Transposons were first discovered as "jumping genes" by Barbara McClintock, who continued to study them in maize through the 1940's and 1950's. Since then, transposons have been shown to make up a large percentage of eukaryotic genomes, including close to half of the human genome, but have been dismissed as simply "junk DNA." Recently, the importance of keeping transposons tightly regulated within the cellular environment has begun to be appreciated; the mechanisms to accomplish this have been studied and the current understanding of pathways governing transposon regulation is discussed within this dissertation. However, recent work presented within the scope of this dissertation in Drosophila melanogaster revealed a previously unknown function for condensin complexes in transposable element regulation. These studies provide a link between pathways governing chromosome pairing and transposon regulation. The potential interplay between these two pathways is intriguing and until now, largely unexplored.Aside from how transposons themselves are regulated, studies into potential roles they may play in the regulation of other protein coding genes within the cell may provide clues into the functionality of these elements within our genome. As a specific example, BRCA1 has a high density of retrotransposon sequences within its primary transcript, and studies of BRCA1 regulation presented within this dissertation has led to the development of a model for a novel gene regulatory mechanism occurring in human cells involving retrotransposons. This mechanism may provide direct relevance to cancer etiology, as retrotransposons have long been known to be misregulated in cancer.As a sum, the work presented within this dissertation extends our knowledge of how transposons are regulated and provides some of the first evidence for their functionality in gene regulatory pathways within human cells.

Thesis advisor: Jeffrey H. Chuang / Gene regulation is perhaps least understood among vertebrate species, where cell differentiation, tissue-types and body-plans indicate a complexity in need of careful coordination to achieve such hierarchical design. Recent studies reveal the intricacy of vertebrate gene regulation through diverse events including transcriptional regulatory histone modifications and non-coding DNA [1-5]. Almost 98% of the human genome is noncoding DNA, much of which may be actively involved in regulating healthy and disease-state gene expression and environmental response [6]. Conserved noncoding elements (CNEs) are sequences of noncoding DNA that are known to regulate gene expression [7-9]. The CNEs identified thus far are a small percentage of the total noncoding DNA in the human genome, and many identified CNEs still lack experimental characterization [10]. Thus, there is a need for functional characterization and streamlined identification of CNEs in order to more fully annotate vertebrate genomes and understand gene expression. The work in this thesis identified over 6000 CNEs and experimentally characterized over 150 CNEs conserved between zebrafish and human (> 60% DNA sequence conservation), using the experimental model Danio rerio (zebrafish). Functional, tissue and time-specific CNEs were identified through analysis of conservation, accelerated evolution, distance, GC content, motifs, transcription factors and gene function. In addition, a searchable database and website was created to host data and facilitate collaborative research between experimental and computational labs. While non-coding DNA is an important area of discovery for gene regulation, protein-coding DNA also has the potential to contain non-coding transcriptional information. DNA is typically conceptualized as either noncoding or protein coding. An underlying assumption to this framework assumes that the function of noncoding DNA is "regulatory" and coding DNA is "protein coding." Consequently, the potential for DNA to harbor both types of information in one sequence has been minimally researched. For the second-half of this thesis, I identified and experimentally tested 31 conserved coding exons ( > 60% zebrafish and human DNA sequence conservation) in zebrafish. To improve annotation of live embryonic expression, a novel voice-recognition expression analysis system was developed that allows quick comparison and annotation of embryonic expression at the microscope. In addition, a website and webtool to calculate significant expression was created as a resource for experimental research on anatomical analysis in whole organisms. The experimental results show that a large number of protein-coding DNA sequences can act as non-coding enhancers. This knowledge may impact methods to identify noncoding signals and, further, the scientific conceptualizations of coding and noncoding DNA in the genome. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Doctor of Philosophy (PhD) / Gene expression is known to be regulated at the level of transcription. Recently, however, there has been a growing realisation of the importance of gene regulation at the post-transcriptional level, namely at the level of pre-mRNA processing (5’ capping, splicing and polyadenylation), nuclear export, mRNA localisation and translation. Erythroid krüppel-like factor (Eklf) is the founding member of the Krüppel-like factor (Klf) family of transcription factors and plays an important role in erythropoiesis. In addition to its nuclear presence, Eklf was recently found to localise to the cytoplasm and this observation prompted us to examine whether this protein has a role as an RNA-binding protein, in addition to its well-characterised DNA-binding function. In this thesis we demonstrate that Eklf displays RNA-binding activity in an in vitro and in vivo context through the use of its classical zinc finger (ZF) domains. Furthermore, using two independent in vitro assays, we show that Eklf has a preference for A and U RNA homoribopolymers. These results represent the first description of RNA-binding by a member of the Klf family. We developed a dominant negative mutant of Eklf by expressing its ZF region in murine erythroleukaemia (MEL) cells. We used this to investigate the importance of this protein in haematopoietic lineage decisions by examining its effect on the multipotent K562 cell line. We provide evidence that Eklf appears to be critical not only for the promotion of erythropoiesis, but also for the inhibition of megakaryopoiesis.

Cellular messenger RNAs (mRNAs) exist almost exclusively in the context of ribonucleoprotein complexes (RNPs), which are largely responsible for the coordinated regulation of mRNA fate, and in particular, the post-transcriptional regulation of mRNA stability and translation. RNA- binding proteins, antisense RNAs, and microRNAs represent three major classes of post- transcriptional regulatory factors that interact with target mRNAs. Significantly, these interactions are dynamically regulated under both basal and stress conditions, such as hypoxia. Given the prominent contributions of post-transcriptional regulation to overall gene expression, a more comprehensive understanding of the underlying mechanisms is required. In this thesis, we present exciting new evidence for the functional importance of post- transcriptional gene regulation, especially in the vascular endothelium. Firstly, we show that the formation of hnRNP E1-containing RNPs contributes significantly to the remarkable basal stability of endothelial nitric oxide synthase (eNOS) mRNAs in endothelial cells by protecting them from inhibitory post-transcriptional forces. However, hypoxia impairs such RNP formation through hnRNP E1 serine phosphorylation and nuclear localization. Together, these mechanisms contribute significantly to decreased eNOS expression and activity in chronic hypoxia. ii Secondly, we reveal an important functional relationship between the microRNA pathway and the HIF-mediated cellular hypoxic response. Specifically, the down-regulation of Dicer and an important number of Dicer-dependent microRNAs in chronic hypoxia represents an important adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, with significant implications for the development of RNAi- based therapies. Finally, we provide evidence that the up-regulation of specific microRNAs in acute hypoxia is a potentially important mechanism that serves to suppress global translation initiation in order to conserve energy and ensure cellular survival. Collectively, the findings presented in this thesis provide important new mechanistic insight into the post-transcriptional regulation of eNOS, as well as the functional integration of the microRNA and the cellular hypoxic response pathways.

Cellular messenger RNAs (mRNAs) exist almost exclusively in the context of ribonucleoprotein complexes (RNPs), which are largely responsible for the coordinated regulation of mRNA fate, and in particular, the post-transcriptional regulation of mRNA stability and translation. RNA- binding proteins, antisense RNAs, and microRNAs represent three major classes of post- transcriptional regulatory factors that interact with target mRNAs. Significantly, these interactions are dynamically regulated under both basal and stress conditions, such as hypoxia. Given the prominent contributions of post-transcriptional regulation to overall gene expression, a more comprehensive understanding of the underlying mechanisms is required. In this thesis, we present exciting new evidence for the functional importance of post- transcriptional gene regulation, especially in the vascular endothelium. Firstly, we show that the formation of hnRNP E1-containing RNPs contributes significantly to the remarkable basal stability of endothelial nitric oxide synthase (eNOS) mRNAs in endothelial cells by protecting them from inhibitory post-transcriptional forces. However, hypoxia impairs such RNP formation through hnRNP E1 serine phosphorylation and nuclear localization. Together, these mechanisms contribute significantly to decreased eNOS expression and activity in chronic hypoxia. ii Secondly, we reveal an important functional relationship between the microRNA pathway and the HIF-mediated cellular hypoxic response. Specifically, the down-regulation of Dicer and an important number of Dicer-dependent microRNAs in chronic hypoxia represents an important adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, with significant implications for the development of RNAi- based therapies. Finally, we provide evidence that the up-regulation of specific microRNAs in acute hypoxia is a potentially important mechanism that serves to suppress global translation initiation in order to conserve energy and ensure cellular survival. Collectively, the findings presented in this thesis provide important new mechanistic insight into the post-transcriptional regulation of eNOS, as well as the functional integration of the microRNA and the cellular hypoxic response pathways.

Doctor of Philosophy (PhD) / Gene expression is known to be regulated at the level of transcription. Recently, however, there has been a growing realisation of the importance of gene regulation at the post-transcriptional level, namely at the level of pre-mRNA processing (5’ capping, splicing and polyadenylation), nuclear export, mRNA localisation and translation. Erythroid krüppel-like factor (Eklf) is the founding member of the Krüppel-like factor (Klf) family of transcription factors and plays an important role in erythropoiesis. In addition to its nuclear presence, Eklf was recently found to localise to the cytoplasm and this observation prompted us to examine whether this protein has a role as an RNA-binding protein, in addition to its well-characterised DNA-binding function. In this thesis we demonstrate that Eklf displays RNA-binding activity in an in vitro and in vivo context through the use of its classical zinc finger (ZF) domains. Furthermore, using two independent in vitro assays, we show that Eklf has a preference for A and U RNA homoribopolymers. These results represent the first description of RNA-binding by a member of the Klf family. We developed a dominant negative mutant of Eklf by expressing its ZF region in murine erythroleukaemia (MEL) cells. We used this to investigate the importance of this protein in haematopoietic lineage decisions by examining its effect on the multipotent K562 cell line. We provide evidence that Eklf appears to be critical not only for the promotion of erythropoiesis, but also for the inhibition of megakaryopoiesis.

No description available.

abstract: Multicellular organisms use precise gene regulation, executed throughout development, to build and sustain various cell and tissue types. Post-transcriptional gene regulation is essential for metazoan development and acts on mRNA to determine its localization, stability, and translation. MicroRNAs (miRNAs) and RNA binding proteins (RBPs) are the principal effectors of post-transcriptional gene regulation and act by targeting the 3'untranslated regions (3'UTRs) of mRNA. MiRNAs are small non-coding RNAs that have the potential to regulate hundreds to thousands of genes and are dysregulated in many prevalent human diseases such as diabetes, Alzheimer's disease, Duchenne muscular dystrophy, and cancer. However, the precise contribution of miRNAs to the pathology of these diseases is not known. MiRNA-based gene regulation occurs in a tissue-specific manner and is implemented by an interplay of poorly understood and complex mechanisms, which control both the presence of the miRNAs and their targets. As a consequence, the precise contributions of miRNAs to gene regulation are not well known. The research presented in this thesis systematically explores the targets and effects of miRNA-based gene regulation in cell lines and tissues. I hypothesize that miRNAs have distinct tissue-specific roles that contribute to the gene expression differences seen across tissues. To address this hypothesis and expand our understanding of miRNA-based gene regulation, 1) I developed the human 3'UTRome v1, a resource for studying post-transcriptional gene regulation. Using this resource, I explored the targets of two cancer-associated miRNAs miR-221 and let-7c. I identified novel targets of both these miRNAs, which present potential mechanisms by which they contribute to cancer. 2) Identified in vivo, tissue-specific targets in the intestine and body muscle of the model organism Caenorhabditis elegans. The results from this study revealed that miRNAs regulate tissue homeostasis, and that alternative polyadenylation and miRNA expression patterns modulate miRNA targeting at the tissue-specific level. 3) Explored the functional relevance of miRNA targeting to tissue-specific gene expression, where I found that miRNAs contribute to the biogenesis of mRNAs, through alternative splicing, by regulating tissue-specific expression of splicing factors. These results expand our understanding of the mechanisms that guide miRNA targeting and its effects on tissue-specific gene expression. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2019

Orientador: Ney Lemke / Resumo: MicroRNAs (miRNAs) são pequenas moléculas de RNA que regulam pós-transcricionalmente a expressão de genes, modelando o transcriptoma e a produção de proteínas. Em geral, os miRNAs são conservados no genoma de eucariotos, sendo considerados elementos vitais em diversos processos biológicos durante o desenvolvimento, tais como crescimento, diferenciação e morte celular. A grande diversidade de miRNAs identificados está restrita a poucas espécies e apenas uma parte do total de alvos de miRNAs preditos foi caracterizada funcionalmente. Nesse contexto, o uso da tecnologia de sequenciamento de alto rendimento (high throughput sequencing) atrelada à análise de nível transcricional por RT-qPCR possibilitam a identificação do microRNoma. A tilápia do Nilo, Oreochromis niloticus, é considerada um excelente modelo biológico para o estudo de miRNAs em vertebrados devido à sua importância econômica e evolutiva. O presente trabalho teve como objetivos: organizar os dados do sequenciamento dos miRNAs da tilapia do Nilo; disponibilizá-los em forma de uma base de dados para a comunidade científica; integrar as informações dos miRNAs identificados com outros bancos de dados de miRNAs; analisar os dados através de análises de bioinformática para determinação de agrupamentos definidos pelo nível de expressão de cada miRNA em seis tipos de tecido (músculo branco, músculo vermelho, testículo, ovário, fígado, olho, cérebro e coração) com distinção entre os gêneros e nas fases do desenvolvimento (2,... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

No description available.