Increasingly the role of epigenetic machinery as a bridge between underlying DNA sequence and cellular phenotype is being discovered. The establishment of a myriad of unique cellular types sharing identical gene sequences in a multicellular organism gives a broad sense for the inherent role of epigenetic influence on cell differentiation. Importantly, the epigenetic mechanisms involved in establishing cell identity unsurprisingly contribute to diseased states, including cancer. Recent research continues to elucidate contributory roles of epigenetic mechanisms, such as DNA methylation, histone modification, and microRNA regulation, in human cancers. Additionally, chromatin remodelers, such as the Nucleosome Remodeling Factor (NURF), have been identified as important regulators for normal cell biology. While much has been done to identify and characterize the role of NURF chromatin remodeling complex as a key regulator of development in a number of model organisms, little has been published on the implications of NURF in diseases such as cancer. Our preliminary data shows dysregulation of E-cadherins, N-cadherins, and MHC-I genes in Bptf (an essential subunit of NURF) knocked down murine breast cancer cell lines. These proteins have well documented roles in the development and metastatic progression of cancers. To study the effect of Bptf knockdown on the development and progression of cancer we injected Bptf knocked down mouse breast cancer cell lines, 4T1, 66cl4, and 67NR, into syngenic BALB/c mice. Our findings reveal decreased tumor growth in 66cl4 and 67NR as measured by tumor weight at 3-4 weeks post injection. Tumor growth did not appear to be significantly affected in 4T1 challenged mice. However, mice inoculated with Bptf knockdown 4T1 cell lines have decreased metastasis to lungs as compared to control while metastasis of 66cl4 tumors to the lungs appear unaffected. To assess the role of the immune system in decreasing tumor growth in BALB/c mice, we injected 66cl4 tumors into NOD-SCID-Gamma (NSG) immune deficient mice. The tumors from these mice show no difference in tumor growth between Bptf knockdown and control tumors, implicating a role for the immune system regulating the decreased tumor weight in BALB/c mice. To delineate which immune cell effector may impede breast cancer carcinogenesis, we performed an in vitro natural killer (NK) cell cytotoxicity assay against 66cl4 tumors and found greater susceptibility to NK killing in Bptf knockdown tumors.

La rapamycine est un immunosuppresseur utilisé pour traiter plusieurs types de maladies dont le cancer du rein. Son fonctionnement par l’inhibition de la voie de Tor mène à des changements dans des processus physiologiques, incluant le cycle cellulaire. Chez Saccharomyces cerevisiae, la rapamycine conduit à une altération rapide et globale de l’expression génique, déclenchant un remodelage de la chromatine. Nous proposons que les modifications des histones peuvent jouer un rôle crucial dans le remodelage de la chromatine en réponse à la rapamycine. Notre objectif principal est d’identifier d’une banque de mutants d’histone les variantes qui vont échouer à répondre à la rapamycine dans une tentative de réaliser une caractérisation des modifications d’histone critiques pour la réponse à cette drogue. Ainsi, nous avons réalisé un criblage d’une banque de mutants d’histone et identifié plusieurs mutants d‘histone dont la résistance à la rapamycine a été altérée. Nous avons caractérisé une de ces variantes d’histone, à savoir H2B, qui porte une substitution de l’alanine en arginine en position 95 (H2B-R95A) et démontré que ce mutant est extrêmement résistant à la rapamycine, et non à d’autres drogues. Des immunoprécipitations ont démontré que H2B-R95A est défectueux pour former un complexe avec Spt16, un facteur essentiel pour la dissociation de H2A et H2B de la chromatine, permetant la réplication et la transcription par les ADN et ARN polymérases, respectivement. Des expériences de ChIP-Chip et de micropuce ont démontré que l’arginine 95 de H2B est requise pour recruter Spt16 afin de permettre l’expression d’une multitude de gènes, dont certains font partie de la voie des phéromones. Des évidences seront présentées pour la première fois démontrant que la rapamycine peut activer la voie des phéromones et qu’une défectuosité dans cette voie cause la résistante à cette drogue. / Rapamycin is an immunosuppressant used for treating many types of diseases such as kidney carcinomas. It works by inhibiting the Tor signaling pathway leading to changes in physiological processes, including cell cycle arrest. In Saccharomyces cerevisiae, rapamycin leads to a rapid and global alteration in gene expression, prompting chromatin remodeling. We propose that histone modification(s) might play a crucial role in remodeling of the chromatin in response to rapamycin. Our main objective is to identify from a histone mutant collection variants that fail to respond to rapamycin in an attempt to characterize histone modifications critical for this drug response. As such, we conducted a screen of the histone mutant collection and identified several hits that showed resistance to rapamycin. We characterized one of the histone variants, namely H2B, carrying alanine substitution at arginine 95 (H2B-R95A) and show that it is extremely resistant to rapamycin, but not to other drugs. Pull downs demonstrated that H2B-R95A was defective in forming a complex with Spt16, an essential factor that is required to disassociate H2A and H2B from the chromatin in order to allow replication and transcription by DNA and RNA polymerases, respectively. ChIP-Chip and microarray experiments showed that arginine 95 of H2B is required to recruit Spt16 to allow expression of several genes, a subset of which are involved in the pheromone signaling pathway. Evidence will be presented to show for the first time that rapamycin can activate the pheromone pathway and that defects in this pathway cause resistance to the drug.

<div>Vertebrate neurogenesis is a multistep process that coordinates complex signaling pathways and chromatin-based regulatory machinery to generate highly specialized cells (Hsieh and Zhao 2016; Urban and Guillemot 2014; Alunni and Bally-Cuif 2016; Yao and Jin 2014; Schmidt, Strahle, and Scholpp 2013). Epigenetic factors play a fundamental role in underwriting neurogenesis in part by contributing to control of gene expression in differentiating neurons. A mechanistic understanding of the epigenetic machinery underlying neurogenesis in vertebrates is necessary both to fully understand biogenesis of neural tissue in this subphylum as well as to develop effective therapeutic strategies to treat diseased or damaged neural tissue. </div><div>An example of an epigenetic factor that is important for both neuronal differentiation and disease states is CHD5, a vertebrate-specific member of the CHD family of ATP-dependent chromatin remodeling proteins. Chromodomain / Helicase / DNA-binding (CHD) proteins play a variety of roles in vertebrate development, and misregulation or loss of CHD proteins has been linked to numerous diseases (Mayes et al. 2014; Marfella and Imbalzano 2007; Bartholomew 2014). CHD5 is expressed primarily in neural tissue, where it is thought to contribute to neurogenesis, and has been strongly linked to tumor suppression (Thompson et al. 2003; Vestin and Mills 2013). Loss of CHD5 plays a significant role in development of neuroblastoma, a devastating tumor that is a leading cause of cancer-related death in children (Jiang, Stanke, and Lahti 2011; Maris and Matthay 1999). Consistent with the disease phenotype associated with loss of CHD5, reduced expression of CHD5 impairs differentiation of neuronal cells (Egan et al. 2013b). However, ablation of CHD5 in mice surprisingly resulted in no detectable defects in brain development (Li et al. 2014; Zhuang et al. 2014). A subsequent report revealed that mice conditionally ablated for CHD5 in neural tissue exhibit symptoms consistent with an autism spectrum disorder (Pisansky et al. 2017). Much remains to be learned about the role of CHD5 in these processes to clarify these observations.</div><div>Further, Chd5 is unique among the family of Chd remodelers in that it provides a biochemical basis for crosstalk between the critical epigenetic marks H3K27me3 and DNA methylation. Chd5 and the closely related remodelers Chd3 and Chd4 are all components of the Mi-2/NuRD histone deacetylase complex that plays a critical role in mediating transcriptional repression in response to DNA methylation in mammals (Allen, Wade, and Kutateladze 2013). Only CHD5 is preferentially expressed in neural tissue, however, and only Chd5 remodelers have biochemical evidence of direct interaction with H3K27me3, which plays a critical role in enabling proper expression of transcriptional programs during neurogenesis (Egan et al. 2013b). Chd5 is thus unique among CHD remodelers in that it is biochemically linked to both DNA methylation and H3K27me3 in addition to being preferentially expressed in neural tissue.</div><div>With regards to mechanism, much remains to be learned regarding how Chd5 remodelers contribute to gene expression and tumor suppression. However, the data to date do not show extensive transcript phenotypes and it is not clear how the biochemical action of CHD5 contributes to the neurological phenotypes ascribed to altered expression of CHD5. Therefore, it is critical to determine a suitable context to study the role of CHD5 in these processes. Identification of CHD5-dependent genes in neurons is likely to generate insight into how loss of CHD5 contributes to tumorigenesis, in particular with regards to development of neuroblastoma. Regulatory pathways that drive neurogenesis have been found to be extensively conserved between humans and zebrafish. Therefore, we have turned to the power of the zebrafish model system to characterize how loss of Chd5 alters brain development during embryogenesis.</div><div>Importantly zebrafish development, and neurogenesis in particular, occurs largely over the first 5-days of development. Zebrafish are born outside of the mother, which can produce large clutches of several hundred embryos per week, providing us with an accessible context to study the role of chd5, the zebrafish homolog of human CHD5. The central nervous system of the zebrafish develops rapidly, and shares many of the organization features of the mammalian brain (Kalueff, Stewart, and Gerlai 2014). In particular, neuroblastoma arises from a population of cells known as sympathetic ganglion cells that are derived from the neural crest (Pei et al. 2013). These cells are conserved in vertebrates, and several models to study how these cells transform into neuroblastoma exist in zebrafish (Zhu et al. 2017; Morrison et al. 2016; Zhu and Thomas Look 2016). However, our understanding of the mechanisms controlling ganglion cell differentiation is incomplete and requires further investigation to understand how epigenetic and transcriptional mechanisms contribute to development of these cells and how failure of these processes leads to cancer. The neural crest undergoes a series of differentiation steps to form mature sympathetic neurons that are guided by bone morphogenic protein signaling, and transcription changes (Ernsberger and Rohrer 2018). These cells express key enzymes for synthesizing dopamine and norephinephrine to control the sympathetic system throughout the central nervous system (Ernsberger and Rohrer 2018).</div><div>To address these questions about Chd5, we have used CRISPR/Cas9 to generate chd5-/- zebrafish that are protein nulls as determined by western blot. These chd5-/- fish are phenotypically indistinguishable from wild-type fish under standard growth conditions as was previously observed for mice lacking CHD5 (Zhuang et al. 2014; Li et al. 2014). By using zebrafish, we are able to perform transcriptome analyses to identify Chd5 target genes at stages much earlier than has previously been performed in mice because we can harvest large amounts of the tissue of interest from the readily accessible embryos. We have therefore undertaken RNA-seq analysis of isolated brains from wild-type and chd5-/- fish to identify chd5-dependent genes in predominantly differentiating (2-day old) and substantially differentiated (5-day old) neural tissue. These data provide a substantively different perspective from previous studies that examine the role of CHD5 in gene expression of differentiated SY-SH5Y cells (Egan et al. 2013a) or in the forebrain of 8-week-old mice (Pisansky et al. 2017). (Jiang, Stanke, and Lahti 2011). One role we identified from this data, is the promotion of development of sympathetic ganglion cells (detailed below), illuminating for the first time a role for chd5 in promoting differentiation of cells directly involved in neuroblatoma.</div><div>We observe not only extensive changes in gene expression, but also identify a novel role for Chd5 in enabling proper splicing during this critical window of neurogenesis in the zebrafish brain. We are further exploring the role of CHD5 in these processes by creating comparable cell culture-based models of loss of CHD5 to determine the conservation of molecular phenotypes observed in zebrafish. Furthermore, this model enables us to leverage the extensive biochemical tools available in cell culture to examine alterations to the chromatin that are difficult to interpret from studies of complex tissues such as the brain. </div><div>Herein I will describe the research progress we have made to understand the role of Chd5 in gene expression and splicing in zebrafish, as well as ongoing work to engineer mouse embryonic stem cells as an additional model to study the transcriptional consequences of loss of CHD5. Critically, the addition of the cell culture model will greatly enable biochemical characterization of the changes that are leading in particular to the changes in gene expression and splicing and will provide us with a context to test for a direct role of CHD5 in these processes. In addition, this thesis will detail the results from ongoing projects using the zebrafish model system, including: development of models in zebrafish to study the tumor suppressive role of Chd5, phenotypes observed using a targeted chemical-genetic screen, and advancement in developing new tools in zebrafish to engineer specific genomic modifications that will greatly expand the power of this vertebrate model.</div><div><br></div>

En la levadura S. cerevisiae, un incremento de la osmolaridad extracelular activa la vía de Hog1, lo que produce una compleja respuesta adaptativa. Entre las respuestas adaptativas que Hog1 coordina, está un importante cambio en el partón de expresión génica. La tesis presentada se centra en la respuesta a nivel de regulación génica, y en ella se ponen de manifiesto nuevos mecanismos por los cuales Hog1 regula la transcripción para inducir genes necesarios para la adaptación celular en respuesta a estrés osmótico. Este trabajo demuestra que Hog1 controla la iniciación y la elongación de la transcripción, interacciona con la RNA polimerasa elongando, y es reclutado en toda la región codificante de los genes que se inducen por estrés osmótico a traves del 3'UTR. Asimismo, Hog1 recluta el complejo remodelador de cromatina RSC para promover un dramático cambio en el posicionamiento de nucleosomas, permitiendo una correcta inducción de la expresión génica. / In the yeast S.cerevisiae, an increase in extra cellular osmolarity activates the Hog1 Pathway, which produces a very complex adaptive response. Among these adaptive responses coordinated by Hog1, there is an important change in the gene expression pattern. The presented Thesis focuses on the response triggered at the genomic level, showing novel mechanisms by which Hog1 regulates transcription to efficiently and properly induce a subset of genes critical for the cellular adaptation to osmotic stress. This work demonstrates that Hog1 promotes and regulates transcription not only at the initiation level, as was previously described, but it also interacts with the RNA Polymerase while elongating, and travels along the coding regions of genes induced upon osmotic stress through recognition of the 3'UTR. Furthermore, Hog1 recruits a chromatin-remodeling complex known as RSC to promote a dramatic change in nucleosome positioning of target genes, allowing a proper induction of the transcription

Le cancer du sein est le cancer qui a la plus forte fréquence au Canada. En 2012, on estime que 23 200 nouveaux cas de cancer du sein seront diagnostiqués. Deux tiers des tumeurs mammaires expriment ou surexpriment le récepteur des oestrogènes α (ERα). De même, les oestrogènes sont importants pour la croissance de ces tumeurs. La présence des récepteurs hormonaux est un critère qui détermine le choix de la thérapie; à cet égard, le ciblage des récepteurs des oestrogènes par les antioestrogènes a pour but d’inactiver ces récepteurs et diminuer leur contribution à la croissance tumorale. Les antioestrogènes sont des inhibiteurs compétitifs de ERα. Tamoxifene est le médicament le plus utilisé pour traiter les tumeurs mammaires ER+ de tous les stades, avant ou après la ménopause. Tamoxifene est antioestrogène partiel ou SERM qui a un profile mixte d’activités agonistes et antagonistes. Fulvestrant ou ICI 182, 780 est un antioestrogène de type total ou SERD dépourvu de toute activité agoniste. Ce composé est utilisé en clinique chez les femmes après la ménopause ayant des tumeurs mammaires avancées. Fulvestrant constitue, donc, une deuxième ligne thérapeutique en cas de rechute après à un traitement par Tamoxifene. Afin de comprendre le potentiel thérapeutique de Fulvestrant, il est primordial d’étudier son impact sur ERα. Actuellement, la polyubiquitination et la dégradation de ERα sont les mécanismes les plus connus pour expliquer l’inactivation de ERα par Fulvestrant. Par ailleurs, en utilisant des modèles cellulaires ER+ et ER-; nous avons montré que les antioestrogènes totaux induisent une insolubilité de ERα indépendamment de leur capacité à induire sa dégradation. L’insolubilité corrèle avec l’association de ERα avec la matrice nucléaire et avec l’inhibition de sa transactivation. L’hélice H12 du domaine de liaison du ligand joue un rôle important dans l’insolubilité et l’inactivation de ERα par les antioestrogènes totaux. Par ailleurs, les antioestrogènes totaux se distinguent par leur capacité à induire la SUMOylation de ERα par SUMO1 et SUMO2/3. La SUMOylation est rapide et précède la dégradation de ERα dans cellules ER+. À l’aide de dérivés de l’antioestrogène total ICI 164, 384, nous avons montré que la chaine latérale des antioestrogènes totaux est à la base de l’induction de la SUMOylation et de l’inactivation de ERα. De plus, la SUMOylation semble être une marque d’inhibition, car la déSUMOylation restaure une activité de ERα en présence des antioestrogènes totaux. L’hélice H12 du LBD et le domaine de liaison à l’ADN sont requis pour l’induction de la SUMOylation. La recherche de protéines impliquées dans l’inactivation et dans la SUMOylation a permis d’identifier le facteur de remodelage de la chromatine ACF dans le même complexe que ERα. De manière similaire à la SUMOylation, le recrutement de ACF est précoce et constitue une propriété spécifique des antioestrogènes totaux. D’autre part, Fulvestrant induit le recrutement de ACF au niveau du promoteur du gène cible des oestrogènes pS2, ce qui suggère une contribution du remodelage de la chromatine dans les mécanismes d’action des antioestrogènes totaux. La surexpression de la DéSUMOylase SENP1 abolit le recrutement de ACF ce qui indique un rôle de la SUMOylation dans le recrutement de ACF. De même, l’hélice H12 du LBD de ERα constitue un lien entre l’inactivation de ERα et le recrutement de ACF. L’insolubilité, la SUMOylation et l'interaction du complexe ACF sont le reflet des mécanismes d’action des antioestrogènes totaux. Ces observations peuvent être utilisées comme des critères fonctionnels pour identifier d’autres composés avec de meilleures propriétés pharmacologiques que Fulvestrant. / Approximately 70% of breast tumors express or overexpress estrogen receptor alpha (ERα) and are treated with antiestrogens (AEs), which act as competitive inhibitors of this receptor. Tamoxifen has been widely used for the treatment of ERα-positive tumors, but intrinsic or acquired resistance can lead to tumor recurrence. Full AEs such as Fulvestrant (ICI182, 780) are currently used to treat postmenopausal women with ERα-positive breast cancers with disease progression following Tamoxifen therapy. Unlike Tamoxifen and other Selective estrogen receptor modulators (SERMs), full AEs (SERDs) are devoid of any agonistic activity. It is currently thought that the capacity of full AEs to induce rapid polyubiquitination and degradation of ERα underlies their complete suppression of ERα signalling. On the one hand, we show a correlation between ICI 182, 780 induced ERα inhibition and its association with the insoluble fraction. This insolubility corresponds to an immobilization within the nuclear matrix and takes place in the absence of an accelerated turn over. The helix 12 in the ligand binding domain is important in the induction of insolubility and inactivation. On the other hand, we identify ERα as a target for Small Ubiquitin-like Modifier (SUMO) posttranslational modification by SUMO1 and SUMO2/3 specifically when liganded with full AEs. Induction of SUMOylation is rapid and precedes receptor degradation in ERα-positive breast cancer cells. On the other hand, the SERMs do not induce SUMOylation. The helix 12 in the ligand binding domain and the DNA binding domain play a role in the induction of SUMOylation in the presence of full AEs. Structure activity relationship experiments with full AE derivatives showed that the induction of SUMOylation is correlated with the degree of inhibition of ERα-mediated transcription. In addition, preventing SUMOylation by overexpression of a SENP1 deSUMOylase abolished the inverse agonist properties of full AEs without increasing activity in the presence of agonists or of Tamoxifen. In our attempt to screen for factors with a possible role in SUMOylation and inactivation, we show that the treatment with SERDs but not SERMs, induces a rapid interaction between ERα and the human ATP-utilizing chromatin assembly and remodeling factor (ACF) in ERα-negative and ERα-positive cell lines. The helix 12 is important since introducing single point mutations in this helix lead to an increased solubility and abrogate ACF recruitment. Using ChIP, we find an increase of ACF1 subunit association with proximal promoter of estrogen target gene pS2 suggesting a possible role of ACF in remodeling in this promoter. ACF recruitment is SUMOylation dependant since the overexpression of DeSUMOylase SENP1 abolishes the interaction between ERα and ACF. Together, induction of insolubility, SUMOylation and ACF recruitment are characteristic properties of full antiestrogens that contribute to their specific activity profile. They can be used to screen for new compounds with an improved therapeutic potential.

La Nucléophosmine (NPM1) est l’une des protéines les plus fréquemment altérée dans les troubles hématopoïétiques. Sa mutation, au niveau de l’exon 12, est impliquée dans un tiers des leucémies myéloïdes aiguës et constitue l’un des premiers évènements dans la leucémogénèse. Elle est caractérisée par la séquestration aberrante de NPM1 au cytoplasme, on parle alors de NPMc+. NPM1 peut également subir plusieurs translocations menant à la synthèse de protéines de fusion dont NPM-MLF1. Cette dernière est impliquée dans les syndromes myéloprolifératifs et en plus faible proportion dans les leucémies myéloïdes aiguës. Cette translocation reste peu documentée. NPM1 est une protéine multifonctionnelle dont le rôle dans la régulation génique reste à être définie. La littérature fait état de quelques études rapportant que NPM1 influencerait l’expression génique mais aucun mécanisme n’a encore été clairement décrit. Nous proposons donc dans ce projet de doctorat, de définir comment NPM1 régule la transcription et nous déterminerons si la translocation NPM-MLF1 a un effet différent de celui de NPM1. Afin de déterminer de nouveaux partenaires d’interaction, nous avons purifié les complexes protéiques de NPM1 et NPM-MLF1 dans la lignée de cellules hématopoïétique K562. Dans une étude protéomique, nous avons mis en évidence par spectrométrie de masse puis par immunoprécipitation, que NPM1 et NPM-MLF1 interagissent avec les complexes de remodelage de la chromatine NuRD, P/BAF et ISWI. Par Ampli-Sequencing, nous avons identifié des gènes dérégulés par la baisse d’expression de NPM1 et anormalement exprimés chez les patients présentant une leucémie myéloïde aiguë avec mutation NPMc+. Nous avons déterminé par immunoprécipitation de la chromatine que NPM1 et NPM-MLF1 peuvent influencer l’expression de gènes cibles via le recrutement du complexe NuRD au niveau du site d’initiation de la transcription. Le profil des modifications d’histone est également perturbé. Nous montrons ici que NPM1 et NPM-MLF1 sont impliqués à la fois dans la répression et l’activation génique. Dans une seconde partie du projet, nous proposons de déterminer l’importance physiologique de la régulation, par NPM1-NuRD, de l’un des gènes cibles mis en évidence : SPARC. La méthode CRISPR-Cas9 a été utilisée pour générer des clones uniques K562 dont l’expression de SPARC est absente et celle de NPM1 est diminuée. Cette étude démontre que la diminution d’expression de NPM1 synergise avec la perte de SPARC afin d’induire la différenciation érythroïde et réduire l’invasion cellulaire. Nous montrons donc pour la première fois que NPM1 et la translocation NPM-MLF1 peuvent interagir avec des complexes de remodelage de la chromatine et peuvent influencer leur recrutement au niveau de gènes cibles afin de réguler la transcription. Nos résultats suggèrent que NPM1 est un facteur important dans le contrôle de la modification du transcriptome associée à la différenciation des cellules hématopoïétiques. Ce projet apporte donc de nouvel éclairage sur la compréhension des mécanismes associés à la mise en place des leucémies myéloïdes aiguës présentant une mutation NPMc+ ou la translocation NPM-MLF1. / Nucleophosmine (NPM1) is one of the most frequent altered proteins in hematopoietic disorders. NPM1 exon 12 mutation characterizes one third of all acute myeloid leukemia and can be fundamental to leukemogenesis. This mutation is characterized by the aberrant sequestration of NPM1 to the cytoplasm, the mutated protein is then called NPMc+. NPM1 can also undergo several translocations leading to the synthesis of fusion proteins including NPM-MLF1. The latter is involved in myeloproliferative disorders and in a lower proportion in acute myeloid leukemia. Little is known about this translocation. NPM1 is a multifunctional protein reported to influence gene regulation. The function(s) of NPM1 in gene regulation remains to be defined. The aim of this doctoral research project was to define how NPM1 regulates transcription and determine whether the NPM-MLF1 translocation could disrupt gene regulation imposed by NPM1. In order to determine new protein interaction partners, we purified NPM1 and NPM-MLF1 complexes in the K562 hematopoietic cell line. In a proteomic analysis performed by tandem immunoaffinity purifications followed by mass spectrometry and immunoprecipitation, we identified multiple nuclear protein partners of NPM1 and NPM-MLF1. Importantly, we found that NPM1 and NPM-MLF1 can interact with the chromatin remodeling complexes NuRD, P/BAF and ISWI. An analysis made by Ampli-Sequencing, indicated that multiple genes dysregulated by the decrease expression of NPM1 were also reported to be abnormally expressed in acute myeloid leukemia cells characterized by the NPMc + mutation. We demonstrate that not only NPM1 but also NPM-MLF1 can be recruited to NPM1 regulated genes and affect the chromatin recruitment of the NuRD complex. The variation in NuRD recruitment imposed by NPM1 knockdown or NPM-MLF1 expression, is associated to the abnormal transcriptional regulation of the target genes. We show here that NPM1 and NPM-MLF1 are involved in both gene repression and activation. In a second part of the project, we investigated the physiological importance of gene regulation imposed by NPM1-NuRD in these cells. Importance of the genetic link between NPM1 and the target gene SPARC was defined. Using the CRISPR-Cas9 methodology, we generated unique K562 clones whereby SPARC expression is abrogated and NPM1 expression is decreased. The decrease of NPM1 expression synergizes with the loss of SPARC expression to induce erythroid differentiation and reduce cell invasion. The results presented provide the first demonstration that NPM1 and NPM-MLF1 translocation can interact with chromatin remodeling complexes, influence their recruitment to target genes and thereby, influence the expression of specific genes. This project provides new information to understanding mechanisms affected in acute myeloid leukemia characterized by NPMc+ mutation or NPM-MLF1 translocation.

No description available.

Mutually linked expression and methylation dynamics in the brain govern genome regulation over the whole lifetime with an impact on cognition, psychological disorders, and cancer. We performed a joint study of gene expression and DNA methylation of brain tissue originating from the human prefrontal cortex of individuals across the lifespan to describe changes in cellular programs and their regulation by epigenetic mechanisms. The analysis considers previous knowledge in terms of functional gene signatures and chromatin states derived from independent studies, aging profiles of a battery of chromatin modifying enzymes, and data of gliomas and neuropsychological disorders for a holistic view on the development and aging of the brain. Expression and methylation changes from babies to elderly adults decompose into different modes associated with the serial activation of (brain) developmental, learning, metabolic and inflammatory functions, where methylation in gene promoters mostly represses transcription. Expression of genes encoding methylome modifying enzymes is very diverse reflecting complex regulations during lifetime which also associates with the marked remodeling of chromatin between permissive and restrictive states. Data of brain cancer and psychotic disorders reveal footprints of pathophysiologies related to brain development and aging. Comparison of aging brains with gliomas supports the view that glioblastoma-like and astrocytoma-like tumors exhibit higher cellular plasticity activated in the developing healthy brain while oligodendrogliomas have a more stable differentiation hierarchy more resembling the aged brain. The balance and specific shifts between volatile and stable and between more irreversible and more plastic epigenomic networks govern the development and aging of healthy and diseased brain.

No description available.

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