Juvenile hormone (JH) is an important insect hormone that controls diverse biological processes in postembryonic development and adult reproduction. JH exerts its effects through the nuclear receptor Methoprene-tolerant (MET). MET is a transcription factor of the basic helix-loop-helix (bHLH)/Per-Arnt-Sim (PAS) family. In the presence of JH, MET forms a heterodimer with its DNA-binding partner Taiman (TAI). The MET-TAI complex directly binds to the regulatory regions of some JH target genes and regulates their transcription. However many questions remain unanswered regarding the JH-regulated gene expression. The work in this report aims to determine the role of protein kinase C (PKC) in JH signaling in adult mosquitoes and to find the direct target genes of Krüppel homolog 1 (Kr-h1), a zinc finger transcription factor encoded by a JH early response gene. We discovered that PKC is an essential component of a membrane-initiated JH signaling pathway. PKC was activated by JH in a phospholipase C (PLC)-dependent manner. Inhibition of PKC activity dramatically decreased the JH-induced gene expression. RNAi experiment indicated that several PKC isoforms were involved in the JH action in adult female mosquitoes. We showed that PKC modulated the transactivation activity of MET by enhancing the binding of MET and TAI to the promoters of JH target genes. Kr-h1 is rapidly upregulated by JH in newly emerged mosquitoes. RNAi-mediated depletion of AaKr-h1 caused a substantial decrease in oviposited eggs, indicating that this protein plays an essential role in mosquito reproduction. We combined chromatin immunoprecipitation (ChIP) with cloning of the generated DNA and have identified chromatin binding sites of AaKr-h1 in Aedes aegypti. After adult emergence, binding of AaKr-h1 to its in vivo targets increased with the JH-induced increase in AaKr-h1. Interestingly, depletion of AaKr-h1 in newly emerged mosquitoes led to considerable upregulation of some AaKr-h1 target genes but downregulation of other target genes. The results suggest that AaKr-h1 acts downstream of AaMET to regulate gene expression in response to JH and that AaKr-h1 can activate or repress the expression of individual target gene. / Ph. D.

Sarcocystis neurona is an apicomplexan parasite that is a major cause of equine protozoal myeloencephalitis (EPM). During intracellular development of S. neurona, many genes are temporally regulated. To better understand gene regulation, it is important to identify and characterize regulatory elements controlling gene expression in S. neurona. To perform this study, it was essential to establish transfection system for this parasite. Hence, the 5 flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules -galactosidase (-gal) and yellow fluorescent protein (YFP) were expressed by electroporated S. neurona, thereby confirming the feasibility of performing molecular genetic experiments in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of T. gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express -gal and YFP. These transgenic clones were shown to be useful for analyzing growth rate of parasites in-vitro and for assessing drug sensitivities. To uncover possible sequence elements involved in promoter activity, the 5 flanking regions of five S. neurona genes were subjected to comparative analysis. This revealed the presence of a 7-base conserved motif GCGTCTC. Using a dual luciferase assay system, the SnSAG1 promoter was subjected to functional analysis. The motif GAGACGC located between -136 and -129 upstream of the transcription start site was found to be essential for SnSAG1 expression. This motif functions in an orientation dependent manner and was shown to play a role in binding nuclear proteins of S. neurona.

In the last decades, the explosion of data from quantitative techniques has revolutionised our understanding of biological processes. In this scenario, advanced statistical methods and algorithms are becoming fundamental to decipher the dynamics of biochemical mechanisms such those involved in the regulation of gene expression. Here we develop mechanistic models and approximate inference techniques to reverse engineer the dynamics of gene regulation, from mRNA and/or protein time series data. We start from an existent variational framework for statistical inference in transcriptional networks. The framework is based on a continuous-time description of the mRNA dynamics in terms of stochastic differential equations, which are governed by latent switching variables representing the on/off activity of regulating transcription factors. The main contributions of this work are the following. We speeded-up the variational inference algorithm by developing a method to compute a posterior approximate distribution over the latent variables using a constrained optimisation algorithm. In addition to computational benefits, this method enabled the extension to statistical inference in networks with a combinatorial model of regulation. A limitation of this framework is the fact that inference is possible only in transcriptional networks with a single-layer architecture (where a single or couples of transcription factors regulate directly an arbitrary number of target genes). The second main contribution in this work is the extension of the inference framework to hierarchical structures, such as feed-forward loop. In the last contribution we define a general structure for transcription-translation networks. This work is important since it provides a general statistical framework to model complex dynamics in gene regulatory networks. The framework is modular and scalable to realistically large systems with general architecture, thus representing a valuable alternative to traditional differential equation models. All models are embedded in a Bayesian framework; inference is performed using a variational approach and compared to exact inference where possible. We apply the models to the study of different biological systems, from the metabolism in E. coli to the circadian clock in the picoalga O. tauri.

<p>Gene regulation is a complex and tightly controlled process that defines cell function in physiological and abnormal states. Programmable gene repression technologies enable loss-of-function studies for dissecting gene regulation mechanisms and represent an exciting avenue for gene therapy. Established and recently developed methods now exist to modulate gene sequence, epigenetic marks, transcriptional activity, and post-transcriptional processes, providing unprecedented genetic control over cell phenotype. Our objective was to apply and develop targeted repression technologies for regenerative medicine, genomics, and gene therapy applications. We used RNA interference to control cell cycle regulation in myogenic differentiation and enhance the proliferative capacity of tissue engineered cartilage constructs. These studies demonstrate how modulation of a single gene can be used to guide cell differentiation for regenerative medicine strategies. RNA-guided gene regulation with the CRISPR/Cas9 system has rapidly expanded the targeted repression repertoire from silencing single protein-coding genes to modulation of genes, promoters, and other distal regulatory elements. In order to facilitate its adaptation for basic research and translational applications, we demonstrated the high degree of specificity for gene targeting, gene silencing, and chromatin modification possible with Cas9 repressors. The specificity and effectiveness of RNA-guided transcriptional repressors for silencing endogenous genes are promising characteristics for mechanistic studies of gene regulation and cell phenotype. Furthermore, our results support the use of Cas9-based repressors as a platform for novel gene therapy strategies. We developed an in vivo AAV-based gene repression system for silencing endogenous genes in a mouse model. Together, these studies demonstrate the utility of gene repression tools for guiding cell phenotype and the potential of the RNA-guided CRISPR/Cas9 platform for applications such as causal studies of gene regulatory mechanisms and gene therapy.</p> / Dissertation

Les protéines des groupes Polycomb et trithorax sont des régulateurs épigénétiques très conservés qui permettent le maintient de l'identité cellulaire en régulant le niveau d'expression des gènes. Ils agissent sur leurs gènes cibles à travers des éléments régulateurs en cis, appelés éléments de réponse aux Polycombs (PRE). Dans des tests transgéniques, il a été montré que deux copies du même PRE sont fréquemment regroupés dans la même région nucléaire. Dans le cas particulier du PRE Fab-7, ce regroupement corrèle avec sa fonction répressive. Durant ma thèse, j'ai tenté de cloner un outil bicolore qui permet la visualisation en 4D de deux PRE Fab-7 stablement intégrés dans le génome de Drosophila melanogaster. De plus, j'ai amélioré le protocole de DNA-FISH du labo. Ceci m'a permis d'identifier vestigial et apterous comme étant des loci qui forment des associations nucléaires, de façon dépendante de la transcription, dans Drosophila melanogaster. / Polycomb- and trithorax-Group proteins are highly conserved epigenetic regulators which maintain cell identities by maintaining states of gene expression. They act on their target genes through /cis/ regulatory elements, named Polycomb Response Elements (PREs). In transgene assays it has been shown that two copies of the same PRE are frequently found clustered in nuclear space and for one particular PRE named Fab-7 clustering is correlated with its repressive function. In the course of this thesis I tried to clone a two colour real-time tool which allows distinguishing in 4D two /Fab-7/s stably integrated into the genome of Drosophila melanogaster. Additionally, I improved the DNA-FISH protocol of the lab and identified vestigial and apterous as potential gene loci forming nuclear associations dependent on transcription in Drosophila melanogaster.

This dissertation sheds light on several novel mechanistic findings in astrocyte specific gene regulation and function by the NFI-X transcription factor which can be further extrapolated to astrocyte differentiation and glial tumor invasion. First, we cloned and analyzed human NFI-X3, a novel splice variant of the nfix gene, which contains a unique transcriptional activation (TA) domain completely conserved in primates. In contrast to previously cloned NFI-X1, overexpression of NFI-X3 potently activates NFI reporters, including GFAP reporter, in astrocytes and glioma cells. The expression of NFI-X3 is dramatically upregulated during the differentiation of neural progenitors to astrocytes and precedes the expression of astrocyte markers such as GFAP and SPARCL1. Overexpression of NFI-X3 dramatically upregulates GFAP and SPARCL1 expression in glioma cells, while the knockdown of NFI-X3 diminishes the expression of both GFAP and SPARCL1 in astrocytes. Although activation of astrocyte-specific genes involves DNA demethylation and subsequent increase of histone acetylation, the TA domain of NFI-X3 activates GFAP expression by inducing alteration in the +1 nucleosome architecture that lead to the increased recruitment of RNA polymerase II. Thus, we propose that NFI-X3 is the major isoform of NFI-X regulating astrocyte specific gene expression during their differentiation, likely via nucleosomal remodeling of the astrocyte specific promoters. NFI-X knock-out animals display severe neuroanatomical defects including partial agenesis of the corpus callosum and hydrocephalus, however the target genes of NFI-X in the CNS remained elusive. Here, we show for the first time that YKL-40 is a novel target gene of NFI-X in astrocytes and controls their migration. In addition, we report that YKL-40 expression is activated during mouse brain development and also during the differentiation of neural progenitors into astrocytes in vitro. In primary astrocytes, YKL-40 expression is controlled by nuclear factor I-X (NFI-X) and signal transducer and activator of transcription 3 (STAT3), which are known to regulate gliogenesis. Indeed, knock-down of NFI-X and STAT3 significantly reduced YKL-40 expression in astrocytes, while overexpression of NFI-X3 (a splice isoform of NFI-X) dramatically induced YKL-40 expression in glioma cells. In addition, activation of STAT3 by oncostatin M induced YKL-40 expression in astrocytes. Interestingly, STAT3 activated YKL-40 expression via its binding site located in the YKL-40 proximal promoter, whereas direct NFI-X binding had not been detected. Surprisingly, NFI-X and STAT3 physically interact and this complex likely regulates YKL-40 expression in astrocytes. We further show that NFI-X controls migration and invasion of astrocytes and glioma cells, respectively, by regulating YKL-40 expression. These novel data suggest that YKL-40 is expressed by astrocytes during brain development and controls astrocyte migration. Since YKL-40 is used as a shared biomarker for ongoing inflammation and oncogenic transformation and its (high) levels correlate to the severity of disease, we have tested its expression in astrocytes and microglia (CNS macrophage) after treatment of various neuro-inflammatory cytokines. Here we report, that IL-1 and IL-6/OSM synergistically activate YKL-40 expression in astrocytes but not in microglia when added together. Furthermore, induced YKL-40 expression can be detected in the media from astrocytes but not from microglia. Since YKL-40 is a secreted molecule and is highly upregulated in CSF of multiple sclerosis patients, we have tested its role in oligodendrocyte differentiation. Preliminary observations clearly demonstrate that YKL-40 inhibits myelin basic protein (MBP) expression during the in-vitro differentiation of oligodendrocyte progenitor cells into myelin producing oligodendrocytes. Thus, we propose that YKL-40 is produced and secreted by reactive astrocytes during various CNS pathologies, and may inhibit MBP expression in MS. In summary, these studies have identified novel mechanisms in astrocyte gene regulation and functions, and provided new insights into astrocyte biology, with the implications for further understanding of the development and progression of CNS pathology.

The chicken embryonic β-type globin gene, ρ, is silenced on day five of embryogenesis. Concomitant with this silencing is methylation of cytosine residues in the promoter and proximal transcribed region of the gene, which is first detected on day seven and is complete in adult cells. Once methylated, expression of the gene cannot be induced unless the methylation is removed by treatment of cells with Sazacytidine. Therefore ρ-globin is a member of a small group of genes whose normal developmentally regulated expression is mediated at least in part by DNA methylation.A methyl-DNA binding complex, termed the MeCPC (Erythroid Methyl Cytosine-binding Protein Complex), has been found to bind to the methylated, but not unmethylated, ρ-globin promoter and proximal transcribed region in nuclear extracts from definitive erythrocytes. This complex has a stronger binding affinity for its cognate binding sequence, the methylated ρ-globin proximal transcribed region (M-ρ248), than for an artificial 5-methylcytosine-rich sequence (M-CG11).To define the components of the MeCPC, we developed two chromatographic procedures to purify the complex from adult chicken red blood cell nuclear extracts (Purification Strategies I and II). Mass spectrometry was performed on the MeCPC obtained by Purification Strategy I and proteins were identified by a novel application of peptide mass fingerprint data fitting. Four components of the previously-purified MeCPl transcriptional repression complex were identified in the sample: MBD2, RbAp48, HDAC2 and MTA1. Another identified protein, MENT, is a factor expressed only in chicken hematopoietic cells. These five proteins, as well as the MeCPl component Mi2, were found to tightly coelute by Western blotting of gel-filtration fractions from Purification Strategy II. Therefore, we conclude that these five proteins are components of the MeCPC.To confirm that MBD2 is associated with the ρ-globin gene in vivo, we perfomed the chromatin immunoprecipitation assay using anti-MBD2 antibodies. In adult erythrocytes, significant enrichment for MBD2 is seen at the transcriptionally inactive ρ-globin gene, but no enrichment is observed at the transcriptionally active βA globin gene. These experiments confirm that MBD2 binds to the methylated p-globin gene in adult chicken erythroid cells.

Human (h) growth hormone (GH) levels decline rapidly in response to excess caloric intake before there is any evidence of obesity. In this thesis, the mechanism underlying this response was examined by manipulating levels of caloric intake and physical activity at the levels of gene expression and chromosomal structure. Transgenic mice containing the intact hGH locus were used as a model system. Briefly, the findings are: (I) High caloric intake (high fat diet) for three days resulted in hyperinsulinemia and a decrease in both hGH synthesis and secretion. (II) Incorporation of physical activity (swim) muted the effects of excess caloric intake on insulin levels as well as hGH production. (III) Human GH RNA accumulation was shown for the first time to be negatively regulated by insulin in pituitary cultures, and (IV) an enhancer box (E-box) DNA element was implicated in this response. (V) Induction of the E-box associated transcription factor HIF-1α with insulin significantly decreased hGH RNA levels, and was accompanied by recruitment of HIF-1α to the hGH gene (hGH) promoter in situ. (VI) Both a reduction in HIF-1α synthesis and HIF-1 DNA binding blunted the negative effect of insulin on hGH RNA levels. (VII) The hGH response to insulin was associated with a decrease in histone H3/H4 hyperacetylation in the proximal hGH promoter region. The same pattern of chromatin remodelling was observed in pituitary cells in vivo in response to excess caloric intake. (IX) Increased recruitment of nuclear receptor co-repressor and decreased association of RNA polymerase II were also observed. Collectively, these effects are consistent with reduced hGH promoter function. (X) This reduction by excess caloric intake was also consistent with changes in the three dimensional-structure of the hGH locus including detected loss of physical interaction between hGH enhancer and promoter regions. (XI) By contrast, physical activity combined with the high caloric intake preserved the chromosomal structure of the hGH locus. These observations are discussed in relation to a physiological requirement for rapid control of hGH levels in response to energy homeostasis, as well as the molecular basis governing this process. / May 2015

Student Number : 9503239E - PhD thesis - Faculty of Science / Malaria is one of the most debilitating pathogenic infections known to man, responsible for approximately three million deaths annually, primarily children in sub-Saharan Africa. The parasite has evaded multiple attempts at eradication, predominantly through the complexity of its life cycle, the ability to elude host immune response, and gametocyte formation to ensure dissemination. The recent completion of the genome sequence has opened up a multitude of avenues for exploration and identification of novel drug and vaccine targets, as well as providing a glimpse into the complex mechanisms that have contributed to the success of this pathogen. The mechanisms of gene regulation, especially those governing gametocytogenesis, have, however, not yet been elucidated. In this research, differential display has been used to identify some of the genes that are differentially expressed between the asexual parasite and gametocyte stages of P. falciparum. Numerous genes involved in diverse aspects of metabolism, protein synthesis and immune evasion were identified. A combination of BLASTN and BLASTX similarity searches was used to categorize and increase the confidence with which a transcript could be identified. Expression data for confidently identified genes were confirmed using reverse slot blot and available microarray data. PfMyb2, a novel transcription factor which may regulate genes involved in gametocytogenesis, was characterized. The DNA binding domains of the protein were cloned and expressed as a histidine fusion protein. Mobility shift assays were used to assess the in vitro binding capability of the recombinant 6xHis-PfMyb2, which bound to oligonucleotides containing the consensus Myb regulatory element. Two of the oligonucleotides represent sequences located within promoters of P. falciparum genes (Pfcrk1 and Pfmap1) known to play a role in regulating the cell cycle, a function ascribed to many members of the vertebrate Myb family. The identification of PfMyb2 as a bona fide transcription factor is a first step into gaining some insight into the many regulatory processes that occur during the life cycle of this complex organism. A better understanding of the molecular mechanisms that govern its survival is essential for the ultimate eradication of this deadly parasite.

Thesis advisor: Michelle M. Meyer / Bacteria commonly utilize cis-acting mRNA structures that bind specific molecules to control gene expression in response to changing cellular conditions. Examples of these ligand-sensing RNA cis-regulators are found throughout the bacterial world and include riboswitches, which interact with small metabolites to modulate the expression of fundamental metabolic genes, and the RNA structures that bind select ribosomal proteins to regulate entire ribosomal protein operons. Despite advances in both non-coding RNA discovery and validation, many predicted regulatory RNA motifs remain uncharacterized and little work has examined how RNA cis-regulators behave within their physiological context in the cell. Furthermore, it is not well understood how structured RNA regulators emerge and are maintained within bacterial genomes. In this thesis, I validate the biological function of a conserved RNA cis-regulator of ribosomal protein synthesis previously discovered by my group using bioinformatic approaches. I then investigate how bacteria respond to the loss of two different cis-regulatory RNA structures. Using Bacillus subtilis as a model organism, I introduce point mutations into the native loci of the ribosomal protein L20-interacting RNA cis-regulator and the tandem glycine riboswitch and assay the strains for fitness defects. I find that disrupting these regulatory RNA structures results in severe mutant phenotypes, especially under harsh conditions such as low temperatures or high glycine concentrations. Together, this body of work highlights the advantages of examining RNA behavior within its biological context and emphasizes the important role RNA cis-regulators play in overall organismal viability. My studies shed light on the selective pressures that impact structured RNA evolution in vivo and reinforce the potential of cis-regulatory RNAs as novel antimicrobial targets. / Thesis (PhD) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.