Cette thèse cherche à discriminer au niveau génomique entre le développement d'embryons vers un mode de reproduction sexué et le développement vers un mode asexué chez le puceron du pois, Acyrthosiphon pisum. Cette discrimination passe par la création du réseau de régulation post-transcriptionnelle des microARN et des ARNm qui possèdent des cinétiques d'expression différentes entre ces deux embryogenèses ainsi que par l'analyse des modules d'interactions de ce réseau par l'utilisation de l'analyse de concepts formels. Pour ce faire, une stratégie en plusieurs étapes a été mise en place : la création d'un réseau d'interactions entre les microARN et les ARNm du puceron du pois ; l'extraction et la réduction du réseau aux microARN et ARNm qui possèdent des cinétiques différentes entre les deux embryogenèses à partir des données d'expression tirées du séquençage haut-débit ; l'analyse du réseau d'interactions réduit aux éléments d’intérêt par l'analyse de concepts formels. L'analyse du réseau a permis l'identification de différentes fonctions potentiellement importantes comme l'ovogenèse, la régulation transcriptionnelle ou encore le système neuroendocrinien. En plus de l'analyse du réseau, l'analyse de concepts formels a été utilisée pour définir une méthode de réparation de graphe biparti basée sur une topologie en "concepts" ainsi qu'une méthode de visualisation de graphes bipartis par ses concepts. / This thesis aims to discriminate between embryos development towards either sexual or asexual reproduction types in pea aphids, Acyrthosiphon pisum, at the genomic level. This discrimination involves the creation of a post-transcriptional regulation network between microRNAs and mRNAs whose kinetic expressions change depending on the embryogenesis. It also involves a study of this network's interaction modules using formal concept analysis. To do so, a three-step strategy was set up. First the creation of an interaction network between the pea aphid's microRNAs and mRNAs. The network is then reduced by keeping only microRNAs and mRNAs which possess differential kinetics between the two embryogeneses, these are obtained using high-throughput sequencing data. Finally the remaining network is analysed using formal concept analysis. Analysing the network allowed for the identification of several functions of potential interest such as oogenesis, transcriptional regulation or even neuroendocrine system. In addition to network analysis, formal concept analysis was used to create a new method to repair a bipartite graph based on its topology and a method to visualise a bipartite graph using its formal concepts.

Genetic discoveries have highlighted the role of gene expression dysregulation in both rare and common diseases. In particular, a large number of chromatin modifiers, transcription factors, and RNA-binding proteins have been implicated in neurodevelopmental diseases, including epilepsy, autism spectrum disorder, schizophrenia, and intellectual disability. Elucidating the disease mechanisms for these genes is challenging, as the encoded proteins often regulate thousands of downstream targets. In Chapter 2 of this thesis, we describe the use of single-cell RNA-sequencing (scRNA-seq) to characterize a mouse model of HNRNPU-mediated epileptic encephalopathy. This gene encodes a ubiquitously expressed RNA-binding protein, yet we demonstrate that reduction in its expression leads to cell type-specific transcriptomic defects. Specifically, excitatory neurons in a region of the hippocampus called the subiculum carried the strongest burden of differential gene expression. In Chapter 3, we use scRNA-seq to identify convergent molecular and transcriptomic features in four different organoid models of a cortical malformation called periventricular nodular heterotopia. In Chapter 4, we build on these successes to propose a high-throughput drug screening program for neurodevelopmental genes that encode regulators of gene expression. This approach—termed transcriptomic reversal—attempts to identify compounds that reverse disease-causing gene expression changes back to a normal state. Finally, in Chapter 5, we focus on the role of synonymous codon usage in human disease. Codon usage can affect mRNA stability, yet its role in human physiology has been historically overlooked. We use population genetics approaches to demonstrate that natural selection shapes codon content in the human genome, and we find that dosage sensitive genes are intolerant to reductions in codon optimality. We propose that synonymous mutations could modify the penetrance of Mendelian diseases through altering the expression of disease-causing mutations. In summary, the work in this thesis broadly focuses on the role of gene expression dysregulation in disease. We provide novel frameworks for interrogating disease gene expression signatures, prioritizing mutations that may alter expression, and identifying targeted therapeutics.

Bacteria regulate gene expression through coordinated interactions between cis-regulatory sequences and trans-regulatory factors. Understanding the molecular basis for the functions of these regulatory components is not only essential for deciphering complex biological processes in diverse bacteria but also critical for rational engineering of microbial phenotypes. However, systematically dissecting the sequence-function relationships of cis and trans regulatory components that underly gene expression is still a key challenge. Recent technological advances have provided novel tools and methods for mapping sequence-function relationships in high-throughput. This dissertation focuses on applying novel methods enabled through increased throughput and scalability of DNA synthesis and sequencing to elucidate the sequence-function relationships of cis and trans components that underlie bacterial gene regulation. In Chapter 2, evolutionary and functional diversity of primary σ70, a universally conserved global regulator in bacteria, is studied through comparative genomics, saturation mutagenesis, and transcriptomics. Through the combined efforts of these approaches, we demonstrate that sequence diversity of σ70 factors reflects functional differences which have been shaped by evolutionary constraints from co-evolving regulatory sequence targets during evolution. Chapter 3 discusses systematically mapping transcriptional activities of cis-regulatory sequences from Biosynthetic Gene Clusters (BGCs). Using a Streptomyces as a host, we found key regulatory features that affected gene expression, such as GC content, transcription start sites, and sequence motifs. We further explored regulation of BGC derived regulatory sequences by expressing global regulatory factors and screening for regulator sequences with altered expression levels. Finally, Chapter 4 highlights recent studies that made key contributions towards elucidating and modulating bacterial gene regulatory networks and reviews the current state of microbial systems biology and gene regulation. Together, the results and discussions presented in this dissertation seeks to further advance the current knowledge of sequence-function relationships of microbial regulatory components to enable better understanding, modeling, and rational engineering of bacterial gene regulation.

The helix-loop-helix (HLH) family of transcription regulatory proteins are fundamental regulators in the processes of cell proliferation and differentiation, cell lineage determination, myogenesis, neurogenesis, and sex determination in a wide range of multicellular organisms. A gene encoding a novel class II HLH protein has recently been identified from a human B-cell eDNA library using a yeast two-hybrid screen. The predicted human ABF -1 polypeptide sequence was used to search the Caenorhabditis elegans genome database for a C. elegans ABF-1 homolog. This bHLH protein, called C. elegans ABF -1 (CeABF -1 ), has a bHLH domain that shares 72% amino acid similarity with its human ABF-1 relative. The expression of the CeABF-1 mRNA has been detected in larval stages L2, L3, L4, and adult, however the mRNA is most highly expressed at the L3 and L4 stages. CeABF -1 protein is capable of heterodimerizing with the human E2A gene product, E4 7. Like human ABF -1, CeABF -1 expression in the presence of the E4 7 protein results in a reduction in E2A mediated gene activation. It has therefore been concluded that CeABF -1 , like human ABF -1 , also acts as a transcriptional repressor. Because C. elegans shares many conserved genes with higher eukaryotic organisms it has become a model organism for in depth genetic studies. It has therefore become increasingly desirable to investigate the possibility of alternative protein-protein interactions that can potentially occur within C. elegans, so it was necessary to construct a C. elegans eDNA library along with the appropriate bait vector expressing the CeABF- 1 protein. The titer ofthe primary library was calculated to be 9.7 x I06 clones, 10-fold greater than minimum titer requirement of I x I 06 clones for a good representational library. Sequencing of the CeABF -I insert confirmed successful construction of a mutation-free bait construct suitable for use in yeast two-hybrid screening. Yeast-two hybrid analysis revealed two new interactors, one of which was identified as an aldose reductase homolog, while the other remains uncharacterized.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / LHX3 is a transcription factor important in pituitary and nervous system development. Patients with mutations in coding regions of the gene have combined pituitary hormone deficiency (CPHD) that causes growth, fertility, and metabolic problems. Promoter and intronic elements of LHX3 important for basal gene expression in vitro have been identified, but the key regulatory elements necessary for in vivo expression were unknown. With these studies, I sought to elucidate how LHX3 gene expression is regulated in vivo. Based on sequence conservation between species in non-coding regions, I identified a 7.9 kilobase (kb) region 3' of the human LHX3 gene as a potential regulatory element. In a beta galactosidase transgenic mouse model, this region directed spatial and temporal expression to the developing pituitary gland and spinal cord in a pattern consistent with endogenous LHX3 expression. Using a systematic series of deletions, I found that the conserved region contains multiple nervous system enhancers and a minimal 180 base pair (bp) enhancer that direct expression to both the pituitary and spinal cord in transgenic mice. Within this minimal enhancer, TAAT/ATTA sequences that are characteristic of homeodomain protein binding sites are required to direct expression. I performed DNA binding experiments and chromatin immunoprecipitation assays to reveal that the ISL1 and PITX1 proteins specifically recognize these elements in vitro and in vivo. Based on in vivo mutational analyses, two tandem ISL1 binding sites are required for enhancer activity in the pituitary and spine and a PITX1 binding site is required for spatial patterning of gene expression in the pituitary. Additional experiments demonstrated that these three elements cannot alone direct gene expression, suggesting a combination of factors is required for enhancer activity. This study reveals that the key regulatory elements guiding developmental regulation of the human LHX3 gene lie in this conserved downstream region. Further, this work implicates ISL1 as a new transcriptional regulator of LHX3 and describes a possible mechanism for the regulation of LHX3 by a known upstream factor, PITX1. Identification of important regulatory regions will also enable genetic screening in candidate CPHD patients and will thereby facilitate patient treatment and genetic counseling.

Cell type specification in the ventral spinal cord is controlled by a suite of transcription factors (TFs) whose expression is induced by opposing gradients of retinoic acid (RA) and sonic hedgehog (SHH). The action of these TFs results in the creation of five distinct progenitor domains, four of which produce interneurons and one that produces motor neurons (MNs). Though the transcription factors (Pax6, Sp8, Nkx2.2, Olig2) involved in progenitor domain boundary setting and in the specification of adult cell types from these domains are known, major questions remain as to how these factors are able to specify specific fates. Questions also remain about the function of the TFs involved in boundary specification. Do any of these factors have additional functions? Are they necessary for adult cell type specification? In this thesis I investigate how the putative domain boundary TF Sp8 affects adult fate choice, and how TF specificity might be achieved in nascent MNs born in the spinal cord. In particular, I examine the role of Sp8, in motor neuron cell type choice. I also investigate candidate co TFs that could generate cell type enhancer specificity. In Chapter 2 I describe a novel function for a domain boundary setting TF, Sp8, in specifying adult MN type. In this chapter I leverage in vitro MN differentiation models to examine KO and overexpression lines to define the action of this TF. In addition to its ability to repress dorsal P3 domain this TF also represses the formation of cranial MNs in favor of skeletal MNs and is able to induce skeletal MN TFs. In Chapter 3 I describe a series of experiments that dissect enhancer TF motif content in a search for a TF that, along with known MN TFs, differentiates between active and inactive enhancers bound by the MN TFs Isl1 and Lhx3. Though neither Klf investigated plays this role, their true role in gene regulation in MNs remains obscure. Though the data are preliminary, the motifs investigated in this chapter seem to have some measure of control over enhancer activity. Overall this thesis paints a picture of novel activity for Sp8, separate from its defined minor function in the developing ventral spinal cord, opening new ways to understand cell fate regulation in the ventral spinal cord. It also rules out two candidate MN master TF co-regulators (Klf6 and Klf7), while suggesting the importance of their binding motifs for enhancer function. I propose that the Sp/Klf family of TFs have novel roles in MN gene regulation.

The cellular slime mold, Dictyostelium discoideum, provides an ideal model system to study eukaryotic development, cell differentiation, and aging. A crucial developmental event in Dictyostelium is glycogen degradation. The degradation of glycogen provides glucose monomers that are used to synthesize structural components necessary for cellular differentiation. Glycogen degradation is catalyzed by glycogen phosphorylase, and two developmentally regulated glycogen phosphorylase activities have been discovered in Dictyostelium. Glycogen phosphorylase 1 (gp-1) activity is predominant early in development, and is dependent upon 5’ AMP as a positive allosteric modifier; glycogen phosphorylase 2 (gp-2) activity peaks late in development and is independent of 5° AMP. I showed that these two glycogen phosphorylase activities are associated with unique proteins that are the products of two distinct, but related, genes. Both genes were observed to be typical Dictyostelium genes in a number of respects. The gp-1 and gp-2 enzymes were also found to be similar to glycogen phosphorylases from other organisms. I also examined the developmental expression of these genes and found that both mRNAs are developmentally regulated; gp-1 mRNA levels fluctuate during development, while gp-2 mRNA levels increase late in development. The expression of the gp-1 and gp-2 genes is regulated by exogenous cAMP. Exogenous cAMP enhances the level of gp-1 mRNA, apparently through a mechanism that requires intracellular cAMP signaling. Specific DNA sequence elements appear to be required for maximal vegetative and late developmental expression of gp-1. Exogenous cAMP induces the appearance of gp-2 mRNA via a mechanism that appears to be independent of intracellular cAMP signaling. Repeated TA-rich sequences located between nucleotides 193 and 305 upstream of the transcriptional start site are necessary for maximal cAMP induction of gp-2. I also examined the cell type specific expression of gp-1 and gp-2. gp-1 is expressed predominantly in pre-stalk cells. gp-2 1s expressed in both cell types in a temporally regulated fashion; this type of expression has not been reported for other Dictyostelium genes, but, given the importance of glycogen degradation in both stalk and spore cells, it is not inconceivable that such regulation 1s necessary. / Ph. D.

Part of work presented in this thesis has been published: Regulation of gene expressions by GC-rich DNA cis-elements / J.P. Hapgood, J. Riedemann and S.D. Scherer in Cell biology international, vol. 25, 2001. / Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: The sea urchin G-string binding factor 1 (suGF1) has previously been shown to bind with high affinity and selectivity to stretches of contiguous deoxyguanosine residues, a DNA motif found in the upstream regions of many unrelated genes from several organisms. It has been proposed that suGF1 plays a role in transcriptional regulation. Homopurine.homopyrimidine stretches have been shown to form unusual DNA structures, in vitro. To investigate the potential of the suGF1 binding site to form unusual structures under certain conditions, synthetic oligodeoxyribonucleotides containing the suGF1 poly(dG).(dC) binding site were subjected to circular dichroism (CD) analyses. The CD results indicate that the suGF1 binding site forms a mixture of unusual DNA structures, as deduced by comparison with the spectra obtained for B-DNA, triplex and quadruplex conformations. These results are consistent with the hypothesis that suGF1 specifically recognises G-strings that exhibit unusual structures. Exhaustive database searches showed that suGF1 has no significant homology with any previously identified proteins or cDNAs from any species. Given the relevance of mammalian models to medical science, and since no sea urchin cell lines are currently available, the identification of a mammalian functional homologue would facilitate determination of the in vivo function of such a potentially important, putative, novel DNAbinding protein in mammalian cell lines. In this study sequence analysis tools were used to identify hORFX, a putative human functional homologue of suGF1. Similarities in the domain organisation of the two proteins, prompted an investigation into the DNA-binding properties of hORFX, as well as a more detailed structure prediction analysis, with a view to determining whether hORFX is a functional homologue of suGF1. hORFX was successfully expressed in vitro, but lacked the ability to specifically bind G-strings. Theoretical predictions suggest that suGF1 has a DNA-binding domain belonging to a different family to that predicted for hORFX, consistent with differences in their respective DNA-binding specificities. suGF1 and hORFX were predicted to have helix-turn-helix and helix-loop-helix DNA-binding domains, respectively. Taken together the results do not support the hypothesis that hORFX is a suGF1 homologue. To date, no direct evidence for the in vivo function of suGF1 has been obtained. With a view to performing transactivation assays in the future, the expression of suGF1 in yeast was investigated in this project. An suGF1 expression construct was engineered and transformed into a protease-deficient yeast strain. Nuclear extracts were prepared and subjected to SOS-PAGE and electrophoretic mobility shift assays (EMSAs). suGF1 was shown to be successfully expressed in yeast cells and exhibited similar G-string-binding properties to that of native and in vitro transcribed and translated (IVT) suGF1. The suGF1 eDNA was also subjected to in si/ico expression, which together with the SDSPAGE results of yeast nuclear extracts and IVT suGF1, indicated that the protein might be expressed as multiple truncated products, due to the utilisation of multiple AUG translation start sites. These in vitro results are crucial for the ultimate outcome and correct interpretation of future transactivation experiments and lay the foundation for further investigation into the possible role of suGF1 in transcriptional regulation. / AFRIKAANSE OPSOMMING: In die verlede is bewys dat die seepampoentjie G-string-bindende faktor (suGF1) hoë affiniteit en spesifisiteit vir aaneenlopende volgordes van deoksiguanosien residue besit. Hierdie DNA motief kom algemeen voor in die stroom-op gebiede van verskeie gene in verskillende organismes. Daar is 'n veronderstelling dat suGF1 betrokke is by die regulering van geenuitdrukking. Vroeër is bewys dat homopurien.homopirimidien-ryke areas die vermoë besit om in vitro ongewone DNA-strukture te vorm. Die potentiaal van die suGF1-bindingsetel om ongewone DNA-strukture te vorm is gevolglik deur sirkulêre dikroïsme (SD) analise ondersoek. Vergelyking van die spektra vir B-DNA-, tripleks- en kwadrupleks-strukture met dié van die suGF1-bindingsetel, toon duidelik dat laasgenoemde 'n mengsel van ongewone DNA konformasies, onder die spesifieke eksperimentele omstandigehede, aanneem. Deeglike inspeksie van die beskikbare geen- en proteïendatabasisse vir alle spesies het aangetoon dat suGF1 geen merkbare kDNA- of proteïenhomoloë besit nie. As gevolg van die belang van soogdiermodelsisteme in die mediese wetenskappe, asook die onbeskikbaarheid van seepampoentjie-sellyne, is 'n soektog na 'n funktionele suGF1 homoloog in soogdiere geloods. Die ontdekking van só 'n homoloog sal dit moontlik maak om die rol van hierdie potensiaal belangrike en unieke DNA-bindingsproteïen te ondersoek. Tydens hierdie soektog is spesiale analise-programme gebruik en 'n potensiële menshomoloog van suGF1, hORFX, is geïdentifiseer. Die mees prominente ooreenkoms tussen die twee proteïene is die soortgelyke rangskikking van funksionele motiewe. Gevolglik is die DNA-bindings eienskappe van die hORFX-proteïen ondersoek, insluitende 'n detaileerde struktuur-funksie-voorspelling ten einde vas te stel of dit wél 'n homoloog van suGF1 is. hORFX is suksesvol uitgedruk in vitro, maar besit nie die vermoë om dieselfde G-string waaraan suGF1 spesifiek bind te herken nie. Teoretiese analise het voorspel dat suGF1 en hORFX aan verskillende DNA-bindings proteïen-families behoort, aangesien suGF1 'n heliks-draai-heliks en hORFX 'n heliks-lus-heliks motief bevat. Hierdie inligting, tesame met die eksperimentele resultate, dui aan dat hORFX nie 'n homoloog van suGF1 is nie. Tot op hede is daar niks bekend aangaande suGF1 se funksie in vivo nie. Met die oog op transaktiveringseksperimente in die toekoms, is die ekspressie van suGF1 in gisselle tydens hierdie navorsingsprojek ondersoek. 'n suGF1 ekspressievektor is berei en gebruik om 'n protease-negatiewe gissellyn te transformeer. Kernekstrakte is ondersoek deur SDS-PAGE en elektroforetiese mobiliteitsessais. Daar is gevind dat suGF1 suksesvol uitgedruk is in die gisselle. Die rekombinante suGF1 besit G-volgorde bindingsaktiwiteite soortgelyk aan dié van suGF1 in kernekstrakte van seepampoentjies, asook in vitro getranskribeerde-en getransleerde suGF1. Die kDNA vir suGF1 is ook in silico uitgedruk. Tesame met die SDS-PAGE-resultate het laasgenoemde aangetoon dat die suGF1-kDNA veelvuldige AUG-kodons bevat vir die inisiasie van proteïentranslasie. Dit lei moontlik tot die translasie van 'n reeks proteïenprodukte wat verkort is aan die N-terminale kant, afgesien van die volledige suGF1-proteïen. Die in vitro resultate in geheel is essensieel vir die toekomstige uitvoering en interpretasie van transaktiveringseksperimente. Hierdie projek lê gevolglik die fondasie vir 'n verdere ondersoek na die rol van suGF1 in die regulering van geenuitdrukking.

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Master / Master of Philosophy