Le remodelage de la chromatine et la modification des histones jouent des rôles très importants dans l’établissement et la reprogrammation de l’état de l’expression génique. Il reste largement inconnu concernant les mécanismes de la régulation de ces processus chromatiniens dans le contrôle de l’expression génique impliquée dans le développement de la plante et son adaptation à l’environnement. Mon sujet de thèse se focalise sur l’analyse fonctionnelle d’un facteur de remodelage de la chromatine de type Chromodomain/Hélicase/DNA-binding 1 (CHD1) d’Arabidopsis, appelé CHR5 et une histone démethylase qui est spécifiquement impliquée dans la démethylation de l’histone H3 lysine 4 (H3K4), appelée JMJ15. Dans la première partie de cette étude, nous avons montré que le gène CHR5 est activé au cours de l’embryogénèse et que son expression se maintient élevé dans les tissues/organes en développement. L’analyse de mutants révèle que la perte de fonction de ce gène fait réprimer l’expression de gènes régulateurs de la maturation de l’embryon tels que LEC1, ABI3 et FUS3 pendant le développement des graines, et fait baisser l’accumulation des protéines de réserve. L’analyse de double mutants a permis de démontrer une fonction antagoniste entre CHR5 et PKL, une protéine du groupe « CHD3 », dans l’activité du promoteur de gènes régulateurs du développement de l’embryon et l’accumulation de réserve de graine. Nous avons montré que la protéine CHR5 s’associe directement avec les promoteurs d’ABI3 et FUS3 et que la mutation du gène CHR5 conduit à l’augmentation de présence de nucléosome dans la région du départ de transcription. Ces résultats suggèrent que CHR5 est impliquée dans le positionnement de nucléosome pour stimuler l’expression de gènes de la maturation de l’embryon, ce qui est contrebalancé par l’action de PKL au cours du développement de l’embryon. La deuxième partie de cette étude a permis de montrer que l’expression du gène de l’histone démethylase JMJ15 manifeste une forte spécificité tissulaire. L’analyse de mutants du gène a permis de l’identification de 2 allèles de gain de fonction (avec surexpression du gène), et un allèle de perte de fonction. La surexpression du gène réduit la croissance d’hypocotyle et de tige de la plante avec accumulation de lignine dans la tige, mais le perte de fonction du gène ne produise pas de phénotype apparent. Par ailleurs, la surexpression du gène renforce la tolérance de la plante au stress salin, alors la perte de fonction du gène rend la plante plus sensible. L’analyse du transcriptome a révélé beaucoup plus de gènes réprimés qu’activés par la surexpression du gène JMJ15. Ces gènes réprimés sont préférentiellement marqué la H3K4me2 ou H3K4me3, parmi lesquels beaucoup codent de facteurs de transcription. Ces données suggèrent que l’induction de JMJ15 pourrait réguler le programme de l’expression génique qui coordonne la restriction de la croissance de la plante et la tolérance au stress. Ces travaux de thèse a permis ‘identifier quelques nouveaux éléments dans la compréhension de la fonction de régulateurs chromatiniens dans l’expression génique de la plante. / Chromatin remodeling and histone modification play important roles in the establishment and dynamic regulation of gene expression states. However, little is known regarding to the regulatory mechanism of chromatin modification and remodeling that control gene expression involved in plant development and responses to environmental cues. My thesis work concerns functional analysis of an Arabidopsis Chromodomain/Helicase/DNA-binding 1 (CHD1) type chromatin remodeling gene known as CHR5 and a histone demethylase gene that specifically removes methyl groups from methylated histone H3 lysine 4 (H3K4me), called JMJ15 in regulating chromatin structure or in resetting chromatin modifications that control the expression of plant developmental and stress responsive genes. In the first part of the study we found that CHR5 expression is activated during embryogenesis and remained to be expressed in developing organs/tissues. Analysis of mutants revealed that loss-of-function of the genes led to decreased expression of key embryo maturation genes LEC1, ABI3 and FUS3 in developing seeds and reduced seed storage protein accumulation. Analysis of double mutants revealed an antagonistic function between CHR5 and PKL, a CHD3 gene, in embryo gene promoter activity and seed storage protein accumulation. CHR5 was directly associated with the promoters of ABI3 and FUS3 and chr5 mutations led to increased nucleosome occupancy near the transcriptional start site. The results suggest that CHR5 is involved in nucleosome occupancy to regulate embryo identity genes expression, which is counterbalanced by PKL during embryo development. The second part of this study showed that expression of JMJ15 was restricted to a few tissues during vegetative growth. The jmj15 gain-of-function mutations reduced the length of seedling hypocotyls and inflorescence stems with higher accumulation of lignin in the stem, while the loss-of-function mutants did not show any visible phenotype. The gain-of-function mutants enhanced salt tolerance, whereas the loss-of-function mutants were more sensitive to salt. Transcriptomic analysis revealed a much higher number of genes down-regulated in JMJ15 over-expression plants, which are highly enriched for H3K4me3 and H3K4me2. Among the down-regulated genes, many encode transcription regulators of stress responsive genes. The data suggest that increased JMJ15 levels may regulate the gene expression program that may coordinate plant growth restrains and enhances stress tolerance. Taken together, my thesis work brought a few new elements to the current understanding of chromatin regulators function in plant gene expression.

<p>In eukaryotic cells, DNA is packaged into a structure named chromatin which contains DNA and proteins. Nucleosomes are building blocks of chromatin and contain DNA wrapped around a histone octamer. Chromatin modifications (histone post-translational modifications and histone variants) play central roles in various cellular processes including gene expression and DNA damage response. Chromatin modifying enzymes use metabolites as co-substrates and co-factors, and changes in metabolic pathways and metabolite availability affects chromatin modifications and chromatin-associated functions. Moreover, recent studies have uncovered direct roles of metabolic enzymes in chromatin-associated functions. Fumarase, a TCA cycle enzyme that catalyzes the reversible conversion of fumarate to malate in mitochondria (a hydration reaction), is an example of an enzyme with dual functions in metabolism and genome integrity. Cytoplasmic fraction of yeast fumarase, Fum1p, localizes to the nucleus and promotes growth upon DNA damage. Fum1p promotes homologous recombination by enhancing DNA end resection. Human fumarase is involved in DNA repair by non-homologous end joining. Here, we provide evidence that yeast Fum1p and the histone variant Htz1p are also involved in DNA replication stress response and DNA repair by non-homologous end joining (NHEJ). Using mutants lacking the histone variant <i>HTZ1</i>, we show that high cellular levels of fumarate, by deletion of <i>FUM1</i> or addition of exogenous fumarate, suppressed the sensitivity to DNA replication stress by modulation of activity of Jhd2p. This suppression required sensors and mediators of the intra-S phase checkpoint, but not factors involved in the processing of replication intermediates. These results imply that high cellular levels of fumarate can confer resistance to DNA replication stress by bypassing or complementing the defects caused by loss of <i>HTZ1</i> and replication fork processing factors. We also show that upon induction of DSBs, exogenous fumarate conferred resistance to mutants with defects in NHEJ, early steps of homologous recombination (DNA end resection pathway) or late steps of homologous recombination (strand invasion and exchange). Taken together, these results link the metabolic enzyme fumarase and the metabolite fumarate to DNA damage response and show that modulation of DNA damage response by regulating activity of chromatin modifying enzymes is a plausible pathway linking metabolism and nutrient availability to chromatin-associated functions like genome integrity.<br><a></a></p>

The amine oxidase family of enzymes has been the center of numerous mechanistic studies because of the medical relevance of the reactions they catalyze. This study describes transient and steady-state kinetic analyses of two flavin amine oxidases, mouse polyamine oxidase (PAO) and human lysine specific demethylase (LSD1), to determine the mechanisms of amine oxidation. PAO is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyzes the oxidation of N1-acetylated polyamines. The pH-dependence of the kcat/Kamine indicates that the monoprotonated form of the substrate is required for catalysis, with the N4 nitrogen next to the site of CH bond cleavage being unprotonated. Stopped-flow spectroscopy shows that the pH-dependence of the rate constant for flavin reduction, kred, displays a pKa of 7.3 with a decrease in activity at acidic pH. This is consistent with an uncharged nitrogen being required for catalysis. Mutating Lys315 to methionine has no effect on the kcat/Kamine-pH profile with the substrate spermine, and the kred value only shows a 1.5-fold decrease with respect to wild-type PAO. The mutation results in a 30- fold decrease in kcat/KO2. Solvent isotope effects and proton inventories are consistent with Lys315 accepting a proton from a water molecule hydrogen-bonded to the flavin N5 during flavin oxidation. Steady-state and transient kinetic studies of para-substituted N,N'-dibenzyl-1,4- diaminobutanes as substrates for PAO show that the kred values for each correlate with the van der Waals volume (VW) and the value. The coefficient for VW is the same at pH 8.6 and 6.6, whereas the p value increases from -0.59 at pH 8.6 to -0.09 at pH 6.6. These results are most consistent with a hydride transfer mechanism. The kinetics of oxidation of a peptide substrate by human lysine specific demethylase (LSD1) were also studied. The kcat/KM pH-profile is bell-shaped, indicating the need for one unprotonated nitrogen next to the site of CH bond cleavage and another protonated nitrogen. The kcat and kred values are equal, and identical isotope effects are observed on kred, kcat, and kcat/KM, indicating that CH bond cleavage is rate-limiting with this substrate.

Hematopoietic stem and progenitor cells proliferate and differentiate to reconstitute all lineages of functional blood cells. They are regulated by intricate cellular and molecular signals, on both genetic and epigenetic levels. Alterations in these regulatory signaling networks can lead to hematopoietic dysfunction, as well as transformation of hematopoietic cells and induction of leukemogenesis. This thesis focuses on uncovering molecular modulators that are crucial for the proper regulation of hematopoietic stem/progenitor cells. In Chapter II, I describe studies investigating functional roles of the histone demethylase UTX in normal and malignant hematopoiesis, using a short hairpin RNA-mediated knockdown approach. My data revealed that UTX is required for proliferation, self-renewal and differentiation of hematopoietic progenitor cells ex vivo through transcriptional regulation of hematopoiesis- specific transcriptional factors. I also discovered that UTX is critical for the proliferation of leukemia cells, implicating UTX as a possible target for clinical therapy. In Chapter III, I focus on understanding the process of leukemogenesis by generating and characterizing a novel model of myeloid sarcoma and acute myeloid leukemia in mice. This model induces these hematopoietic malignancies by introduction of multiple oncogenetic lesions (specifically, p16/p19-/-;Kras(G12V)) into bone marrow cells, and subsequent transplantation of these gene-modified cells into immunodeficient NOD.SCID mice. This model is very rapid and reproducible, and represents the first transplantable myeloid sarcoma model reported. Moreover, the disease induced in mice recapitulates the pathological progression of myeloid sarcoma in patients, providing a powerful model for dissection of critical leukemogenic events and discovery of new candidate therapeutic targets. Together, these studies help to reveal novel molecular modulators required for normal hematopoiesis, and offer potential animal model and drug target for therapeutic applications.

The epigenetic mechanisms that connect hormone signaling to chromatin remain largely unknown. Here we show that LSD1/KDM1A is a critical glucocorticoid receptor (GR) coactivator and report a previously unexplored mechanism where LSD1 activates gene transcription through H3K4me2 demethylation. We demonstrate that direct interaction of GR with LSD1 primarily inhibit its activity against H3K4me1 in vitro. While this interaction enables GR to recruit LSD1 in vivo and allows loss of H3K4me2, it impedes further demethylation. Thus resulting in conversion of H3K4me2 to H3K4me1 at enhancers and promotes H3K27 acetylation and gene activation. We also find that H3K4me2 is an early enhancer mark predicting GR and LSD1 recruitment. These findings differ from the reported mechanism for ER and AR-mediated gene activation, providing a novel mechanism for LSD1 coactivator function as well as shed light on the role of H3K4me2 and enhancers in hormone-mediated gene regulation. In addition we present evidence supporting never before characterized H3K79me3 demethylase activity by members of the JMJD2 family of proteins.

A novel caffeine-degrading bacterium, Pseudomonas putida CBB5 was isolated from the soil by an enrichment procedure using caffeine as the sole source of carbon and nitrogen. CBB5 grew not only on caffeine, theobromine, paraxanthine, and 7-methylxanthine as sole carbon and nitrogen sources, but also on theophylline and 3-methylxanthine. Analyses of metabolites in spent media, resting cell suspensions, and crude cell extracts confirmed that CBB5 degraded caffeine via N-demethylation to theobromine (major metabolite) and paraxanthine (minor metabolite). These dimethylxanthines were further N-demethylated to xanthine via 7-methylxanthine. A previously unreported pathway for N-demethylation of theophylline to 1- and 3-methylxanthines, followed by further N-demethylation to xanthine, was also discovered in CBB5. A 240 kDa, Fe2+-dependent N-demethylase (Ndm) was purified from CBB5 by traditional chromatographic techniques. Ndm was composed of NdmA (40 kDa) and NdmB (35 kDa), which could not be resolved further. Ndm was active only in the presence of a partially purified protein which exhibited cytochrome c reductase activity (Ccr). Ccr transfered reducing equivalents from NAD(P)H to Ndm, which catalyzed an oxygen-dependent N-demethylation of methylxanthines to xanthine, formaldehyde and water. Ndm displayed N-demethylation activity toward all substrates in the caffeine and theophylline metabolic pathways. Ndm was deduced to be a Rieske [2Fe-2S]-domain-containing non-heme iron oxygenase base on its distinct absorption spectrum and significant identity of NdmA and NdmB sequences of other Rieske non-heme iron proteins. The ndmA- and ndmB- gene sequences were determined and cloned individually into the pET32a expression vector as C-terminal His-tagged proteins. Both NdmA-His and NdmB-His proteins were purified using a Ni-NTA column. NdmA-His, in conjunction with Ccr, was capable of N-demethylating caffeine, theophylline, paraxanthine, and 1-methylxanthine to theobromine, 3-methylxanthine, 7-methylxanthine, and xanthine, respectively, suggesting that NdmA-His is a specific N-1-demethylase. Similarly, NdmB-His was determined to be a specific N-3-demethylase, as it was capable of N-demethylating caffeine, theophylline, theobromine, and 3-methylxanthine to paraxanthine, 1-methylxanthine, 7-methylxanthine, and xanthine, respectively. N-demethylation activity of 7-methylxanthine to xanthine (putative NdmC) co-eluted with the partially purified Ccr fraction. This is the first report of multiple, highly positional-specific, Rieske, non-heme iron N-demethylase enzymes for bacterial metabolism of purine alkaloids.

Les chondrosarcomes sont des tumeurs malignes osseuses, considérés comme radio- et chimio-résistants, du fait de leur environnement hypoxique. Dans ce contexte, cette étude vise à mieux comprendre le rôle de l’hypoxie dans la résistance de ces tumeurs à la chimiothérapie (cisplatine) et à la radiothérapie (rayons X) et à identifier de nouvelles stratégies thérapeutiques permettant de sensibiliser les chondrosarcomes aux traitements, par un ciblage épigénétique de la méthylation de la lysine 27 de l’histone H3 (H3K27).Dans un premier temps, nous avons montré que, contrairement à ce qui est communément admis, l’hypoxie n’a pas d’effet sur la sensibilité au cisplatine ou aux rayons X dans certains chondrosarcomes alors qu’il augmente la résistance au cisplatine et la sensibilité aux rayons X uniquement dans une lignée de chondrosarcome. Dans un second temps, nous avons montré que le 3-deazaneplanocine A (DZNep) induit l’apoptose dans ces tumeurs, par un mécanisme indépendant de la méthylation de H3K27 et de sa méthylase EZH2 et semblerait agir par la voie Rhoβ/EGFR. Cependant, il provoque des effets secondaires sur la fertilité masculine. Par ailleurs, son association avec le cisplatine potentialise ses effets toxiques sur les chondrosarcomes. Le GSK-J4, quant à lui ralentit la croissance cellulaire des chondrosarcomes et son association avec le cisplatine augmente cet effet. Cette étude souligne que les chondrosarcomes possèdent des mécanismes de régulation cellulaires différents, d’où l’importance de mener des études sur plusieurs lignées cellulaires afin de mieux prédire la réponse aux traitements. De plus, ces travaux démontrent les propriétés anti-tumorales du DZNep et du GSK-J4 dans le traitement de ces tumeurs. / Chondrosarcomas are bone malignant tumors, considered as radio- and chemo-resistant, due to their hypoxic environment. In this context, this study aimed to better understand the role of hypoxia in the resistance of these tumors to chemotherapy (cisplatin) and radiotherapy (X-rays) and to identify new therapeutic strategies to re-sensitize chondrosarcomas by epigenetic targeting of H3K27 methylation. First, we showed that, contrary to what is commonly accepted, hypoxia has differential effect on cisplatin or X-ray sensitivity in chondrosarcomas, while it increases cisplatin resistance and X-ray sensitivity only in one cell line. Secondly, 3-deazaneplanocin A (DZNep) induces apoptosis in these tumors by a mechanism independent of H3K27 methylation and its methylase EZH2 and seems to act through the Rhoβ / EGFR pathway. However, it causes side effects on male fertility. In addition, its association with cisplatin potentiates its toxic effects on chondrosarcomas. The GSK-J4, on the other hand, decreases cell growth and its association with cisplatin increases this effect.This study highlights that chondrosarcomas use different cellular regulation mechanisms, showing the importance of conducting studies on several cell lines in order to better predict the response to treatments. In addition, these studies demonstrate the anti-tumoral properties of DZNep and GSK-J4 in the treatment of these tumors.

Epithelial cells that lose attachment to the extracellular matrix (ECM) undergo a specialized form of apoptosis called anoikis. Anoikis has an important role in preventing oncogenesis, particularly metastasis, by eliminating cells that lack proper ECM cues. The basis of anoikis resistance remains to be determined and to date has not been linked to alterations in expression or activity of previously identified anoikis effector genes. Here, I utilized two different screening strategies to identify novel anoikis effector genes and miRNAs in order to gain a deeper understanding of anoikis and the potential mechanisms of anoikis resistance in cancer. Using large-scale RNA interference (RNAi) screening, I found that KDM3A, a histone H3 lysine 9 (H3K9) mono- and di-demethylase plays a pivotal role in anoikis induction. In attached breast epithelial cells, KDM3A expression is maintained at low levels by integrin signaling. Following detachment, integrin signaling is decreased resulting in increased KDM3A expression. RNAi-mediated knockdown of KDM3A substantially reduces apoptosis following detachment and, conversely, ectopic expression of KDM3A induces cell death in attached cells. I found that KDM3A promotes anoikis through transcriptional activation of BNIP3 and BNIP3L, which encode pro-apoptotic proteins. Using mouse models of breast cancer metastasis I show that knockdown of Kdm3a enhances metastatic potential. Finally, I find defective KDM3A expression in human breast cancer cell lines and tumors. Collectively, my results reveal a novel transcriptional regulatory program that mediates anoikis. Next, I sought to discover miRNAs involved in anoikis by investigated changes in miRNA expression during anoikis using small RNA sequencing technology. Through this approach I discovered that miR-203 is an anoikis effector miRNA that is also highly down-regulated in invasive breast cancer cells. In breast epithelial cells, miR-203 is induced upon the loss of ECM attachment and inhibition of miR-203 activity leads to a resistance to anoikis. I utilized a dual functional- and expression- based RNA sequencing approach and found that miR-203 directly targets a network of pro-survival genes to induce cell death upon detachment. Finally, I found that the loss of miR-203 in invasive breast cancer leads to the elevation of several anoikis-related pro-survival target genes to contribute to anoikis resistance. Taken together, my studies reveal novel pathways through which cell death is induced upon detachment from the ECM and provide insight into potential mechanisms of anoikis resistance in cancer.

2. Abstract Histone methylation plays an important role in almost all cellular processes and its homeostasis is maintained by histone methyltransferases and histone demethylases. Misregulation of histone methylation levels is associated with gene expression misregulation and consequently also with various developmental defects and diseases. In this thesis we focus on the lysine demethylases KDM2A and KDM2B and on their demethylation deficient isoforms KDM2A-SF and KDM2B-SF. The lysine specific demethylases KDM2A and KDM2B have been predominantly studied for their demethylation function on CpG island-rich gene promoters. However, KDM2A-SF and KDM2B-SF have not been studied in detail. Therefore, the main goal of this thesis was to characterize KDM2A-SF more in detail and to focus on the role that KDM2A/B-SF might potentially play in canonical Wnt signaling pathway. We found that the KDM2A-SF mRNA arises through the action of an alternative intronic promoter and not by alternative splicing. We showed that the KDM2A-SF start codon is located in the exon that corresponds to KDM2A exon 14 and we thus determined the exact amino acid sequence of the KDM2A-SF protein. Furthermore, using an isoform specific knockdown assay we showed that KDM2A-SF, unlike KDM2A-LF, forms distinct nuclear foci on pericentromeric...

2. Abstract Histone methylation plays an important role in almost all cellular processes and its homeostasis is maintained by histone methyltransferases and histone demethylases. Misregulation of histone methylation levels is associated with gene expression misregulation and consequently also with various developmental defects and diseases. In this thesis we focus on the lysine demethylases KDM2A and KDM2B and on their demethylation deficient isoforms KDM2A-SF and KDM2B-SF. The lysine specific demethylases KDM2A and KDM2B have been predominantly studied for their demethylation function on CpG island-rich gene promoters. However, KDM2A-SF and KDM2B-SF have not been studied in detail. Therefore, the main goal of this thesis was to characterize KDM2A-SF more in detail and to focus on the role that KDM2A/B-SF might potentially play in canonical Wnt signaling pathway. We found that the KDM2A-SF mRNA arises through the action of an alternative intronic promoter and not by alternative splicing. We showed that the KDM2A-SF start codon is located in the exon that corresponds to KDM2A exon 14 and we thus determined the exact amino acid sequence of the KDM2A-SF protein. Furthermore, using an isoform specific knockdown assay we showed that KDM2A-SF, unlike KDM2A-LF, forms distinct nuclear foci on pericentromeric...