L’Ostéoarthrose (OA) est une maladie articulaire entrainant une dégénérescence du cartilage et une ossification de l’os sous-chondral. Elle touche un Canadien sur 10 et pourtant l’origine de cette pathologie est encore inconnue. Dans le cadre de ce projet, la contribution de deux facteurs de transcription, NFAT1 et PITX1, dans la régulation transcriptionnelle du promoteur d’IHH a été examiné compte tenu de l’implication potentielle de la voie hedgehog (Hh) et de ces facteurs dans la pathogenèse de l’OA. La voie de signalisation Hh régule la croissance et la différenciation des chondrocytes. Indian hedgehog (IHH), l’un des trois membres de la famille Hh, contrôle leur prolifération et leur différenciation. / Osteoarthritis (OA) is the most common joint disorder and is characterized by cartilage degradation and endochondral ossification. One in every ten Canadians is affected, yet its aetiopathogenesis remains unknown. In this present study, two new regulators of the IHH promoter, NFAT1 and PITX1, were studied. The downregulation of IHH expression by these factors could contribute to the OA pathogenesis. The Hedgehog (Hh) signaling pathway regulates chondrocyte growth and differentiation in the growth plate. Indian hedgehog (IHH), one of its members, stimulates chondrocyte proliferation and osteoblast differentiation. IHH is essential in skeletogenesis, osteoblastogenesis and cartilage growth.

Alginate overproduction by Pseudomonas aeruginosa, which causes a mucoid phenotype, is a major virulence factor associated with chronic pulmonary infections in cystic fibrosis patients. Expression of the algD operon for alginate biosynthesis requires three major regulators in association with the ECF sigma factor, σ22, in mucoid strains that are typically defective in anti-sigma factor, MucA. One such algD regulator is AlgB, a member of the NtrC family of two-component systems, which typically utilize σ54. However, neither σ54 nor the cognate sensor kinase (KinB) of AlgB are required for algD expression in such mucoid strains. I hypothesized that KinB-phosphorylated AlgB must play some role in gene regulation, and so I sought to construct a constitutively active AlgB that simulated kinase-phosphorylation. I took a predictive approach and genetically introduced substitutions in AlgB that had been shown to activate DctD, a close homologue of AlgB in Rhizobium (52). When one such substitution, AlgBE125K, was transferred to a nonmucoid P. aeruginosa PAO ΔalgB-kinB (JK159) strain, alginate overproduction was observed. Interestingly, introduction of an algT mutation to remove σ22 did not block alginate production induced by AlgBE125K; although, it did stimulate the production of alginate in the presence of AlgBwt in trans to similar levels induced by the constitutive mutant. In contrast, introduction of an rpoN mutation showed that alginate production mediated by AlgBwt and AlgBE125K was σ54 dependent. The increase in expression of alginate by AlgBwt in the presence of σ54 and the absence of σ22 suggested a competition between the sigma factors for binding to PalgD. Biochemical assays were conducted to assess the constitutive property of AlgBE125K. For the ATPase assay, an equivalent amount of ATP hydrolysis was observed between the mutant and the wild type AlgB proteins. Slight differences seen for the EMSA data suggested possible higher order complex formation for AlgBE125K compared to AlgBwt. Collectively, these results suggested that in wild-type (MucA+) P. aeruginosa, expression of the algD operon is dependent on the phosphorylation of AlgB by KinB in a typical two-component fashion that is triggered by some as yet unknown environmental stimulus.

Hemoglobinopathies are some of the most common monogenic disorders in the world, affecting millions of people and representing a growing burden on health systems worldwide. Although the pathophysiology of sickle cell anemia and beta-thalassemia, two of the most common hemoglobinopathies, have been the focus of much research over the last century, patients affected by these diseases still lack a widely applicable and easily available cure. Sickle cell anemia and beta-thalassemia are caused by defects in the structure and production of the beta-globin chains that, along with the alpha-globin chains make up the heterotetrameric hemoglobin molecule. Studies geared towards re-expression of the silenced fetal gamma-globin gene in adult erythroid cells as a therapeutic strategy to alleviate the symptoms of beta-globin deficiencies have met with some success for the treatment of sickle cell anemia but not for beta-thalassemia. A better understanding of normal gamma-globin gene regulation will undoubtedly advance the development of more effective therapeutic strategies. Because many of the potential targets that may be modulated to achieve gamma-globin re-expression also have functions in erythroid cells other than regulating the gamma-globin gene, it is imperative to understand their role in all aspects of erythropoiesis before they are used for therapy. The current study focuses on the role of two Krüppel-like transcription factors, KLF1 and KLF2, which have known roles in the processes of primitive and definitive erythropoiesis as well as globin gene regulation. The regulation of primitive erythropoiesis by KLF1 and KLF2 is studied using the mouse as a model system because it is not possible to study primitive erythropoiesis in humans. Previous studies have shown that KLF1 and KLF2 are essential for and have overlapping roles in primitive erythropoiesis. Simultaneous ablation of KLF1 and KLF2 results in a severely anemic embryonic phenotype that is not evident in KLF1 or KLF2 single knockout embryos. In this study, we show that this anemia is caused by a paucity of blood cells, and exacerbated by diminished beta-like globin gene expression. The anemia phenotype is dose-dependent, and interestingly, can be ameliorated by a single copy of the KLF2, but not the KLF1 gene. The roles of KLF1 and KLF2 in maintaining both normal peripheral blood cell numbers and globin mRNA amounts are erythroid cell-specific. It was discovered that KLF2 has an essential function in erythroid precursor maintenance. KLF1 can partially compensate for KLF2 in this role, but is uniquely crucial for erythroid precursor proliferation, through its regulation of G1- to S-phase cell cycle transition. A more drastic impairment of primitive erythroid colony formation from embryonic progenitor cells occurs with simultaneous deficiency of KLF1 and KLF2, than with loss of a single factor. The regulation of human beta-like globin gene expression is studied using a recently developed in vitro system for the production of erythroid cells from umbilical cord blood hematopoietic precursor cells, representing a more “fetal” model of globin gene expression. Previous studies have shown that KLF1 binds to the promoters of the gamma- and beta-globin genes, while KLF2 binds to the promoter of the gamma-globin gene in cord blood-derived erythroid cells. Studies using transgenic mice carrying the entire human beta-globin locus had indicated that KLF1 and KLF2 positively regulate gamma-globin expression in mouse embryonic erythroid cells. We demonstrate in this study that KLF1 appears to have dual roles in the regulation of gamma-globin expression in human cord blood-derived definitive erythroid cells. Partial depletion of KLF1 causes elevated gamma-globin expression, while nearly complete depletion of KLF1 results in a down-regulation of gamma-globin expression. Of particular interest was the observation that KLF2 positively regulates gamma-globin expression in cord blood-derived erythroid cells. Surprisingly, KLF2 also positively regulates beta-globin expression in these cells. If regulation of gamma-globin by KLF2 proves to be a direct effect, KLF2 will join a very small group of factors known to directly activate gamma-globin expression.

Lysophosphatidic acid (LPA) is a potent bioactive phospholipid mediator that functions through multiple G protein couple receptors (GPCRs). LPA is elevated in ascites of ovarian cancer patients and is involved in growth, survival and metastasis of ovarian cancer cells. Gene promoter analyses revealed that some LPA-target genes share similar sets of binding sites for prominent transcription factors posing the possibility of a general mechanism for activation of their expression by LPA. Detailed investigation of the mechanisms of regulation of cyclooxygenase 2 (Cox-2), a paradigm of LPA-regulated genes, showed that LPA robustly upregulated the expression of Cox-2 in ovarian cancer cells through multiple receptors. LPA induced rapid increase in Cox-2 mRNA and significantly enhanced the stability of Cox-2 transcript with the support of mRNA binding protein HuR. The effects of LPA on Cox-2 transcriptional activation include essential involvement of transcription factor, C/EBP-b. Further studies on mechanisms of activation of C/EBP-b demonstrated that LPA increased phosphorylation, binding and transcriptional activities of C/EBP-b. In addition, activation of C/EBP-b and LPA-target genes required contribution from EGFR. This novel crosstalk between LPA GPCRs and EGFR in mediating transcription factors activation was further explored by investigating the mechanisms of activation of AP-1 and NF-kB by LPA. Activation of AP-1 family of proteins by LPA relied heavily on basal inputs from EGFR as inhibition of EGFR kinase activity with AG1478 caused significant loss of LPA-induced AP-1 expression, binding and transcription activities. Although HGF and other agonists of RTK only weakly stimulate LPA-target genes and transcription factors in ovarian cancer cells, costimulation with HGF in the presence of AG1478 restored LPA signals to both C/EBP-b and AP-1. This suggests an obligatory role for a RTK in LPA-induced transcriptional activation, not necessarily inputs from EGFR. Interestingly, inhibition of EGFR with AG1478 did not interfere with LPA-induced NF-kB activation. Pharmacological inhibition and molecular targeting revealed that only a subset of G proteins participate in the crosstalk between LPA receptors and EGFR. Collectively, these results demonstrate the presence of at least two signals downstream of LPA receptors: one dependent on basal RTK activity and another mediated directly by LPA GPCRs.

Transcription of human protein-coding genes and most small nuclear RNA genes is mediated by RNA Polymerase II (Pol II). During a cycle of transcription, Pol II recruits a variety of factors that facilitate transcription elongation, RNA processing and termination, through its long, unstructured C-terminal domain (CTD). The CTD in humans comprises 52 tandem heptapeptide repeats with the consensus sequence Y₁S₂P₃T₄S₅P₆S₇. Each amino acid of the heptapeptide can be chemically modified, which influences the recruitment of other protein factors to the transcription machinery. Not all enzymes that modify the CTD have been discovered. Recent studies have identified a novel CTD phosphatase: RPAP2 in humans and its yeast homologue Rtr1, which dephosphorylate phospho-Ser5 of the heptapeptide repeats. RPAP2 has been shown to stimulate 3' end cleavage of nascent snRNAs through recruitment of the Integrator complex, and unpublished work suggests the involvement of RPAP2 in regulating vertebrate developmental programs. However, the exact mechanisms that regulate the function of human RPAP2, and thus impact on CTD modification, are not well-understood. This thesis presents a novel mechanism whereby RPAP2 recruits protein phosphatase 1 (PP1) to snRNA genes, where PP1 is postulated to activate P-TEFb to phosphorylate Ser2 of the CTD. At the same time, P-TEFb may have a role in activating the phosphatase activity of RPAP2. Furthermore, RPAP2 itself is shown to be recruited to a number of gene promoters by the RPRD1A protein, which also stimulates its phosphatase activity. RPAP2 was shown to have another role in regulating transcription termination: by recruiting the Integrator complex, which is shown here to mediate termination of snRNA genes, and by a so far unknown mechanism on a long protein-coding gene. An attempt was made to purify and crystallise the human RPAP2 to obtain a crystal structure, however the crystallisation trials were not successful. Finally, a correlation was found in human embryonic stem cells and induced pluripotent stem cells between low levels of RPAP2 and high levels of CTD Ser5P, suggesting a potential involvement of RPAP2 in regulating transcription at a key developmental stage. The results presented here contribute to the understanding of human transcriptional mechanisms and the numerous interactions within the transcription machinery. In particular, the mechanism of terminating the transcription of snRNA genes is identified. An interesting possibility is the regulation of development and stem cell differentiation by RPAP2; however the exact pathways by which this occurs are yet to be discovered.

Cardiac fibrosis contributes to heart failure by dramatically impairing cardiac function, increasing patient morbidity and mortality. The primary fibrillar collagen expressed in the heart is type I, and increased collagen synthesis is the hallmark of cardiac fibrosis. Our laboratory has shown that the transcription factor scleraxis is sufficient to regulate the gene encoding collagen Iα2. The present thesis identifies and focuses on three key functions of scleraxis in the heart. First, we show that scleraxis is required for production of the cardiac extracellular matrix. Using in vitro and in vivo models, we observed a significant upregulation/reduction of matrix genes in response to induction/loss of scleraxis gene function respectively. In fact, scleraxis overexpression was sufficient to rescue matrix synthesis in scleraxis-null cells. In a murine model of cardiac pressure overload, scleraxis gene deletion blunted the induction of fibrotic collagen gene expression. Second, we provide evidence that scleraxis governs fibroblast-myofibroblast phenotype transition and fibroblast number. Scleraxis gene induction promoted cardiac myofibroblast phenoconversion while knockdown reduced myofibroblast marker gene expression. Scleraxis exerts direct transcriptional control on the a-smooth muscle actin gene-an established marker of myofibroblasts. Scleraxis null mice exhibited a dramatic reduction in cardiac fibroblast numbers- this is attributed to impairment of the epithelial-to-mesenchymal transition program which was marked by a corresponding loss of mesenchymal markers and increased epithelial markers. Loss-of-function experiments using primary cardiac proto-myofibroblasts recapitulated this paradigm, whereas scleraxis gene induction showed a reciprocal effect on mesenchymal markers. Third, data from this study supports the required role of scleraxis in the TGFb/Smad signaling pathway. Scleraxis is strongly upregulated by the potent pro-fibrotic cytokine TGFb, and works synergistically with the canonical Smad signaling pathway to increase Col1a2 expression by cardiac fibroblasts and myofibroblasts. Smad3 induced expression of the fibrillar collagens – an effect that was significantly attenuated following scleraxis knockdown. Smad3 binding to the Col1a2 gene promoter was significantly reduced in scleraxis null hearts. This study involved a comprehensive series of in vitro and in vivo experiments, and is the first to identify scleraxis as a key regulator of multiple fibroblast functions and a potential future target for therapeutic intervention in cardiac fibrosis. / May 2016

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Klára Dohnalová Supervisor: prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Inhibitory effect of SPA70 on hPXR activation This work focuses on pregnane X receptor (PXR) and its antagonists. PXR is a ligand-activated nuclear receptor that plays a major role in detoxification of xenobiotics and protecting the organism from their toxic effects. Recent evidence also shows endogenous action of PXR in the metabolism of lipids, glucose and bile acids. However, PXR activation could be harmful, since induction of biotransformation enzymes by PXR agonists may result in reduced treatment efficacy, increased toxicity of drug metabolites and resistance to chemotherapeutic agents. Recent research has been intensively focused on PXR antagonists capable of abolishing these unfavourable effects. Recently discovered human PXR antagonist SPA70 has a promising potential for future usage. In this study, we investigated the inhibitory effect of SPA70 on activated PXR. To activate PXR we used agonists binding directly to PXR (rifampicin, hyperforin, SR12813) and also agonists activating PXR indirectly via cell signalling pathways (U0126, PD184352, PD0325901). Experiments were performed using luciferase...

Although skeletal myogenesis is largely controlled by myogenic regulatory factors, epigenetic modifications have recently emerged as an essential regulatory mechanism of gene expression. Molecular regulation of stem cell differentiation is exerted through both genetic and epigenetic factors over distal enhancer regions. Understanding the mechanistic action of active or poised enhancers is therefore, imperative for the control of stem cell differentiation. Based on the genome-wide co-occurrence of different epigenetic marks in proliferating myoblasts, we have generated a chromatin state model to profile differentiation- and rexinoid-responsive histone acetylation in early myoblast differentiation. Here, we delineate the functional mode of transcription regulators during early myogenic differentiation using genome-wide chromatin state association. We define a role of transcriptional coactivator p300, when recruited by muscle master regulator MyoD, in the establishment and regulation of myogenic loci at the onset of myoblast differentiation. In addition, we reveal an enrichment of loci-specific histone acetylation at p300 associated active or poised enhancers, mainly when enlisted by MyoD. We have previously established that bexarotene, a clinically approved agonist of retinoid X receptor (RXR), promotes the specification and differentiation of skeletal muscle lineage. Hence, we investigated the genome-wide impact of rexinoids on myogenic differentiation and uncovered a new mechanism of rexinoid action, which is mediated by the nuclear receptor and largely reconciled through direct regulation of MyoD gene expression. In addition, we determined rexinoid-responsive residue-specific histone acetylation at a distinct chromatin state associated with MyoD and myogenin. Finally, through ChIP-seq and RNA-seq analyses, we have identified dystroglycan (Dag1) as a differentiation-dependent and a rexinoid-responsive model target, and we revealed a possible co-regulation of Dag1 by p300 and MyoD accompanied by enrichment of loci-specific histone acetylation. Taken together, we provide novel molecular insights into the regulation of myogenic enhancers by p300 in concert with MyoD. Furthermore, we provide novel mechanistic perceptions into the interplay between RXR signaling and chromatin states pertinent to myogenic programs in early myoblast differentiation. Our studies present a valuable insight for driving condition-specific chromatin state or enhancers pharmacologically to treat muscle-related diseases and for the identification of additional myogenic targets and molecular interactions for therapeutic development.

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.

Influência dos sistemas de Quorum Sensing AI-1/AI-2/AI-3 nos fatores de virulência de EPEC atípica de origem animal. A secreção de moléculas sinalizadoras de baixo peso molecular que se acumulam no meio extracelular até atingir um limite crítico de concentração para sua detecção, acarretando em sinalizações intracelulares e respostas efetoras, define o sistema de comunicação bacteriano chamado Quorum Sensing. Este sistema, que regula comportamentos coletivos mostrou-se um mecanismo disseminado em diversas espécies bacterianas. Diversos estudos descreveram a existência de pelo menos 3 sistemas relacionados ao Quorum Sensing em bactérias Gram-negativas, LuxIR/AI-1, LuxS/AI-2 e QseBC/AI-3/Epinefrina/Norepinefrina. Fatores de virulência como formação de biofilme, motilidade e adesão ao epitélio do hospedeiro devem ser controlados de maneira adequada para tirar o melhor proveito da situação em que a bactéria se encontra. Este trabalho teve como objetivo analisar a influência e a correlação dos genes luxS, qseC e sdiA, relacionados ao sistema de comunicação bacteriana Quorum Sensing, nos principais fatores de virulência de uma amostra de EPEC atípica de origem animal. Foram construídos mutantes pela metodologia baseada na recombinação homóloga mediada pelo sistema Lambda Red, que foram submetidos a ensaios fenotípicos. A sinalização por AI-2 e luxS desempenham papéis na motilidade, formação de biofilme e adesão a células epiteliais. QseC influencia a produção de biofilme pela regulação de componentes da matriz extracelular, e participa de processos de motilidade e adesão. O hormônio epinefrina contribui na alteração de processos de motilidade, formação de biofilme e desenvolvimento da lesão A/E. O gene sdiA também tem um papel importante na regulação de fatores de virulência, mesmo na ausência de AHL. Uma interação antagônica parece existir entre os genes qseC e luxS. A ausência de sistemas de Quorum Sensing promove a produção de um biofilme robusto na amostra AP155. / The secretion of low molecular weight signaling molecules that accumulate in the extracellular medium until reaching a critical concentration limit for its detection, leading to intracellular signaling and effector responses, defines the bacterial communication system called Quorum Sensing. This system regulates collective behaviors and has been proven to be a widespread mechanism in several bacterial species. Several studies have described the existence of at least 3 Quorum Sensing-related systems in Gram-negative bacteria, LuxIR/AI-1, LuxS/AI-2 and QseBC/AI-3/Epinephrine/Norepinephrine. Virulence factors such as biofilm formation, motility and adhesion to the host epithelium should be adequately regulated to make the most of the situation in which the bacterium is found. The aim of this work was to analyze the influence and correlation of luxS, qseC and sdiA genes, related to the bacterial communication system Quorum Sensing, on the main virulence factors of an atypical EPEC strain isolated from an animal. Mutants were obtained through homologous recombination mediated by the Lambda Red system, and then were submitted to phenotypic assays. AI-2 signaling and luxS play roles in motility, biofilm formation, and adhesion to epithelial cells. QseC influences the production of biofilm by the regulation of components of the extracellular matrix, and participates in the processes of motility and adhesion as well. The epinephrine hormone alters the processes of motility, biofilm formation and development of the A/E lesion. The sdiA gene also plays an important role in the regulation of virulence factors, even in the absence of AHL. An antagonistic interaction seems to exist between the qseC and luxS genes. The absence of Quorum Sensing systems promotes the production of a robust biofilm in the AP155 strain.