<p>Lysine specific demethylase 1 (LSD1) is a FAD-dependent amine oxidase enzyme responsible for removing methyl groups from the side chain nitrogen of lysine within histones in order to regulate gene transcription. By its interaction with various transcriptional complexes, including those containing estrogen receptor &alpha; (ER&alpha;), LSD1 mediates expression of many genes important in cancer proliferation and progression. Herein, we report our efforts towards understanding the function of LSD1 in breast cancer. We have developed a straightforward method for the syntheses of 2-arylcyclopropylamines as irreversible mechanism-based inactivators of LSD1. We employed these small molecules as probes of LSD1 activity, and together with experiments involving the knockout of LSD1 by small interfering RNA (siRNA), we have shown that LSD1 activity is essential for both ER&alpha;-postive and ER&alpha;-negative breast cancer proliferation. LSD1 inhibitors induce a dramatic cell cycle arrest without causing apoptosis. </p><p>Furthermore, we observe that LSD1 and ER&alpha; work cooperatively to express certain estrogen-target genes through simultaneous recruitment to promoters; LSD1 inhibition diminishes ER&alpha; recruitment. Similarly, knockdown of CoREST, a binding partner of LSD1, results in comparable changes in gene expression. Although, we have not observed a direct interaction between LSD1 and ER&alpha;, we believe that CoREST may be facilitating this interaction. We have made efforts to inhibit the interaction between LSD1 and CoREST <italic>in vitro</italic> in hopes of targeting this interface in breast cancer cells in order to disrupt the necessary functional complex and prevent LSD1 activity.</p> / Dissertation

In the natural environment plants are confronted to a multitude of biotic and abiotic stress factors that must be perceived, transduced, integrated and signaled in order to achieve a successful acclimation that will secure survival and reproduction. Plants have to deal with excess excitation energy (EEE) when the amount of absorbed light energy is exceeding that needed for photosynthetic CO2 assimilation. EEE results in ROS formation and can be enhanced in low light intensities by changes in other environmental factors. The lesions simulating disease resistance (lsd1) mutant of Arabidopsis spontaneously initiates spreading lesions paralleled by ROS production in long day photoperiod and after application of salicylic acid (SA) and SA-analogues that trigger systemic acquired resistance (SAR). Moreover, the mutant fails to limit the boundaries of hypersensitive cell death (HR) after avirulent pathogen infection giving rise to the runaway cell death (rcd) phenotype. This ROS-dependent phenotype pointed towards a putative involvement of the ROS produced during photosynthesis in the initiation and spreading of the lesions. We report here that the rcd has a ROS-concentration dependent phenotype and that the light-triggered rcd is depending on the redox-state of the PQ pool in the chloroplast. Moreover, the lower stomatal conductance and catalase activity in the mutant suggested LSD1 was required for optimal gas exchange and ROS scavenging during EEE. Through this regulation, LSD1 can influence the effectiveness of photorespiration in dissipating EEE. Moreover, low and high SA levels are strictly correlated to lower and higher foliar H2O2 content, respectively. This implies an essential role of SA in regulating the redox homeostasis of the cell and suggests that SA could trigger rcd in lsd1 by inducing H2O2 production. LSD1 has been postulated to be a negative regulator of cell death acting as a ROS rheostat. Above a certain threshold, the pro-death pathway would operate leading to PCD. Our data suggest that LSD1 may be subjected to a turnover, enhanced in an oxidizing milieu and slowed down in a reducing environment that could reflect this ROS rheostat property. Finally, the two protein disulphide isomerase boxes (CGHC) present in the protein and the down regulation of the NADPH thioredoxin reductase (NTR) in the mutant connect the rcd to a putative impairment in the reduction of the cytosolic thioredoxin system. We propose that LSD1 suppresses the cell death processes through its control of the oxidation-reduction state of the TRX pool. An integrated model considers the role of LSD1 in both light acclimatory processes and in restricting pathogen-induced cell death.

<p>In the natural environment plants are confronted to a multitude of biotic and abiotic stress factors that must be perceived, transduced, integrated and signaled in order to achieve a successful acclimation that will secure survival and reproduction. Plants have to deal with excess excitation energy (EEE) when the amount of absorbed light energy is exceeding that needed for photosynthetic CO2 assimilation. EEE results in ROS formation and can be enhanced in low light intensities by changes in other environmental factors.</p><p>The lesions simulating disease resistance (lsd1) mutant of Arabidopsis spontaneously initiates spreading lesions paralleled by ROS production in long day photoperiod and after application of salicylic acid (SA) and SA-analogues that trigger systemic acquired resistance (SAR). Moreover, the mutant fails to limit the boundaries of hypersensitive cell death (HR) after avirulent pathogen infection giving rise to the runaway cell death (rcd) phenotype. This ROS-dependent phenotype pointed towards a putative involvement of the ROS produced during photosynthesis in the initiation and spreading of the lesions.</p><p>We report here that the rcd has a ROS-concentration dependent phenotype and that the light-triggered rcd is depending on the redox-state of the PQ pool in the chloroplast. Moreover, the lower stomatal conductance and catalase activity in the mutant suggested LSD1 was required for optimal gas exchange and ROS scavenging during EEE. Through this regulation, LSD1 can influence the effectiveness of photorespiration in dissipating EEE. Moreover, low and high SA levels are strictly correlated to lower and higher foliar H2O2 content, respectively. This implies an essential role of SA in regulating the redox homeostasis of the cell and suggests that SA could trigger rcd in lsd1 by inducing H2O2 production.</p><p>LSD1 has been postulated to be a negative regulator of cell death acting as a ROS rheostat. Above a certain threshold, the pro-death pathway would operate leading to PCD. Our data suggest that LSD1 may be subjected to a turnover, enhanced in an oxidizing milieu and slowed down in a reducing environment that could reflect this ROS rheostat property. Finally, the two protein disulphide isomerase boxes (CGHC) present in the protein and the down regulation of the NADPH thioredoxin reductase (NTR) in the mutant connect the rcd to a putative impairment in the reduction of the cytosolic thioredoxin system. We propose that LSD1 suppresses the cell death processes through its control of the oxidation-reduction state of the TRX pool. An integrated model considers the role of LSD1 in both light acclimatory processes and in restricting pathogen-induced cell death.</p>

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.

Lysine acetyltransferases (KATs) and lysine deacetylases (KDACs) are known to cooperate with the glucocorticoid receptor (GR) to regulate transcription. The current model of GR-mediated transcription classifies KATs as coactivators as they acetylate histones to form an open chromatin conformation and casts KDACs as corepressors that deacetylate histones and condense chromatin. Our recent studies have challenged this long-standing model. In the current study, we show that KDACs act as versatile coregulators, facilitating both the onset and maintenance of GC-induced transcriptional activation and repression. Through siRNA depletion studies, we define KDAC1 as the predominant Class I KDAC for efficient transactivation of a majority of the GR-target genes tested. KDACs 1 and 2 co-operate with each other to activate and repress a few target genes, however KDAC2 alone is not sufficient for activation or repression of the genes, thus questioning the functional redundancy of KDACs 1 and 2. Additionally, we found that there is a unique population of KDAC2 that does not associate with KDAC1 in our cell line. Through a series of siRNA depletion studies, steroid receptor coactivator proteins (SRCs) were shown to be dispensable for GC-induced gene activation and SRC2 was not required for Dex-induced transcriptional repression. We performed ChIP assays to address the mechanism by which Class I KDACs facilitate transactivation and transrepression. At GC-activated genes we found that KDACs are constitutively present at the gene enhancers and that KDAC inhibition does not affect the binding of GR or SRC proteins to chromatin. However, KDACs do influence the histone methylation status of H3K4 at GREs of activated genes and TSSs of repressed genes. To explain the change in the methylation status of this marker, we depleted LSD1, the specific demethylase for mono- and demethylation of H3K4, and found that LSD1 action is required for GC-mediated transrepression. However it is unlikely that KDAC inhibition impairs GR transactivation through effects on LSD1. Glucocorticoid signaling regulates multiple vital biological processes. Glucocorticoids play a major role in regulating carbohydrate, protein and lipid metabolism. They increase hepatic gluconeogenesis to maintain blood glucose concentration in the fasting state. GCs also act as potent anti-inflammatory molecules, stimulate lung maturation in the developing fetus, and affect bone metabolism. Additionally, excess or deficiency of GCs can lead to a variety of psychological abnormalities, indicating their role in CNS functions. Our results indicate that pharmaceutical modulation of KDACs may impair proper glucocorticoid signaling and disrupt vital biological processes. Other steroid hormone receptors function similarly to GR in regulating gene expression and could also be impacted by KDAC inhibition, thus suggesting serious physiological implications in patients. Therefore, the possibility of endocrine modulation should be taken into account when using KDAC inhibitors in the clinic.

Les multi-thérapies actuelles permettent de maintenir l’infection au VIH-1 sous contrôle, mais malheureusement n’entraînent pas l’éradication du virus du fait de l’existence de réservoirs cellulaires, où le virus est intégré de façon latente. Les cellules microgliales, cibles privilégiées du VIH-1 dans le cerveau, sont les macrophages résidents du système nerveux central et ont été décrites comme un réservoir cellulaire avec une longue durée de vie. Ce genre de cellule, infectée de façon latente, apparaît comme un des principaux obstacles à l’éradication. Ainsi, la compréhension des mécanismes sous-jacents impliqués dans l’extinction de la transcription virale, semble une étape cruciale afin de parvenir à purger ces réservoirs. Notre laboratoire à déjà montré l’importance du répresseur transcriptionnel CTIP2 dans l’établissement et le maintien de la latence dans ces cellules. Dans le cadre de ma thèse je me suis intéressé à deux autres facteurs cellulaires, LSD1 et HIC1. Au cours de mes travaux, j’ai mis en évidence le rôle répresseur de ces protéines sur la transcription virale dans les microglies. LSD1 coopère avec CTIP2 pour promouvoir l’établissement de marques épigénétiques au niveau du promoteur viral pour induire la mise en place d’hétérochromatine. LSD1 est à l’origine du recrutement de CTIP2, mais aussi d’un autre complexe multiprotéique, COMPASS. A la différence de CTIP2 et LSD1, le suppresseur de tumeur HIC1 est un perturbateur du transactivateur viral TAT. HIC1 est préalablement modifié post-traductionnellement par la déacétylase SIRT1 et va ensuite contrecarrer l’activité de TAT afin d’empêcher la réactivation de la transcription du virus. Ainsi, tandis que LSD1 et CTIP2 favorise l’établissement de la latence, HIC1 permet quant à lui d’entretenir cet état du provirus dans les cellules microgliales. Les travaux présentés ici mettent en évidence deux nouveaux facteurs de la restriction de l’expression virale et permettent de définir de nouvelles cibles thérapeutiques potentielles pour les stratégies de purge des réservoirs. / Even though current multitherapies maintain HIV infection under control, they unfortunately do not achieve viral eradication due to the existence of latently infected cell reservoirs. Microglial cells are resident macrophages and the main HIV-1 target in brain. They have been described as a long-lived HIV-1 cell reservoir, and so appear as a one of the main obstacle to viral clearance. Thus, understanding of the mechanisms implicated in the establishment and maintaining of viral latency in these cells is a critical step on the way towards an HIV cure. Our team already demonstrated the implication of the cellular transcription factor CTIP2 in the establishment of HIV-1 silencing. In this work, I present two other cellular factors, LSD1 and HIC1 as new HIV-1 transcriptionnal expression inhibitors in microglial cells. LSD1 cooperates with CTIP2 to promote the establishment of epigenetic marks associated to heterochromatin structure at the viral promoter. LSD1 act as an anchoring plateform for CTIP2, and so the CTIP2-associated mutli-enzymatic chromatin remodeling complex, but also recruits the COMPASS methyltransferase complex. Unlike CTIP2 and LSD1, the tumor suppressor HIC1 disrupts the TAT-mediated transactivation cycle. HIC1 is beforehand modified post-translationnaly by the deacetylase SIRT1 and then counteracts TAT activity in order to limit viral transcription reactivation. Thus, while CTIP2 and LSD1 favour latency establishment, HIC1 maintains provirus silencing in microglial cells. All together, the results presented in this work introduce two new viral expression restriction factors and new potential therapeutic targets in reservoir purge strategies.