapeutic Potential of the Zinc Finger Protein ZNF827." Thesis, The University of Sydney, 2019. http://hdl.handle.net/2123/21100.
Full textAbstract:
The ends of linear eukaryotic chromosomes resemble double strand breaks, susceptible to the vigilant cellular DNA damage response machinery. Telomeres are specialised nucleoprotein structures found at the ends of human chromosomes that function as a protective cap safeguarding the integrity of the genome. In normal human somatic cells, telomeres shorten with each replicative cell division. Eventually, shortened telomeres trigger cellular senescence and apoptosis. Cancer cells overcome this proliferative barrier by acquiring a telomere maintenance mechanism. Alternative Lengthening of Telomeres (ALT) is a homologous recombination (HR) mediated telomere maintenance mechanism utilised by approximately 10-15% of all cancers. ALT status in certain cancer subtypes, such as osteosarcomas and astrocytomas, often predicts aggressive nature and poor prognosis. Therefore, the ALT pathway presents an attractive avenue for developing novel cancer therapeutics. To date, ALT specific therapeutic targets are lacking. Our lab has previously identified the zinc finger protein ZNF827 as a promising molecular target in the ALT pathway. ZNF827 recruits the nucleosome remodelling and histone deacetylase (NuRD) complex to ALT telomeres, and collaboratively facilitates multifaceted functions to promote HR-mediated telomere synthesis. However, the precise molecular mechanisms underlying the important roles of ZNF827 at ALT telomeres have not been fully elucidated. Prior to the study by our lab, ZNF827 was a protein of unknown function. This thesis has focused on the functional characterisation of ZNF827, with an ultimate goal of validating it as a therapeutic target in cancers utilising the ALT pathway. We have characterised ZNF827 from several perspectives. First, we have explored the biological functions of ZNF827 as a transcription factor and discovered novel roles of ZNF827 in cellular processes including embryonic development and immune response. Second, we have gained new insights into the recruitment of ZNF827 to ALT telomeres, characterised structurally and functionally the interaction interface between ZNF827 and the NuRD complex, and implicated ZNF827 SUMOylation in promoting ALT activity. We have also discovered that ZNF827 is novel ssDNA binding protein implicated in HR-directed repair of replication-associated DNA damage at telomeres as well as genome-wide, and that its role as a DNA damage response and repair protein is likely to be mediated through the ATR-mediated DNA signalling pathway. Finally, we have provided preliminary evidence that supports synthetic lethality between ZNF827 inhibition and the topoisomerase I inhibitor topotecan, implicating ZNF827 as a potential molecular target for ATR inhibition. Taken together, our findings have substantially expanded our knowledge on the zinc finger protein ZNF827 and provided further support for its therapeutic potential in the development of novel cancer therapies.
33
Kentner, Jeffrey Louis. "Engineering the zinc finger recombinase for use in targeted genomic editing." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6910/.
Full text
34
Bird, Amanda Jane. "Zinc homeostasis in Synechococcus PCC 7942." Thesis, University of Newcastle Upon Tyne, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.245707.
Full text
35
Hamilton, Tatyana. "Protein-nucleic acid interactions of Wilms' tumor and TFIIIA zinc finger proteins." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ34266.pdf.
Full text
36
Green, Andrew F. D. "Metal ligation in ZIF268, a zinc finger protein, effects on DNA binding." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1996. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ45850.pdf.
Full text
37
Pelletier, Nadine. "The monocytic Leukemia zinc finger protein MOZ and its related factor MORF /." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=84309.
Full textAbstract:
Regulation of chromatin structure involves histone modifications such as acetylation. Since 1996, the identification and characterization of histone acetyltransferases have had tremendous impact on our understanding of the molecular mechanisms related to eukaryotic gene regulation and human diseases associated with abnormal chromatin functions. The MYST family of histone acetyltransferases is very interesting because of their various biological functions. In agreement with the correlation between aberrant histone acetylation and cancer, the MYST family proteins MOZ and MORF are linked to leukemogenesis.Identification and characterization of a gene encoding a novel histone acetyltransferase were the goals of this thesis project. Human MORF gene was cloned and the encoded protein, MORF, was shown to be very similar to MOZ. Biochemical studies demonstrated that both MOZ and MORF possess intrinsic histone acetyltransferase activities. The amino- and carboxy-terminal regions of MOZ and MORF contain transcriptional repression and activation domains, respectively.
Runx2, an osteoblast-specific transcription factor, binds to the activation domains of MOZ and MORF and thus recruits them to the osteocalcin promoter for transcriptional activation. TAZ, a WW-domain transcriptional coactivator of Runx2, potentiates the transcriptional activation of the osteocalcin promoter by MOZ and Runx2. Interestingly, treatment of cells with PMA enhances the synergy between MOZ and TAZ in activating the osteocalcin promoter. Consistent with this, PMA treatment strengthens the interaction of Runx2 with MOZ and TAZ.
This study, therefore, identified the histone acetyltransferase MORF and demonstrated that MOZ and MORF are transcriptional coactivators, thus providing new insights into how histone acetyltransferases are implicated in cell differentiation and leukemogenesis.
38
Lin, Ying [Verfasser]. "Isolation and characterization of DNA aptamers for zinc finger proteins / Ying Lin." Berlin : Freie Universität Berlin, 2009. http://d-nb.info/1023581051/34.
Full text
39
Kim, Juhwa. "Multiplexed Detection of Double-Stranded Pathogenic DNA with Engineered Zinc Finger Proteins." TopSCHOLAR®, 2016. http://digitalcommons.wku.edu/theses/1616.
Full textAbstract:
The development of a new technology for the detection of doublestranded (ds) DNA enables multiple biomedical applications including identifying multiple pathogens simultaneously. We previously employed colorimetric SEquence-Enabled Reassembly with TEM-1 β-lacatamase (SEER-LAC) to detect specific bacterial DNA sequence. SEER-Lac consists of the two inactive β-lactamase fragments which of each attached to a zinc finger protein (ZFP) would reassemble into an active full-length enzyme upon ZFPs binding to its target DNA. Here, we engineered two pairs of ZFPs which of each recognizes shiga toxin in E. coli O157 and staphylococcal enterotoxin B in Staphylococus Aureus, respectively. Biotin was simply conjugated to the detection probe ZFP, which allows for generating chemiluminescent signal in streptavidin-HRP (Horseradish peroxidase) assay upon ZFPs binding to their target DNA. Our assay generates DNA-dependent signal and allows for a detection limit of 0.5 nM without DNA amplification or DNA labeling. Our system can be developed into a simple multiplexed detection diagnostic for multiplexed detection of dsDNA.
40
Bahadoran, Mahshid. "Role of the Transcription Factor Zinc Finger Protein 521 on Runx2 Acetylation." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17331945.
Full textAbstract:
Runx2 is a transcription factor that has a crucial role in the development of bone; haploinsufficiency of Runx2 leads to the autosomal-dominant disorder, cleidocranial dysplasia (CCD) characterized by various skeletal abnormalities. Zinc finger protein 521 (Zfp521) is a transcription factor that is expressed in several cell types including bone. Recent studies demonstrated that Zfp521 interacts with Runx2 and regulates osteoblast maturation at least in part by repressing the transcriptional activity of Runx2; furthermore, it was demonstrated that the repression of Runx2 by Zfp521 involves the recruitment of HDAC3. The interaction of Runx2 with HDAC3 is strongly enhanced by Zfp521. Zfp521 may regulate osteoblast commitment and differentiation by modulating the Runx2 transcriptional activity by decreasing the levels of Runx2 acetylation.
Objective: Runx2 is a key regulator of osteoblast differentiation; Zfp521 may regulate osteoblast commitment and differentiation, at least in part by decreasing the levels of Runx2 acetylation. We investigated 1) Effect of Zfp521 on Runx2 acetylation in HEK293 cells 2) The endogenous Runx2 acetylation levels in MC3T3-E1 cells during osteoblast differentiation.
Results: These studies demonstrated that Runx2 acetylation is decreased when Zfp521 is stable expressed in HEK293 cells. Runx2 acetylation levels were detected using immunoprecitation analyses. Sodium butyrate (NaB) prevents protein deacetylation by inhibiting HDACs. The treatment of cells with NaB increased global protein acetylation levels. Importantly, stable expression of Zfp521 did not change global protein acetylation. Therefore, this study suggested that Zfp521 specifically influences Runx2 acetylation. In addition, our findings suggest that Zfp521 impairs Runx2 acetylation by HDACs. Preliminary results show that transient transfection of P300 in HEK293 increased Runx2 acetylation levels. However, stable expression of Zfp521 can still partially decrease Runx2 acetylation levels. This study suggests that Zfp521 function can be linked to P300.
Runx2 acetylation levels were then assessed in MC3T3-E1 cells during osteoblast differentiation. While Runx2 protein levels increase by 7 days in culture, and gradually decreases by days 14, Runx2 acetylation was undetectable.
Conclusion: Runx2 is the transcription factor that has essential role in osteoblast commitment and differentiation. Zfp521 represses the transcriptional activity of Runx2 by recruiting HDAC3. These studies suggest that Zfp521 modulates Runx2 activity by decreasing Runx2 acetylation level. These studies have extended our knowledge of the mechanisms by which Zfp521 regulates osteoblast differentiation and bone formation, which could have important implications for on the development of future osteo-anabolic treatments.
41
PIERACCIOLI, MARCO. "Functional role of the zinc finger factor ZNF281 in DNA damage response." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2015. http://hdl.handle.net/2108/203092.
Full textAbstract:
La sopravvivenza degli organismi dipende dall'accurata trasmissione dell'informazione genetica durante i processi che portano alla divisione cellulare. Questa fedele trasmissione richiede non soloun'elevata precisione nella replicazione del DNA e nella perfetta segregazione cromosomica, ma anche la capacità di sopravvivere ai danni al DNA spontanei o indotti minimizzando il numero di mutazioni ereditabili dalle cellule figlie. Le cellule sono constantemente soggette agli effetti citotossici e mutageni degli agenti in grado di causare danno al DNA. Per rispondere e difendersi da questa minaccia, le cellule eucariotiche hanno evoluto dei processi che sono collettivamente catalogati come risposta al danno al DNA (DDR). La DDR è una complessa via di trasduzione del segnale che ha la capacità di riconoscere la presenza di danni al DNA e trasdurre questa informazione nella cellula al fine di attivare i processi di riparo. Infatti, le cellule possiedono diversi enzimi capaci di rimodellare e riparare il DNA, comunque la loro attività deve essere spazialmente e temporalmente regolata per ottimizzare il riparo del DNA e prevenire potenziali e deleterie alterazioni nella struttura del DNA. In questi ultimi anni considerevoli progressi sono stati fatti per delucidare le componenti e i processi che caratterizzano la DDR eucariotica. Nonostante questo, un punto centrale in questo campo di studio rimane l'identificazione dei geni e delle proteine che controllano l'espressione delle proteine coinvolte nella DDR. Di interesse è la recente scoperta di un numero crescente di fattori di trascrizione che in grado di regolare direttamente la riparazione del DNA e che fanno parte come componenti integrali del macchinario di riparo stesso con meccanismi indipendenti dalla loro abilità di promuovere e/o reprimere la trascrizione. Infatti, gli agenti di danno al DNA (radiazioni, chemioterapia, agenti genotossici) sono in grado di promuovere la traslocazione di alcuni fattori di trascrizione direttamente sulle lesioni al DNA, dove partecipano all'attivazione del riparo al DNA. ZNF281 è un fattore di trascrizione appartenente alla tipologia degli "zinc finger" coinvolto nel mantenimento della staminalità cellulare e nei processi di transizione epiteliale-mesenchimale (EMT). In questo studio vengono analizzati i ruoli di ZNF281 durante la DDR. Infatti, riportiamo che l'espressione di ZNF281 aumenta sia a livello di mRNA, che di proteina dopo stress genotossico indotto da agenti chimici che causano danno al DNA (etoposide, doxorubicina, camptotecina) in diversi sistemi quali le cellule tumorali, cellule di cheratinociti primari e nell’epidermide del Mus musculus. Il comet assay dimostra che il riparo al DNA è ritardato nelle cellule silenziate per l'espressione di ZNF281 e trattate con etoposide. Inoltre, l'analisi con RT profiler array ha dimostrato che l'espressione di dieci geni coinvolti nella risposta al danno al DNA è diminuita nelle cellule trattate con etoposide e silenziate per l'espressione di ZNF281. In linea con questa scoperta, XRCC2 e XRCC2, due geni che prendono parte rispettivamente ai processi di ricombinazione omologa (HR) e Non Homologous End Joining (NHEJ), sono trascrizionalmente attivati da ZNF281 attraverso un meccanismo dipendente dal legame al DNA come dimostrato da esperimenti di saggio della luciferasi e di immunoprecipitazione della cromatina (ChIP). Inoltre, ZNF281 è un co-fattore della proteina c-Myc per l'attivazione dell'espressione della nucleolina e della ciclina B1; mentre c-Myc, anche se in grado di legare promotori di XRCC2 e XRCC4, non è capace di promuovere la trascrizione o di modificare l'attività di ZNF281 sui promotori di questi due geni. L'analisi bioinformatica di una coorte di 1971 pazienti affetti da cancro alla mammella rivela una correlazione statisticamente significativa tra l'espressione di ZNF281 e di XRCC2. Inoltre l'analisi proteomica e il proximity ligation assay (PLA) dimostrano che ZNF281 interagisce con DNA-PK, un'importante proteina del processo di DDR, suggerendo per ZNF281, un ruolo indipendente dalla trascrizione. In sintesi, i risultati discussi in questo lavoro, evidenziano per la prima volta il coinvolgimento di ZNF281 nella risposta cellulare a stress genotossici attraverso un controllo esercitato sull'espressione di geni che agiscono in differenti meccanismi di riparo al danno e attraverso la sua interazione con DNA-PK.The survival of organisms depends on the accurate transmission of genetic information from one cell to its daughters. Such faithful transmission requires not only extreme accuracy in replication of DNA and precision in chromosome distribution, but also the ability to survive spontaneous and induced DNA damage while minimizing the number of heritable mutations. Therefore, cells are constantly under threat from the cytotoxic and mutagenic effects of DNA damaging agents. To respond to these threats, eukaryotes have evolved the DNA damage response (DDR). The DDR is a complex array of different mechanisms that have the ability to sense DNA damage and transduce this information to the cell in order to modulate cellular responses to DNA damage. Cells possess several enzymatic tools capable of remodeling and repairing DNA; however, their activities must be tightly regulated in a temporal, spatial, and DNA lesion-appropriate fashion to optimize repair and prevent unnecessary and potentially deleterious alterations in the structure of DNA during normal cellular processes. During the past several years, considerable progress has been made in elucidating the components and the processes of the eukaryotic DDR. A central issue in this field, which remains to be understood in greater detail, is the identification of the controllers of the expression of DDR proteins. Interestingly, in recent years an increasing number of studies have revealed that several TFs regulate DNA repair directly and can function as integral components of the repair machinery itself in a transcription independent fashion. In fact, DNA damage-inducing insults (irradiation, chemotherapy drugs) promote translocation of some TFs directly to DNA lesions, where they actively facilitate DNA repair. ZNF281 is a zinc finger transcription factor involved in the control of cellular stemness and Epithelial Mesenchymal Transition (EMT). In this study we analyze the roles of ZNF281 during DDR. We report that ZNF281 expression increased after genotoxic stress caused by DNA damaging drugs (Etoposide, Doxorubicin, Camptothecin) in cancer cell lines, normal keratinocytes and in mouse skin in vivo. Comet assays demonstrated that DNA repair was delayed in cells silenced for the expression of ZNF281 and treated with Etoposide. Furthermore, RT profiler array analysis demonstrated that the expression of ten DDR genes was down-regulated in cells treated with Etoposide and silenced for ZNF281. In line with these findings, XRCC2 and XRCC4, two genes that take part in Homologous Recombination (HR) and Non Homologous End Joining (NHEJ) respectively, were transcriptionally activated by ZNF281 through a DNA binding-dependent mechanism as demonstrated by luciferase assays and Chromatin crosslinking ImmunoPrecipitation (ChIP) experiments. In addition, ZNF281 works as a c-Myc co-factor to stimulate the expression of nucleolin and cyclin B1; instead c-Myc, which also binds to the promoters of XRCC2 and XRCC4, was unable to promote their transcription or to modify ZNF281 activity. Bioinformatic analysis of 1971 breast cancer patients disclosed a significant correlation between the expression of ZNF281 and XRCC2. Moreover proteomic analysis and Proximity Ligation Assay (PLA) demonstrated that ZNF281 interacts with DNA-PK, an important protein of DDR, suggesting a transcription-independent role of ZNF281 in DDR. Our data highlight, for the first time, the involvement of ZNF281 in the cellular response to genotoxic stress through the control exercised on the expression of genes that act in different repair mechanisms and through interaction with with corecomponents of DNA repair pathways.
42
Gaither, L. Alex. "Molecular and biochemical characterization of the human zinc transport proteins hZip1 & hZip2 /." free to MU campus, to others for purchase, 2001. http://wwwlib.umi.com/cr/mo/fullcit?p3025618.
Full text
43
Czerny, Florian. "Development of Zinc-Finger-Based Artificial Restriction Endonucleases and Fluorescent Peptidyl Metal Sensors." Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2016. http://hdl.handle.net/11858/00-1735-0000-002B-7CAB-3.
Full text
44
Chung, Ho Ryun. "The zinc finger associated domain of Drosophila melanogaster, its evolution and phylogenetic restriction." [S.l. : s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=974179248.
Full text
45
Mark, Charlotta. "Three Subfamilies of KRAB Zinc Finger Proteins : A Structural, Functional and Evolutionary Analysis." Doctoral thesis, Uppsala University, Department of Cell and Molecular Biology, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3512.
Full textAbstract:
Krüppel-related zinc finger proteins constitute the largest single class of transcription factors within the human genome. Members of this protein family have the ability to either activate or repress transcription depending on the presence of specific activator or repressor domains within the protein. Approximately one third of the Krüppel-related zinc finger proteins contain an evolutionarily well-conserved repressor domain termed the KRAB domain. This domain acts as a potent repressor of transcription by interacting with the co-repressor protein, TIF1β. TIF1β then, in turn, recruits HP1 proteins, HDACs and probably other proteins involved in gene silencing. In order to identify novel KRAB-containing zinc finger proteins, one mouse monocytic cDNA library and two testis cDNA libraries were screened for novel members of this multigene family. Six novel KRAB-ZNF cDNAs, four mouse and two human, were isolated. The corresponding proteins were all shown to contain N-terminally located KRAB domains as well as varying numbers of C-terminally located zinc finger motifs. An extensive comparative sequence analysis of the KRAB domains of these proteins together with KRAB domains from a large number of previously identified KRAB-ZNF proteins resulted in a clear subdivision into three different subfamilies, A+B, A+b and A. Later, we also isolated a fourth KRAB box, which is present downstream of the KRAB A box in a few proteins of the KRAB A family. This module was named KRAB C. Potential functional differences between these different subfamilies were investigated. In line with previous observations, the KRAB A box was shown to repress transcription, an activity which was enhanced by the presence of the KRAB B box. However, addition of neither the KRAB b box nor the KRAB C box had any effect on repression. Moreover, all KRAB A motifs had the ability to bind TIF1β, and this binding was increased both by the presence of the KRAB B box and by the KRAB C box. The KRAB b box, however, did not seem to contribute to TIF1β-binding. One of the novel human cDNAs, HKr19, was found to be a member of the large ZNF91 family of KRAB zinc finger genes. Interestingly, the expression of HKr19 and a number of other closely related genes were restricted to lymphoid cells, indicating that these genes may be involved in regulating lineage commitment. The effect of HKr19 on cell viability was investigated by transfection into human embryonic kidney cells (HEK 293). The results indicated that HKr19, or its zinc finger domain in isolation, were toxic to these cells when expressed at high levels. The MZF6D protein, on the other hand, showed a testis-specific expression. In situ hybridization analysis located this expression to meiotic germ cells, suggesting a role for this protein in spermatogenesis. Further, the evolutionary perspectives of this large gene family were addressed, and its enormous expansion throughout evolution probably includes numerous duplication events. The results from two extensive sequence analyses give clues to how the repetitive nature of the ZNF motif has given rise to both internal duplications of single motifs as well as duplications of entire genes resulting in gene clusters.
46
Yan, Wei. "Design of artificial 6-zinc finger peptides : linker alteration and DNA binding selectivity." 京都大学 (Kyoto University), 2008. http://hdl.handle.net/2433/137164.
Full text
47
Kulczyk, Arkadiusz Wojciech. "NMR and biochemical studies of novel zinc-finger domains binding to nicked DNA." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.619875.
Full text
48
Özkan, Burak. "Role of zinc finger protein WIZ in the recruitment of histone methylase G9a." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28731.
Full textAbstract:
The N-terminal tails of histones are subject to many chemical modifications that are involved in a variety of biological functions. Histone methylation is a major epigenetic modification found in both single and multicellular organisms and is directly involved in the regulation of gene expression. Methylation of lysine 9 of histone 3 (H3K9) has been shown to have diverse functions depending on the number of methyl groups added; H3K9me1 marks the active promoters, while H3K9me2 and H3K9me3 are present within inactive gene promoters and pericentric heterochromatin. G9a, also known as euchromatic histone-lysine N-methyltransferase 2 (Ehmt2), is a histone methylase that catalyses addition of mono- and dimethyl groups to H3K9 in euchromatic regions of the genome to silence genes. Therefore, it is a vital component of the gene expression regulation machinery. In mouse embryonic stem (ES) cells, G9a forms a stable heterodimer with the G9a-like protein (GLP or Ehmt1), which is further stabilised by the C2H2-type zinc finger protein, widely interspaced zinc finger protein (WIZ). These three proteins form the core G9a complex, which is essential for mouse development. Lack of any G9a complex member leads to embryonic lethality at E9.5 with severe growth defects. The ankyrin repeat domain of G9a/GLP can bind to H3K9me1/2 with high affinity in vitro (Collins et al. 2008). This enables the self-recruitment of the G9a complex to sites with H3K9me1/2 and maintenance of the mark. However, the initial recruitment of the G9a complex to sites lacking H3K9me1/2 mark during differentiation is poorly understood. Neither G9a nor GLP has a DNA/RNA binding domain, so recruitment of the G9a complex to specific sites must be mediated by other binding partners of the G9a complex. Using mass spectrometry, I was able to identify a number of zinc finger proteins as binding partners of G9a. Among these, WIZ was identified in stoichiometric amounts to G9a and GLP, and is a potential DNA binding protein similar to other C2H2-type zinc fingers. The aim of this study was to determine the role of WIZ in the recruitment of the G9a complex to specific sites. I showed that knockdown of WIZ had no significant effect on the chromatin binding of G9a in undifferentiated mouse ES cells, which indicates WIZ is dispensable in the maintenance of H3K9me2. However, I observed a 30% decrease in the G9a levels upon WIZ knockdown, which shows that WIZ might have a role in stabilising G9a. Using recombinant WIZ zinc finger pairs, I was able to show that the 3rd and 4th zinc finger of WIZ bind DNA in vitro. Furthermore, using the systematic evolution of ligands exponential enrichment (SELEX) approach I demonstrated that the zinc fingers of WIZ preferentially bind to G-rich double-stranded DNA sequences. Binding site analysis with synthetic DNA indicated that WIZ ZF3-4 require two binding sites that are a certain distance apart from each other for efficient binding. In addition, ZF3-4 binds ssDNA with higher affinity than dsDNA, and binding to ssDNA is sequence-independent. This study shows for the first time that mouse WIZ zinc finger pairs can bind DNA and RNA in vitro. Therefore, sequence-specific recruitment of G9a might be mediated by WIZ during differentiation. Furthermore, DNA binding preference of WIZ might suggest that WIZ-mediated recruitment of G9a to establish H3K9me2 could occur at the R-loops where G-rich DNA forms a hybrid with newly transcribed RNA or at the G-rich repetitive sequences.
49
Oliveira, Alessandra Rejane Ericsson de. "Identificação e caracterização de uma proteína com motivos ZINC FINGER de Trypanosoma cruzi." reponame:Repositório Institucional da UnB, 2006. http://repositorio.unb.br/handle/10482/3320.
Full textAbstract:
Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2006.Submitted by Larissa Ferreira dos Angelos (ferreirangelos@gmail.com) on 2009-11-14T12:47:05Z No. of bitstreams: 1 2006_Alessandra Rejane Ericsson de Oliveira.pdf: 1765643 bytes, checksum: 9a175047678704e91e49247033e86e47 (MD5)
Approved for entry into archive by Marília Freitas(marilia@bce.unb.br) on 2010-01-19T16:03:40Z (GMT) No. of bitstreams: 1 2006_Alessandra Rejane Ericsson de Oliveira.pdf: 1765643 bytes, checksum: 9a175047678704e91e49247033e86e47 (MD5)
Made available in DSpace on 2010-01-19T16:03:40Z (GMT). No. of bitstreams: 1 2006_Alessandra Rejane Ericsson de Oliveira.pdf: 1765643 bytes, checksum: 9a175047678704e91e49247033e86e47 (MD5) Previous issue date: 2006
Proteínas zinc finger são compostas por domínios compactados contendo α- hélices e folhas β unidos e estabilizados por um ou dois átomos de zinco. Arranjos repetidos de zinc fingers são comumente utilizados para reconhecimento de ácidos nucléicos. Dentre outras atividades, eles estão envolvidos em replicação, transcrição e reparo de DNA. No nucleocapsídeo do vírus HIV tipo 1 foi identificada uma proteína contendo o motivo zinc finger CX2CX4HX4C, estando esta proteína envolvida em várias etapas do ciclo de vida viral, incluindo a propriedade de encapsidação do RNA viral. Em tripanosomatídeos, somente poucas proteínas contendo o motivo zinc finger já foram identificadas até o presente momento. Em um fragmento genômico de 17 kb da banda XX de T. cruzi, nós identificamos três genes in tandem codificando para proteínas zinc finger do tipo CX2CX2HX4C. Nós também demonstramos que genes similares estão presentes em T. brucei e L. major como três definidos grupos monofiléticos entre esses tripanosomatídeos. Em T. cruzi, TcZFP8 corresponde a um novo gene codificando para uma proteína com oito motivos zinc finger. O homólogo de TcZFP8 em T. brucei é aparentemente ausente, enquanto um candidato foi identificado em L. major. A clonagem molecular e a expressão heteróloga de TcZFP8 foi realizada para produção de anticorpos e procedimentos como imunocitolocalização, SELEX e EMSA. Análise por Western blotting revelou a presença dessa proteína nas três formas do parasita. Análises usando extratos protéicos nucleares e citoplasmáticos de T.cruzi mostraram que essa proteína está presente na porção nuclear. Esse resultado foi confirmado através de análise de microscopia por imunofluorescência indireta. Experimento de SELEX demonstrou quatro diferentes populações com uma região interna rica em C e/ou G, porém sem seqüências consenso específicas. Análises preliminares de EMSA de uma das quatro populações selecionadas revelaram evidências de que TcZPP8 possa ter afinidade de ligação à fita simples de DNA. __________________________________________________________________________________________ ABSTRACT
Zinc fingers are compact protein domains composed of a α-helix and a β-sheet held together by a zinc ion. Tandem arrays of zinc fingers are commonly used to recognize nucleic acids. Among other activities, they are involved in the processes of replication, transcription, and DNA repair. The nucleocapsid protein of HIV-1 contains a zinc finger motif CX2CX4HX4C that contributes to multiple steps of the viral life cycle, including the proper encapsidation of HIV RNA. In trypanosomatids, only a few of the proteins that contain such fingers were identified. In a 17-kb genomic fragment of Trypanosoma cruzi chromosome XX we identified three tandemly linked genes coding for CX2CX4HX4C zinc finger proteins. We also showed that similar genes are present in Trypanosoma brucei and Leishmania major sharing three monophyletic groups among these trypanosomatids. In T. cruzi, TcZFP8 corresponds to a novel gene coding for a protein containing eight zinc finger motifs. Homologous of TcZFP8 in T. brucei is apparently absent, while one candidate in L. major was identified. Molecular cloning of gene TcZFP8 and heterologous expression were performed in Escherichia coli. The purified recombinant protein His6x-TcZFP8 was used to produce antibody in rabbits and GST-TcZFP8 in SELEX and EMSA procedures. Using Western blot analysis, we observed the presence of this protein in all three forms of the parasite: amastigote, trypomastigote and epimastigote. Analysis using cytoplasm and nuclear cell extracts showed that this protein is present in the nuclear extracts and indirect immunofluorescence microscopy analysis confirmed the nuclear localization of the TcZFP8. SELEX experiment showed four different populations rich in C and/or G nucleotides, but with none consensus sequence. Preliminary EMSA from one population gave evidence that TcZFP8 has affinity to bind to singlestranded DNA.
50
Del, Rio Samuel. "Structural and functional studies of Xenopus laevis transcription factor IIIA zinc finger mutants." Case Western Reserve University School of Graduate Studies / OhioLINK, 1992. http://rave.ohiolink.edu/etdc/view?acc_num=case1056548235.
Full text