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Maeder, Morgan Lee. "Engineered DNA-Binding Proteins for Targeted Genome Editing and Gene Regulation." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10770.
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Engineered DNA-binding proteins enable targeted manipulation of the genome. Zinc fingers are the most well characterized DNA-binding domain and for many years research has focused on understanding and manipulating the sequence-specificities of these proteins. Recently, major advances in the ability to engineer zinc finger proteins, as well as the discovery of a new class of DNA-binding domains - transcription activator-like effectors (TALEs), have made it possible to rapidly and reliably engineer proteins targeted to any sequence of interest. With this capability, focus has shifted to exploring the applications of this powerful technology. In this dissertation I explore three important applications of engineered DNA-binding proteins.
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Yi, Jia. "The Role of Convergent Transcription in Regulating Alternative Splicing : Targeted Epigenetic Modification via Repurposed CRISPR/Cas9 System and Its Impact on Alternative Splicing Modulation." Electronic Thesis or Diss., Sorbonne université, 2020. http://www.theses.fr/2020SORUS382.
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L'épissage alternatif de l'ARN précurseur est un processus co-transcriptionnel qui affecte la grande majorité des gènes humains et contribue à la diversité des protéines. Le dérèglement d'un tel processus est impliqué dans diverses maladies, y compris la tumorigènes. Cependant, les mécanismes qui régulent ces processus restaient à caractériser. Dans cette étude, nous avons montré que les perturbations de l'épissage alternatif étaient corrélées aux dérèglements de la transcription convergente et de la méthylation de l'ADN. Une transcription convergente peut être générée entre des paires de gènes voisins en en orientation opposée, ou entre des amplificateurs intragéniques et leur gène hôte. CENPO et ADCY3 ont été identifiés comme une paire de gènes de transcription convergentes. Nous avons constaté, dans un modèle de progression tumorale du cancer du sein, que le changement d'épissage de l'exon22 variant d'ADCY3 était corrélé à une augmentation de sa transcription qui allait contre celle de CENPO. En utilisant le système de répression ciblée de la transcription CRISPRi, nous avons démontré que l’inhibition de la transcription de CENPO ne pouvait pas inverser l'altération d'épissage alternatif d'ADCY3 dans les cellules cancéreuses (DCIS). Un activateur intragénique actif a été identifié dans l'intron16 du gène CD44, en aval de ses exons alternatifs. En utilisant le système d'activation ciblée de transcription CRISPRa, nous avons montré que l’augmentation de la transcription de CD44 ne pouvait pas modifier l'épissage alternatif de CD44 dans les cellules DCIS. Ces résultats suggèrent que la modification de transcription convergente par des changements d’activité des promoteurs ne permet pas d’altérer l'épissage alternatif de ADCY3 et CD44 dans les cellules DCIS. Cependant, en remplaçant l'amplificateur intragénique par un promoteur inductible, nous avons constaté que l'activation de la transcription intragénique augmentait le niveau d'inclusion de plusieurs exons alternatifs de CD44 dans les cellules HCT116. Ce résultat suggère que la transcription convergente local pourrait avoir un impact direct sur l'épissage alternatif de CD44. De plus, en utilisant le système de méthylation de l'ADN ciblée CRISPR/dCas9-DNMT3b, nous avons démontré que la méthylation de l'ADN au niveau des exons variants pouvait modifier l'épissage alternatif de CD44. Ce travail de thèse a également exploré les limites et la faisabilité de l'étude de l'épissage alternatif avec des outils moléculaires basés sur le système CRISPRAlternative splicing of precursor RNA is a co-transcriptional process that affects the vast majority of human genes and contributes to protein diversity. Dysregulation of such process is implicated in various diseases, including tumorigenesis. However, the mechanisms regulating these processes were still to be characterized. In this study, we showed that perturbations of alternative splicing correlated with dysregulations of convergent transcription and DNA methylation. Convergent transcription could be generated between pairs of neighboring genes in opposite orientation, or between intragenic enhancers and their host gene. CENPO and ADCY3 was identified as a convergent transcription gene pair. We found, in a tumor progression model of breast cancer, that the splicing change of the ADCY3 variant exon22 correlated with an increase of its transcription that went against that of CENPO. By using targeted transcription repression system CRISPRi, we demonstrated that downregulating the transcription of CENPO could not reverse the alternative splicing alteration of ADCY3 in cancer cells (DCIS). An active intragenic enhancer was identified in the intron16 of CD44, at the downstream of its alternative exons. By using targeted transcription activation system CRISPRa, we showed that upregulating the transcription of CD44 could not alter the alternative splicing of CD44 in DCIS cells. These results suggest that convergent transcription modulation through changes of promoter activity does not alter the alternative splicing of ADCY3 and CD44 in DCIS cells. However, through replacing the intragenic enhancer by an inducible promoter, we found that intragenic transcription activation increased the inclusion level of several alternative exons of CD44 in HCT116 cells. This result suggested that local convergent transcription could have a direct impact on the alternative splicing of CD44. Furthermore, by using targeted DNA methylation system CRISPR/dCas9-DNMT3b, we showed that DNA methylation at variant exons could directly modify CD44 alternative splicing. This thesis work also explored the limitation and feasibility of studying alternative splicing with repurposed CRISPR systems
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Carvin, Christopher Dumas. "Transcriptional regulation and chromatin remodeling mechanisms at PHO5." Diss., Texas A&M University, 2003. http://hdl.handle.net/1969.1/2193.
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Regulation of gene expression is vital for proper growth and prevention of disease states. In eukaryotes this regulation occurs in the context of chromatin which creates an inherent barrier for the binding of trans-acting factors, such as transcription factors and RNA polymerase. This dissertation focuses on the role of transcriptional activators and chromatin remodeling coactivators in the regulation of the repressible acid phosphatase gene PHO5. Our studies show that histone methylation at lysine 4 of histone H3 is required for the full repression of PHO5and GAL1-10. We show that bromodomains, a domain conserved in chromatin remodeling coactivators, may function to stabilize binding. Finally, we present a strategy using DNA methyltransferases as in vivo probes to detect DNA-protein interactions and examine chromatin structure. We extend this strategy to zinc-finger proteins which can be engineered to bind to any desired DNA sequence as a means of targeting methylation with potential use in epigenetic silencing.
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MARIANI, JESSICA. "Transcriptional regulation, target genes and functional roles of the SOX2 transcription factor in mouse neural stem cells maintenance and neuronal differentiation." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2009. http://hdl.handle.net/10281/8321.
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The aims of this PhD research were: to examine molecular mechanisms underlying the transcriptional regulation of the Sox2 gene during forebrain development; to examine the role of Sox2 for the proper neuronal differentiation of neural stem cells; and to examine the role of Sox2 in controlling the maintenance of neural stem cells (in vivo and in vitro).
The aim of the first work (Chapter 1) was to investigate the transcription factors and the regulatory sequences that control transcription of the Sox2 gene in the developing brain and neural stem cells. Our laboratory previously identified Sox2 regulatory sequences able to drive expression of a reporter β-geo transgene to neural stem cells of the brain in transgenic mice. I focused on two mouse forebrain-specific enhancers able to recapitulate Sox2 telencephalic expression throughout forebrain development, also active in neural stem cells of the adult and embryonic brain (Sox2 5’ and 3’ enhancers). This work showed that Emx2 acts as a direct transcriptional repressor of both Sox2 telencephalic enhancers, acting in two different ways to repress their transcriptional activity: by directly binding to a specific site within these regulatory elements, thus preventing the binding of activators, or possibly by protein to protein interaction sequestring the activators, thus antagonizing their activity. By the study of double mutant mice (expressing reduced levels of Sox2 and Emx2) we further found that Emx2 deficiency counteracts (at least in part) the deleterious effects of Sox2 deficiency on neural stem cell proliferation ability in the postnatal hippocampus, and also rescued other brain morphological abnormalities of Sox2-deficient mutants. It is likely possible that a simultaneous decrease of Emx2 levels (a Sox2 repressor) may antagonize these defects, by restoring Sox2 levels.
In the second line of my research (Chapter 2) we performed in vitro differentiation studies on neural stem cells cultured from embryonic and adult brains of Sox2 “knockdown” mutants (expressing reduced levels of Sox2) where Sox2 deficiency impairs neuronal differentiation. In particular, my contribution to this work was to evaluate the in vitro differentiation defects of Sox2 mutant neurospheres by immunofluorescence staining for different glial and neuronal markers. Strikingly, I observed that mutant cells produce reduced numbers of mature neurons (in particular GABAergic neurons), but generate normal glia. Most of the cells belonging to the neuronal lineage failed to progress to mature neurons showing morphological abnormalities. To evaluate if restoration of Sox2 levels is able to rescue the differentiation defects of mutant cells, I engineered Sox2-expressing lentiviral vector, which I used to infect neural cells at early or late differentiation stages. I found that, Sox2 overexpression is able to rescue the neuronal maturation defects of mutant cells only if administered at early stages of differentiation. Further, I observed that Sox2 suppresses the endogenous GFAP gene, a marker of glial differentiation. These results suggests that Sox2 is required in early in vitro differentiating neuronal cells, for maturation and for suppression of alternative lineage markers.
The third research (Chapter 3) investigated neurogenesis and neural stem cells properties in mice carrying a conditional mutation in the Sox2 gene (Sox2flox). Here, Sox2 was deleted via a nestin-Cre transgene that leads to complete Sox2 loss in the central nervous system by 12.5 dpc. These studies showed that embryonic neurogenesis was not importantly defective, however shortly after birth, NSC and neurogenesis are completely lost in the hippocampus. The expression of cytokine-encoding genes, essential for stem cell niche, is also strongly perturbed and leads to impaired stem cell maintenance (in vivo and in vitro). In vitro, NSC cultures derived from Sox2-deleted forebrain become rapidly exhausted, losing their proliferation and self-renewal properties. In Sox2-deleted neurospheres, Shh is extremely downregulated. However, the conditioned medium from wild type NSC cultures or the administration of a Shh agonist efficiently rescue the proliferation defects. These results suggest that the effect of Sox2 on neural stem cells growth and maintenance is partially mediated by Shh secretion, and that the Shh gene must be a direct target of Sox2. To confirm this hypothesis, I infected Sox2-deleted NSC with a Sox2-IRES-GFP expressing lentivirus just prior to the beginning of the growh decline, and I observed that the re-expression of Sox2 induces the ability to re-express Shh and rescues the formation of neurosphere. These findings indicate that NSC control their status, at least in part, through non cell-autonomous mechanisms (such as activation of important cytochine-encoding genes) which depend on Sox2.
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ZHAO, CHEN. "DECIPHERING TRANSCRIPTIONAL ACTIVITY OF DROSOPHILA BICOID MORPHOGEN: SELECTIVITY AND REGULATION." University of Cincinnati / OhioLINK, 2001. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1006196849.
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Akhmetova, Laila. "Transcriptional Regulation of Nodal Target Genes in Early Zebrafish Development." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493493.
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Nodal signaling is one of the principal players in the process of gastrulation, during which the primary germ layers (endoderm, mesoderm, and ectoderm) are formed and organized in their proper locations. During my PhD I studied how Smad2 and FoxH1 transcription factors regulate the expression of Nodal target genes, and the relationship between the chromatin state of target genes and their expression.
In order to carry out in depth analysis of FoxH1 function I generated a complete mutant of this transcription factor and used deep sequencing to identify which genes FoxH1 regulates in zebrafish development. Using ChIP-Seq experiments, I also found the binding sites of FoxH1 and found that FoxH1 is capable of binding to genomic DNA in the absence of Nodal signaling. This finding suggests that it may act as a pioneer factor, preparing target genes for rapid activation when gastrulation starts. I also identified 54 direct FoxH1 target genes that are not Nodal-dependent, as well as 13 genes that are repressed, rather than activated, by FoxH1.
To identify Smad2 binding sites, I carried out ChIP-Seq in embryos overexpressing Nodal signal Squint, thus detecting loci that bind Smad2 after exposure to high Nodal levels. I tested the identified Smad2-bound DNA elements for their gene regulatory potential, and discovered that they are sufficient to drive gene expression in a Nodal-dependent manner. I also identified 26 previously unpublished Nodal target genes, and 55 genes that are bound by Smad2 upon exposure to high, but not low levels of Nodal signaling.
In the last chapter I describe the study of the interaction between Nodal signaling in early zebrafish development and chromatin marks. I found that exposure of embryonic cells to high levels of Nodal is associated with low levels of H3K27me3 and high levels of H3K4me3 marks on Nodal target genes, compared to unexposed cells. I also describe a Cas9-based system that we used to change H3K27me3 levels in a targeted manner, and tested it in a developing embryo on a Nodal-responsive fgf8a gene. Our results suggest that reduction of H3K27me3 mark on its own is not sufficient to affect the expression of this gene, and additional mechanisms are involved in target gene activation by Smad2.Biology, Molecular and Cellular
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Bolick, Sophia C. E. "Regulation of transcription and analysis of drug targets in lymphoma and myeloma cells." [Tampa, Fla] : University of South Florida, 2006. http://purl.fcla.edu/usf/dc/et/SFE0001750.
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MARKEY, MICHAEL PATRICK. "TRANSCRIPTIONAL REGULATION BY THE RETINOBLASTOMA TUMOR SUPPRESSOR: NOVEL TARGETS AND MECHANISMS." University of Cincinnati / OhioLINK, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1092243630.
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Markey, Michael P. "Transcriptional regulation by the retinoblastoma tumor suppressor novel targets and mechanisms /." Cincinnati, Ohio : University of Cincinnati, 2004. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=ucin1092243630.
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Ratanamart, Jarupa. "Immunogenicity, efficiency and transcriptional regulation of plasmin-mediated muscle-targeted insulin gene therapy for diabetes." Thesis, University of Newcastle Upon Tyne, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.431132.
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Winther, Kristoffer Skovbo. "Molecular target of enteric VapC toxins and regulation of vapBC transcription by conditional cooperativity." Thesis, University of Newcastle upon Tyne, 2012. http://hdl.handle.net/10443/1384.
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The uibiquitous Type II toxin – antitoxin (TA) loci encode two proteins, a toxin and an antitoxin. The antitoxin combines with and neutralizes a cognate toxin. Usually, the TA genes form an operon that is transcribed by a single promoter located upstream of the genes. In most cases, the antitoxin autoregulates the TA operon via binding to operator sites in the promoter region. In almost all such cases, the toxin act as a co-repressor of transcription as the toxin enhances the DNA binding of the antitoxin. Recently, it has been shown that toxins play an additional role in stimulating transcription, as the antitoxin and toxin ratio is important for cooperative binding of the complex to DNA. The antitoxin is rapidly degraded by cellular proteases under conditions of stress and treatment with antibiotics, which leads to activation of the toxin. The toxins of TAs belong to different gene families. The most abundant TA gene family is vapBC that, in some organisms, have expanded into cohorts of genes. For example, the major human pathogen Mycobacterium tuberculosis contains at least 88 TAs, 45 of which are vapBC loci. VapC toxins encoded by vapBC loci are PIN domain proteins (PilT N-terminal). Eukaryotic PIN domain proteins are site-specific ribonucleases involved in quality control, metabolism and maturation of mRNA and rRNA. From in vitro experiments it has been postulated that VapC toxins are RNases or DNases but their exact cellular target has remained elusive. Here I show that VapC encoded by Shigella flexneri 2a virulence plasmid pMYSH6000 and the chromosome of Salmonella enterica serovar Typhimurium LT2 are site-specific endoribonucleases that specifically cleave tRNAfMet in the anticodon stem-loop in vivo and in vitro. Furthermore, I show that VapC dependent depletion of tRNAfMet leads to bacteriostatic inhibition of global translation, which surprisingly induces low-level initiation of translation at elongator codons that are correctly positioned relative to a Shine & Dalgarno sequence. I also show that VapC forms a complex with VapB and acts as a co-repressor of vapBC transcription. During steady state growth VapB is in excess of VapC. However, nutrient stress or treatment with antibiotics leads to Lon protease dependent decrease in VapB levels. Furthermore, I show that VapC in excess of VapB directly interferes with cooperative DNA binding of the VapBC complex, which is dependent on the dimerisation of the VapC toxin. 2 In conclusion, I show that enteric VapCs not only regulate global cellular translation by tRNAfMet cleavage, but also regulate vapBC transcription by conditional cooperativity.
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Lopes, Jared Emery. "Amino terminal region of FOXP3 coordinates the regulation of transcriptional targets in regulatory and effector T cell lineages /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/8354.
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Lorenzin, Francesca [Verfasser], and Martin [Gutachter] Eilers. "Regulation of transcription by MYC - DNA binding and target genes / Francesca Lorenzin ; Gutachter: Martin Eilers." Würzburg : Universität Würzburg, 2017. http://d-nb.info/1136272682/34.
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Fernández, Coll Llorenç. "Secondary channel of the RNA polymerase, a target for transcriptional regulation in bacteria." Doctoral thesis, Universitat de Barcelona, 2015. http://hdl.handle.net/10803/298719.
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Gene expression begins by an enzymatic complex known as RNA polymerase (RNApol). The basic unit (core) of RNApol in bacteria is formed by 5 protein subunits (α2ββ’ω). The three-dimensional structure of the RNApol defines two spaces that play a relevant role during transcription, defined as primary and secondary channel. The holoenzyme needs the binding of a σ subunit to recognise promoter sequences and initiate the transcription process.
Transcription is a dynamic process controlled at different steps. Genetic regulation during transcription initiation has been highly studied, and several mechanisms of regulation exist. However, the aim of this project is to study some aspects of the regulation during transcription elongation. It has been described that the alamone ppGpp, as well as several proteins, such as GreA, GreB or DksA, enter within the secondary channel and interact directly with the catalytic centre of the RNApol. The swap between the different factors that bind to the secondary channel of the RNApol may cause changes in the expression pattern.
It has been postulated that DksA and ppGpp act as cofactors, however, a previous study performed in our research group, indicated that the phenotype of ppGpp and DksA deficiencies were not always identical, letting us suggest that the occupancy degree of the secondary channel of the RNApol may have significant impact in the expression pattern in E. coli. The data obtained clearly indicate that upregulation of some genes, such as fliC, that occurs in absence of DksA, was the result of the vacancy of the secondary channel generated in a dksA strain rather than being the result of DksA having a direct repressor effect. We suggested that in the absence of DksA, the interactions of other proteins, such as GreA, are promoted and responsible of the upregulation observed.
In this project, functional, structural and phylogenetical studies of the protein GreA were performed to determine which amino acids are important for i) the functionality of GreA, ii) the ability to bind to the secondary channel of the RNApol or iii) the capacity to compete with other factors, such as DksA. We have determined that greA overexpression produces a negative effect of the bacterial growth. Moreover, this negative effect is enhanced in absence of DksA, highlighting the hypothesis of a competition between factors that bind into the secondary channel.
The effect of this competition between GreA and DksA was also determined studying the expression of the fliC gene. Our data showed that both, GreA and DksAare required for fliC expression but act at different levels in the regulatory cascade of flagella expression regulation. GreA may control fliC expression during transcription elongation whereas DksA may act during transcription initiation.
Changes in the amount of GreA, could affect the competition between factors that bind to the secondary channel of the RNApol. Therefore, we have determined the expression pattern of greA. Transcriptional studies showed a crosstalk between the different factors that bind into the secondary channel of the RNApol exists.
Finally, transcriptomic studies were performed to determine the effect of ppGpp and DksA on the expression pattern of Salmonella enterica serovar Typhimurium. The results obtained indicate : i) the effect of the possible competence between the factors that interact into the secondary channel of the RNApol and ii) the effect of ppGpp and DksA on the expression of several virulence factors as well as different mobile elements present in Salmonella.El control de l’expressió gènica en bacteris recau principalment sobre un complex enzimàtic anomenat ARN polimerasa (ARNpol). A procariotes, la seva unitat bàsica (core) està formada per 5 subunitats proteiques (a2bb’w). S’han determinat dos canals entre les diferents subunitats de l’ARNpol: el canal primari, on es desenvolupa la transcripció, i el canal secundari, que comunica el medi exterior amb el centre catalític de l’ARNpol. Tot i així, aquest holoenzim necessita la unió d’una subunitat σ per ser capaç de reconèixer una seqüència promotora i iniciar la transcripció. S’han descrit diferents factors, tant proteics com no proteics, que poden interaccionar amb el canal secundari de l’ARNpol i causar alteracions a l’expressió gènica. En aquesta tesi ens hem centrat en la possible competència entre els diferents factors que poden interaccionar amb el canal secundari de l’ARNpol. Estudis anterior duts a terme en el nostre grup d’investigació, ens van permetre postular una possible competència entre els diferents factors que interaccionen amb el canal secundari de l’ARNpol, més concretament entre les proteïnes GreA i DksA. Aquesta competència provocaria alteracions en el patró d’expressió gènica d’Escherichia coli. En aquest treball s’han dut a terme estudis funcionals, estructurals i filogenètics de la proteïna GreA que ens han permès determinar quins aminoàcids, i com a conseqüència quins dominis, podrien ser importants per la funcionalitat de la proteïna, la seva capacitat d’unir-se a l’ARNpol i la seva capacitat de competir amb altres factors. A més, hem estudiat quin efecte té la competència entre els diferents factors que interaccionen amb el canal secundari sobre l’expressió d’un gen diana. Canvis en els nivells de la proteïna GreA, poden afectar la competència pel canal secundari de l’ARNpol Per això hem determinat el patró d’expressió del gen greA, així com l’existència d’una regulació creuada entre les diferents proteïnes que interaccionen amb el canal secundari. Finalment, hem dut a terme un estudi transcriptòmic en Salmonella enterica serovar Typhimurium, amb l’objectiu de determinar quin és l’efecte d’aquesta competència en l’expressió de factors de virulència.
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Cunningham, Katherine Ann. "The transcriptional regulation and target binding site of the sporulation kinase inhibitor, Sda /." May be available electronically:, 2008. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
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GIACOBBE, ARIANNA. "Two novel p63 transcriptional target genes regulating cell metabolism and metastasis." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2014. http://hdl.handle.net/2108/204221.
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La famiglia di p53, costituita dai tre fattori di trascrizione p53, p63 e p73, assume un ruolo chiave nel direzionare il destino di una cellula verso la sopravvivenza o attivando meccanismi di morte cellulare programmata. Sebbene la loro struttura genica sia simile, p63 e p73 codificano per varie isoforme generate a partire da promotori diversi all’estremità 5’ e da meccanismi di splicing alternativo al 3’, a seguito del quale derivano isoforme TA complete del dominio di trans-attivazione all’N-terminale o isoforme tronche denominate ΔN. Il capostipite della famiglia, p53, è principalmente descritto come il guardiano del genoma ed è noto agire come soppressore dei tumori, essendo mutato in più del 50% dei tumori umani. Nell’ultimo decennio, l’identificazione di nuovi geni bersaglio di p53 implicati nella regolazione della glicolisi, come SCO2 e PGM, e nella difesa antiossidante come TIGAR, sestrine, ALDH4 e GPX1 sottolinea l’importanza di p53 nella regolazione del metabolismo energetico e dello stress ossidativo. Inoltre, recentemente, l’altro membro della famiglia, p73, è stato mostrato essere coinvolto nel metabolismo energetico attraverso il controllo trascrizionale del gene COX4A. Il fattore di trascrizione p63 svolge un ruolo chiave nello sviluppo e nel mantenimento dei tessuti epidermici e di tutti quelli di origine epiteliale. Nonostante p63 condivida alcuni ruoli biologici con gli altri membri della famiglia, come la regolazione del ciclo cellulare o della morte cellulare, il suo ruolo in alcuni processi cellulari basilari come il metabolismo energetico o in alcuni stadi patologici, come nei processi tumorigenici o metastatici non è stato ancora chiarito. In questo studio abbiamo dimostrato il coinvolgimento di p63 nei meccanismi di regolazione di due nuovi geni bersaglio: la glutaminasi mitocondriale 2 (GLS2) e il gene soppressore delle metastasi (MTSS1). La glutaminasi GLS2 idrolizza la glutammina in glutammato, un importante metabolita coinvolto nella via energetica di sintesi dell’ATP e nei processi di difesa cellulare contro le specie ossidanti. Abbiamo dimostrato che l’espressione di GLS2 aumenta in seguito all’espressione ectopica del’isoforma TAp63 in varie linee cellulari. Tale aumento è confermato anche dall’induzione endogena di TAp63 in seguito all’inibizione delle deacetilasi istoniche. Attraverso saggi di immunoprecipitazione della cromatina e saggi della luciferasi è stato confermato il legame diretto di TAp63 sulla sequenza di DNA consenso per p53/p63 localizzata all’interno del promotore di GLS2. Abbiamo inoltre osservato che l’espressione di GLS2 e di TAp63 aumenta contemporaneamente sia durante il differenziamento in vitro di cheratinociti primari umani, e sia in cellule tumorali esposte a stress ossidativo. I nostri risultati dimostrano che la deplezione di GLS2, da una parte inibisce il differenziamento dei cheratinociti umani in vitro e, dall’altra, aumenta la morte cellulare indotta da ROS in cellule tumorali. Questi dati mostrano che l’asse TAp63/GLS2 può essere funzionalmente importante sia nei processi fisiologici che patologici, rappresentando una via cellulare anti-ossidante in grado di compensare l’attività di p53 quando assente. Infatti, l’espressione di p63 correla con quella di GLS2 nei tumori umani del carcinoma del colon e nei carcinomi a cellule squamose della testa e del collo, suggerendo che in alcuni tumori questo sistema potrebbe essere importante nel proteggere le cellule tumorali dallo stress ossidativo. Un altro gene che abbiamo identificato essere regolato da p63 è MTSS1. Il gene MTSS1 è stato originariamente identificato come soppressore delle metastasi, la cui espressione era assente nel carcinoma metastatico della vescica e della prostata. Tuttavia, recentemente è stato dimostrato che la proteina MTSS1 è in grado di agire come un oncogene e come un fattore pro-migratorio nel melanoma. I nostri risultati caratterizzano il gene MTSS1 come un nuovo bersaglio trascrizionale di p63, implicato nella regolazione dei processi di migrazione e invasione cellulare. Abbiamo trovato che sia in cellule normali che in cellule tumorali, l’espressione ectopica di p63, ed in particolare delle isoforme di ΔNp63, è in grado di guidare l’espressione di MTSS1. Inoltre abbiamo confermato che ques11ta regolazione è possibile grazie al legame diretto di p63 a un elemento di risposta nel DNA, avente un motivo di sequenza specifico per p63 e localizzato nelle regioni introniche del locus genico di MTSS1. Inoltre, da un punto di vista biologico, l’espressione di MTSS1 ΔNp63-dipendente, sembra regolare le proprietà migratorie e invasive di cellule tumorali. Da sottolineare, i nostri dati indicano che l’espressione di MTSS1 correla positivamente con quella di ΔNp63 in 3 datasets di tumori alla mammella, alla vescica e alla prostata. Inoltre, in tre datasets di tumori umani della mammella, la co-espressione di MTSS1 e p63 è un fattore prognostico negativo per la sopravvivenza del paziente, suggerendo che questo asse p63/MTSS1 possa essere funzionalmente importante nel regolare la progressione del tumore. In conclusione, attraverso l’individuazione e la caratterizzazione di due nuovi geni bersaglio di p63, abbiamo sottolineato l’importanza di p63 in due processi funzionalmente interconnessi quali il metabolismo cellulare e i processi di migrazione e invasione cellulare, che sono a loro volta fondamentali per la corretta regolazione della proliferazione e della sopravvivenza cellulare sia in sistemi fisiologici che patologici.The transcription factor p63 is critical for the development and maintenance of epidermal tissues and its role in other processes such as cell energy metabolism, or in pathological conditions, including tumorigenesis and metastasis, is far to be completely characterized. Here, we report the characterization of two novel p63 target genes: the mitochondrial Glutaminase 2 (GLS2), and the Metastasis Suppressor 1 (MTSS1) gene. GLS2 converts glutamine into glutamate, an important metabolite involved in ATP production and anti-oxidant cellular defense. We found that the p63 TA-isoforms induce GLS2 expression in several cell lines and, by ChIP analysis and luciferase assay, we demonstrated the direct binding of TAp63 to the p53/p63 consensus DNA responsive sequence localized within the GLS2 promoter. GLS2 and TAp63 expression concomitantly increases both during the in vitro differentiation of primary human keratinocytes, and in cancer cells exposed to oxidative stresses, while depletion of GLS2 inhibits skin differentiation and sensitizes tumor cells to ROS-induced apoptosis. Our findings show that TAp63/GLS2 axis may be functionally important as a cellular anti-oxidant pathway in the absence of p53 for both physiological and pathological processes. Metastasis Suppressor 1 (MTSS1) was originally identified as a metastasis suppressor protein whose expression is missing in metastatic bladder carcinoma and prostate cancer. However, recent findings indicate that MTSS1 acts as oncogene and pro-migratory factor in melanoma tumors. Here, we characterized MTSS1 as a new p63 transcriptional target gene involved in the regulation of the migration/invasion processes. We found that in normal and in cancer cell lines p63 is able to drive the expression of MTSS1 through binding to one p63 binding element localized in the MTSS1 locus. We also found that the p63-mediated expression of MTSS1 plays a critical role in the regulation of the migration and invasion properties of tumor cells. Importantly, the expression of MTSS1 positively correlates with that of ΔNp63 isoforms in breast, bladder and prostate human tumors datasets. Furthermore, in three human breast tumors datasets the MTSS1-p63 co-expression is a negative prognostic factor on patient survival, suggesting that the MTSS1/p63 axis might be functionally important to regulate tumor progression. In conclusion, by identifying and characterizing two novel p63 target genes, we have unveiled the importance of p63 in two functionally interconnected pathways, cell metabolism and migration/invasion processes, which are critical for the proper regulation of cell proliferation and survival in both physiological and pathological process.
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Abdel, Samad Omar. "Hox genes and the specification of neuronal phenotype in the vertebrate hindbrain : transcriptional regulation of the Hox target gene Phox2b." Université Louis Pasteur (Strasbourg) (1971-2008), 2004. http://www.theses.fr/2004STR13027.
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Pastic, Alyssa. "LRRK2 Phosphorylates HuD to Affect the Post-Transcriptional Regulation of Parkinson's Disease-Linked mRNA Targets." Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/38593.
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Parkinson's Disease (PD) is a late-onset neurodegenerative disease characterized by progressive motor dysfunction caused by a loss of dopaminergic neurons for which there is no known cure. Among the most common genetic causes of PD are mutations in the leucine-rich repeat kinase 2 gene (LRRK2), encoding a multi-domain protein with kinase activity. The LRRK2 G2019S mutation causes hyperactivity of the kinase domain and is the most frequent LRRK2 mutation in patients with familial PD, though its role in PD pathology remains unclear. Preliminary data from the lab of our collaborator, Dr. David Park, demonstrated through a genetic screen in Drosophila melanogaster that the deletion of rbp9 encoding an RNA-binding protein prevented pathology induced by PD-relevant mutations in the LRRK2 kinase domain. The neuronal homolog of RBP9 in humans is HuD, a member of the Hu family of RNA-binding proteins that regulates the expression of many transcripts involved in neuronal development, plasticity, and survival. In addition, HuD has been shown to modify the age-at-onset or risk of developing PD. Here, we studied the effect of LRRK2 on the post-transcriptional regulation of mRNAs bound by HuD in the context of PD. Our findings showed that HuD is a substrate for LRRK2 phosphorylation in vitro, and that LRRK2 G2019S hyperphosphorylates HuD. We demonstrated that LRRK2 kinase activity is required for the binding of several transcripts by HuD that encode PD-relevant proteins such as α-synuclein and neuronal survival factor BDNF. Our findings in human neuroblastoma cells indicated that LRRK2 regulates the protein levels of HuD mRNA targets α-synuclein and BDNF in a mechanism that can by modified by HuD. Finally, we showed that the combination of HuD knockout with LRRK2 G2019S expression in mice rescues aberrant expression of HuD targets in mice with only the LRRK2 G2019S mutation or the knockout of HuD alone. Together, our findings demonstrate that LRRK2 affects the post-transcriptional regulation of HuD-bound mRNAs, and suggest the use of HuD as a potential therapeutic target in patients with PD caused by the LRRK2 G2019S mutation.
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Stephenson, Troy James. "Characterisation of the TaNF-Y family of transcription factors in wheat." Thesis, Queensland University of Technology, 2011. https://eprints.qut.edu.au/47525/1/Troy_Stephenson_Thesis.pdf.
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Light plays a unique role for plants as it is both a source of energy for growth and a signal for development. Light captured by the pigments in the light harvesting complexes is used to drive the synthesis of the chemical energy required for carbon assimilation. The light perceived by photoreceptors activates effectors, such as transcription factors (TFs), which modulate the expression of light-responsive genes. Recently, it has been speculated that increasing the photosynthetic rate could further improve the yield potential of three carbon (C3) crops such as wheat. However, little is currently known about the transcriptional regulation of photosynthesis genes, particularly in crop species. Nuclear factor Y (NF-Y) TF is a functionally diverse regulator of growth and development in the model plant species, with demonstrated roles in embryo development, stress response, flowering time and chloroplast biogenesis. Furthermore, a light-responsive NF-Y binding site (CCAAT-box) is present in the promoter of a spinach photosynthesis gene. As photosynthesis genes are co-regulated by light and co-regulated genes typically have similar regulatory elements in their promoters, it seems likely that other photosynthesis genes would also have light-responsive CCAAT-boxes. This provided the impetus to investigate the NF-Y TF in bread wheat. This thesis is focussed on wheat NF-Y members that have roles in light-mediated gene regulation with an emphasis on their involvement in the regulation of photosynthesis genes.
NF-Y is a heterotrimeric complex, comprised of the three subunits NF-YA, NF-YB and NF-YC. Unlike the mammalian and yeast counterparts, each of the three subunits is encoded by multiple genes in Arabidopsis. The initial step taken in this study was the identification of the wheat NF-Y family (Chapter 3). A search of the current wheat nucleotide sequence databases identified 37 NF-Y genes (10 NF-YA, 11 NF-YB, 14 NF-YC & 2 Dr1). Phylogenetic analysis revealed that each of the three wheat NF-Y (TaNF-Y) subunit families could be divided into 4-5 clades based on their conserved core regions. Outside of the core regions, eleven motifs were identified to be conserved between Arabidopsis, rice and wheat NF-Y subunit members. The expression profiles of TaNF-Y genes were constructed using quantitative real-time polymerase chain reaction (RT-PCR). Some TaNF-Y subunit members had little variation in their transcript levels among the organs, while others displayed organ-predominant expression profiles, including those expressed mainly in the photosynthetic organs.
To investigate their potential role in light-mediated gene regulation, the light responsiveness of the TaNF-Y genes were examined (Chapters 4 and 5). Two TaNF-YB and five TaNF-YC members were markedly upregulated by light in both the wheat leaves and seedling shoots. To identify the potential target genes of the light-upregulated NF-Y subunit members, a gene expression correlation analysis was conducted using publically available Affymetrix Wheat Genome Array datasets. This analysis revealed that the transcript expression levels of TaNF-YB3 and TaNF-YC11 were significantly correlated with those of photosynthesis genes. These correlated express profiles were also observed in the quantitative RT-PCR dataset from wheat plants grown under light and dark conditions. Sequence analysis of the promoters of these wheat photosynthesis genes revealed that they were enriched with potential NF-Y binding sites (CCAAT-box).
The potential role of TaNF-YB3 in the regulation of photosynthetic genes was further investigated using a transgenic approach (Chapter 5). Transgenic wheat lines constitutively expressing TaNF-YB3 were found to have significantly increased expression levels of photosynthesis genes, including those encoding light harvesting chlorophyll a/b-binding proteins, photosystem I reaction centre subunits, a chloroplast ATP synthase subunit and glutamyl-tRNA reductase (GluTR). GluTR is a rate-limiting enzyme in the chlorophyll biosynthesis pathway. In association with the increased expression of the photosynthesis genes, the transgenic lines had a higher leaf chlorophyll content, increased photosynthetic rate and had a more rapid early growth rate compared to the wild-type wheat.
In addition to its role in the regulation of photosynthesis genes, TaNF-YB3 overexpression lines flower on average 2-days earlier than the wild-type (Chapter 6). Quantitative RT-PCR analysis showed that there was a 13-fold increase in the expression level of the floral integrator, TaFT. The transcript levels of other downstream genes (TaFT2 and TaVRN1) were also increased in the transgenic lines. Furthermore, the transcript levels of TaNF-YB3 were significantly correlated with those of constans (CO), constans-like (COL) and timing of chlorophyll a/b-binding (CAB) expression 1 [TOC1; (CCT)] domain-containing proteins known to be involved in the regulation of flowering time.
To summarise the key findings of this study, 37 NF-Y genes were identified in the crop species wheat. An in depth analysis of TaNF-Y gene expression profiles revealed that the potential role of some light-upregulated members was in the regulation of photosynthetic genes. The involvement of TaNF-YB3 in the regulation of photosynthesis genes was supported by data obtained from transgenic wheat lines with increased constitutive expression of TaNF-YB3. The overexpression of TaNF-YB3 in the transgenic lines revealed this NF-YB member is also involved in the fine-tuning of flowering time. These data suggest that the NF-Y TF plays an important role in light-mediated gene regulation in wheat.
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Sugimoto, Yoichiro. "Understanding the role of Staufen 1 in post-transcriptional regulation via the global characterization of its target RNA structures." Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708017.
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Fielhaber, Jill. "Regulation of signal tranducer and activator of transcription-1 and cell death by mammalian target of Rapamycin." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=104692.
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Mammalian target of rapamycin (mTOR) is a highly-conserved serine/threonine kinase that permits anabolic responses to mitogens and metabolic substrates (e.g., insulin, amino acids, ATP). When its activity is reduced, TOR can also initiate catabolic responses. In S. cerevisiae, nutrient or physical stress initiates a transcriptional stress response that requires the TOR-associated phosphatases and the nuclear translocation of stress transcription factors. In this thesis, I explore a novel physical and functional relationship between mammalian TOR, its associated phosphatases, and 'signal transducer and activator of transcription -1' (STAT1), a key transcription factor that controls the induction of apoptosis genes. I hypothesized that inactivation of mTOR favours the nuclear import of STAT1 in a mechanism that requires its associated phosphatases alpha4 and ‘protein phosphatase 2A catalytic subunit' (PP2Ac), as well as the STAT1 importin karyopherin alpha-1 (KPNA1). Here, I show that mTOR, alpha4, PP2Ac, KPNA1, and STAT1 are physically associated in a dynamic macromolecular protein complex. The mTOR inhibitor rapamycin promotes STAT1 nuclear import and the induction of STAT1-dependent apoptosis genes in a mechanism that requires alpha4, PP2Ac, and KPNA1. I further show that STAT1 is required for the enhancing effect of rapamycin on apoptosis per se in cultured cells exposed to pro-inflammatory STAT1 agonists (i.e., bacterial lipopolysaccharide, interferon-beta). Finally, I demonstrate that rapamycin enhances the levels of activated STAT1, STAT1-dependent apoptosis genes, cellular apoptosis, and tissue injury in the lungs of mice exposed to intratracheal lipopolysaccharide. Thus, in mammalian cells, reduction of mTOR activity favours cellular apoptosis and organ injury via a mechanism that involves a physical and functional interaction with STAT1. The molecular mechanisms that mediate the regulation of STAT1 by mTOR are targets for the modulation of mammalian apoptosis and tissue injury.mTOR (mammalian target of rapamycin), la cible de la rapamycine chez les mammifères, est une kinase de type sérine-thréonine très bien conservée à travers les phyla, qui est responsable de la réponse aux mitogènes anabolisants et aux substrats métaboliques tel que l'insuline, les acides aminés et l'ATP. Une réduction de l'activité de TOR peut également permettre l'initiation de réponses cataboliques. Chez S. cerevisiae, le stress physique et la déficience en éléments nutritifs induisent une réponse transcriptionnelle qui nécessite des phosphatases associées à TOR ainsi que la translocation nucléaire de facteurs de transcriptions dépendant de la réaction aux éléments de stress. Dans cette thèse, j'explore une nouvelle interaction physique et fonctionnelle entre mTOR, ses phosphatases associées et le transducteur de signalisation et activateur de transcription-1 (STAT1), un facteur de transcription contrôlant l'induction des gènes de l'apoptose. J'émets l'hypothèse que l'inactivation de mTOR promeut l'importation de STAT1 au noyau en adoptant un mécanisme nécessitant l'interaction avec ses phosphatases associées; la phosphatase alpha4 et la phosphatase 2A sous-unité catalytique (PP2Ac), ainsi que STAT1 kariophérine importine-alpha1 (KPNA1). Ces résultats démontrent que mTOR, alpha4, PP2Ac, KPNA1 et STAT1 interagissent physiquement et forment un complexe protéique macromoléculaire dynamique. La rapamycine, l'inhibiteur de mTOR, promeut la translocation nucléaire de STAT1 et l'induction de la transcription des gènes de l'apoptose STAT1-dépendant selon un mécanisme nécessitant alpha4, PP2Ac et KPNA1. De plus je démontre que STAT1 est nécessaire pour renforcer l'effet de la rapamycine sur l'apoptose sur des cellules en culture exposées à des agonistes de STAT1 à caractère pro-inflammatoire (c.-à-d lipopolysaccharide bactérien et interféron-beta). Enfin, je démontre que la rapamycine augmente le niveau de STAT1 actif, l'expression des gènes de l'apoptose STAT1-dépendant, l'apoptose cellulaire et les lésions tissulaires dans les poumons de souris auxquelles du lipopolysaccharide a été administré par voie intratrachéale. Ainsi, dans les cellules mammifères, la réduction de l'activité de mTOR résulte en une augmentation de l'apoptose cellulaire et des lésions tissulaires par l'intermédiaire d'un mécanisme impliquant une interaction physique et fonctionnelle avec STAT1. Les mécanismes moléculaires contrôlant la régulation de STAT1 par mTOR sont des cibles importantes pour la modulation de l'apoptose et des lésions tissulaires chez les mammifères.
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Wu, Qianchao [Verfasser], and Frank [Akademischer Betreuer] Lyko. "Regulation of p53 target gene transcription by a TBL1-mediated epigenetic mechanism / Qianchao Wu ; Betreuer: Frank Lyko." Heidelberg : Universitätsbibliothek Heidelberg, 2019. http://d-nb.info/119334736X/34.
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Ravanpay, Ali Cyrus. "Insights into the molecular interactions of the neurogenic basic helix-loop-helix transcription factor, neuroD2, and the mechanism of regulation of a key target, RE-1 silencing transcription factor /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/10628.
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Chow, Christine 1974. "Identification of target DNA binding sites for a yeast zinc cluster transcriptional regulator." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=30813.
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Zinc cluster proteins represent a subclass of zinc finger proteins and function as transcriptional regulators. An in vivo genetic screening system was developed in yeast to identify DNA binding sites and specificities for these proteins.An oligonucleotide library of 200 000 clones was constructed. Control screening trials with Hap1p and Gal4p demonstrated effectiveness in recovering binding sites. Sequencing of isolated clones showed correlation with published target sequences and binding was confirmed by electrophoretic mobility shift assay (EMSA).
Screening over 100 000 clones of the library with the YLR228c gene product allowed the isolation of 10 clones. Mutational EMSA studies were performed to identify nucleotides important for binding to derive a consensus sequence. A CGG triplet was found to be significant for binding. It can be hypothesized that Ylr228p may bind as a monomer to its targets.
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Glaser, Laura Viola [Verfasser], and Bettina [Akademischer Betreuer] Kempkes. "Target gene regulation by EBV latent transcription factors - exploiting cellular enhancer elements / Laura Viola Glaser ; Betreuer: Bettina Kempkes." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2017. http://d-nb.info/1156173256/34.
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Wang, Gang. "Functional dissection of homeodomain transcription factors HoxA9 and Meis1 in myeloid leukemia their epigenetic regulation and their downstream targets /." Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2006. http://wwwlib.umi.com/cr/ucsd/fullcit?p3232136.
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Thesis (Ph. D.)--University of California, San Diego, 2006.Title from first page of PDF file (viewed December 4, 2006). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 163-175).
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Alvarez-Saavedra, Matias Alberto. "MicroRNA-132-Dependent Post-Transcriptional Regulation of Clock Entrainment Physiology Via Modulation of Chromatin Remodeling and Translational Control Gene Targets." Thesis, University of Ottawa (Canada), 2010. http://hdl.handle.net/10393/28722.
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Mammalian circadian rhythms of behaviour are synchronized to external time by daily resetting of the master pacemaker, the suprachiasmatic nucleus (SCN), in response to light, in a process known as light-induced clock entrainment. microRNA-132 (miR-132) is induced by light within the SCN via a MAPK-CREB-dependent mechanism and has the capacity to attenuate the entraining effects of light. However, the identity of the genes that miR-132 regulates and their roles in the light-entrainment process have not yet been characterized. This thesis describes that 2 gene clusters involved in chromatin remodeling (i.e. Mecp2, Ep300, Jarid1a) and translational control (i.e. Btg2, Paip2a) are under the regulation of miR-132 in the SCN and coordinated regulation of these genes underlies miR132-dependent modulation of mPeriod1 (mPer1) and mPeriod2 (mPer2) and the light-entrainment process. I find that the Period genes are bound and transcriptionally modulated by MeCP2. In addition, Paip2a acts as a repressor of Period translation. This work further proposes that miR-132 is enriched for chromatin and translation-associated target genes-and, thus, miR-132 is an important orchestrator of chromatin remodeling and protein translation within the SCN clock, thereby fine-tuning clock entrainment.
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Bauer, Tobias Hartmut [Verfasser], and Rainer [Akademischer Betreuer] König. "Deciphering transcriptional regulation in cancer cells and development of a new method to identify key transcriptional regulators and their target genes / Tobias Hartmut Bauer ; Betreuer: Rainer König." Heidelberg : Universitätsbibliothek Heidelberg, 2011. http://d-nb.info/1179782380/34.
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Collins, Kelly J. "Structure-Function Analysis of the Notch Signaling CSL-KyoT2 and SPOC-NCoR Corepressor Complexes: understanding how corepressor assembly is regulated at Notch target genes." University of Cincinnati / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1393236995.
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Mehanovic, Samir. "Molecular mechanisms of nuclear receptor COUP-TFII action in the regulation of Amhr2 and identification of additional target genes in MA-10 Leydig cells." Doctoral thesis, Université Laval, 2021. http://hdl.handle.net/20.500.11794/69443.
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On estime qu'environ 5 millions d'hommes américains souffrent de taux de testostérone réduits ou d'hypogonadisme. Chez les hommes, les cellules de Leydig sont les principales productrices de testostérone et d'insuline-like 3, deux hormones essentielles à la différenciation sexuelle masculine, aux fonctions reproductives et à la santé globale de l’homme. Les facteurs de transcription sont des protéines qui régulent la transcription des gènes en se liant à des séquences d'ADN spécifiques. Le facteur de transcription “chicken ovalbumin upstream promoter type 2” (COUP-TFII) est un facteur de transcription qui appartient à la superfamille des récepteurs nucléaires. Dans le testicule, COUP-TFII est exprimé dans les cellules se différenciant en cellules de Leydig adulte (ALCs) pleinement fonctionnelles et joue un rôle majeur dans leur différenciation et leur fonction. La synthèse des stéroïdes est réduite dans des cellules de Leydig appauvries en COUP-TFII, ce qui suggère que ce facteur joue un rôle important dans la stéroïdogenèse. Cependant, le mécanisme d'action de COUP-TFII dans les cellules de Leydig demeuraient largement méconnus. Dans cette thèse, une analyse des données de puces à ADN de cellules MA-10 Leydig appauvries en COUP-TFII a été effectuée afin d’identifier le rôle global de ce facteur dans les cellules de Leydig. Cette étude a permis d’identifier 262 gènes différentiellement exprimés, notamment Hsd3b1, Cyp11a1, Prlr, Pdgfra, Shp, Ear1, Amhr2, Fdx1, Inha et Gsta3. De plus, l’étude du promoteur proximal d’Amhr2 par des études de gène rapporteur a permis de démontrer que COUP-TFII est recruté au promoteur proximal d’Amhr2 et coopère avec SP1 et GATA4 afin de réguler son expression. Les résultats présentés dans cette thèse fournissent une meilleure compréhension du mécanisme d'action de COUP-TFII dans les cellules de Leydig, et des preuves supplémentaires renforçant l'importance du récepteur nucléaire COUP-TFII dans la stéroïdogenèse, l'homéostasie androgénique, la défense cellulaire et la différenciation des cellules de Leydig.It is estimated that about 5 million American men have low testosterone levels, hypogonadism, and infertility problems. Leydig cells are the primary producers of testosterone and insulin-like 3 hormones in males, both essential for male sex differentiation, reproductive roles, and overall health. Transcription factors are proteins that bind to various DNA sequences to control DNA transcription. Chicken ovalbumin upstream promotertranscription factors II (COUP-TFII) belongs to the steroid/thyroid hormone nuclear receptor superfamily of transcription factors. COUP-TFII is expressed in the cells that give rise to fully functional steroidogenic Adult Leydig cells in the testis and plays an important role in their function and differentiation. Steroid synthesis is reduced in COUP-TFII-depleted Leydig cells, suggesting that this protein plays a vital role in steroidogenesis. However, the mechanisms of action of COUP-TFII in Leydig cells were largely unknown. The analysis of microarray data from COUP-TFII-depleted MA-10 Leydig cells identified 262 differentially expressed genes, including Hsd3b1, Cyp11a1, Prlr, Pdgfra, Shp, Ear1, Amhr2, Fdx1, Inha, and Gsta3. Anti-Müllerian hormone (AMH), which is expressed in Sertoli cells, is essential for the regression of the Müllerian ducts during male embryonic development. In Leydig cells, AMH signals through the anti-Müllerian hormone type II receptor (AMHR2). In male mammals, mutations affecting AMH or AMHR2 expression cause Persistent Müllerian Duct Syndrome (PMDS), characterized by infertility, inguinal hernias, cryptorchidism, and reduced serum testosterone levels. COUP-TFII was found to cooperate with specificity protein 1 (SP1) and GATA-binding factor 4 (GATA4) in the regulation of the Amhr2 promoter using reporter promoter assays. COUP-TFII and GATA4 were found to be recruited to the same region of the Amhr2 gene via chromatin immunoprecipitation assay (ChIP), further strengthening their cooperative roles in the regulation of this gene. Furthermore, COUP-TFII and GATA4 were found to associate in the Leydig cells molecularly. The results presented in this thesis provide a better understanding of the mechanism of COUP-TFII action in Leydig cells, and additional evidence strengthening the importance of COUP-TFII in steroidogenesis, androgen homeostasis, cellular defense, and differentiation.
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Ding, Shuai. "Characterization of P.falciparum histone methyltransferases : biological role and possible targets for new intervention strategies." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066578/document.
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On a montré que les PTM jouaient un rôle significatif de P. falciparum dans l'année de contrôle de la régulation transcriptionnelle, de l'expression monoaléique et de la différenciation sexuelle. Dix SET contenant des HKMTs contenant du domaine-ont été prédits; Six d'entre eux être essentiels pour le développement de stages de sang asexués. Le projet de thèse est centré sur la caractérisation biologique de PfSET7 et PfSET6. Nous avons observé l'échange de localisation cellulaire dynamique Pendant le cycle de vie: PfSET7 se trouvent dans de multiples foyers cytoplasmiques dans les stades érythrocytaires asexués et le stage de foie, et plus frappante, dans la membrane du parasite enrichie en gamétocytes. PfSET6 EXPOSÉ une localisation nucléaire dans les anneaux et un modèle ponctué dans le cytoplasme des trophozoites matures et schizontes, et est enrichi dans les structures de foyers dans le cytoplasme des gamétocytes. Pris ensemble, notre étude suggère que la méthylation non histone est beaucoup plus significative chez P. falciparum que précédemment attendu. La méthylation à médiation par PfSET7 Peut être une extension du code histone à - d'autres protéines cytosoliques; Partiellement PfSET6 s'associe à des voies de répression transcriptionnelle dans le noyau et des régulateurs post-transcriptionnels dans le cytoplasme. Une étude plus approfondie vise à identifier des cibles de domaine SET contenant des protéines dans le gène inducible knockout mutant parasite lignes. Le fait que PfSET7 et PfSET6 sont exprimés à différents stades du cycle de vie, les fait comme de nouvelles cibles pour le développement de médicaments contre cette maladie grave et de bloquer la transmissionIn P. falciparum, PTMs have been shown to play an important role in the control of transcriptional regulation, monoallelic expression, and sexual differentiation. Ten SET domain-containing HKMTs have been predicted; six of them appear to be essential for asexual blood stage development. My lab has expressed and purified two enzymatically active recombinant methyltransferase PfSET7 and PfSET6. In vitro enzyme kinetics assays shows they can methylate histones. The dissertation project is centered around the biological characterization of PfSET7 and PfSET6. We observed the dynamic changes of cellular localization during life cycle: PfSET7 are found in multiple cytoplasmic foci in asexual erythrocytic stages and liver stage, and more strikingly, enriched in parasite membrane in gametocytes. PfSET6 exhibited a nuclear localization in rings and a punctuated pattern in the cytoplasm of mature trophozoites and schizonts, and is enriched within foci-like structures in the cytoplasm of gametocytes. Taken together, our study suggests that non-histone methylation is much more important in P. falciparum than previously anticipated. PfSET7-mediated methylation may be an extension of the histone code to other cytosol proteins; PfSET6 partially associates with transcriptional repression pathways in the nucleus and post-transcriptional regulators in the cytoplasm. Further study aims to identify targets of SET domain containing proteins within the inducible gene knock out mutant parasite lines. The fact that PfSET7 and PfSET6 are expressed in different life cycle stages, makes them as novel targets for drug development that could against severe disease and to block pathogen transmission
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Zhang, Minlu. "Discovery and Analysis of Patterns in Molecular Networks: Link Prediction, Network Analysis, and Applications to Novel Drug Target Discovery." University of Cincinnati / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1330024618.
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Ding, Shuai. "Characterization of P.falciparum histone methyltransferases : biological role and possible targets for new intervention strategies." Electronic Thesis or Diss., Paris 6, 2016. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2016PA066578.pdf.
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On a montré que les PTM jouaient un rôle significatif de P. falciparum dans l'année de contrôle de la régulation transcriptionnelle, de l'expression monoaléique et de la différenciation sexuelle. Dix SET contenant des HKMTs contenant du domaine-ont été prédits; Six d'entre eux être essentiels pour le développement de stages de sang asexués. Le projet de thèse est centré sur la caractérisation biologique de PfSET7 et PfSET6. Nous avons observé l'échange de localisation cellulaire dynamique Pendant le cycle de vie: PfSET7 se trouvent dans de multiples foyers cytoplasmiques dans les stades érythrocytaires asexués et le stage de foie, et plus frappante, dans la membrane du parasite enrichie en gamétocytes. PfSET6 EXPOSÉ une localisation nucléaire dans les anneaux et un modèle ponctué dans le cytoplasme des trophozoites matures et schizontes, et est enrichi dans les structures de foyers dans le cytoplasme des gamétocytes. Pris ensemble, notre étude suggère que la méthylation non histone est beaucoup plus significative chez P. falciparum que précédemment attendu. La méthylation à médiation par PfSET7 Peut être une extension du code histone à - d'autres protéines cytosoliques; Partiellement PfSET6 s'associe à des voies de répression transcriptionnelle dans le noyau et des régulateurs post-transcriptionnels dans le cytoplasme. Une étude plus approfondie vise à identifier des cibles de domaine SET contenant des protéines dans le gène inducible knockout mutant parasite lignes. Le fait que PfSET7 et PfSET6 sont exprimés à différents stades du cycle de vie, les fait comme de nouvelles cibles pour le développement de médicaments contre cette maladie grave et de bloquer la transmissionIn P. falciparum, PTMs have been shown to play an important role in the control of transcriptional regulation, monoallelic expression, and sexual differentiation. Ten SET domain-containing HKMTs have been predicted; six of them appear to be essential for asexual blood stage development. My lab has expressed and purified two enzymatically active recombinant methyltransferase PfSET7 and PfSET6. In vitro enzyme kinetics assays shows they can methylate histones. The dissertation project is centered around the biological characterization of PfSET7 and PfSET6. We observed the dynamic changes of cellular localization during life cycle: PfSET7 are found in multiple cytoplasmic foci in asexual erythrocytic stages and liver stage, and more strikingly, enriched in parasite membrane in gametocytes. PfSET6 exhibited a nuclear localization in rings and a punctuated pattern in the cytoplasm of mature trophozoites and schizonts, and is enriched within foci-like structures in the cytoplasm of gametocytes. Taken together, our study suggests that non-histone methylation is much more important in P. falciparum than previously anticipated. PfSET7-mediated methylation may be an extension of the histone code to other cytosol proteins; PfSET6 partially associates with transcriptional repression pathways in the nucleus and post-transcriptional regulators in the cytoplasm. Further study aims to identify targets of SET domain containing proteins within the inducible gene knock out mutant parasite lines. The fact that PfSET7 and PfSET6 are expressed in different life cycle stages, makes them as novel targets for drug development that could against severe disease and to block pathogen transmission
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Ogba, Ndiya. "Transcriptional Regulation Of Estrogen Receptor Alpha Target Genes By Hexamethylene Bisacetamide-Inducible Gene 1 (HEXIM1) And Its Role In Mammary Gland Development And Breast Cancer." Cleveland, Ohio : Case Western Reserve University, 2010. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1258406511.
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Ganguly, Atish. "Wnt8 Is a Novel Target of the Dorsal/Twist/Snail Network and an Inhibitor of Dorsal in the Gastrulating Drosophila Embryo: A Dissertation." eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/201.
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The work in presented in this thesis identifies Drosophila Wnt8 as a novel zygotic target of the Dorsal/Twist/Snail network using a microarray analysis to identify differentially expressed genes in maternal dorsal mutant and gain-of-function Toll10b embryos as compared to wild type. In-situ hybridization with a Wnt8 antisense RNA probe revealed a fairly complex expression pattern in the early embryo. No maternal expression was observed and the first zygotic expression appeared at stage 4 at the poles. This was followed by patchy and relatively weak expression in the presumptive mesoderm with stronger expression in the mesectoderm and later the neuroectoderm. These expression required the Dorsal/Twist/Snail network with some input from Delta in the neuroectoderm. All embryonic Wnt8 expression ceased after late stage 10. Snail was found to repress Wnt8 in the presumptive mesoderm. The relevance of Wnt8 as a Snail target was tested by bypassing this repression in wild type embryos using a maternal Gal4 driver to drive UAS-Wnt8. This led to a loss of ventral furrow formation and a phenocopy of the snail mutant phenotype, thereby indicating that the repression of Wnt8 by Snail is important for gastrulation. Further investigation into the mechanism revealed a reduction in the expression of multiple target genes of Dorsal (including snail) in these Wnt8 overexpressing embryos. Ventral nuclear Dorsal protein was reduced as compared to wild type, suggesting that high levels of Wnt8 can antagonize Dorsal nuclear localization. Deficiency embryos lacking Wnt8 showed the opposite phenotype of expanded anterior and posterior snail RNA staining, as well as an expanded nuclear Dorsal signal in the posterior. This could be phenocopied using dsRNA against Wnt8, and was fully rescueable in the deficiency background using a Wnt8 genomic fragment. It has been reported that loss-of-function snail embryos lose the sharp lateral boundaries and high levels of snail expression more rapidly as compared to wild type. We hypothesize that this loss is due to derepressed Wnt8 antagonizing Dorsal and consequently its target, snail. In support of our hypothesis, double mutants of snail and the Wnt8 deficiency show a rescue of the snail pattern, though not a rescue of ventral furrow formation. Western blot analysis reveals a decrease in the levels of phosphorylation of Dorsal in Wnt8 overexpressing embryos as compared to wild type. Phosphorylation of Dorsal is required for its nuclear translocation. Hence, these data corroborate the observation of reduced nuclear Dorsal in embryos overexpressing Wnt8. Together, these data point to Wnt8 being an important target and a feedback inhibitor of the Dorsal/Twist/Snail pathway that achieves its effect by the inhibition of Dorsal.
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Argai, Aisha Ayad [Verfasser], Thomas [Akademischer Betreuer] Hollemann, Harald [Akademischer Betreuer] Loppnow, and Alexandra [Akademischer Betreuer] Schambony. "Identification of new target genes of the transcriptional regulator Ets-related protein 71 / Aisha Ayad Argai. Betreuer: Thomas Hollemann ; Harald Loppnow ; Alexandra Schambony." Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2015. http://d-nb.info/1065670141/34.
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Mahgoub, Hany. "Régulation post-transcriptionnelle dans l'adaptation des plantes genes aux stress abiotiques." Thesis, Aix-Marseille 2, 2011. http://www.theses.fr/2011AIX22030/document.
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Les plantes sont ancrées au sol pendant la majorité de leur cycle de vie et doivent donc constamment adapter leur croissance et leur métabolisme aux stress abiotiques. Ainsi, la subsistance des plantes dépend de leur capacité à réguler rapidement l’expression des gènes afin d’adapter leur physiologie à l’environnement. L’expression d’un gène peut être contrôlé à plusieurs niveaux; transcriptionnel, post-transcriptionnel, traductionnel et post-traductionnel.De nombreux processus cellulaires vitaux tels que la réplication de l’ADN, la transcription, la synthèse protéique, et la dégradation des protéines, sont régulés par les signaux environnementaux. Des études chez la levure, la drosophile et les animaux ont montré que la protéine kinase TOR (Target Of Rapamycin) est impliquée dans le contrôle de la croissance cellulaire et de la prolifération en réponse à différents signaux tels que les nutriments, les acides aminés, les hormones et les facteurs de croissance. Chez Arabidopsis thaliana, TOR est nécessaire au développement de l’embryon et de l’endosperme. De plus, des modifications du niveau de protéine AtTOR affectent la croissance végétative et la reproduction.Le principal objectif de cette thèse est de caractériser les mécanismes qui contrôlent l’expression de AtTOR en déterminant les éléments de régulation situés sur le la région 5’ non traduite (5′UTR) de l’ARNm de AtTOR, puis de manipuler ces éléments de régulation afin d étudier leur rôle. Nous avons choisi de nous focaliser sur la région 5′UTR de AtTOR, et sur une microORF (uORF) située en amont de l’ORF principale de AtTOR. Il s’agit de la première tentative d’étude de la régulation de l’expression de TOR par ces éléments chez les eucaryotes.Trois constructions chimériques ont été réalisées pour cette étude et transformée transitoirement est de manière stable dans des plantes. La première construction (contrôle positif) incluse le promoteur de AtTOR, la région 5′UTR, le premier intron et le début du premier exon fusionné au gène rapporteur GUS. La seconde construction (microORF mutée) est présente une mutation du codon start de la microORF (ATG changé en TTG). Enfin, la troisième construction (5′UTR délétée) contient la même séquence que le contrôle positif mais sans la région 5′UTR. Ces constructions ont également été placée sous le contrôle du promoteur 35S au lieu du promoteur de AtTOR afin d’étudier un lien éventuel entre la 5′UTR et la microRF et le promoteur de AtTORNos résultats indiquent une régulation généralement négative exercée par la 5′UTR, et dans une moindre mesure par la microORF, sur l’expression de AtTOR. Cette régulation semble avoir lieu au niveau transcriptionnel ou au niveau de la stabilité de l’ARNm, mais pas au niveau de la traduction. En effet, les modifications du niveau de transcrit GUS sont suivie d’un changement équivalent de l’activité GUS. De plus, nous avons observé que l’auxine et le sucrose ont un effet positif sur l’expression de AtTOR. Dans le cas de l’auxine, cet effet semble lié à la présence de la région 5′UTR de AtTOR.D’autres études de la fonction de la région 5’UTR et de la microORF de AtTOR, ainsi que de leur relation avec d’autres éléments régulateurs localisée dans le promoteur de AtTOR, permettront de mieux comprendre comment ces éléments régulateurs contrôlent finement l’expression de AtTORLand plants are anchored in one place for most of their life cycle and therefore must constantly adapt their growth and metabolism to abiotic stresses. Thus, plants’ subsistence depends on their ability to regulate rapidly gene expression in order to adapt their physiology to their environment. The expression of a gene can be controlled at many levels, including transcription, post-transcription, translation, and post-translation.Many vital cellular processes like DNA replication, transcription, protein synthesis, and protein degradation are regulated by environmental signals. Studies in yeast, Drosophila, and mammals showed that the target of rapamycin (TOR) protein is involved in control of cell growth and cell proliferation in response to different types of environmental signals such as nutrients, amino acids, hormones, and growth factors. In Arabidopsis thaliana, TOR is necessary for both embryo and endosperm development in, and changes of TOR protein level affect both vegetative and reproductive growth.The main purpose from this thesis is to highlight the mechanisms that control AtTOR expression at the post-transcriptional level through determination of the possible regulatory elements within the 5′ untranslated region (5′UTR) or the first intron of AtTOR mRNA itself, and through manipulation of these regulatory elements to study their precise role. We have chosen to focus on the small upstream open reading frame (uORF) as well as the 5′UTR region. This is the first attempt to study the regulation of TOR kinase expression in eukaryotes through these small uORF or the sequence of 5′ untranslated region (5′UTR).To achieve this purpose, three chimeric constructs have been established and transformed in Nicotiana benthamiana leaves and Arabidopsis thaliana plants. The first construct (the positive control) contains the AtTOR promoter, the 5′UTR, the first intron, and the beginning of the second exon fused to the GUS reporter gene. The second construct (mutated uORF) have the same sequence as the positive control construct except the start codon of uORF was changed from ATG to TTG. The third construct (deleted 5′UTR) have the same sequence as the positive control construct without the 5′UTR. These constructs have also been placed under the activity of CaMV 35S promoter instead of AtTOR promoter to investigate whether there is a link between the 5′UTR/or uORF and the promoter.Our work show an overall negative regulation exerted by the 5′UTR and, to a lesser extent, by the uORF on AtTOR gene regulation. This regulation is likely at the level of transcription or mRNA stability, since the changes in GUS transcript level was followed by the same changes in GUS activity. In addition we found that external inducers like auxin or sucrose exert a positive effect on AtTOR expression. This effect appears somehow linked to the presence of the 5′UTR of AtTOR mRNA.Greater insight into the molecular mechanisms of AtTOR 5′UTR/or uORF function and its relationship with other regulatory elements located in AtTOR promoter will be required to understand how these regulatory elements work either individually or in combination to achieve the fine and accurate regulation of their gene expression
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Wolf, Horrell Erin M. "Regulation of UV-Protective Pathways Downstream of the Melanocortin 1 Receptor in Melanocytes." UKnowledge, 2016. http://uknowledge.uky.edu/physiology_etds/29.
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Malignant cutaneous melanoma is the deadliest form of skin cancer, and a majority of melanoma diagnoses are a result of exposure to ultraviolet (UV) radiation. UV radiation causes DNA damage, which if not repaired correctly via nucleotide excision repair (NER) can result in mutations and melanomagenesis. The melanocortin 1 receptor (MC1R) is a Gs protein coupled receptor located on melanocyte plasma membranes and is involved in protecting the skin from UV induced damage. MC1R signaling results in the activation of two protective pathways: 1) induction of eumelanin synthesis downstream of micropthalmia-associated transcription factor (MITF) and 2) acceleration of NER downstream of ataxia telangiectaseia mutated and Rad3 related (ATR). MC1R signaling, however, also promotes melanocyte proliferation, therefore, the activation of the MC1R pathway must be regulated. The overall hypothesis of this dissertation is that the pathways downstream of MC1R can be manipulated to protect against UV induced damage.
Chapter 2 investigates the regulation of the MC1R neutral antagonist human β-defensin 3 (βD3). UV damage did not induce βD3 mRNA expression in ex vivo human skin explants. The induction of βD3 expression instead correlated with inflammatory cytokines including TNF.
Chapter 3 investigates the interdependence and cross talk between the two protective pathways downstream of MC1R. We directly tested the effect of MITF on the acceleration of NER and the effect of ATR on the induction of eumelanin synthesis following MC1R activation. MITF was not required for the acceleration of NER as mediated by ATR, however, the induction of transcription of enzymes involved in eumelanin synthesis was dependent upon ATR kinase activity.
Finally, Chapter 4 investigates the mechanism by which MC1R promoted proliferation and whether the two UV protective pathways downstream of MC1R could be selectively activated without the risk of melanocyte proliferation. MC1R signaling resulted in activation of the mechanistic target of rapamycin complex 1 (mTORC1), a major regulator of cell growth and proliferation. Inhibition of mTORC1 signaling via rapamycin prevented MC1R induced proliferation in vitro. Rapamycin, however, did not prevent MC1R induced eumelanin synthesis or the acceleration of NER in vitro or in vivo suggesting it is possible to selectively activate the beneficial signaling pathways without the risk of melanocyte proliferation.
The results of this dissertation suggest that MC1R signaling could be augmented in individuals to prevent UV induced damage.
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Kelley, Kevin Daniel. "Understanding the Molecular Dynamics of YPEL3 and FHIT Gene Expression." Wright State University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=wright1279328219.
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Guidi, Mònica. "Micro RNA-Mediated regulation of the full-length and truncated isoforms of human neurotrophic tyrosine kinase receptor type 3 (NTRK 3)." Doctoral thesis, Universitat Pompeu Fabra, 2009. http://hdl.handle.net/10803/7114.
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Neurotrophins and their receptors are key molecules in the development of thenervous system. Neurotrophin-3 binds preferentially to its high-affinity receptor
NTRK3, which exists in two major isoforms in humans, the full-length kinaseactive
form (150 kDa) and a truncated non-catalytic form (50 kDa). The two
variants show different 3'UTR regions, indicating that they might be differentially
regulated at the post-transcriptional level. In this work we explore how
microRNAs take part in the regulation of full-length and truncated NTRK3,
demonstrating that the two isoforms are targeted by different sets of microRNAs.
We analyze the physiological consequences of the overexpression of some of the
regulating microRNAs in human neuroblastoma cells. Finally, we provide
preliminary evidence for a possible involvement of miR-124 - a microRNA with no
putative target site in either NTRK3 isoform - in the control of the alternative
spicing of NTRK3 through the downregulation of the splicing repressor PTBP1.
Las neurotrofinas y sus receptores constituyen una familia de factores cruciales
para el desarrollo del sistema nervioso. La neurotrofina 3 ejerce su función
principalmente a través de una unión de gran afinidad al receptor NTRK3, del cual
se conocen dos isoformas principales, una larga de 150KDa con actividad de tipo
tirosina kinasa y una truncada de 50KDa sin dicha actividad. Estas dos isoformas
no comparten la misma región 3'UTR, lo que sugiere la existencia de una
regulación postranscripcional diferente. En el presente trabajo se ha explorado
como los microRNAs intervienen en la regulación de NTRK3, demostrando que las
dos isoformas son reguladas por diferentes miRNAs. Se han analizado las
consecuencias fisiológicas de la sobrexpresión de dichos microRNAs utilizando
células de neuroblastoma. Finalmente, se ha estudiado la posible implicación del
microRNA miR-124 en el control del splicing alternativo de NTRK3 a través de la
regulación de represor de splicing PTBP1.
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Ho, Rita Sam Man. "Disruption of imprinted transcription regulation of the Mash2 gene by targeted DNA insertion." 2004. http://link.library.utoronto.ca/eir/EIRdetail.cfm?Resources__ID=81172&T=F.
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Ademi, Irsa. "The Nickel-responsive Binding and Regulation of Two Novel Helicobacter pylori NikR–targeted Genes." Thesis, 2013. http://hdl.handle.net/1807/35577.
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Nickel is an essential transition metal for the virulence and survival of Helicobacter pylori in the acidic human stomach. The nickel– and proton– dependent transcriptional regulator HpNikR is important for maintaining nickel homeostasis inside the cytosol by regulating multiple H. pylori genes. A previous ChIP-sequencing experiment with H. pylori G27 and HpNikR identified two novel genes currently annotated as putative iron-transporters, HpG27_866 and HpG27_1499. In vitro DNA-binding assays with the promoter sequences of the two genes revealed nickel-dependent HpNikR binding with an affinity of ~10-7 M. The recognition site of HpNikR was identified on HpG27_1499 by footprinting assays, which loosely correlates with the HpNikR pseudo-consensus sequence. Furthermore, HpG27_1499 transcription showed nickel-dependent repression in WT H. pylori, and no changes in an isogenic ΔnikR strain. These data suggest that HpG27_1499 could be a nickel importer that is regulated by HpNikR in a nickel-responsive manner.
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Tripathi, Anamika. "Weighted gene co-expression network analysis of colorectal patients to identify right drug-right target for potent efficacy of targeted therapy." Thesis, 2017. https://doi.org/10.7912/C22Q09.
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Indiana University-Purdue University Indianapolis (IUPUI)Colon rectal cancer (CRC) is one of the most common cancers worldwide. It is characterized by the successive accumulation of mutations in genes controlling epithelial cell growth and differentiation leading to genomic in-stability. This results in the activation of proto-oncogene(K-ras), loss of tumor suppressor gene activity and ab-normality in DNA repair genes. Targeted therapy is a new generation of cancer treatment in which drugs attack targets which are specific for the cancer cell and are critical for its survival or for its malignant behavior. Survival of metastatic CRC patients has approximately doubled due to the development of new combinations of stan-dard chemotherapy, and the innovative targeted therapies, such as monoclonal antibodies against epidermal growth factor receptor (EGFR) or monoclonal antibodies against vascular endothelial growth factor (VEGFR).The study is to exhibit the need for right drug-right target and provides a proof of principle for potent efficacy of molecular targeted therapy for CRC. We have performed the weighted gene co-expression network analysis for three different patient cohort treated with different targeted therapy drugs. The results demonstrates the variation across different treatment regime in context of transcription factor networks. New significant tran-scription factors have been identified as potential biomarker for CRC cancer including EP300, STAT6, ATF3, ELK1, HNF4A, JUN, TAF1, IRF1, TP53, ELF1 and YY1. The results provides guidance for future omic study on CRC and additional validation work for potent biomarker for CRC.
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Prouse, Michael B. "Examination of the Transcriptional Regulation and Downstream Targets of the Transcription Factor AtMYB61." Thesis, 2013. http://hdl.handle.net/1807/43703.
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The mechanisms behind how a transcription factor elicits a given phenotype can be complex. The aim of the research presented herein was to provide experimental evidence to characterise the upstream and downstream regulation of the Arabidopsis thaliana R2R3-MYB transcription factor, AtMYB61. To address these aims, three separate experiments were undertaken.
First, three direct downstream target genes of AtMYB61 were predicted based on a two-stage complete transcriptome analysis, using publicly available microarray datasets in combination with a custom microarray dataset comparing the transcriptomes of WT, atmyb61 and 35S::MYB61 plants. These candidate target genes encode the following proteins: a KNOTTED1-like transcription factor, a caffeoyl-CoA 3-O-methyltransferase and a pectin-methylesterase. AtMYB61 bound the 5’ non-coding regulatory regions of these target genes, as determined by electrophoretic mobility shift assay.
Second, the preferred DNA-binding sites of recombinant AtMYB61 protein were assessed with a cyclic amplification and selection of targets (CASTing) assay. Key interactions between amino acids in the AtMYB61 DNA-binding site and nucleotides in the preferred DNA targets were predicted by molecular modeling. While recombinant AtMYB61 was sufficient to drive gene expression from CASTing-identified target DNA sequences in yeast, it did so in a manner that was not entirely consistent with predicted DNA-binding affinities determined by a nitrocellulose filter binding assay.
Finally, the molecular components that function upstream to modulate AtMYB61 expression were determined. AtMYB61 was determined to be de-repressed by sucrose in a mechanism involving its second intron. An over-represented motif was conserved within the second intron of Brassicaceae AtMYB61 homologues and this motif functioned as a binding target for a putative sugar-mediated repressor, as determined by EMSA. Putative AtMYB61 repressor proteins that bound this motif in the absence of sucrose were affinity purified and characterised using LC-MS/MS, and the proteins identified based on their MS fingerprints.
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Lorenzin, Francesca. "Regulation of transcription by MYC - DNA binding and target genes." Doctoral thesis, 2016. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-150766.
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MYC is a transcription factor, whose expression is elevated or deregulated in many human cancers (up to 70%) and is often associated with aggressive and poorly differentiated tumors. Although MYC is extensively studied, discrepancies have emerged about how this transcription factor works. In primary lymphocytes, MYC promotes transcriptional amplification of virtually all genes with an open promoter, whereas in tumor cells MYC regulates specific sets of genes that have significant prognostic value. Furthermore, the set of target genes that distinguish MYC’s physiological function from the pathological/oncogenic one, whether it exists or not, has not been fully understood yet. In this study, it could be shown that MYC protein levels within a cell and promoter affinity (determined by E-box presence or interaction with other proteins) of target genes toward MYC are important factors that influence MYC activity. At low levels, MYC can amplify a certain transcriptional program, which includes high affinity binding sites, whereas at high levels MYC leads to the specific up- and down regulation of genes with low affinity. Moreover, the promoter affinity characterizes different sets of target genes which can be distinguished in the physiological or oncogenic MYC signatures. MYC-mediated repression requires higher MYC levels than activation and formation of a complex with MIZ1 is necessary for inhibiting expression of a subset of MYC target genesMYC ist ein Transkriptionsfaktor, dessen Expression in vielen humanen Tumoren (bis zu 70 %) erhöht oder dereguliert ist. Die Tumore, in denen viel MYC hergestellt wird, zeichnen sich durch einen geringen Differenzierungsgrad aus und verhalten sich sehr aggressiv. Obwohl das biologische Verhalten des MYC Proteins intensiv untersucht wurde, sind unterschiedliche Modelle, wie dieser Transkriptionsfaktor funktioniert, entwickelt worden. In primären Lymphozyten verstärkt MYC die Expression fast aller Gene mit offener Chromatinstruktur, während MYC in Tumorzellen spezifische Gengruppten reguliert, deren Expression mit der Prognose von Patienten korreliert. Es ist also unklar, ob sich die Zielgene der physiologischen Funktion von Myc von den oncogenen/pathophysiologischen Zielgenen unterscheidet und um welche Gene es sich bei letzteren handelt. In dieser Arbeit konnte gezeigt werden, dass Expressionsniveau von MYC und unterschiedliche Promotoraffinitäten zu MYC (charakterisiert durch den Ebox-Gehalt und Interaktionen zu anderen Proteinen) wichtig für die Aktivität des MYC Proteins sind. So kann Myc bei niedrigen Konzentrationen ein bestimmtes transkriptionelles Programm amplifizieren, das sich aus hochaffinen Promotoren zusammensetzt. Bei hohen Konzentrationen hingegen führt MYC zur transkriptionellen Aktivierung und Repression bestimmter Zielgengruppen, die sich durch niedrige Affinität zu MYC auszeichnen. Somit ist die Promotoraffinität ein Parameter, der physiologische von oncogenen MYC Signaturen trennen kann. Darüberhinaus konnte gezeigt werden, dass MYC-vermittelte Repression höhere MYC Mengen benötigt, als MYC-vermittelte Transaktivierung und die Komplexbildung mit MIZ1 für die Repression einer Gruppe an MYC Zielgenen nötig ist
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Patel, Rushang Dilipkumar Perdew Gary H. "Regulation of gene transcription by the aryl hydrocarbon receptor -new targets and mechanisms of regulation." 2008. http://etda.libraries.psu.edu/theses/approved/WorldWideIndex/ETD-2823/index.html.
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Qadi, AA. "Regulation of the LIFR and gp130 genes by the RUNX1 transcription factor." Thesis, 2012. https://eprints.utas.edu.au/15940/2/whole-qadi-thesis-2012.pdf.
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The RUNX1 transcription factor is an important regulator of haematopoiesis and has been found to influence gene activity at both the transcriptional and chromatin levels. In leukaemia, particularly Acute Myeloid Leukaemia, RUNX1 activity is frequently altered by point mutations and chromosomal rearrangements, the most common being the t(8;21) translocation that produces a RUNX1-ETO fusion protein. While RUNX1 is generally associated with gene activation, RUNX1-ETO mainly acts as a transcriptional repressor and its presence is therefore associated with altered expression of RUNX1 target genes.
Previous microarray analysis performed in our laboratory identified both the LIFR and gp130 genes as novel putative RUNX1 targets. LIFR together with gp130 forms a heterodimeric receptor complex that mediates LIF signalling, and in doing so controls various cellular processes including cellular development, differentiation and inflammatory responses. However, despite their important biological roles little is known about regulation of the LIFR and gp130 genes. Bioinformatic analysis identified potential RUNX1 binding sites in the promoters of both the LIFR and gp130 genes, and this study therefore examined the hypothesis that LIFR and gp130 are RUNX1 target genes and their altered expression in leukemic cells in which RUNX1 is disrupted may therefore contribute to leukaemia.
The LIFR gene is regulated by alternate promoters, with a so called ‘general’ and ‘placental’ promoter previously described. Analysis of LIFR mRNA across a number of cell types demonstrated that activity of the placental promoter is limited to cell lines of placental origin, while the general promoter is active in a range of cell types, including myeloid cells. This was confirmed by analysis of the chromatin status of the two promoters, which found that the general, but not placental promoter is assembled into highly acetylated histones in myeloid cells. The expression of the gp130 mRNA was detected across all cells lines examined.
Reporter analysis demonstrated that both the placental and general promoters can be activated by RUNX1 in placental and myeloid cell lines respectively. In addition, the gp130 promoter was activated by RUNX1 in myeloid cell lines. In contrast, RUNX1-ETO repressed both the LIFR and gp130 promoters in myeloid cells. Further, binding of RUNX1 to the endogenous LIFR and gp130 promoters was confirmed by chromatin immunoprecipitation, suggesting that the endogenous promoters are targeted by RUNX1. In support of this RUNX1 knockdown reduced LIFR and gp130 expression in myeloid cell lines, and decreased expression of the placental LIFR transcript in placental cells.
Put together, the data presented in this thesis demonstrates that RUNX1 regulates expression of the LIFR and gp130 promoters, suggesting that activity of the LIFR/gp130 receptor complex is likely to be altered in leukaemic cells in which RUNX1 is altered.
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Gupta, Nimish. "Target(s) of transcriptional regulators ppGpp and DksA." 2009. http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.000051014.
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Raimondo, Anne. "Identification of downstream target genes and analysis of obesity-related variants of the bHLH/PAS transcription factor single-minded 1." Thesis, 2011. http://hdl.handle.net/2440/73199.
Full textAbstract:
Single-minded 1 (SIM1) is a basic Helix-Loop-Helix/PER-ARNT-SIM (bHLH/PAS) transcription factor essential for survival in mice. The early post-natal lethality exhibited by Sim1⁻ʹ⁻ mice is believed to be the consequence of severely compromised hypothalamus development, although the contribution of reduced SIM1 signalling in the numerous other tissues in which it is expressed has never been formally investigated. The presence of a single Sim1 allele is sufficient to avoid this perinatal lethality, and instead confers an early onset, hyperphagic obesity phenotype, potentially via disruption of critical intracellular signalling pathways that are activated in Sim1-expressing hypothalamic neurons in response to food intake. Similar correlations between reduced SIM1 gene dosage and severe obesity have also been documented in humans. Alterations in SIM1 expression and/or function therefore have important implications in health and disease, and warrant a detailed investigation into the downstream target genes and regulatory behaviours of this critical transcription factor, which are thus far almost entirely lacking in the literature. The studies presented in this thesis describe a twofold approach to dissecting the gene regulatory properties of the SIM1 protein. Firstly, we optimised and performed a range of functional assays, including a cell-based luciferase reporter gene assay, a subcellular localisation assay, a co-immunoprecipitation assay, and an electrophoretic mobility shift assay, which were designed to assess the contribution of nineteen unique point mutations within the SIM1 protein sequence to altered SIM1 expression and behaviour. These nineteen mutations were identified in multiple cohorts of severely obese humans, and therefore represent potentially pathogenic alterations in the SIM1 sequence. Indeed, we observed a significant loss of function for many of these variants in luciferase reporter gene assays relative to wild type SIM1. The severe loss of function observed for one of these variants, SIM1 T292A, could be further attributed to altered subcellular localisation, thus impacting on its ability to form a stable heterodimer with ARNT2 in co-immunoprecipitation experiments. Secondly, we performed microarray studies on cultured kidney-derived cells inducibly overexpressing Myc-tagged SIM1 and its obligate partner factor ARNT2, and subsequently identified several genes that selectively responded to SIM/ARNT2 overexpression in this context. Further validation in hypothalamus-derived cultured cells highlighted Myomesin 2 (Myom2) as a potentially genuine downstream SIM1 target gene in both kidney and hypothalamus. We also present data that are the first to indicate Somatostatin (Ss) as a hypothalamic target gene regulated by SIM1 in a cell-autonomous manner. These data are among the first to dissect the downstream target genes and regulatory properties of the SIM1 protein, and therefore make an important contribution to our understanding of the molecular basis to the hyperphagic obesity exhibited by Sim1⁺ʹ⁻ mice. They are also the first to link reduced activities of mis-sense mutations in the SIM1 coding sequence to increased weight gain in humans, and give further credence to the possibility that SIM1 represents a novel genetic contributor to obesity disorders in the wider population. This knowledge may inform future attempts to develop therapies for obese phenotypes in humans, and broaden our understanding of the molecular events that underpin Sim1-mediated survival and maintenance of homeostasis.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2011
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Bhattacharya, Surajit. "Computational Interrogation of Transcriptional and Post-Transcriptional Mechanisms Regulating Dendritic Development." 2017. http://scholarworks.gsu.edu/biology_diss/190.
Full textAbstract:
The specification and modulation of cell-type specific dendritic morphologies plays a pivotal role in nervous system development, connectivity, structural plasticity, and function. Regulation of gene expression is controlled by a wide variety of cellular and molecular mechanisms, of which two major types are transcription factors (TFs) and microRNAs (miRNAs). In Drosophila, dendritic complexity of dendritic arborization (da) sensory neurons of the peripheral nervous system are known to be regulated by two transcription factors Cut and Knot, although much remains unknown about the molecular mechanisms and regulatory networks via which they regulate the final arbor shape through spatio-temporal modulation of dendritic development and dynamics. Here we use bioinformatics analysis of transcriptomic data to identify putative genomic targets of these TFs with a particular emphasis on those that effect neuronal cytoskeletal architecture. We use transcriptomic, as well as data from various genomic and protein interaction databases, to build a weighted functional gene regulatory network for Knot, to identify the biological pathways and downstream genes that this TF regulates. To corroborate bioinformatics network predictions, knot putative targets, which classify into neuronal and cytoskeletal functional groups, have been experimentally validated by in vivo genetic perturbations to elucidate their role in Knot-mediated Class IV (CIV) dendritogenesis. MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of gene expression, however identification of biologically-relevant target genes for this epigenetic regulatory mechanism remains a significant challenge. To address this knowledge gap, we have developed a novel R based tool, IntramiR-ExploreR, that facilitates integrated discovery of miRNA targets by incorporating target databases and novel target prediction algorithm to arrive at high confidence intragenic miRNA target predictions. We have explored the efficacy of this tool using D.melanogaster as a model organism for bioinformatics analyses and functional validation, and identified targets for 83 intragenic miRNAs. Predicted targets were validated, using in vivo genetic perturbation. Moreover, we are constructing interaction maps of intragenic miRNAs focusing on neural tissues to uncover regulatory codes via which these molecules regulate gene expression to direct cellular development.