Fragile X syndrome (FXS), the most common cause of inherited mental impairment, is caused by the loss of expression of the fragile X mental retardation protein (FMRP). As an RNA binding protein, FMRP has been proposed to regulate the transport and translation of specific message RNA (mRNA). It has been reported that FMRP uses its RGG box domain to bind mRNA targets that form a G-quadruplex structure, structure believed to be important for FMRP recognition of at least a subclass of its mRNA targets. We have hypothesized that the interaction of FMRP with selected relevant mRNA targets occurs in a G-quadruplex dependent manner. By analyzing the structure of two FMRP in vivo mRNA targets, Shank1 mRNA and BASP1 mRNA, and their interactions with FMRP, we showed a high-affinity interaction between Shank1 RNA G-quadruplex and FMRP. The other G-quadruplex forming mRNA BASP1, however, interacts with FMRP using other structural elements. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Pharmaceutics / MS; / Thesis;

G-quadruplex structures are highly stable alternative DNA structures that can, when not properly regulated, impede replication fork progression and cause genome instability (Castillo Bosch et al, 2014; Crabbe et al, 2004; Koole et al, 2014; Kruisselbrink et al, 2008; London et al, 2008; Lopes et al, 2011; Paeschke et al, 2013; Paeschke et al, 2011; Piazza et al, 2015; Piazza et al, 2010; Piazza et al, 2012; Ribeyre et al, 2009; Sabouri et al, 2014; Sarkies et al, 2012; Sarkies et al, 2010; Schiavone et al, 2014; Wu & Spies, 2016; Zimmer et al, 2016). The aim of this thesis was to identify novel G-quadruplex interacting proteins in Saccharomyces cerevisiae and to unravel their regulatory function at these structures to maintain genome integrity. Mms1 and Rtt101 were identified as G-quadruplex binding proteins in vitro via a pull-down experiment with subsequent mass spectrometry analysis. Rtt101, Mms1 and Mms22, which are all components of an ubiquitin ligase (Rtt101Mms1/Mms22), are important for the progression of the replication fork following fork stalling (Luke et al, 2006; Vaisica et al, 2011; Zaidi et al, 2008). The in vivo binding of endogenously tagged Mms1 to its target regions was analyzed genome-wide using chromatin-immunoprecipitation followed by deep-sequencing. Interestingly, Mms1 bound independently of Mms22 and Rtt101 to G-rich regions that have the potential to form G-quadruplex structures. In vitro, formation of G-quadruplex structures could be shown for the G-rich regions Mms1 bound to. This binding was observed throughout the cell cycle. Furthermore, the deletion of MMS1 caused replication fork stalling as evidenced by increased association of DNA Polymerase 2 at Mms1 dependent sites. A gross chromosomal rearrangement assay revealed that deletion of MMS1 results in a significantly increased genome instability at G-quadruplex motifs compared to G-rich or non-G-rich regions. Additionally, binding of the helicase Pif1, which unwinds G4 structures in vitro (Paeschke et al, 2013; Ribeyre et al, 2009; Sanders, 2010; Wallgren et al, 2016), to Mms1 binding sites was reduced in mms1 cells. The data presented in this thesis, together with published data, suggests a novel mechanistic model in which Mms1 binds to G-quadruplex structures and enables Pif1 association. This allows for replication fork progression and genome integrity. / Bei G-quadruplex Strukturen handelt es sich um stabile Sekundärstrukturen der DNA, welche das Fortschreiten der Replikationsgabel behindern und Genominstabilität verursachen können, falls sie nicht konsequent reguliert werden (Castillo Bosch et al, 2014; Crabbe et al, 2004; Koole et al, 2014; Kruisselbrink et al, 2008; London et al, 2008; Lopes et al, 2011; Paeschke et al, 2013; Paeschke et al, 2011; Piazza et al, 2015; Piazza et al, 2010; Piazza et al, 2012; Ribeyre et al, 2009; Sabouri et al, 2014; Sarkies et al, 2012; Sarkies et al, 2010; Schiavone et al, 2014; Wu & Spies, 2016; Zimmer et al, 2016). Ziel dieser Doktorarbeit war es, neue Proteininteraktionspartner dieser Strukturen in Saccharomyces cerevisiae zu identifizieren und zu untersuchen, wie diese Proteine die Strukturen regulieren um Genomstabilität zu gewährleisten. Mit Hilfe eines Pulldown Assays und anschließender massenspektrometrischer Analyse wurden Mms1 und Rtt101 in vitro als Interaktionspartner von G-quadruplex Strukturen identifiziert. Rtt101, Mms1 und Mms22, Komponenten der Ubiquitinligase Rtt101Mms1/Mms22, spielen eine wichtige Rolle beim Fortschreiten der Replikationsgabel, falls dieses durch Agenzien gehemmt wurde (Luke et al, 2006; Vaisica et al, 2011; Zaidi et al, 2008). Durch Chromatin-Immunpräzipitation mit anschließender Hochdurchsatzsequenzierung wurden die Bindestellen von Mms1 identifiziert. Interessanterweise hat Mms1 genomweit an G-reiche Sequenzen gebunden. Diese G-reichen Sequenzen bildeten G-quadruplex Strukturen in vitro aus. Die Bindung von Mms1 erfolgte unabhängig von Rtt101 und Mms22 sowie während des gesamten Zellzyklus. Außerdem kam es zu einer Verlangsamung der Replikationsgabel in mms1 Zellen, was durch eine verstärkte Bindung der DNA Polymerase 2 nachgewiesen wurde. Ein gross chromsomal rearrangement assay zeigte, dass die Genominstabilität in mms1 Zellen signifikant erhöht ist, wenn G-quadruplex Motive, im Vergleich zu nicht-G-reichen oder G-reichen Kontrollregionen, vorhanden sind. Zudem war die Bindung der Helikase Pif1, welche G-quadruplex Strukturen in vitro entwindet (Paeschke et al, 2013; Ribeyre et al, 2009; Sanders, 2010; Wallgren et al, 2016), stark reduziert, wenn Mms1 fehlte. Mit Hilfe der in dieser Doktorarbeit gewonnenen Ergebnisse, sowie mit Hilfe publizierter Daten, lässt sich ein Model postulieren, in welchem Mms1 an G-quadruplexe bindet und somit die Bindung von Pif1 ermöglicht. Dadurch werden das Fortschreiten der Replikationsgabel und die Genomstabilität gewährleistet.

No description available.

Aberrant expression of Platelet-derived growth factor A (PDGF-A) and PDGF receptor-β (PDGFR-β) play critical roles in the angiogenesis and proliferation of several malignancies. In this dissertation I explore the transcriptional regulatory role of the Gquadruplex- forming regions in the promoters of human PDGF-A and PDGFR-β, and identify new targets for developing small molecules to modulate their expression in tumors. For PDGF-A promoter, our studies focus on two essential nuclease hypersensitive elements, NHE(PDGF-A) and 5´-end far upstream 5´-SHS. The structural aspects of the intramolecular G-quadruplexes formed in NHE(PDGF-A) and the ligands to stabilize these secondary DNA structures have been investigated by using singlestranded and duplex DNA of the NHE(PDGF-A). We demonstrate that the G-quadruplexinteractive compound, TMPyP4, can selectively inhibit the basal promoter activity of PDGF-A, suggesting that the NHE(PDGF-A) G-quadruplex acts as a repressor in PDGF-A transcription. We also found that the 5´-SHS G-rich strand oligomer can invade the NHE(PDGF-A) and form a unique three-stranded complex in supercoiled plasmids, which is facilitated by potassium ions and TMPyP4. Therefore, we propose a novel molecular mechanism for transcriptional silencing of the NHE(PDGF-A) by 5´-SHS in the PDGF-A promoter, in that the formation of G-quadruplex in the NHE(PDGF-A) provides a platform for the G-rich strand of 5´-SHS to invade and form a partial duplex DNA with the C-rich strand of the NHE(PDGF-A), resulting in displacement of hnRNP K and thus transcription silencing. Prior to the studies describe here, the promoter of human PDGFR-β had not been identified. Herein, we have cloned and characterized the first functional promoter of human PDGFR-β gene. A crucial highly GC-rich region (NHE(PDGFR-β)) in the human PDGFR-β promoter has been identified by its hypersensitivity to the S1 nuclease. Further studies demonstrate that stable G-quadruplex structures can form in the G-rich strand of NHE(PDGFR-β). The G-quadruplex-interactive molecule, telomestatin, can selectively stabilize G-quadruplexes formed in the human PDGFR-β promoter and inhibit its expression in Daoy cells. On the basis of these results, we propose that ligandmediated stabilization of the G-quadruplex structure in the proximal promoter region of human PDGF-A or PDGFR-β can be used to modulate the expression of these protooncogenes.

Diarrheagenic Escherichia coli (E. coli) and Neisseria sp. are Gram-negative pathogens that cause high disease burden, especially in low-income countries.Enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) are a subset of E. coli that can cause disease. The sequence of E. coli genomes revealed the presence of at least 16 putative pili operons, it is still unknown if they encode functional pili. Several adhesins have been described in EPEC; however it is still an enigma if EHEC produces pili. In this dissertation the identification and characterization of a new pilus in EHEC is described. The main pilin subunit is encoded in the yagZ gene (renamed ecpA) and is present in all E. coli. We demonstrate ECP production in 137 (70%) of a total of 197 ecpA+ strains representing different categories of E. coli. Isogenic ecpA mutants of EHEC O157:H7 and fecal commensal E. coli showed significant reduction in adherence to cultured epithelial cells. Adherence levels were not hampered after single mutation of ecpA in EPEC. Only after the removal of the known EPEC adhesins such as BFP and intimin we were able to see significant reduction in adherence levels. In sum, ECP is the first pilus of EHEC O157:H7 with a potential role in host epithelial cell colonization. However, EPEC-ECP plays a secondary role in adherence.Since 2007 the CDC recommends only third generation cephalosporins as the elected treatment for Neisseria gonorrhoeae infections. There is an urgent need to search for new drug targets and to development new drugs. Regions rich in guanine in the DNA are able to form secondary structures known as G-quadruplexes. It has been shown that G-quadruplexes are involved in control of transcription, translation and telomere elongation in mammalian cells. G-quadruplex interactive compounds are being developed for cancer therapy. G-quadruplex motifs are also present in bacteria. The fact that G-quadruplex interactive compounds can impair cancer development leads us to hypothesize that these drugs can be used as antimicrobials. This work presents evidence for the potential of G-quadruplex interactive compounds as broad-spectrum antimicrobials.

Thesis (Ph. D.)--University of Idaho, 2006. / Abstract. "May 2006." Includes bibliographical references. Also available online in PDF format.

Genomic complexity in non-Hodgkin’s Diffuse Large B-cell Lymphoma (DLBCL) leads to a treatment failure in ~40% of patients. Activation-Induced Cytosine Deaminase (AID), one of the enzymes involved in generating antibody diversity via class switching recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes in activated B-cells is one mechanism for the introduction of genomic lesions. In previous studies, AID was shown to preferentially bind to super-enhancer (SE) regions within the genome, but 26% of AID targets were not within the SE regions. The mechanism by which AID interacts with SE elements and its off-target interactions still remains a mystery. Recent evidence suggests that AID may cause genomic lesions in DLBCL via interaction with oncogenes such as MYC and BCL2 resulting in mutations and translocations. Sequences within the MYC promoter contain the four-nucleotide AID target sequence (WRCY) and highly G-rich sequences known to form G-quadruplex DNA secondary structures. We hypothesize that key DNA secondary structures act as recruiting elements for aberrant AID activity at promoters and SEs of key genes involved in the development of DLBCL. Here, we first sought to determine whether known AID DNA targets have the potential to form G-quadruplex DNA secondary structures. The data collected from activated mouse B-cells showed 90% of the AID targets contained sequences that could potentially form G-quadruplexes and the data collected from the human Ramos cell line showed 100% of the sequences had the potential to form G-quadruplexes. To further study our hypothesis we used the techniques circular dichroism (CD) and the electrophoresis motility shift assay (EMSA) to explore the potential interaction between AID and the BCL2 and MYC G-quadruplexes. We observed no significant interactions between AID and these two G-quadruplexes, however further experimentation with different conditions and molecular techniques may show interaction. Additional studies will not only provide key insight into the genomic instability within DLBCL, but will also provide a potential mechanism by which AID is recruited to its DNA targets.

Dominant-activating mutations in the RET (rear-ranged during transfection) proto-oncogene, which encodes a receptor tyrosine kinase, is often associated with the development of medullary thyroid carcinoma (MTC). The proximal promoter region of the RET gene consists of a guanine-rich sequence containing five runs of three consecutive guanine residues that serve as the binding site for transcriptional factors. As we have recently shown, this stretch of nucleotides in the promoter region is highly dynamic in nature and tend to form non-B DNA secondary structures called G-quadruplexes, which suppress the transcription of the RET gene. In the present study, ellipticine and its derivatives were identified as excellent RET G-quadruplex stabilizing agents. Circular dichroism (CD) spectroscopic studies revealed that the incorporation of a piperidine ring in an ellipticine derivative, NSC311153 improves its binding with the G-quadruplex structure and the stability induced by this compound is more potent than ellipticine. Furthermore, this compound also interfered with the transcriptional mechanism of the RET gene in an MTC derived cell line, TT cells and significantly decreased the endogenous RET protein expression. We demonstrated the specificity of NSC311153 by using papillary thyroid carcinoma (PTC) cells, the TPC1 cell line which lacks the G-quadruplex forming sequence in the promoter region due to chromosomal rearrangement. The RET downregulation selectively suppresses cell proliferation by inhibiting the intracellular Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways in the TT cells. In the present study, we also showed that the systemic administration of a water soluble NSC311153 analog in a mouse MTC xenograft model inhibited the tumor growth through RET downregulation.

Des séquences compatibles avec la formation de G4 sont présentes au niveau de certaines régions clés du génome telles que les extrémités des chromosomes, mais également les régions de commutation de classe des immunoglobulines, les promoteurs de certains gènes dont des oncogènes et des séquences transcrites. Plus de 370 000 cibles potentielles ont été prédites lors des analyses bioinformatiques du génome humain. Cependant, ces prédictions ne sont pas exhaustives étant limitées par la formulation des algorithmes de prédiction utilisés. En effet, les séquences recherchées suivent la formule consensus suivante G3+N(1−7)G3+N(1−7)G3+N(1−7)G3+. Ainsi, en apportant plus de souplesse dans la description du quadruplex nous pourrons identifier et localiser plus de cibles potentielles. C’est pourquoi, nous proposons un nouvel algorithme G4-Hunter qui permettra l’identification la plus exhaustive possible de séquences cibles en prenant en compte la totalité de la région et non plus uniquement la cible potentielle. Par ailleurs, une étude expérimentale à grande échelle (sur une centaine de séquences cibles) a été menée afin de valider et tester la robustesse de G4-Hunter. A l’aide de ce nouvel outil, nous avons pu identifier de nouvelles séquences cibles non identifiées par les approches déjà existantes au sein des génomes humain, HIV et Dictyostelium discoideum. / Biologically relevant G4 DNA structures are formed throughout the genome including immunoglobulin switch regions, promoter sequences and telomeric repeats. They can arise when single-stranded G-rich DNA or RNA sequences are exposed during replication, transcription or recombination. Computational analysis using predictive algorithms suggests that the human genome contains approximately 370 000 potential G4-forming sequences. These predictions are generally limited to the standard G3+N(1−7)G3+N(1−7)G3+N(1−7)G3+ description. However, many stable G4s defy this description and escape this consensus; this is the reason why broadening this description should allow the prediction of more G4 loci. We propose an objective score function, G4- hunter, which predicts G4 folding propensity from a linear nucleic acid sequence. The new method focus on guanines clusters and GC asymmetry, taking into account the whole genomic region rather than individual quadruplexes sequences. In parallel with this computational technique, a large scale in vitro experimental work has also been developed to validate the performance of our algorithm in silico on one hundred of different sequences. G4- hunter exhibits unprecedented accuracy and sensitivity and leads us to reevaluate significantly the number of G4-prone sequences in the human genome. G4-hunter also allowed us to predict potential G4 sequences in HIV and Dictyostelium discoideum, which could not be identified by previous computational methods.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第21584号 / 理博第4491号 / 新制||理||1645(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 秋山 芳展, 准教授 竹田 一旗 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM