Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

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.

Thesis (Ph. D.)--Georgia State University, 2008. / Title from file title page. Markus W. Germann, committee chair; Kathryn B. Grant , W. David Wilson, committee members. Electronic text (147 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Oct. 6, 2008. Includes bibliographical references.

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 158-168). Also available on the World Wide Web.

ZNF292 is a gene that encodes for a large multifunctional zinc finger protein. ZNF292 has a role in Growth Hormone transcription, developmental disorders on the autism spectrum, and in the initiation of tumorigenesis. Cancer cells have revealed ZNF292 as a gene with unique features: it is present in both linear and circular RNA (circRNA) forms. Circular ZNF292 RNAs vary in size depending on the number of exons that are back-spliced together forming a nested set of babushkas or “Russian dolls” – larger forms add an exon to a smaller circle. To determine whether anti-cancer treatments change the expression of circRNA forms as well as the linear form of ZNF292, we performed quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) analysis. Primers used were designed to amplify only the specified form of ZNF292, either the linear form or one of four targeted circular forms. Control and flavone (3,5 dihydroxy-7-methoxyflavone)-treated cell lines were grown, harvested, and total RNA extracted. Then, samples were analyzed by qRT-PCR with specific ZNF292 primer sets for each product using a standard curve for comparisons. All results were normalized to actin levels in each sample prior to statistical analysis. When compared to untreated controls, two linear ZNF292 RNAs were each reduced to 52% of control levels (p Funded by the Bill Gatton College of Pharmacy.

No description available.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24947号 / 生博第509号 / 新制||生||68(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 原田 浩, 教授 松本 智裕, 教授 鈴木 淳 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Les gènes IMA (Inhibitor of Meristem Activity) et MIF2 (MIni zinc Finger 2) codent des protéines de la famille des « MIni zinc Finger » impliquées dans le développement et la réponse hormonale. Leur fonction est intimement liée à leur capacité d’interaction avec des protéines partenaires au travers de leur unique domaine, un doigt de zinc non-canonique. La génération et la caractérisation de plantes gain- et perte-de-fonction pour ces gènes chez Solanum lycopersicum et Arabidopsis thaliana nous a permis de mettre en évidence leur homologie fonctionnelle dans le mécanisme d’arrêt de la prolifération des cellules souches méristématiques : la terminaison florale. Au cours du développement floral précoce, leur expression est induite par le facteur de transcription à MADS-BOX AGAMOUS. Les protéines codées sont capables d’intéragir avec le facteur de transcription à doigt de zinc C2H2 KNUCKLES (KNU) et de recruter le co-facteur TOPLESS (TPL) ainsi que l’HISTONE DEACETYLASE19 (HDA19) pour former un complexe multimérique impliqué dans le remodelage de la chromatine. Ce complexe intervient ensuite afin de réprimer l’expression du régulateur majeur de la prolifération des cellules souches méristématiques WUSCHEL. Les travaux qui ont suivi ont permis de montrer qu’au-delà de leur intervention dans ce processus, les protéines IMA et MIF2 sont impliquées d’une manière plus générale dans le développement floral et l’organogenèse. Leur fonction de protéine adaptatrice est sans doute permise par le désordre intrinsèque qui les caractérise et qui leur permettrait une spécificité d’interaction avec divers partenaires protéiques. / IMA (Inhibitor of Meristem Activity) and MIF2 (MIni zinc Finger 2) are two members of the MIni zinc Finger family (MIF) involved in the regulation of floral development and hormonal signaling pathways. Their ability to control physiological events is linked to their unique domain, a non canonical zinc finger, which confers to MIF the capacity to interact with other proteins. The characterization of gain- and loss-of-function lines for these genes in Solanum lycopersicum and Arabidopsis thaliana allowed us to unravel their functional homology in the termination of floral stem cell maintenance. During early floral development, IMA and MIF2 gene expression is induced by the MADS-Box transcription factor AGAMOUS. Then, IMA and MIF2 proteins recruit the C2H2 zinc finger KNUCKLES (KNU), in a transcriptional repressor complex together with TOPLESS (TPL) and HISTONE DEACETYLASE19 (HDA19). This complex binds to the WUSCHEL (WUS) locus leading to WUS repression via a chromatin deacetylation mechanism. Further work allowed us to demonstrate that IMA and MIF2 play a wider role in floral development and organogenesis. Their function as adaptor protein is probably linked to their intrinsic disorder leading to structural flexibility and interaction specificity with a large range of partners.

Methoden zur direkten Detektion oder Anreicherung von doppelsträngiger DNA (dsDNA) bieten ein hohes Potential zum Einsatz in der molekularen Diagnostik. Bereits etablierte Methoden für die Nukleinsäure - Detektion (NAD) basieren in der Regel auf der Hybridisierung des komplementären Stranges gefolgt von der optischen Detektion oder enzymatischer Amplifikation. DNA - bindende oder organisierende Proteine (z.B. endogene Transkriptionsfaktoren) bieten im Kontrast zu den Hybridisierungsreaktionen eine überaus interessante Alternative um dsDNA direkt und zugleich spezifisch zu detektieren oder diese aus einem komplexen Gemisch heraus anzureichern. Im Rahmen der Entwicklung von neuartigen NAD - Assays zur direkten Detektion oder Anreicherung von Nukleinsäuren wurden vier DNA - bindende Proteine kloniert und in HEK293 und E. coli exprimiert. Der Cys2His2 - Zinkfinger (ZFD) vom humanen Transkriptionsfaktor Sp1 wurde mit MBP und 9×Lys - MBP fusioniert. Das MBP - Derivat 9×Lys - MBP ist eine erweiterte Variante mit neun aufeinanderfolgenden Lysinen im N - terminalen Bereich, welche eine regioselektive Immobilisierung ermöglichen soll. Der humane Sp3 - ZFD wurde mit EGFP fusioniert. Die Mitglieder der Sp - Familie binden spezifisch die Konsensussequenz 5’ - GGG GCG GGG - 3’ (GC - Box). Zusätzlich wurde die C - terminale DNA - bindende Domäne der E. coli DNA - Gyrase Untereinheit A (gyrA - CTD) ebenfalls mit MBP fusioniert. Die Domäne bindet spezifisch repetitive extragene Palindrome (REP), welche bislang nur auf bakteriellen Chromosomen vorkommen. Sämtliche MBP - Fusionsproteine liegen nach der Expression löslich vor und konnten über eine native Strategie aufgereinigt werden. Transiente Transfektionsexperimente in HEK293 zeigten einen destabilisierenden Effekt der Sp3 - ZFD und eine massive einhergehende Degradierung des EGFP - Fusionsproteins nach 120 h. Die Analyse der mRNA - Integrität nach Transfektion des Expressionsplasmids, sowie zellbiologische und proteinbiochemische Untersuchungen mit Durchflusszytometrie bzw. Western Blots deuten auf eine posttranslationale Modulierung von EGFP - Sp3 hin. Um die Hypothese der proteasomalen Degradierung von EGFP - Sp3 zu belegen, wurden transfizierte HEK293 mit dem reversiblen Proteasominhibitor MG132 behandelt. In Gegenwart von 1 µM MG132 konnte das zytosolische Fusionsprotein stabilisiert werden. Die hier präsentierten Daten offenbaren die humane Sp3 - ZFD als ein neues Substrat für das 26S - Proteasom. Lediglich die SUMOylierung von Wildtyp - Sp3 im Bereich der inhibitorischen Domäne (ID) ist bislang beschrieben worden. Die Funktionalität, Affinität und kinetische Parameter der mit MBP fusionierten Sp1 - ZFD und gyrA - CTD wurden anhand von Oberflächenplasmonresonanz (BIAcore) bzw. EMSAs analysiert. Sämtliche gewonnenen MBP - Fusionsproteine sind funktionell und interagieren mit dsDNA. Fusionsproteine mit Sp1 - Domäne zeigten in EMSAs ebenso eine Bindung an unspezifische dsDNA. In sensitiveren BIAcore - Assays mit immobilisierter dsDNA wurden (um den Faktor 2) geringere Assoziations (ka) - und Dissoziationsraten (kd) von MBP - Sp1 ermittelt, wenn bestimmte Basen innerhalb der GC - Box ausgetauscht wurden. Die Affinität (Kd) von MBP - Sp1 mit 4×10 - 9 M zur GC - Box und deren Derivate ist vergleichbar mit der Kd von nativem Sp1. Die EMSA - Experimente für MBP - gyrA zeigen eine deutliche Präferenz zum spezifischen dsDNA - Oligo in Gegenwart von humaner gDNA, eine interessante Eigenschaft die durchaus zur Anwendung in einem Assay zur Anreicherung von bakterieller DNA dienen kann. Nach der vorausgehenden Charakterisierung der MBP - Fusionsproteine wurden diese auf verschiedenen gängigen festen und semifesten Substraten über physische Adsorption, kovalent oder Affinität immobilisiert um das Konzept der direkten Detektion von dsDNA mit funktionellen Proteinen als neuartige Komponente in NAD - Assays umzusetzen. Lediglich MBP - Sp1 zeigte auf Glas und Polystyren - Mikrotiterplatten nach kovalenter oder adsorptiver Immobilisierung eine ausgeprägte Funktionalität hinsichtlich der Bindung von dsDNA. Die Immobilisierung von 9×Lys - MBP - Sp1 über identische Strategien führten zum massiven Verlust der ZFD - Funktion. Aus dieser Datenlage heraus wurde erfolgreich ein simples Lumineszenz - basiertes Mikrotiterplatten - Assay mit MBP - Sp1 entwickelt um PCR - Amplikons direkt aus einer analytischen PCR auf gDNA von S. aureus, welche die GC - Box beinhalten, nachzuweisen. Das spezifische Amplikon konnte mittels des simplen Assays in Gegenwart von 100fachem Überschuss an humaner gDNA nachgewiesen werden. Mit einem höheren Anteil an humaner gDNA wurde die PCR massiv inhibiert, ein negativer Effekt der bislang im Bereich der diagnostischen NAD - Assays nicht optimal adressiert wurde. Die magnetische Separation von bakterieller und humaner gDNA wurde dazu mit MBP - gyrA umgesetzt. Zunächst erfolgte die regioselektive Immobilisierung von MBP - gyrA auf Protein A - funktionalisierte magnetische Nanopartikel mittels MBP - Antikörper, wodurch die Funktionalität hinsichtlich der Bindung von dsDNA gewährleistet werden konnte. Dieses System eignet sich insbesondere für die Separation von bakterieller DNA (E. coli oder S. aureus) aus einem komplexen Gemisch mit bis zu 100fachem Überschuss an humaner gDNA. Die Kombination von MBP - gyrA - basierter magnetischer Separation mit NAD - Assays könnte deren Sensitivität signifikant erhöhen. Durch simple Verfahrensweise bietet das System einen wesentlichen Beitrag zur Verringerung des zeitlichen Aufwands für die Generierung therapierelevanter Resultate. / Methods for direct detection or enrichment of double - stranded DNA (dsDNA) possess tremendous potential for use in molecular diagnostics. Already established methods for nucleic acid detection (NAD) are generally based on the hybridization of two complementary strands followed by optical detection or enzymatic amplification. In contrast, DNA - binding or organizing proteins (e.g. endogenous transcriptions factors) are able to read the sequence information directly from dsDNA without prior denaturation of the double strand and subsequent hybridization. In order to develop novel NAD assays or assays for sample preparation, four artificial DNA - binding proteins were cloned, expressed and purified in HEK293 cells or E. coli. The Cys2His2 zinc finger domains (ZFD) from human Sp1 were fused to maltose binding protein (MBP) and its derivate 9×Lys - MBP, an extended variant with nine successive lysine residues in the N - terminal region of the protein to facilitate site - directed immobilization. The human Sp3 - ZFD was fused to green fluorescent protein (EGFP). The family of Sp - transcription factors was known to bind specifically the consensus sequence 5\' - GGG GCG GGG - 3 \'(GC - box). Moreover, the C - terminal DNA - binding domain of E. coli DNA Gyrase subunit A (gyrA - CTD) was fused to MBP. The CTD binds specifically repetitive extragenic palindromes (REP), which were only found on prokaryotic chromosomes. All MBP fusion proteins were soluble after expression and could be purified to homogeneity. Surprisingly, transient transfection experiments in HEK293 revealed a destabilizing effect of the Sp3 - ZFD accompanied by massive degradation of the EGFP fusion protein after 120 h post transfection. Analysis of mRNA integrity in combination with western blots indicates a posttranslational modulation of EGFP - Sp3. To confirm the hypothesis of proteasomal degradation of EGFP - Sp3, transfected cells were treated with the reversible proteasome inhibitor MG132. In the presence of 1µM MG132 the fusion protein could be stabilized. Taken together, the data presented here identified the human Sp3 - CTD as a new substrate for the 26S proteasome. Only SUMOylation of wild type human Sp3 within the inhibitory domain (ID) has been described so far. Initial EMSA experiments showed that purified MBP - ZFD fusion proteins were functional in terms of interacting with dsDNA containing the specific sequence motiv. However, all proteins bound to unspecific dsDNA as well. Therefore MBP - Sp1 was subjected to BIAcore analysis to determine the rate constants for association ka, dissociation kd and the dissociation constant Kd of the GC - Box - Protein complex as well as mutants of the GC - Box. The determined Kd (4 × 10 - 9 M) for MBP - Sp1 associated with GC - box or its derivatives were found to be comparable with the Kd of native Sp1, however the rate constants were reduced 2 fold in presence of the modified GC - boxes. EMSA experiments with MBP - gyrA revealed functionality and a clear preference for specific dsDNA in the presence of unspecific human genomic DNA (gDNA). After preliminary functional characterization, MBP fusion proteins were immobilized by physical adsorption, covalent or by affinity on various solid substrates or nanoscaled magnetic beads to implement the concept of direct detection of dsDNA or specific enrichment of bacterial DNA, respectively. MBP - Sp1 remains functional after adsorptive or covalent immobilization on different chemical modified glas surfaces. 9×Lys - MBP - Sp1 shows significantly reduced functionality after immobilization on the same glas substrates by similar strategies. Moreover, a simple NAD - assay with adsorptive immobilized MBP - Sp1 on polystyrene in microtiter format was established for direct detection of GC - boxes within PCR - products from S. aureus gDNA. By using the assay, specific PCR - products could be detected in presence up to 100 - fold excess of human gDNA in relation to 10 ng bacterial DNA. Separation of bacterial DNA from human DNA from clinical samples may have an important impact on downstream applications, involving NAD assays. To address this often underestimated technical problem, a new functional protein MBP - gyrA was introduced to overcome some limitations of already established methods. MBP - gyrA was site - directed coupled on nanoscaled magnetic beads by affinity. This system enabled the fast and specific separation of gDNA of E. coli or S.aureus from a huge background of human gDNA. The combination of MBP - gyrA - based magnetic separation with NAD assays could significantly increase the sensitivity and shorten the time for initiation of effective treatment.

<p>Duchenne muscular dystrophy (DMD) is a severe hereditary disorder caused by a loss of dystrophin, an essential musculoskeletal protein. Decades of promising research have yielded only modest gains in survival and quality of life for these patients and there have been no approved gene therapies for DMD to date. There are two significant hurdles to creating effective gene therapies for DMD; it is difficult to deliver a replacement dystrophin gene due to its large size and current strategies to restore the native dystrophin gene likely require life-long administration of a gene-modifying drug. This thesis presents a novel method to address these challenges through restoring dystrophin expression by genetically correcting the native dystrophin gene using engineered nucleases that target one or more exons in a mutational hotspot in exons 45-55 of the dystrophin gene. Importantly, this hotspot mutational region collectively represents approximately 62% of all DMD mutations. In this work, we utilize various engineered nuclease platforms to create genetic modifications that can correct a variety of DMD patient mutations.</p><p>Initially, we demonstrate that genome editing can efficiently correct the dystrophin reading frame and restore protein expression by introducing micro-frameshifts in exon 51, which is adjacent to a hotspot mutational region in the dystrophin gene. Transcription activator-like effector nucleases (TALENs) were engineered to mediate highly efficient gene editing after introducing a single TALEN pair targeted to exon 51 of the dystrophin gene. This led to restoration of dystrophin protein expression in cells from DMD patients, including skeletal myoblasts and dermal fibroblasts that were reprogrammed to the myogenic lineage by MyoD. We show that our engineered TALENs have minimal cytotoxicity and exome sequencing of cells with targeted modifications of the dystrophin locus showed no TALEN-mediated off-target changes to the protein coding regions of the genome, as predicted by in silico target site analysis. </p><p>In an alternative approach, we capitalized on the recent advances in genome editing to generate permanent exclusion of exons by using zinc-finger nucleases (ZFNs) to selectively remove sequences important in specific exon recognition. This strategy has the advantage of creating predictable frame restoration and protein expression, although it relies on simultaneous nuclease activity to generate genomic deletions. ZFNs were designed to remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript, a method that can potentially restore the dystrophin reading frame in up to 13% of DMD patients. Nucleases were assembled by extended modular assembly and context-dependent assembly methods and screened for activity in human cells. Selected ZFNs had moderate observable cytotoxicity and one ZFN showed off-target activity at two chromosomal loci. Two active ZFN pairs flanking the exon 51 splice acceptor site were transfected into DMD patient cells and a clonal population was isolated with this region deleted from the genome. Deletion of the genomic sequence containing the splice acceptor resulted in the loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin expression in vitro. Furthermore, transplantation of corrected cells into the hind limb of immunodeficient mice resulted in efficient human dystrophin expression localized to the sarcolemma. </p><p>Finally, we exploited the increased versatility, efficiency, and multiplexing capabilities of the CRISPR/Cas9 system to enable a variety of otherwise challenging gene correction strategies for DMD. Single or multiplexed sgRNAs were designed to restore the dystrophin reading frame by targeting the mutational hotspot at exons 45-55 and introducing either intraexonic small insertions and deletions, or large deletions of one or more exons. Significantly, we generated a large deletion of 336 kb across the entire exon 45-55 region that is applicable to correction of approximately 62% of DMD patient mutations. We show that, for selected sgRNAs, CRISPR/Cas9 gene editing displays minimal cytotoxicity and limited aberrant mutagenesis at off-target chromosomal loci. Following treatment with Cas9 nuclease and one or more sgRNAs, dystrophin expression was restored in Duchenne patient muscle cells in vitro. Human dystrophin was detected in vivo following transplantation of genetically corrected patient cells into immunodeficient mice. </p><p>In summary, the objective of this work was to develop methods to genetically correct the native dystrophin as a potential therapy for DMD. These studies integrate the rapid advances in gene editing technologies to create targeted frameshifts that restore the dystrophin gene around patient mutations in non-essential coding regions. Collectively, this thesis presents several gene editing methods that can correct patient mutations by modification of specific exons or by deletion of one or more exons that results in restoration of the dystrophin reading frame. Importantly, the gene correction methods described here are compatible with leading cell-based therapies and in vivo gene delivery strategies for DMD, providing an avenue towards a cure for this devastating disease.</p> / Dissertation