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1
Blancafort, Pilar. "Making conformation-specific RNA-binding zinc fingers." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0023/NQ47598.pdf.
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Giesecke, Astrid. "Protein-protein interactions mediated by Cys2His2 zinc-fingers." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=981809715.
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Isalan, Mark David. "Selection of zinc fingers with novel DNA-binding specificities." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621667.
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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.
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Hallal, Samantha. "Characterisation of the zinc fingers of erythroid krüppel-like factor." Connect to full text, 2008. http://ses.library.usyd.edu.au/handle/2123/4030.
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Thesis (Ph. D.)--University of Sydney, 2009.Title from title screen (viewed February 10, 2009). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Molecular and Microbial Biosciences, Faculty of Science. Degree awarded 2009; thesis submitted 2008. Includes bibliographical references. Also available in print form.
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Hallal, Samantha. "Characterisation of the zinc fingers of Erythroid Kruppel-Like Factor." Thesis, The University of Sydney, 2008. http://hdl.handle.net/2123/4030.
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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.
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Hallal, Samantha. "Characterisation of the zinc fingers of Erythroid Kruppel-Like Factor." University of Sydney, 2008. http://hdl.handle.net/2123/4030.
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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.
8
Simpson, Raina Jui Yu. "The multiple roles of zinc finger domains." Thesis, The University of Sydney, 2004. http://hdl.handle.net/2123/655.
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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.
9
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.
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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.
10
Garcia, Anderson. "Peptídeos derivados da proteína bacteriana YacG : síntese e estudos de estrutura-função /." Araraquara : [s.n.], 2010. http://hdl.handle.net/11449/87996.
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Resumo: YacG é uma pequena proteína (65 resíduos de aminoácidos) ligada ao zinco codificada pelo gene yacG de Escherichia coli. Seu papel fisiológico não está bem caracterizado, porém acredita-se que ela exerça ação inibitória sobre a atividade catalítica da DNA girase, enzima responsável por alterações no estado topológico do DNA bacteriano. Com base nas informações da estrutura primária desta proteína, uma série constituída de oito seqüências peptídicas foram projetadas e sintetizadas pela metodologia da fase sólida, objetivando-se avaliar e melhor entender o efeito da coordenação do íon zinco no seu mecanismo de ação. As sequências foram projetadas de maneira a resultar em uma substituição parcial ou integral dos resíduos de cisteína da sequência nativa da YacG, por resíduos de serina, além da variação da carga efetiva da molécula, por amidação ou acetilação das extremidades C e N terminais, respectivamente. Os peptídeos obtidos e purificados foram ensaiados quanto à estequiometria de coordenação empregando titulação com íon cobalto, bem como na capacidade inibitória frente à DNA girase, empregando eletroforese em gel de agarose. YacGAG4, inibiu a atividade de superenovelamento do DNA, catalisada pela girase, somente na ausência de íons zinco em concentrações inferiores a 120 μmol.L-1. Os demais peptídeos não apresentaram capacidade inibitória, tanto na presença quanto na ausência de zinco. Ensaios de susceptibilidade bacteriana, empregando algumas espécies de bactérias da família Enterobacteriaceae, confirmaram os resultados in vitro,com exceção das sequências YacGAG1-AC e YacGAG2-AC que mostraram inibição no crescimento bacteriano, sem porém resultarem em atividade in vitro. Com base nos resultados obtidos, é possível concluir que o domínio estrutural relacionado à coordenação do zinco, bem como a presença... (Resumo completo, clicar acesso eletrônico abaixo)Abstract: YacG is a small protein (65 amino acid residues) bounded to zinc and encoded by the Escherichia coli yacG gene. Its physiological role is not well characterized, but it is believed that YacG is an inhibitor of the catalytic activity of DNA gyrase, an enzyme responsible for changes in the topological state of bacterial DNA. Based on information from the primary structure of this protein, a series of eight peptide sequences were designed and synthesized by solid phase methodology, aiming to evaluate and better understand the effect of zinc coordination of in their mechanism of action. The sequences were designed so as to result in a partial or full replacement of the cysteine residues of the native YacG sequence by serine residues and to change the effective charge of the molecule by amidation or acetylation of C and N terminal ends, respectively. The obtained peptides were purified and tested by titration with cobalt ion (coordination stoichiometry), as well as by inhibitory effect against the DNA gyrase, using agarose gel electrophoresis. YacGAG4 inhibited DNA supercoiling activity catalyzed by gyrase only in the zinc ions absence at concentrations below of 120 μmol.L-1. The other peptides showed no inhibitory effect in both the presence and absence of zinc. Bacterial susceptibility tests, using some species of bacteria of the Enterobacteriaceae, confirmed in vitro results, with the exception of the sequences YacGAG1-AC and YacGAG2-AC that showed inhibition of bacterial growth, but no in vitro activity. Based on these results, we conclude that the structural matters related to the coordination of zinc as well as the presence of this ion, showed no significant importance in the activity of DNA gyrase inhibition. In this case, the inhibition of activity recently proposed, should be linked to any other region of the protein molecule, structurally organized when the zinc ion is bound... (Complete abstract click electronic access below)
Orientador: Reinaldo Marchetto
Coorientador: Saulo Santesso Garrido
Banca: Clarice Queico Fujimura Leite
Banca: Vani Xavier de Oliveira Junior
Mestre
11
Wadhwa, Vibhuti. "Biophysical approaches towards greater understanding of eukaryotic zinc sensing." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1593621283583113.
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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.
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Baxley, Ryan M. "Understanding the contribution of individual zinc fingers to a multi-functional, polydactyl transcription factor." Diss., University of Iowa, 2013. https://ir.uiowa.edu/etd/1290.
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Suppressor of Hairy-wing [Su(Hw)] is a twelve zinc-finger (ZF), DNA binding transcription factor. Su(Hw) has been well characterized as critical component of the gypsy insulator complex, required for the enhancer blocking and the barrier activity of the insulator. In addition to gypsy, Su(Hw) localizes to ~3,000 binding sites in the Drosophila genome, with association to a subset of sites required for female germline development. Loss of Su(Hw) results in activation of a developmental checkpoint and apoptosis at mid- oogenesis, with a critical role during oogenesis in down-regulation of neural genes. Studies of Su(Hw) function have identified transcriptional activator, repressor and insulator roles at distinct binding sites. Current investigations aim to understand the factors that dictate the regulatory output of Su(Hw) at individual sites in the Drosophila genome, with a focus on the ZF domain.
A genetic screen was completed to generate novel mutations in su(Hw). After screening more than 8,000 mutagenized chromosomes, we identified four new su(Hw) alleles, including two deletion mutations and two amino acid substitutions disrupting individual ZFs (ZF4 and ZF8). Studies of the ZF4 mutant, Su(Hw)M4M393, revealed that Su(Hw) requires this ZF for female fertility, but notgypsy insulator function. To achieve a comprehensive understanding of the Su(Hw) ZF domain, we generated Su(Hw) mutant proteins carrying disruptions in individual ZFs. Analyses utilizing these proteins have defined the requirement for each ZF in DNA association in vitro. To complement extant ZF alleles, Su(Hw) ZF mutants were expressed in vivo. Analyses of these mutants established how each ZF contributes to SBS occupancy, gypsy insulator function and female fertility. Gene expression and ChIP analyses suggest that some Su(Hw) ZFs may execute roles apart from direct DNA recognition. Genome-wide binding analyses of Su(Hw)M4M393, combined with previous studies, found that the SBS binding motif contains three DNA sequence cores (termed upstream, central and downstream). Analyses of these sequence cores in combination with Su(Hw) ZF mutants have outlined which ZFs associate with each core. Interestingly, the class containing all three sequence cores represents high occupancy SBSs that are enriched for protein factors from functional classes including transcriptional repression, nucleosome remodeling and DNA replication. The class containing the upstream and central core correlates with insulator function, while the class containing the central and downstream cores correlates with activation or repression of Su(Hw) target genes. Finally, in vitro studies of Su(Hw) ZF mutants revealed a DNA bound conformation distinct from wild type Su(Hw).
Su(Hw) is a versatile transcription factor able to act as an insulator, activator and repressor. Analyses of SBSs with these functions suggest that DNA sequence, ZF usage, protein partnership and Su(Hw) conformation, combine to dictate regulatory output. Together, these studies provide insight into how discrete ZFs contribute to the roles of a multifunctional, polydactyl transcription factor.
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.
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Meagher, Madeleine Joy. "Identification and characterization of ZFR, a zinc finger protein required for murine embryonic cell survival and growth /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/10291.
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Hellborg, Fredrik. "Identification, cloning and characterization of the p53 induced gene human wig-1 /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-190-3/.
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Squillace, Rachel Marguerite. "Inhibition of muscle differentiation by the novel muscleblind-related protein CHCR /." Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/15468.
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Kwan, Ann Hau Yu. "Protein Design Based on a PHD Scaffold." Thesis, The University of Sydney, 2004. http://hdl.handle.net/2123/564.
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The plant homeodomain (PHD) is a protein domain of ~45�100 residues characterised by a Cys4-His-Cys3 zinc-binding motif. When we commenced our study of the PHD in 2000, it was clear that the domain was commonly found in proteins involved in transcription. Sequence alignments indicate that while the cysteines, histidine and a few other key residues are strictly conserved, the rest of the domain varies greatly in terms of both amino acid composition and length. However, no structural information was available on the PHD and little was known about its function. We were therefore interested in determining the structure of a PHD in the hope that this might shed some light on its function and molecular mechanism of action. Our work began with the structure determination of a representative PHD, Mi2b-P2, and this work is presented in Chapter 3. Through comparison of this structure with the two other PHD structures that were determined during the course of our work, it became clear that PHDs adopt a well-defined globular fold with a superimposable core region. In addition, PHDs contain two loop regions (termed L1 and L3) that display increased flexibility and overlay less well between the three PHD structures available. These L1 and L3 regions correspond to variable regions identified earlier in PHD sequence alignments, indicating that L1 and L3 are probably not crucial for the PHD fold, but are instead likely to be responsible for imparting function(s) to the PHD. Indeed, numerous recent functional studies of PHDs from different proteins have since demonstrated their ability in binding a range of other proteins. In order to ascertain whether or not L1 and L3 were in fact dispensable for folding, we made extensive mutations (including both insertions and substitutions) in the loop regions of Mi2b-P2 and showed that the structure was maintained. We then went on to illustrate that a new function could be imparted to Mi2b-P2 by inserting a five-residue CtBP-binding motif into the L1 region and showed this chimera could fold and bind CtBP. Having established that the PHD could adopt a new binding function, we next sought to use combinatorial methods to introduce other novel functions into the PHD scaffold. Phage display was selected for this purpose, because it is a well-established technique and has been used successfully to engineer zinc-binding domains by other researchers. However, in order to establish this technique in our laboratory, we first chose a control system in which two partner proteins were already known to interact in vitro. We chose the protein complex formed between the transcriptional regulators LMO2 and ldb1 as a test case. We have examined this interaction in detail in our laboratory, and determined its three-dimensional structure. Furthermore, inappropriate formation of this complex is implicated in the onset of T-cell acute lymphoblastic leukemia. We therefore sought to use phage display to engineer ldb1 mimics that could potentially compete against wild-type ldb1 for LMO2, and this work is described in Chapter 4. Using a phage library containing ~3 x 10 7 variants of the LMO2-binding region of ldb1, we isolated mutants that were able to interact with LMO2 with higher affinity and specificity than wild-type ldb1. These ldb1 mutants represent a first step towards finding potential therapeutics for treating LMO-associated diseases. Having established phage display in our laboratory, we went on to search for PHD mutants that could bind selected target proteins. This work is described in Chapter 5. We created three PHD libraries with eight randomized residues in each of L1, L3 or in both loops of the PHD. These PHD libraries were then screened against four target proteins. After four rounds of selection, we were able to isolate a PHD mutant (dubbed L13-FH6) that could bind our test protein Fli-ets. This result demonstrates that a novel function can be imparted to the PHD using combinatorial methods and opens the way for further work in applying the PHD scaffold to other protein design work. In summary, the work detailed in Chapters 3 and 5 demonstrates that the PHD possesses many of the properties that are desirable for a protein scaffold for molecular recognition, including small size, stability, and a well-characterised structure. Moreover, the PHD motif possesses two loops (L1 and L3) of substantial size that can be remodeled for target binding. This may lead to an enhancement of binding affinities and specificities over other small scaffolds that have only one variable loop. In light of the fact that PHDs are mainly found in nuclear proteins, it is reasonable to expect that engineered PHDs could be expressed and function in an intracellular environment, unlike many other scaffolds that can only function in an oxidizing environment. Therefore, our results together with other currently available genomic and functional information indicate PHD is an excellent candidate for a scaffold that could be used to modify cellular processes. Appendices 1 and 2 describe completed bodies of work on unrelated projects that I have carried out during the course of my PhD candidature. The first comprises the invention and application of DNA sequences that contain all N-base sequences in the minimum possible length. This work is presented as a reprint of our recently published paper in Nucleic Acids Research. The second Appendix describes our structural analysis of an antifreeze protein from the shorthorn sculpin, a fish that lives in the Arctic and Antarctic oceans. This work is presented as a manuscript that is currently under review at the Journal of the American Chemical Society.
19
Kwan, Ann Hau Yu. "Protein Design Based on a PHD Scaffold." University of Sydney. Molecular and Microbial Biosciences, 2004. http://hdl.handle.net/2123/564.
Full textAbstract:
The plant homeodomain (PHD) is a protein domain of ~45�100 residues characterised by a Cys4-His-Cys3 zinc-binding motif. When we commenced our study of the PHD in 2000, it was clear that the domain was commonly found in proteins involved in transcription. Sequence alignments indicate that while the cysteines, histidine and a few other key residues are strictly conserved, the rest of the domain varies greatly in terms of both amino acid composition and length. However, no structural information was available on the PHD and little was known about its function. We were therefore interested in determining the structure of a PHD in the hope that this might shed some light on its function and molecular mechanism of action. Our work began with the structure determination of a representative PHD, Mi2b-P2, and this work is presented in Chapter 3. Through comparison of this structure with the two other PHD structures that were determined during the course of our work, it became clear that PHDs adopt a well-defined globular fold with a superimposable core region. In addition, PHDs contain two loop regions (termed L1 and L3) that display increased flexibility and overlay less well between the three PHD structures available. These L1 and L3 regions correspond to variable regions identified earlier in PHD sequence alignments, indicating that L1 and L3 are probably not crucial for the PHD fold, but are instead likely to be responsible for imparting function(s) to the PHD. Indeed, numerous recent functional studies of PHDs from different proteins have since demonstrated their ability in binding a range of other proteins. In order to ascertain whether or not L1 and L3 were in fact dispensable for folding, we made extensive mutations (including both insertions and substitutions) in the loop regions of Mi2b-P2 and showed that the structure was maintained. We then went on to illustrate that a new function could be imparted to Mi2b-P2 by inserting a five-residue CtBP-binding motif into the L1 region and showed this chimera could fold and bind CtBP. Having established that the PHD could adopt a new binding function, we next sought to use combinatorial methods to introduce other novel functions into the PHD scaffold. Phage display was selected for this purpose, because it is a well-established technique and has been used successfully to engineer zinc-binding domains by other researchers. However, in order to establish this technique in our laboratory, we first chose a control system in which two partner proteins were already known to interact in vitro. We chose the protein complex formed between the transcriptional regulators LMO2 and ldb1 as a test case. We have examined this interaction in detail in our laboratory, and determined its three-dimensional structure. Furthermore, inappropriate formation of this complex is implicated in the onset of T-cell acute lymphoblastic leukemia. We therefore sought to use phage display to engineer ldb1 mimics that could potentially compete against wild-type ldb1 for LMO2, and this work is described in Chapter 4. Using a phage library containing ~3 x 10 7 variants of the LMO2-binding region of ldb1, we isolated mutants that were able to interact with LMO2 with higher affinity and specificity than wild-type ldb1. These ldb1 mutants represent a first step towards finding potential therapeutics for treating LMO-associated diseases. Having established phage display in our laboratory, we went on to search for PHD mutants that could bind selected target proteins. This work is described in Chapter 5. We created three PHD libraries with eight randomized residues in each of L1, L3 or in both loops of the PHD. These PHD libraries were then screened against four target proteins. After four rounds of selection, we were able to isolate a PHD mutant (dubbed L13-FH6) that could bind our test protein Fli-ets. This result demonstrates that a novel function can be imparted to the PHD using combinatorial methods and opens the way for further work in applying the PHD scaffold to other protein design work. In summary, the work detailed in Chapters 3 and 5 demonstrates that the PHD possesses many of the properties that are desirable for a protein scaffold for molecular recognition, including small size, stability, and a well-characterised structure. Moreover, the PHD motif possesses two loops (L1 and L3) of substantial size that can be remodeled for target binding. This may lead to an enhancement of binding affinities and specificities over other small scaffolds that have only one variable loop. In light of the fact that PHDs are mainly found in nuclear proteins, it is reasonable to expect that engineered PHDs could be expressed and function in an intracellular environment, unlike many other scaffolds that can only function in an oxidizing environment. Therefore, our results together with other currently available genomic and functional information indicate PHD is an excellent candidate for a scaffold that could be used to modify cellular processes. Appendices 1 and 2 describe completed bodies of work on unrelated projects that I have carried out during the course of my PhD candidature. The first comprises the invention and application of DNA sequences that contain all N-base sequences in the minimum possible length. This work is presented as a reprint of our recently published paper in Nucleic Acids Research. The second Appendix describes our structural analysis of an antifreeze protein from the shorthorn sculpin, a fish that lives in the Arctic and Antarctic oceans. This work is presented as a manuscript that is currently under review at the Journal of the American Chemical Society.
20
Blottière, Louise. "Etude des genes ozf (only zinc fingers) humain et murin : incidence dans les cancers du sein." Paris 6, 1999. http://www.theses.fr/1999PA066064.
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Ces travaux concernent l'isolement et l'etude des genes murins mozf et zfp260. Le premier est l'homologue murin du gene humain hozf (only zinc fingers), surexprime dans des cancers du pancreas. Le second code une proteine identique a 81% a la proteine mozf et dotee d'un domaine supplementaire. Ces genes participeraient a l'initiation ou a la progression tumorale. Le gene hozf, localise en 19q13. 1, a une expression non ubiquitaire. Il est amplifie dans 15 a 25% des lignees cellulaires et tumeurs du pancreas exocrine analysees, et surexprime en arnm et en proteine dans plus de 50% des cas. La proteine ozf est constituee uniquement de dix motifs doigt a zinc de type c 2h 2 et possede les caracteristiques structurales et biochimiques des proteines kruppel, dont plusieurs sont impliquees dans la regulation de la transcription. Pour aborder la fonction de ce facteur de transcription potentiel, son expression a ete etudiee chez la souris et dans des tumeurs humaines du sein. Le gene murin mozf a ete isole et caracterise. Il est localise en 7 b1-b3 et code une proteine identique a 95% a la proteine ozf humaine. Il est exprime specifiquement dans certains tissus. Pour etudier les consequences de la surexpression du gene mozf dans la glande mammaire, des experiences de transgenese sont en cours, en collaboration avec l'inra. L'etablissement d'un phenotype associe a cette surexpression nous renseignera sur la fonction de ce gene. L'expression du gene hozf dans les cancers du sein a ete abordee par western blot. De fortes variations de la quantite de proteine ozf ont ete observees dans les cellules epitheliales de certaines tumeurs de grades eleves. Des experiences d'immunohistochimie sont en cours afin de completer cette approche. Le second gene murin, zfp260, presente la particularite de coder une proteine qui possede les dix motifs doigt a zinc de la proteine ozf, precedes d'un domaine n-terminal constitue de trois motifs a doigt a zinc degeneres. Le gene zfp260 est localise en 7 b1-b3 et presente une specificite tissulaire. Les genes mozf et zfp260 pourraient, par leur domaine doigt a zinc identique, partager une fonction commune. Des experiences de transgenese permettront d'elucider la fonction de ce gene en associant un phenotype a une alteration de son niveau d'expression.
21
Drolet, Jessica Ann. "Evidence for the involvement of the zinc cluster protein Asg1p in the transcriptional regulation of some stress response genes in Saccharomyces cerevisiae." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=112617.
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Saccharomyces cerevisiae has developed mechanisms in order to survive harsh environmental conditions. This species responds to stresses such as ethanol, heat, and weak acid exposure via two well-characterized stress response pathways. These typically involve either the Hsf1p or the Msn2/4p transcriptional regulators. Recently, our lab has begun to characterize a member of the zinc cluster protein family: Asg1p (Activator of Stress Genes, systematic name: YIL130W), which is presumed to stimulate stress response genes independently of the Hsflp and Msn2/4p pathways. Previous work has revealed five target genes of Asg1p (HSP30, STP4, YER130C, TPO2, YRO2) thought to be involved in this novel stress response pathway. In this study, we attempted to better characterize the role of Asg1p and its target genes during stress induction. We first determined if the induction of certain Asg1p target genes by stress is strain specific. HSP30 induction by heat shock is specific to the W303 strain as shown by primer extension analysis. We then generated the deletion strains Deltaasg1 and Astp4 in W303. We observed a loss of induction of HSP30 in the Deltaasg1 deletion strain when cells were exposed to ethanol. This led us to believe that Asg1p does play a role in the stress response pathway. Also, we attempted to globally define the target sites of Asg1p in vivo on a genome-wide scale by combining Chromatin Immuno Precipitation with microarrays (ChIP-chip). We identified eight putative Asg1p target genes: YRO2, HSP78, ZRT2, ZRT1, MSN4, STP4, TPO2, and HSP30.
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Rao, Harita. "Metal containing peptides as specific DNA binders." Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2015. http://hdl.handle.net/11858/00-1735-0000-0028-86AC-9.
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Nail, Alexandra Nichole. "EVOLUTION OF THE ZHX TRANSCRIPTION FACTOR FAMILY AND ANALYSIS OF ZHX2 TARGET GENES CYP2A4 AND CYP2A5 IN MOUSE LIVER." UKnowledge, 2019. https://uknowledge.uky.edu/microbio_etds/20.
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The liver is the largest internal organ and performs a wide variety of functions to maintain organismal homeostasis. While some liver functions are carried out by all hepatocytes, other functions are restricted to certain populations of hepatocytes within the liver. This phenomenon, called zonal gene regulation or liver zonation, controls may metabolic processes within the liver including ammonia detoxification; glucose homeostasis; bile acid and glutamine synthesis; and metabolism of xenobiotics, lipids, and amino acids. The liver also expresses many genes in a developmental or sex-biased manner. Some genes are expressed at higher levels early or late in development, or alternatively, in male or female liver.
Several years ago, our lab identified a transcription factor called Zinc finger and homeoboxes 2 (Zhx2) based on its ability to control the silencing of genes that are normally expressed in the fetal liver. Zhx2 belongs to a small gene family that also includes Zhx1 and Zhx3. These four exon genes have a rather unique structure in that their entire protein coding region is located on an unusually large third exon. Preliminary studies indicate that these proteins are found only in vertebrates. I have performed a comprehensive analysis of Zhx proteins across a number of chordate species to determine their relationship throughout chordate evolution. Using multiple sequence alignment and phylogenetic tree-building, my studies have found that the primordial Zhx gene is most related to Zhx3 and that this gene exists in lower chordates including lancelet, sea squirt, and sea lamprey.
Additional studies from our lab showed that Zhx2 regulates numerous hepatic genes in the adult liver, including cytochrome p450 (Cyp) genes as well as other genes that exhibit sex-biased expression. Previous studies have demonstrated that female-biased expression of Cyp2a4, is controlled, in part, by Zhx2. I have extended these studies to perform a comprehensive analysis of Cyp2a4 and the highly related Cyp2a5 gene. Despite the high similarity of these two Cyp genes, my data indicate that these genes exhibit different zonal expression patterns and are differentially regulated in the regenerating liver. In the course of these studies, I discovered and characterized antisense transcripts for both Cyp2a4 and Cyp2a5. Both Cyp2a4as and Cyp2a5as have positively correlated expression patterns compared to their sense counterparts. In contrast to Cyp2a4 and Cyp2a5, Cyp2a4as and Cyp2a5as show sex-biased expression patterns earlier in development, suggesting that they might contribute to later sex-biased patterns established for Cyp2a4 and Cyp2a5.
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Gupta, Ankit. "Getting a Tight Grip on DNA: Optimizing Zinc Fingers for Efficient ZFN-Mediated Gene Editing: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/637.
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The utility of a model organism for studying biological processes is closely tied to its amenability to genome manipulation. Although tools for targeted genome engineering in mice have been available since 1987, most organisms including zebrafish have lacked efficient reverse genetic tools, which has stymied their broad implementation as a model system to study biological processes. The development of zinc finger nucleases (ZFNs) that can create double-strand breaks at desired sites in a genome has provided a universal platform for targeted genome modification. ZFNs are artificial restriction endonucleases that comprise of an array of 3- to 6-C2H2-zinc finger DNA-binding domains fused with the dimeric cleavage domain of the type IIs endonuclease FokI. C2H2-zinc fingers are the most common, naturally occurring DNA-binding domain, and their specificity can be engineered to recognize a variety of DNA sequences providing a strategy for targeting the appended nuclease domain to desired sites in a genome. The utility of ZFNs for gene editing relies on their activity and precision in vivo both of which depend on the generation of ZFPs that bind desired target sites high specificity and affinity.
Although various methods are available that allow construction of ZFPs with novel specificities, ZFNs assembled using existing approaches often display negligible in vivo activity, presumably resulting from ZFPs with either low affinity or suboptimal specificity. A root cause of this deficiency is the presence of interfering interactions at the finger-finger interface upon assembly of multiple fingers. In this study we have employed bacterial-one-hybrid (B1H)-based selections to identify two-finger zinc finger units (2F-modules) containing optimized interface residues that can be combined with published finger archives to rapidly yield ZFNs that can target more than 95% of the zebrafish and human protein-coding genes while maintaining a success rate higher than that of ZFNs constructed using available methods. In addition to genome engineering in model organisms, this advancement in ZFN design will aid in the development of ZFN-based therapeutics.
In the process of creating this archive, we have undertaken a broader study of zinc finger specificity to better understand fundamental aspects of DNA recognition. In the process we have created the largest protein-DNA interaction dataset for zinc fingers to be described that will facilitate the development of better predictive models of recognition. Ultimately, these predictive models would enable the rational design of synthetic zinc finger proteins for targeted gene regulation or genomic modification, and the prediction of genomic binding sites for naturally occurring zinc finger proteins for the construction of more accurate gene regulatory networks.
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Garcia, Anderson [UNESP]. "Peptídeos derivados da proteína bacteriana YacG : síntese e estudos de estrutura-função." Universidade Estadual Paulista (UNESP), 2010. http://hdl.handle.net/11449/87996.
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YacG é uma pequena proteína (65 resíduos de aminoácidos) ligada ao zinco codificada pelo gene yacG de Escherichia coli. Seu papel fisiológico não está bem caracterizado, porém acredita-se que ela exerça ação inibitória sobre a atividade catalítica da DNA girase, enzima responsável por alterações no estado topológico do DNA bacteriano. Com base nas informações da estrutura primária desta proteína, uma série constituída de oito seqüências peptídicas foram projetadas e sintetizadas pela metodologia da fase sólida, objetivando-se avaliar e melhor entender o efeito da coordenação do íon zinco no seu mecanismo de ação. As sequências foram projetadas de maneira a resultar em uma substituição parcial ou integral dos resíduos de cisteína da sequência nativa da YacG, por resíduos de serina, além da variação da carga efetiva da molécula, por amidação ou acetilação das extremidades C e N terminais, respectivamente. Os peptídeos obtidos e purificados foram ensaiados quanto à estequiometria de coordenação empregando titulação com íon cobalto, bem como na capacidade inibitória frente à DNA girase, empregando eletroforese em gel de agarose. YacGAG4, inibiu a atividade de superenovelamento do DNA, catalisada pela girase, somente na ausência de íons zinco em concentrações inferiores a 120 μmol.L-1. Os demais peptídeos não apresentaram capacidade inibitória, tanto na presença quanto na ausência de zinco. Ensaios de susceptibilidade bacteriana, empregando algumas espécies de bactérias da família Enterobacteriaceae, confirmaram os resultados in vitro,com exceção das sequências YacGAG1-AC e YacGAG2-AC que mostraram inibição no crescimento bacteriano, sem porém resultarem em atividade in vitro. Com base nos resultados obtidos, é possível concluir que o domínio estrutural relacionado à coordenação do zinco, bem como a presença...
YacG is a small protein (65 amino acid residues) bounded to zinc and encoded by the Escherichia coli yacG gene. Its physiological role is not well characterized, but it is believed that YacG is an inhibitor of the catalytic activity of DNA gyrase, an enzyme responsible for changes in the topological state of bacterial DNA. Based on information from the primary structure of this protein, a series of eight peptide sequences were designed and synthesized by solid phase methodology, aiming to evaluate and better understand the effect of zinc coordination of in their mechanism of action. The sequences were designed so as to result in a partial or full replacement of the cysteine residues of the native YacG sequence by serine residues and to change the effective charge of the molecule by amidation or acetylation of C and N terminal ends, respectively. The obtained peptides were purified and tested by titration with cobalt ion (coordination stoichiometry), as well as by inhibitory effect against the DNA gyrase, using agarose gel electrophoresis. YacGAG4 inhibited DNA supercoiling activity catalyzed by gyrase only in the zinc ions absence at concentrations below of 120 μmol.L-1. The other peptides showed no inhibitory effect in both the presence and absence of zinc. Bacterial susceptibility tests, using some species of bacteria of the Enterobacteriaceae, confirmed in vitro results, with the exception of the sequences YacGAG1-AC and YacGAG2-AC that showed inhibition of bacterial growth, but no in vitro activity. Based on these results, we conclude that the structural matters related to the coordination of zinc as well as the presence of this ion, showed no significant importance in the activity of DNA gyrase inhibition. In this case, the inhibition of activity recently proposed, should be linked to any other region of the protein molecule, structurally organized when the zinc ion is bound... (Complete abstract click electronic access below)
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Schmiedeskamp, Mia Ruth. "NMR studies of the DNA-binding domain of ADR1 /." Thesis, Connect to this title online; UW restricted, 1996. http://hdl.handle.net/1773/9208.
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Frank, Deborah Jean. "Regulation of cell growth in C. elegans and D. melanogaster by ncl-1/brat /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/5029.
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Patel, Reena. "Mediation of pleiotropic drug resistance by zinc cluster transcriptional regulators in Saccharomyces cerevisiae." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:8881/R/?func=dbin-jump-full&object_id=32366.
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Spell, Sarah. "The Study of Au(III) Compounds and their Interaction with Zinc Finger Proteins." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/634.
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Gold compounds have been used in medicine dating back as early as 2500 BC. Over the years gold(I) and gold(III) compounds have been used and designed to target rheumatoid arthritis, cancer, and viral diseases. New drug targets have been found for gold compounds that give insight into their mechanisms of action. Here we focus on the synthesis of Au(III) compounds designed to selectively target zinc finger (ZF) proteins. ZF proteins exhibit a variety of functions, including transcription, DNA repair, and apoptosis. Displacement of the central zinc ion, along with mutation of coordinated amino acids can result in a loss of biological function. Synthesis of complexes that selectively target zinc finger proteins, in turn inhibiting DNA/ZF interactions and therefore resulting in loss of protein function, is of great interest. Of particular interest here is the Cys3His (Cys = cysteine, His = histidine) HIV nucleocapsid zinc finger protein, NCp7. NCp7 is involved in multiple steps of the HIV life cycle, thus making it a desirable drug target. Previous studies from our group show platinated nucleobases such as [Pt(dien)(9-EtG)]2+ (dien = diethylenetriamine; 9-EtG = 9-ethylguanine) to stack effectively in a non-covalent manner with tryptophan of the C-terminal finger of HIV Nucleocapsid, NCp7(F2), a key residue involved in nucleic acid recognition. Due to the isoelectronic and isostructural relationship of Au(III) to Pt(II), we have expanded this system to Au(III)-(nucleobase/N-heterocycle) compounds. Novel Au(III)(dien)(N-heterocycle) compounds, including the first Au(III)N3(N-purine) examples, were synthesized. As previously reported for [AuCl(dien)]Cl2, these compounds exhibit pH dependency of the 1H NMR chemical shifts of the dien ligand. The acidity of the dien ligand is affected by the nature of the fourth ligand as a leaving group. The presence of an inert nitrogen donor, compared to that of the more labile Cl-, as the leaving group stabilizes the Au(III) metal center towards reduction, resulting in significant enhancement of π−π stacking interactions with tryptophan relative to platinum(II) and palladium(II) compounds. The presence of a more inert N-donor as the leaving group slows down the reaction with the sulfur-containing amino acid N-Acetylmethionine (N-AcMet); essentially no reaction was observed for the Au(III)-N-heterocycle compounds. All compounds react readily with N-Acetylcysteine (N-AcCys), however lack of N-heterocycle ligand dissociation indicates, to our knowledge, the first long-lived N-heterocycle-Au-S species in solution. Electrospray ionization mass spectrometry (ESI-MS) studies with NCp7(F2) indicate [Au(dien)(DMAP)]3+ (DMAP = 4-dimethylaminopyridine) to be the least reactive of the Au(III) compounds studied, showing the presence of intact NCp7(F2) zinc finger at initial reaction times. Reactivity of the Au-compounds was compared with that of Sp1(F3), a Cys2His2 ZF; in contrast, no intact ZF was observed for any of the compounds studied, suggesting the mode of action of these compounds is dependent on the nature of the zinc binding core. ESI-MS studies were expanded to that of the full HIV NCp7 zinc finger. [Au(dien)(9-EtG)]3+ reacts quickly with NCp7, resulting in immediate zinc ejection and replacement with up to three gold ions. Unlike with [Au(dien)(DMAP)]3+, no intact NCp7 was observed. Addition of [Au(dien)(9-EtG)]3+ to preformed NC-SL2 complex results in release of free RNA; based on EMSA (electrophoretic mobility shift assay) studies, [Au(dien)(9-EtG)]3+ disrupts the NCp7-RNA complex with an IC50 of ~450 µM. It is possible that this HIV nucleocapsid-nucleic acid antagonism may result in a loss of viral activity.
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Szkudlarek-Mikho, Maria. "Characterizing the function of extracellular protein kinase A in angiogenesis and the effects of Zfp68 and pharmacological inibitors in adipogenesis." Toledo, Ohio : University of Toledo, 2010. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=mco1270565982.
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Dissertation (Ph.D.)--University of Toledo, 2010."Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Science." Title from title page of PDF document. "A Dissertation entitled"--at head of title. Bibliography: p. 54-56, 81-82, 113-128.
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Zhou, Shengli. "ZNF451 is a novel binding partner of the bHLH transcription factor E₁₂." Connect to full text in OhioLINK ETD Center, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=mco1225219996.
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Thesis (M.S.)--University of Toledo, 2008."In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Sciences." Title from title page of PDF document. Bibliography: pages 49-62.
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Parker, Tory L. "The Effects of Selenium on Estrogen-regulated Gene Expression in LNCaP Prostate Cancer Cells." BYU ScholarsArchive, 2004. https://scholarsarchive.byu.edu/etd/637.
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Prostate cancer is the most frequently diagnosed cancer in American men and the second leading cause of cancer deaths. Supplementation with Se has reduced the incidence of prostate cancer and Se status is inversely correlated with prostate cancer risk. One molecular mechanism by which high Se concentrations may affect cancer risk is by catalyzing disulfide bond formation or otherwise complexing with reactive sulfhydryl groups in cellular proteins. The estrogen receptor (ER) contains cysteines in zinc (Zn) fingers that are susceptible to oxidation and internal disulfide formation, which can prevent DNA binding. We examined ER binding to its DNA response element and gene expression levels for estrogen-regulated genes in human prostate cancer cells (LNCaP) treated with control (50 nM) or high (5 ìM) concentrations of Se. High Se treatment resulted in a non-significant 16 % decrease in ER binding to the estrogen response element (ERE), and no significant changes were found in expression levels of estrogen-regulated genes for either run-on nuclear transcripts or total mRNA. The well documented reaction of Se with reactive sulfhydryl groups, if it occurs in the ER in vivo, has a minimal effect on the binding of ER to DNA and its regulation of gene expression.
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Lebrun, Vincent. "Doigts de zinc et stress oxydant : réactivité vis-à-vis de l'oxygène singulet et l'acide hypochloreux." Thesis, Grenoble, 2014. http://www.theses.fr/2014GRENV068/document.
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Très répandues dans le monde vivant, les protéines à doigt de zinc constituent une super-famille dont les membres possèdent un site à zinc de formule générale [ZnII(Cys)4-X(His)X] (x=0, 1 or 2). Tandis que la majorité de ces sites joue un rôle purement structural, certains présentent une fonction réactive, comme la détection de stress oxydant par exemple. En effet, les sites doigt de zinc de Hsp33 et de RsrA ont été décrits comme des interrupteurs rédox[1,2] : transmettant l'information « stress oxydant » sous forme d'un signal structural, via l'oxydation/réduction des cystéines coordonnées au zinc, détruisant/reformant le domaine doigt de zinc. Cependant, certains aspects de l'étape d'oxydation restent mal compris.Étant donné le grand nombre des protéines à doigt de zinc et leurs rôles clés, il est de tout intérêt d'identifier les facteurs contrôlant leur réactivité afin de comprendre pourquoi certaines espèces réactives de l'oxygène (ERO) sont capable d'oxyder des doigts de zinc in vivo, contrairement à H2O2. Durant ce projet, nous avons décidé de nous focaliser sur deux ERO très puissantes et jouant un rôle important en biologie : l'acide hypochloreux (HOCl) et l'oxygène singulet (1O2). Nous étudierons la réactivité des doigts de zinc vis-à-vis de ces deux oxydant en utilisant des modèles peptidiques, reproduisant parfaitement la structure de doigts de zinc courantsWidely spread in the living world, zinc finger proteins constitute a large superfamily, with a zinc site of general formula [ZnII(Cys)4-X(His)X] (x=0, 1 or 2) as a common feature. Whereas the majority of such sites plays a purely structural role, a few of them exhibit a reactive function (e.g. oxidative stress detection). Indeed, zinc finger sites of Hsp33 and RsrA have been shown to act as redox switches[1,2]: transmitting the information “oxidative stress” as a structural signal, by means of oxidation/reduction of its ZnII-coordinating cysteines. However, the precise mechanism of the oxidation step remains poorly understood.Given the occurrence of zinc finger proteins and their key roles, it is of high biological interest to identify factors controlling their reactivity and to understand why some ROS are able to oxidize them in vivo, on the contrary to H2O2. In this project, we decided to focus on two major ROS: hypochlorous acid (HOCl), a key player of the immune response, and singlet oxygen (1O2), produced in significant amount by photosynthetic organisms. By use of peptide model complexes, reproducing perfectly the structure of some archetypal zinc fingers, we investigated the reactivity of zinc fingers toward those ROS
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Runko, Alexander Peter. "Function of the Zinc-Finger Repressor NLZ in the Developing Zebrafish Hindbrain: a Dissertation." eScholarship@UMMS, 2003. https://escholarship.umassmed.edu/gsbs_diss/74.
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Generation of the primitive neuroectoderm into specialized brain subdivisions, such as the hindbrain primordium, involves the regulated coordination of complex morphogenetic and molecular mechanisms. These processes are evident in the segregation of the zebrafish hindbrain into seven distinct lineage-restricted compartments, termed rhombomeres (r), which are established by the interplay of several spatially-restricted expressed genes. These include transcription factors, members of specific signaling pathways and specialized molecules that mediate cell adhesion and identity. Despite their extensive characterization, it is evident that other genes are involved to mediate the proper specification and segregation of individual rhombomeres. One candidate that likely fits this role is related to the no ocelli/l(2)35Ba gene in Drosophila, termed nlz (nocA-like zinc-finger). Nlz-related proteins behave as transcriptional repressors and are related to the vertebrate Sp1-like family of transcription factors. nlz is dynamically expressed in the zebrafish hindbrain, residing in the caudal hindbrain at gastrula stages and rostrally expanding from presumptive r3/r4 boundary to encompass r3 and r2 at segmentation stages. Nlz localizes to the nucleus and associates with the co-repressors Groucho and histone deacetylases, suggesting that Nlz acts as a repressor. Consistent with this, misexpression of nlz into zebrafish embryos results in a loss of gene expression in the rostral hindbrain (rl-r3). Taken together, the findings in this thesis suggest that Nlz functions as a transcriptional repressor to control segmental gene expression in the rostral hindbrain.
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Purcell, Jamie, and Jamie Purcell. "Investigating the RNA Binding Domains of MBNL1 and the Alternative Splicing Motifs They Recognize." Thesis, University of Oregon, 2012. http://hdl.handle.net/1794/12331.
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Muscleblind-like 1 (MBNL1) is a ubiquitously expressed RNA binding protein that regulates the alternative splicing of a variety of transcripts. In Myotonic Dystrophy (DM) aberrant cellular localization of MBNL1 results in disease-associated mis-splicing of several MBNL1 target pre-mRNAs. Due to its role in DM pathogenesis, MBNL1 has been a topic of intense study for the last decade, however many open mechanistic questions remain regarding how MBNL1 recognizes RNA substrates to mediate splicing.
The RNA recognition motif for MBNL1, 5'-YGCY-3', was defined herein. This motif was used to identify novel MBNL1 binding sites within regulated transcripts and create synthetic MBNL1-regulated splicing reporters. MBNL1 contains four zinc finger (ZF) RNA binding domains arranged into two pairs of two ZFs. A comprehensive, combinatorial mutagenic study of MBNL1 was conducted to determine the role of each ZF in RNA binding and splicing activity. Functional analysis of the mutant proteins in cellular splicing assays and assessment of RNA binding activity demonstrated that the ZF pairs (i.e. ZF1-2 or ZF3-4) do not have equivalent activity. The ZF1-2 pair is responsible for MBNL1's high affinity RNA binding and splicing activity, whereas the ZF3-4 pair has reduced affinity for RNA and impaired ability to regulate splicing of some transcripts. Hierarchical clustering analysis revealed that two distinct classes of MBNL1-regulated splicing events exist within the small set of splicing events examined. For Class II splicing events the binding and splicing activity for the ZF mutants correlated well. However, for Class I events there was no significant correlation between RNA binding and splicing activity. For pre-mRNAs in the latter class it appears that MBNL1 exerts surprisingly robust splicing activity in the absence of strong RNA binding, suggesting that MBNL1 may be recruited to some pre-mRNA substrates through protein-protein interactions. This study provides the first demonstration that functionally distinct classes of MBNL1-mediated splicing events exist in terms of requirements for different ZFs and the importance of RNA binding.
This dissertation includes previously published and unpublished co-authored material as well as recently co-authored material that has been submitted for publication.
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Frémy, Guillaume. "Synthèse de sondes luminescentes pour la détection séquence spécifique d'ADN double brin." Thesis, Université Grenoble Alpes (ComUE), 2019. http://www.theses.fr/2019GREAV073.
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L’ADN est le support de l’information de tout être vivant. La possibilité de pouvoir cibler et visualiser in vivo une séquence spécifique d’ADN et plus particulièrement un gène est un enjeu de taille pour le suivi médical aussi bien que pour la compréhension du vivant. Pour y parvenir, la détection par luminescence est particulièrement attrayante de par sa facilité de visualisation avec des outils simples.L’objet de cette thèse était d’apporter la preuve de concept de sondes luminescentes pour la détection séquence spécifique d’ADN double brin, basées d’une part sur les propriétés de luminescence des lanthanides, particulièrement intéressantes pour la détection en milieu biologique, et d’autre part sur les propriétés de reconnaissance de l’ADN par les protéines à doigts de zinc. Nous nous sommes intéressé au ciblage d’un duplex d’ADN palindromique de 12 paires de bases par un couple de protéines à doigts de zinc intégrant un système FRET, où un complexe de lanthanide(III) sur une protéine joue le rôle de donneur et un fluorophore organique sur l’autre protéine joue celui d’accepteur.Pour cela, une nouvelle famille de complexes de lanthanide(III) bioconjugables a été élaborée et des protéines à doigts de zinc fonctionnalisée par différents chromophores ont été synthétisés chimiquement par synthèse peptidique supportée sur résine et assemblage par ligation chimique native de trois fragments. Les caractérisations spectroscopiques des systèmes développés ont permis de mettre en évidence l’interaction des sondes avec la séquence d’ADN palindromique et de valider la preuve de concept d’une détection de cette séquence par un FRET basé sur des lanthanidesDNA is the carrier of genetic information in living organisms. Targeting and visualizing in vivo a specific DNA sequence is of particular interest for medical diagnosis and biological research. In this purpose, luminescent detection is very attractive because it can be easily observed with simple tools.The aim of this work was to establish the proof of concept of luminescent probes for the sequence-specific detection of double stranded DNA based on lanthanide luminescence, which is attractive for biological applications, and on zinc finger proteins for their DNA binding properties. We focused on the detection of a 12-base pair palindromic DNA using a pair of zinc finger proteins, one bearing a lanthanide(III) complex as a the FRET donor and the other an organic fluorophore as an acceptor.In this purpose, a new family of bioconjugatable lanthanide(III) complexes was developed and zinc finger proteins with various chromophores were synthesized chemically, by combination of solid phase peptide synthesis and assembly of peptidic segments by native chemical ligation. The spectroscopic characterizations of these systems have evidenced the interaction of the probes with the palindromic DNA, thereby validating the proof of concept of luminescence detection of this DNA sequence by a lanthanide-based FRET system
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Wilhelm, Margareta. "The p53-induced gene wig-1 : regulation of expression and role in embryonic development /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-728-2.
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Deveau, Laura M. "Characterizing the Disorder in Tristetraprolin and its Contribution to Post-Transcriptional Gene Regulation: A Dissertation." eScholarship@UMMS, 2016. http://escholarship.umassmed.edu/gsbs_diss/855.
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RNA-binding proteins (RBPs) are important for a wide variety of biological processes involved in gene regulation. However, the structural and dynamic contributions to their biological activity are poorly understood. The tristetraprolin (TTP) family of RBPs, including TTP, TIS11b and TIS11d, regulate the stability of mRNA transcripts encoding for key cancer-related proteins, such as tumor necrosis factor- and vascular endothelial growth factor. Biophysical studies have shown that the RNA binding domain, consisting of two CCCH zinc fingers (ZFs), is folded in the absence of RNA in TIS11d and TIS11b. In TTP, however, only ZF1 adopts a stable fold, while RNA is required to completely fold the tandem zinc finger (TZF). The focus of this research was to understand the origin and biological significance of the structural differences observed for the TZF domains of TTP and TIS11d. Three residues were shown to control the affinity for the structural Zn2+ and determine the folding of ZF2 in the absence of RNA. The partially-folded TZF domain of TTP has greater selectivity for RNA sequences than the fully folded TZF domain of TIS11d. The mRNA destabilizing activity of TTP was increased when the partially disordered RBD of TTP was replaced with the fully structured TZF domain of TIS11d. Disruption of the structure and/or dynamics of the TZF domain observed in the disease-associated mutations of TIS11d, P190L and D219E, results in aberrant cytoplasmic localization. This work demonstrates that the extent of RBD folding in the TTP family is important for differential RNA recognition, mRNA turnover, and protein localization in vivo.
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Heath, Megan Joy. "Human immunodeficiency virus nucleocapsid protein analysis of the mechanism of strand exchange and the role of the zinc fingers in nucleic acid chaperone activity /." College Park, Md. : University of Maryland, 2004. http://hdl.handle.net/1903/1690.
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Thesis (Ph. D.) -- University of Maryland, College Park, 2004.Thesis research directed by: Molecular and Cell Biology. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Wilson, Stevin. "Understanding Zinc Homeostasis using Loz1 from the Fission Yeast." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1565902886663748.
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Jacques, Aurélie. "Interaction de l'or avec des peptides modèles de doigts de zinc : caractérisation de la complexation des sels d'or et application à la toxicologie des nanoparticules d'or." Thesis, Grenoble, 2013. http://www.theses.fr/2013GRENV075/document.
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Les complexes d'or(I) tels que l'auranofine et l'aurothiomalate ont été utilisés dans le traitement de la polyarthrite rhumatoïde depuis plusieurs décennies. Plus récemment, de nombreux complexes d'or(I) et d'or(III) ont révélé des activités anti-cancéreuses in vitro importantes. Cependant, du fait de la forte affinité de l'or pour les thiols et les sélénols, ces complexes interagissent dans les cellules avec de nombreuses protéines de façon non spécifique. Ce qui explique les effets secondaires observés chez les patients soumis à des traitements prolongés de chrysothérapie et rend plus difficile la compréhension des mécanismes d'actions de ces complexes. Parmi les cibles thérapeutiques proposées, les protéines à doigts de zinc représentent des cibles très intéressantes. En effet, ces protéines sont très abondantes (environ 8 % du protéome humain) et se retrouvent dans des processus clés de la machinerie cellulaire (régulation de gènes, transcription, apoptose, réparation de l'ADN, résistance au stress oxydant…). Ces protéines possèdent un site Zn(Cys)4-x(His)x (x = 0-2) où le zinc permet d'assurer le bon repliement de la protéine et son bon fonctionnement. Nous nous sommes donc intéressés à déterminer l'interaction de complexes d'or(I) et d'or(III) avec les protéines doigts de zinc à l'aide de modèles peptidiques de ces sites à zinc. Nous avons caractérisé les produits issus de la réaction entre les peptides et l'or et déterminé les cinétiques de déplacement du zinc par l'orGold(I) complexes such as auranofine and aurothiomalate have been used as therapeutic agents for the treatment of rheumatoid arthritis for several decades. More recently, several gold(I) and gold(III) complexes have shown important in vitro anticancer properties. However, because of the high affinity of gold for sulfur and selenium ligands, these complexes interact with lots of proteins in a non-specific manner. This explains some of the side effects observed in patients treated with gold complexes and also why the mechanism of action of these complexes remains misunderstood. Among all the therapeutic target of gold salts that has been proposed, zinc fingers proteins are very interesting targets. Indeed, these proteins are very abundant (ca. 8 % of human proteome) and are involved in key processes of the cell (genes regulation, transcription, apoptose, DNA repair, resistance to oxidant stress…). These proteins contains a Zn(Cys)4-x(His)x (x = 0-2) center where zinc assure the proper fold and function of the protein. We were interested to study the interaction of some gold(I) and gold(III) complexes with zinc finger proteins by using peptidic models these zinc sites. By using different spectroscopic methods, we have characterized the products obtained by reaction between the zinc fingers and the gold salts and determined the kinetics of zinc displacement by gold
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Creasy, Kate Townsend. "ZHX2 REGULATION OF LIPID METABOLISM AND THE BALANCE BETWEEN CARDIOVASCULAR AND HEPATIC HEALTH." UKnowledge, 2015. http://uknowledge.uky.edu/pharmacol_etds/10.
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The growing obesity epidemic in America carries with it numerous health risks, including diabetes, increased serum lipid levels, and excess fat accumulation in the liver. If these conditions persist or become exacerbated, they may lead to the development of cardiovascular disease, the current leading cause of death among Americans, or to nonalcoholic fatty liver disease (NAFLD) which can progress to hepatocellular carcinoma (HCC), one of the deadliest forms of cancer. Better understanding of the genes involved in these diseases can lead to improved identification of at-risk individuals and treatment strategies.
Our lab previously identified zinc fingers and homeoboxes 2 (Zhx2) as a regulator of hepatic gene expression. The BALB/cJ mouse strain has a hypomorphic mutation in the Zhx2 gene, causing a 95% reduction in Zhx2 protein expression. The near ablation of Zhx2 in BALB/cJ mice confers protection from cardiovascular disease when fed a high fat diet, yet these mice show increased hepatic lipid accumulation and liver damage. Microarray data indicates Zhx2 may be involved in the regulation of numerous genes involved in lipid metabolism. Recent GWAS studies indicate ZHX2 may contribute to the risk of cardiovascular disease and liver damage in humans as well.
In this dissertation, I characterize the role of Zhx2 expression in the liver and how it affects the risk of both cardiovascular disease and liver damage. I generated liver-specific Zhx2 knockout mice and confirmed Zhx2 regulates several novel targets that could contribute to the fatty liver phenotype seen in BALB/cJ mice. Further studies revealed that hepatic Zhx2 expression is necessary for proper sex-specific expression of several Cyptochrome P450 (CYP) genes and could contribute to gender differences in disease susceptibility. Lastly, I performed studies into the functional role of the Zhx2 target gene Elovl3. A mouse model of HCC revealed that Elovl3 is completely repressed in HCC tumors. Cell viability and cell cycle assays indicate that Elovl3 expression slows cell proliferation and may be important for proper cell cycle checkpoints. Together, these data indicate that Zhx2 and/or its targets could be clinically relevant in the detection, prevention, or treatment of cardiovascular disease, fatty liver, and HCC.
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Tucker, Sean Newton. "Ikaros affects the expression of the interleukin-2 receptor beta chain and lymphoid cell potential /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/8338.
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Bessa, Danielle de Souza. "Análise de metilação global em pacientes com puberdade precoce central familial." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/5/5135/tde-31102018-120553/.
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A idade normal para início da puberdade em meninas varia bastante, de 8 a 13 anos, e os genes envolvidos nesse controle são parcialmente conhecidos. Fatores ambientais, como alimentação e exposição a disruptores endócrinos, contribuem para essa variabilidade, de modo que genes modulados epigeneticamente podem justificar parte da complexidade desse processo. O termo epigenética se refere às modificações na expressão gênica que não são causadas por alterações na sequência do DNA. A metilação do DNA é o mecanismo epigenético mais bem estudado. Na última década surgiram evidências demonstrando a relação entre metilação do DNA e desenvolvimento puberal. Em fêmeas de roedores, a hipermetilação do DNA levou à puberdade precoce. Em humanos, a puberdade precoce central (PPC) familial causada por mutações nos genes MKRN3 e DLK1 é considerada um defeito do imprinting, fenômeno epigenético no qual apenas um dos alelos parentais é expresso, estando o outro metilado e inativo. Além disso, um conceito atual propõe que o início da puberdade requer a repressão epigenética de fatores inibidores do eixo gonadotrófico. Recentemente, genes zinc finger (ZNF) foram relacionados ao processo puberal, e muitos deles codificam repressores transcricionais. Neste trabalho, estudamos a metilação do DNA do sangue periférico de 10 pacientes do sexo feminino com PPC familial (casos índices) e 33 meninas com desenvolvimento puberal normal (15 pré-púberes e 18 púberes), usando a plataforma Human Methylation 450 BeadChip. Duas pacientes tinham PPC de causa genética (uma com mutação no MKRN3 e outra com deleção no DLK1) e oito tinham PPC idiopática, sem mutações identificadas pelo sequenciamento exômico global. Cento e vinte regiões diferencialmente metiladas foram identificadas entre as meninas saudáveis pré-púberes e púberes, estando 74% delas no cromossomo X. Apenas uma região mostrou-se hipometilada no grupo púbere e, de maneira importante, contém a região promotora do ZFP57, fator necessário para manutenção do imprinting. Uma vez que a hipermetilação nas regiões promotoras dos genes é relacionada à inibição transcricional, o achado de hipermetilação global do DNA na puberdade sugere que haja inibição de fatores inibidores do eixo gonadotrófico, o que resultaria no início do processo puberal. O receptor estrogênico destacou-se como um fator transcricional que se liga a sete genes diferencialmente metilados entre os controles pré-púberes e púberes. As pacientes com PPC apresentaram mais sítios CpG hipermetilados tanto na comparação com as meninas pré-púberes (81%) quanto púberes (89%). Há doze genes ZNF contendo sítios CpG hipermetilados na PPC. Não foram encontradas anormalidades de metilação nos genes MKRN3 e DLK1 nem em suas regiões regulatórias. Em conclusão, este estudo evidenciou hipermetilação global do DNA em meninas com puberdade normal e precoce, sugerindo que esse padrão é uma marca epigenética da puberdade. Pela primeira vez, mudanças no metiloma de pacientes com PPC foram descritas. Modificações na metilação de vários genes ZNF parecem compor a complexa rede de mecanismos que leva ao início da puberdade humanaNormal puberty initiation varies greatly among girls, from 8 to 13 years, and the genetic basis for its control is partially known. Environmental factors, such as nutrition and exposure to endocrine disruptors, contribute to this variance, and epigenetically modulated genes may justify some of the complexity observed in this process. Epigenetics refers to alterations in gene expression that are not caused by changes in DNA sequence itself. DNA methylation is the best studied epigenetic mechanism. In the last decade, evidence has emerged showing the relationship between DNA methylation and pubertal development. In female mice, DNA hypermethylation led to precocious puberty. In humans, familial central precocious puberty (CPP) caused by mutations in the MKRN3 and DLK1 genes is considered a disorder of imprinting, an epigenetic phenomenon in which only one parental allele is expressed, and the other allele is methylated and inactive. In addition, animal studies indicated that pubertal timing requires epigenetic repression of inhibitory factors of the gonadotrophic axis. Recently, zinc finger genes (ZNF) were related to pubertal development, many of which encode transcriptional repressors. In the present study, we analyzed the DNA methylation of peripheral blood samples from 10 female patients with familial CPP (index cases) and 33 girls with normal pubertal development (15 pre-pubertal and 18 pubertal), using the Human Methylation 450 BeadChip assay. Genetic CPP was diagnosed in two patients (one with a MKRN3 mutation and the other with a DLK1 deletion). The remaining eight cases with idiopathic CPP were previously evaluated by whole exome sequencing and no causative mutations were identified so far. We evidenced 120 differentially methylated regions between pre-pubertal and pubertal healthy girls, and 74% of them were located at the X chromosome. Only one genomic region was hypomethylated in the pubertal group. Of note, it contains the promoter region of ZFP57, an important factor for imprinting maintenance. As DNA hypermethylation in gene promoters is related to gene silencing, the finding of global DNA hypermethylation in puberty suggests inhibition of inhibitory factors of the hypothalamic-pituitary-gonadal axis that results in puberty onset. Importantly, the estrogen receptor was identified as a transcriptional factor that binds to seven differentially methylated genes associated with pubertal process. Patients with CPP exhibited more hypermethylated CpG sites compared to both pre-pubertal (81%) and pubertal (89%) controls. Twelve ZNF genes were recognized as having hypermethylated CpG sites in CPP. The methylation analyses of MKRN3 and DLK1 genes showed no abnormalities. In conclusion, this study revealed a widespread DNA hypermethylation in girls with normal and precocious puberty, suggesting that this pattern can be an epigenetic signature of puberty. For the first time, changes in the methylome of patients with CPP were described. We highlight that alterations in methylation levels of several ZNF genes may impact the onset of human puberty
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Harris, John E. "The Molecular Mechanisms of T Cell Clonal Anergy: A Dissertation." eScholarship@UMMS, 2003. https://escholarship.umassmed.edu/gsbs_diss/6.
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A side effect of generating an immune system for defense against invading pathogens is the potential to develop destructive cells that recognize self-tissues. Typically, through the "education" of developing immune cells, the organism inactivates potentially self-destructive cells, resulting in what is called self-tolerance. I proposed to explore the molecular mechanisms responsible for the induction and maintenance of tolerance. Our lab has developed a model of induced immune tolerance to skin and islet allografts utilizing a donor-specific transfusion of spleen cells and a brief course of anti-CD40L antibody. Because the difficulty in isolation of tolerant T cells from this system is prohibitive to performing large screens on these cells directly, I have chosen to study an in vitro CD4+Th1 cell line, A.E7, which can be made anergic via stimulation through the T cell receptor in the absence of costimulation. I hypothesized that anergized T cells upregulate genes that are responsible for the induction and maintenance of anergy and therefore exhibit a unique RNA expression profile. I have screened anergic cells using Affymetrix GeneChips and identified a small number of genes that are differentially expressed long-term in the anergic population compared to mock-stimulated and productively activated controls. The results have been confirmed by quantitative RT-PCR for each of the candidates. One of the most promising, the zinc-finger transcription factor Egr-2, was verified to be expressed long-term by western blotting, demonstrating perfect correlation between Egr-2 protein expression and the anergic phenotype. Silencing Egr-2 gene expression by siRNA in A.E7 T cells prior to anergy induction rescues the cells from the inability to phosphorylate ERK-1 and ERK-2 and also results in increased proliferation in response to antigen rechallenge. In this study I report that Egr-2 is specifically expressed long-term in anergic cells, protein expression correlates inversely with responsiveness to antigen rechallenge, and that Egr-2 is required for the full induction of anergy in T cell clones.
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Valsecchi, Isabel. "AtZDP, a Plant 3' DNA Phosphatase, Involved in DNA Repair." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8907.
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Valeije, Ana Claudia Mancusi. "Utilização de informações termodinâmicas e estruturais na predição de sítios de ligação de receptores nucleares ao DNA: uma abordagem computacional." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/95/95131/tde-23042015-133407/.
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Os projetos genoma têm fornecido uma grande quantidade de informação sobre a arquitetura gênica e sobre a configuração física de suas respectivas regiões flanqueadoras (RF). Estas RF contêm informações com o potencial de auxiliar na elucidação de vários processos biológicos, como os mecanismos de expressão gênica e de sua regulação. Estes mecanismos são de extrema importância para a compreensão do correto funcionamento dos organismos e das patologias que os afetam. Uma parte significativa dos mecanismos de controle de expressão gênica atuam na fase transcricional. Na base destes mecanismos está o recrutamento de proteínas que se ligam às regiões promotoras da transcrição, as quais são segmentos específicos de DNA que podem estar localizados tanto próximos à região de início da transcrição (TSS) quanto a centenas ou até a milhares de pares de bases dela. Essas proteínas compõem a maquinaria transcricional e podem ativar ou inibir o processo de transcrição. Experimentalmente, os segmentos regulatórios podem ser identificadas utilizando métodos complexos de biologia molecular, tais como SELEX, ChiP-ChiP, ChIP-Seq, dentre outros. Uma estratégia alternativa aos métodos experimentais é a utilização de metodologias computacionais. Análises computacionais tendem a ser mais rápidas, baratas e flexíveis do que protocolos experimentais, além de poderem ser utilizadas em larga escala. Atualmente, os métodos computacionais disponíveis necessitam de informações experimentais para a definição de padrões globais de preferências de sequências de DNA para a ligação de fatores de transcrição (TFBS, em inglês transcription factor binding sites). Entretanto, esses métodos apresentam uma elevada taxa de falso positivos e, por vezes, apresentam também taxas significativas de falso negativos, além de serem limitados ao estudo de fatores de transcrição de espécies bem conhecidas, o que diminui a área de aplicação dos mesmos. Diante deste cenário, o uso de métodos computacionais que não necessitem da informação referente aos sítios de ligação, bem como os que utilizem parâmetros mais robustos de detecção dos resultados, em detrimento dos escores de pontuação provindos de alinhamentos, podem acrescentar uma sensível melhoria ao processos de predição de regiões regulatórias. Neste projeto, foi desenvolvido um novo modelo computacional (TFBSAnalyzer) para análise e identificação de TFBS em elementos regulatórios, que utiliza técnicas de modelagem molecular para a construção de complexos entre um fator de transcrição ancorado a estruturas de DNA com sequências variáveis de bases e, através de cálculos termodinâmicos de entalpia de ligação, determina uma função de pontuação baseada na energia de ligação e realiza a predição de sítios de ligação ao DNA para o fator de transcrição em análise. Esta abordagem foi testada com três fatores de transcrição como sistemas-modelo, pertencentes à família dos receptores nucleares, a saber: o receptor de estrógeno ER-alfa (Estrogen Receptor Alpha), o receptor de ácido retinoico RAR-beta (Retinoid Acid Receptor Beta) e o receptor X retinóico RXR (Retinoid X Receptor). Os modelos previstos computacionalmente foram comparados aos dados experimentais disponíveis para estes receptores nucleares, os quais apresentaram as seguintes taxas de FP/FN: 10%/0 para RAR-beta e RXR, 21%/6% para ER-alfa. Também simulamos um experimento de ChIP-seq do ER-alfa no genoma humano, cujos genes selecionados foram submetidos a uma análise de enriquecimento de fatores de transcrição curados experimentalmente, que fazem sua regulação, revelando que o receptor de estrógeno está realmente envolvido no processo. Para mostrar a aplicabilidade geral de nosso método, nós modelamos a distribuição de energia de ligação para o receptor NHR-28 isoforma a de Caenorhabditis elegans com DNA . Obtivemos distribuições de energia semelhantes àquelas encontradas para os NRs modelos, portanto seria possível aplicar o método para buscar possíveis TFBSs para este receptor no genoma de C. elegans. Os dados gerados e as metodologias desenvolvidas neste projeto devem acrescentar uma sensível melhoria aos processos de predição de regiões regulatórias e consequentemente auxiliar no entendimento dos mecanismos envolvidos no processo de expressão gênica e de sua regulação.The genome projects have provided a lot of information about the genetic architecture, as well as on the physical configuration of their flanking regions (FR). These FR have the potential to aid in the elucidation of many biological processes, such as the mechanisms involved in gene expression and its regulation. These mechanisms are extremely important for undeerstanfind the correct functioning of organisms as well as the pathologies that affect them. A significant part of the control mechanisms of gene expression act during transcription. On the basis of this mechanisms is the recruitment of proteins that bind to promoter regions of transcription, which are specific segments of DNA that can be located either near the transcription start site or at hundreds or even thousands of base pairs away. These proteins form the transcription machinery, which can activate or inhibit the transcription process. The regulatory segments can be identified experimentally using complex methods of molecular biology, such as SELEX, ChIP-chip, ChIP-seq, among others. An alternative strategy to these experimental methods is the use of computational methodologies for predicting regulatory regions. Computational analysis tend to be faster, cheaper and more flexible than the experimental protocols, and can be used on a larger scale. Currently, the available computational methods require information previously obtained from experiments in order to define global standards of preference of DNA-Binding sequences for transcription factors (TFBS - Transcription Factor Binding Sites). However, these methods have a high rate of false positives and sometimes also have significant rates of false negatives, besides being limited to the study of transcription factors of well-known species, which decreases their application area. In this scenario, the use of computational methods that do not require previous information concerning the binding sites and use more robust parameters of results detection, instead of alignment scores, may add significant improvement to the processes of predicting regulatory regions. In this project, we developed a new computational model TFBSAnalyzer) for analysis and identification of regulatory elements using molecular modeling techniques for the construction of complexes between a transcription factor bound to specific DNA structures with variable sequences of bases and, by means of thermodynamic calculations of bond enthalpy, provides a scoring function based on the binding energy and predicts the DNA binding sites for the transcription factor in analysis. This approach was tested initially with three transcription factors as models, belonging to the nuclear receptor family, namely estrogen receptor ER-alpha (Estrogen Receptor Alpha), the retinoic acid receptor RAR-beta (Retinoid Acid Receptor Beta) and the retinoic X receptor RXR (Retinoid X Receptor). The computationally predicted models were compared to experimental data available for these nuclear receptors, and presented the following rates of FP/FN: 10%/0 for RAR-beta and RXR, 21%/6% for ER-alpha. We also simulated an experiment of ChIP-seq with ER-alpha with the human genome, where the selected genes were subjected to a transcription factor enrichment analysis, with curated information, revealing that the estrogen receptor is indeed involved in their regulation. To show that our method has a general applicability, we modeled the binding energy distribution for the NHR-28 receptor, isoform a, from Caenorhabditis elegans. The energy distributions obtained were similar to the ones obtained for the model NR, so it would be possible to use the method and search for possible TFBS in the C. elegans genome. The data generated and the methodologies developed in this project should add a significant improvement to the prediction processes of regulatory regions and, consequently, help to understand the mechanisms involved in the gene expression process and its regulation.
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Wang, Zhonghua Laity John H. "Characterization of novel structure-regulatory relationships within interacting two-finger Cys₂His₂ zinc finger protein motifs." Diss., UMK access, 2008.
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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.
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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.
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Crawford, Catherine. "Characterisation of endogenous KRAB zinc finger proteins." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4225.
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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.