ERRα est un récepteur nucléaire dont l’activité est controlée par des co-régulateurs transcriptionnels. La forte expression de ERRα dans les cancers est corrélée à un mauvais pronostic. Les mécanismes par lesquels ERRα régule la migration des cellules cancéreuses sont mal compris, tout comme les co-régulateurs impliqués. Nous avons identifié deux enzymes modificatrices d’histone, LSD1 et SET7, agissant comme régulateurs positifs de ERRα.I. ERRα modifie les activités biochimiques de la déméthylase LSD1 vers la déméthylation (activatrice) de H3K9me2. L’activation des cibles de ERRa-LSD1 (identifiées par RNA-Seq) requiert le recrutement de ce complexe aux sites d’initiation de la transcription (TSSs), réalisé par le facteur de transcription NRF1 qui, lui, ne régule pas l’activité enzymatique de LSD1.II. Un autre groupe de cibles de ERRα (identifié par RNA-Seq) est sous le contrôle de l’histone méthyltransférase SET7 qui mono-méthyle H3K4. Le recrutement de SET7 aux TSSs est contrôlé par le facteur de transcription ETS1, qui promeut les interactions entre SET7 et ERRα, conduisant à l’activation de l’expression des gènes en aval.Des analyses par Gene Ontology ont montré que les cibles communes de ERRα/LSD1 et de ERRα/SET7 sont fortement enrichies en termes d’invasion cellulaire. De manière cohérente, la déplétion individuelle de chacun de ces facteurs (et également celle de NRF1 ou ETS1) réduit les capacités d’invasion, observée en tests in vitro (transwell) ou in vivo par xénogreffe sur embryons de poisson-zèbre.En résumé, nos résultats montrent deux réseaux de régulation impliquant des modifications d’histone induites par ERRα, conduisant à l’invasion cellulaire. / ERRα is a nuclear receptor whose activity mainly depends on its interaction with transcription co-regulators. High levels of ERRα are found in various cancer types and correlate with poor prognosis. However, the mechanisms linking ERRα to cancer cell migration as well as the coregulators involved are unclear. In our study, we found two histone-modifying enzymes, LSD1 and SET7, acting as positive regulators of ERRα.I. ERRα impacts the biochemical activities of the LSD1 demethylase. Activation of ERRα -LSD1 targets (identified by RNA-Seq) requires the recruitment of this complex at Transcriptional Start Sites (TSSs), which is achieved by the NRF1 transcription factor. In our study, we have shown several points: NRF1, but not ERRα , is involved in positioning LSD1 to TSS, whereas ERRα , but not NRF1, regulates LSD1 enzymatic activities towards demethylating H3K9me2.II. A distinct group of ERRa target genes (identified by RNA-Seq) is under the control of the histone methyltransferase SET7 which mono-methylates H3K4. Appropriate recruitment of SET7 at TSSs is controlled by the ETS1 transcription factor, promoting the interaction between SET7-ERRa, leading to target gene expression.Gene Ontology analysis revealed that ERRa-LSD1 co-targets, as well as ERRa-SET7 co-targets, are enriched in terms of cell invasion. Consistently, depletion of each of these factors, as well as depletion of NRF1 or ETS1, leads to reduced cell invasion capacities as observed in transwell assays or in vivo, using xenotransplantation in the zebrafish embryo.Altogether, our results show two regulatory networks involving histone modifications induced by nuclear receptors, leading to increased cell invasion.

Metabolites and peptides have a central role in biology that is often overlooked. Despite the importance of metabolites in key protein-metabolite interactions (PMIs), the extent and identity of these interactions is not known. Likewise, the extent to which short open reading frames (sORFs) in the genome are translated into peptides has also been an elusive question. This dissertation describes the development and application of methods to elucidate unknown molecules and interactions critical to understanding biology, and the subsequent characterization of the biological roles of these discoveries in cells and mice. / Chemistry and Chemical Biology

Embryonic stem (ES) cell pluripotency is sustained by a network of transcription factors centred on Oct4, Sox2 and Nanog. Whilst Oct4 and Sox2 expression is relatively uniform, ES cells fluctuate between states of high Nanog expression possessing high self-renewal efficiency, and low Nanog expression exhibiting increased differentiation propensity. Moreover, modulation in the level of Nanog expression determines the efficiency of ES cell self-renewal. To identify genes regulated by Nanog, genome-wide transcriptional profiling was performed on ES cells expressing different Nanog levels and Nanog-null ES cells expressing a Nanog-ERT2 fusion protein in which nuclear Nanog activity can be regulated by tamoxifen. Surprisingly, only a minor fraction of the genes to which Nanog binds showed significant changes in response to Nanog induction. Prominent amongst Nanog-responsive genes is Estrogen-related receptor b (Esrrb). Nanog binds directly to Esrrb, enhances binding and pause-release of RNAPolII from the Esrrb promoter and stimulates Esrrb transcription. Consistent with these findings, elevation of Nanog produces a cell population that expresses uniformly high Esrrb levels. Moreover, double fluorescent reporter lines show that Esrrb and Nanog levels are strongly correlated in individual cells. Loss of Nanog is required for downregulation of Esrrb, which coincides with commitment to differentiate. Esrrb overexpression results in LIF independent self-renewal, and blocks neural differentiation, even in the absence of Nanog. Cell fusion experiments between ES and neural stem (NS) cells show that elevated Esrrb levels allow the reprogramming of the NS cell genome in the absence of Nanog. Esrrb can rescue stalled reprogramming during the derivation of Nanog-/- induced pluripotent stem (iPS) cells. Moreover, targeted knock-in of Esrrb at the Nanog locus rescues the ability of Nanog null ES cells to maintain germ cell development beyond E12. Finally, Esrrb deletion abolishes the defining ability of Nanog to confer LIF-independent selfrenewal to ES cells. Together these data identify Esrrb as a critical downstream mediator of Nanog function.

In multicellular organism, bioenergetic metabolism is strictly regulated toward efficient generation of ATP. However, in certain situations, such as in limiting oxygen or in the rapidly proliferating system like growing juvenile or cancer cells, organisms apply the metabolic strategy that favors the production of biomass (e.g., nucleotides, amino acids, and lipids) over efficiency of ATP generation. The conserved estrogen-related receptors (ERRs) are master regulators in controlling metabolic homeostasis, and good candidates for mediating the metabolic transition induced by hypoxia and development. First, we investigate how dERR influences hypoxic adaptation in Drosophila melanogaster. We find that dERR is required for a competent hypoxic response alone, or together with hypoxia inducible factor (HIF), which is the main transcription factor modulating the hypoxic adaptation. We show that dERR binds to dHIFα and participates in the HIF-dependent transcriptional program in hypoxia. In addition, dERR acts in the absence of dHIFα in hypoxia and a significant portion of HIF-independent transcriptional responses can be attributed to dERR actions, including up-regulation of glycolytic transcripts. These results indicate that competent hypoxic responses arise from complex interactions between HIF-dependent and -independent mechanisms, and that dERR plays a central role in both of these programs. Secondly, we examine how dERR modulates metabolic transition toward the fatty acid oxidation at late L3 larva stage. We show that dERR is essential for the expression of an uncharacterized long-chain-fatty-acid acyl-CoA synthetase, CG4500, which is subject to induction by starvation. Furthermore, late L3 larvae of dERR mutants exhibit altered lipid profiles with elevated medium-chain and long-chain fatty acids. Together, with the previous finding that ERR directs an early switch toward glycolysis in the embryo, our studies indicate that ERR is a master regulator of programmed metabolic shifts through Drosophila development.

Billions of pounds of bisphenol A (BPA) are produced annually around the globe for the manufacture of numerous consumer products, including polycarbonate food and water containers, the protective resin linings of food cans, thermal printing paper, and dental fillings. BPA exposure during nervous system development has been associated with learning and behavioral impairments in animal models. The mode of action for these effects is not clearly defined. While BPA is a weak estrogen receptor (ER) agonist, it is also an estrogen-related receptor gamma (ERR��) agonist. ERR�� binds BPA with 100 times greater affinity than ERs. This study was designed to test the hypothesis that exposure to human-relevant BPA concentrations impacts nervous system development and behavior through ERR�� activation. To examine whether BPA behaves more like an ER or ERR�� ligand, two positive control compounds were used throughout the study: 17��-estradiol (E2) and GSK4716, ER and ERR�� agonists, respectively. Initial behavior testing results included the observation that neurodevelopmental exposure to 0.01 or 0.1 ��M BPA led to hyperactivity in larvae, while exposure to 0.1 or 1 ��M BPA led to learning deficits in adult zebrafish. Exposure to 0.1 ��M E2 or GSK4716 also led to larval hyperactivity. To identify early molecular signaling events that lead to the observed neurobehavioral phenotypes, a global gene expression analysis using a 135K zebrafish microarray was conducted. The concentrations of compounds tested were anchored to the common larval hyperactivity phenotype they elicited. Gross abnormalities, in the case of higher concentrations of BPA and E2, were also anchored phenotypes included in the analysis. Functional pathway analysis of the BPA versus E2 results predicted an impact on prothrombin signaling from the two lower concentrations of BPA and E2. Both BPA and GSK4716 were also predicted to impact nervous system development, potentially involving inhibition of the upstream regulator, SIM1. Additionally, GSK4716 exposure was predicted to inhibit neuron migration. There were fewer similarities in transcriptional responses between BPA and E2 when the lower versus higher concentrations were compared, suggesting different mechanisms operated at the higher concentrations. Subsequent experiments were focused on the role of ERR�� in the larval hyperactivity phenotype. Transient ERR�� knockdown by antisense oligonucleotide morpholino during the first 24 hours of development abrogated the hyperactive phenotype induced by 0.1 ��M BPA exposure. Transient ERR�� knockdown during the first 48 hours of development resulted in developmental delays, craniofacial defects, pericardial edema, and severe body axis curvature. This work is the first to identify behavioral effects in a fish from developmental BPA exposure. It is also the first study to confirm a role for ERR�� in mediating BPA's neurobehavioral effects in any animal model. The global gene expression analysis identified similarities between BPA, E2, and GSK4716, suggesting that BPA's mode of action may involve crosstalk between ERR�� and other ERs. These results from human-relevant BPA exposures help explain the widely documented in vivo effects of BPA, despite low binding affinity exhibited by nuclear ERs. ERR�� is an evolutionarily conserved vertebrate receptor and the developmental impacts of BPA in the zebrafish are an indication of hazard potential to vertebrates. They are also an important translational step toward knowing the hazard potential from human developmental exposure to BPA and yet unknown environmental ligands of ERR��. / Graduation date: 2013 / Access restricted to the OSU Community, at author's request, from Dec. 21, 2012 - June 21, 2013

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