Tesis sobre el tema "Epigenetic enzymes"

3,4-methylenedioxymethamphetamine (MDMA) is a psychoactive substance used for recreational purposes. Possible clinical use of MDMA, in combination with psychotherapy, has been considered for the treatment of PTSD. However, MDMA causes neurotoxic effects and its use was associated with psychiatric symptoms, memory and cognitive deficits. To elucidate these aspects, the first aim of this study was to investigate the effects of acute and repeated MDMA treatment on BDNF/TrkB and HDACs in animal models. According to recent evidence about HDAC inhibitors, we used sodium butyrate to investigate its ability to affect MDMA-induced molecular and behavioral alterations. Moreover, considering that an alteration of BDNF has been reported in the brain of animals treated with psychoactive substances and in the blood of substance abusers, possible alterations of this neurotrophin levels were investigated in blood samples of MDMA users. Since different BDNF pools exist in plasma and serum, distinct determination of the neurotrophin were evaluated in both matrices. Furthermore, recent evidence has shown that neurofilaments can represent valid biomarkers of neural damage. Given the neurotoxic effects of ecstasy, we investigated neurofilaments in serotonergic neuronal cells. Particularly, we assessed MDMA effects on neurofilament proteins in differentiated serotonergic cells and we investigated if BDNF could protect serotonergic neurons from MDMA effects. Data showed that MDMA alters different crucial genes as well as the proteins involved in both substance use disorders and psychiatric conditions. Animal studies showed alterations both in BDNF pathways and in HDACs. Moreover, investigation in humans brought into view that peripheral BDNF could not reflect central BDNF and serum and plasma BDNF can express different types of this neurotrophin. Furthermore, data obtained in the differentiated serotonergic cell line highlight the useful role of NF-L as a biomarker of neuronal damage induced by MDMA, confirming the importance of studying NFs in the field of neuropsychiatric disorders.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007.
Additional advisors: Vithal K. Ghanta, J. Michael Ruppert, Theresa V. Strong, R. Douglas Watson. Description based on contents viewed Oct. 3, 2008; title from PDF t.p. Includes bibliographical references.

Schizophrenia is a neurodevelopmental disorder affecting 1% of the general population. Dysfunction of the prefrontal cortex (PFC) is associated with the etiology of schizophrenia. Moreover, a substantial deficit of GAD1mRNA in schizophrenic PFC has been reported by different groups. However, the underlying molecular mechanisms are still unclear. Interestingly, epigenetic factors such as histone modifications and DNA methylation could be involved in the pathogenesis of schizophrenia during the maturation of the PFC. In my work, I identified potential epigenetic changes in schizophrenic PFC and developmental changes of epigenetic marks in normal human PFC. Furthermore, mouse and neuronal precursor cell models were used to confirm and investigate the molecular mechanisms of the epigenetic changes in human PFC. My initial work examined whether chromatin immunoprecipitation can be applied to human postmortem brain. I used micrococcal nuclease (MNase)-digested chromatin instead of cross-linked and sonicated chromatin for further immunoprecipitation with specific anti-methyl histone antibodies. Surprisingly, the integrity of mono-nucleosomes was still maintained at least 30 hrs after death. Moreover, differences of histone methylation at different genomic loci were detectable and were preserved within a wide range of autolysis times and tissue pH values. Interestingly, MNase-treated chromatin is more efficient for subsequent immunoprecipitation than crosslinked and sonicated chromatin. During the second part of my dissertation work, I profiled histone methylation at GABAergic gene loci during human prefrontal development. Moreover, a microarray analysis was used to screen which histone methyltransferase (HMT) could be involved in histone methylation during human prefrontal development. Mixed-lineage leukemia 1 (MLL1), an HMT for methylation at histone H3 lysine 4 (H3K4), appears to be the best candidate after interpreting microarray results. Indeed, decreased methylation of histone H3 lysine 4 at a subset of GABAergic gene loci occurred in Mll1 mutant mice. Interestingly, clozapine, but not haloperidol, increased levels of trimethyl H3K4 (H3K4me3) and Mll1 occupancy at the GAD1 promoter. I profiled histone methylation and gene expression for GAD1 in schizophrenics and their matched controls. Interestingly, there are deficits of GAD1 mRNA levels and GAD1 H3K4me3 in female schizophrenics. Furthermore, I was also interested in whether the changes of GAD1 chromatin structure could contribute to cortical pathology in schizophrenics with GAD1 SNPs. Remarkably, homozygous risk alleles for schizophrenia at the 5’ end of the GAD1 gene are associated with a deficit of GAD1 mRNA levels together with decreased GAD1 H3K4me3 and increased GAD1H3K27me3 in schizophrenics. Finally, I shifted focus on whether DNA methylation at the GAD1 promoter could contribute to a deficit of GAD1 mRNA in schizophrenia. However, no reproducible techniques are available for extracting genomic DNA specifically from GABAergic neurons in human brain. Therefore, I used an alternative approach that is based on immunoprecipitation of mononucleosomes with anti-methyl-histone antibodies differentiating between sites of active and silenced gene expression. The methylation frequencies of CpG dinucleotides at the GAD1 proximal promoter and intron 2 were determined from two chromatin fractions (H3K4me3 and H3K27me3) separately. Consistently, the proximal promoter region of GAD1 is more resistant to methylation in comparison to intron 2 of GAD1 in either open or repressive chromatin fractions. Interestingly, overall higher levels of DNA methylation were seen in repressive chromatin than in open chromatin. Surprisingly, schizophrenic subjects showed a significant decrease of DNA methylation at the GAD1proximal promoter from repressive chromatin. Taken together, my work has advanced our knowledge of epigenetic mechanisms in human prefrontal development and the potential link to the etiology of schizophrenia. It could eventually provide a new approach for the treatment of schizophrenia, especially in the regulation of methylation at histone H3 lysine 4.

The precise regulation of Major Histocompatibility class II (MHC-II) genes plays an important role in the control of the adaptive immune response. MHC-II genes are expressed constitutively in only a few cell types, but their expression can be induced by the inflammatory response cytokine interferon gamma (INF-γ). The regulation of MHC-II is controlled by a Master Regulator, the class II transactivator (CIITA). Multiple studies have shown that CIITA regulated expression of MHC-II is controlled and induced by INF-γ. It has been also shown that a functional CIITA gene is necessary for the expression of MHC-II genes. CIITA is thus a general regulator of both constitutive and inducible MHC-II expression. Although much is known about the transcription factors necessary for CIITA expression, there is little information as to the epigenetic modifications and the requisite enzymes needed to provide these transcription factors access to DNA. Previous studies in the Greer lab have shown that increased levels of acetylation of histones H3 upon INF-γ stimulation, as does tri-methylation of H3K4 upon prolonged cytokine stimulation. Similar observations were made at early time points post IFN-γ stimulation, where there is an instantaneous increase in the levels of H3K18ac and H3K4me3. In contrast to this, the levels of silencing modifications begin to drop with in the first 20 minutes of IFN-γ stimulation. The binding of STAT1 reaches its peak at about 60 minutes and the first transcripts for the protein start to appear as early as 40 minutes post the cytokines stimulation. Our study is the first to link the rapidly occurring epigenetic changes at the CIITA promoter pIV to EZH2

Major Histocompatibility Complex Class-II (MHC-II) molecules are critical regulators of adaptive immunity that present extracellular antigens required to activate CD4+ T cells. MHC-II are regulated at the level of transcription by master regulator, the Class II Transactivator (CIITA), whose association with the MHC-II promoter is necessary to initiate transcription. Recently, much research focused on novel mechanisms of transcriptional regulation of critical genes like MHC-II and CIITA; findings that the macromolecular complex of the 26S-proteasome is involved in transcription have been perhaps the most exciting as they impart novel functions to a well studied system. Proteasome is a multi-subunit complex composed of a 20S-core particle capped by a 19S-regulatory particle. The 19S contains six ATPases which are required for transcription initiation and elongation. We demonstrate that 19S ATPase-S6a inducibly associates with CIITA promoters. Decreased expression of S6a negatively impacts recruitment of the transcription factors STAT-1 and IRF-1 to the CIITA due to significant loss in histone H3 and H4 acetylation. S6a is robustly recruited to CIITA coding regions, where S6a binding coordinates with that of RNA polymerase II. RNAi mediated S6a knockdown significantly diminishes recruitment of Pol II and P-TEF-b components to CIITA coding regions, indicating S6a plays important roles in transcriptional elongation. Our research is focused on the ways in which accessibility to and transcription of DNA is regulated. While cancers are frequently linked to dysregulated gene expression, contribution of epigenetics to cancers remains unknown. To achieve metastatic ability, tumors alter gene expression to escape host immunosurveilance. MHC-II and CIITA expression are significantly downregulated in highly metastatic MDA-MB-435 breast cancer cells. This suppression correlates with elevated levels of the silencing modification H3K27me3 at CIITA and a significant reduction in Pol II recruitment. We observe elevated binding of the histone methyltransferase to CIITApIV and demonstrate this enzyme is a master regulator of CIITA gene expression. EZH2 knockdown results in significant increases in CIITA and MHC-II transcript levels in metastatic cells. In sum, transcriptional regulation by the 19S-proteasome and histone modifying enzymes represents novel mechanisms of control of mammalian gene expression and present novel therapeutic targets for manipulating MHC expression in disease.

Recent advances in high throughput technology have opened the door to systematic studies of epigenetic mechanisms. One of the key components in the regulation of the cell cycle and differentiation is the retinoblastoma protein (pRB), a component of the RB/E2F tumor suppressor pathway that is frequently deregulated in cancer. The RBP2/KDM5A histone demethylase was shown to interact with pRB and regulate pRB function during differentiation. However, how precisely differentiation is coupled with halted cell cycle progression and whether an epigenetic mechanism is involved remain unknown. In the present study, I analyzed gene expression levels of human histone methyltransferases (HMT) and demethylases (HDM), as well as their targets in human cancers; and focused on RB/KDM5A connection in control of cell cycle and differentiation. In particular, I used Drosophila as a model to describe a novel mechanism where the RB/E2F pathway interacts with the Hippo tumor suppressor pathway to synergistically control cell cycle exit upon differentiation. Studying the role of miR-11, I found that the inhibition of dE2F1-induced cell death is its highly specialized function. Furthermore, I studied the induction of differentiation and apoptosis as the consequences of KDM5A deletion in cells derived from Rb knockout mice. I concluded that during differentiation, KDM5A plays a critical role at the enhancers of cell type-specific genes and at the promoters of E2F targets; in cooperation with other repressor complexes, it silences cell cycle genes. I found that KDM5A binds to transcription start sites of the majority of genes with H3K4 methylation. These are highly expressed genes, involved in certain biological processes, and occupied by KDM5A in an isoform-specific manner. KDM5A plays a unique and non-redundant role in histone demethylation and its promoter binding pattern highly overlaps with the opposing enzyme, MLL1. Finally, I found that HMT and HDM enzymes exhibit a distinct co-expression pattern in different cancer types, and this determines the level of expression of their target genes.
Los avances recientes en las tecnologías de alto flujo han abierto el camino a los estudios sistemáticos de los mecanismos epigenéticos. La proteína retinoblastoma (pRB), uno de los elementos de la ruta de supresión de tumores RB/E2F que se encuentra desregulado con frecuencia en el cáncer, es uno de los componentes esenciales de la regulación del ciclo celular y la diferenciación. Sin embargo, aún no se conoce de qué manera precisa la diferenciación se acopla a la detención del avance del ciclo celular y si hay algún mecanismo epigenético vinculado a este proceso. En este estudio, he analizado los niveles de expresión de histona metiltransferasas (HMT) y desmetilasas humanas (HDM), así como sus dianas en cánceres humanos, y me he centrado en la conexión de RB/KDM5A en el control del ciclo celular y la diferenciación. Específicamente, utilicé Drosophila como modelo para describir un mecanismo nuevo mediante el cual RB/E2F interactúa con la ruta Hippo de supresión de tumores para controlar de manera sinérgica la detención del ciclo celular relacionada con la diferenciación. Mediante la investigación del papel de miR-11, determiné que su función altamente especializada es la inhibición de la muerte celular inducida por dE2F1. Además, estudié la inducción de la diferenciación y la apoptosis como consecuencia de la pérdida de KDMA5 en células obtenidas a partir de ratones sin Rb. Extraje como conclusión que, durante la diferenciación, KDMA5 desempeña un papel esencial sobre los estimuladores de los genes específicos de los tipos celulares, así como en los promotores de las dianas de E2F; en cooperación con otros complejos represores silencia a los genes del ciclo celular. Investigué el mecanismo de reclutamiento de KDM5A y encontré que se une al sitio de inicio de la transcripción de la mayoría de los genes que poseen metilación en H3K4. Estos genes tienen elevados niveles de expresión, están involucrados en determinados procesos biológicos y están ocupados por diferentes isoformas de KDM5A. KDM5A desempeña un papel único y no redundante en la desmetilación de las histonas y que en gran medida se solapa con la enzima con la función opuesta, MLL1. Para terminar, encontré que las enzimas HMT y HDM muestran patrones de co-expresión distintos en diferentes tipos de cáncer, y que este hecho determina los niveles de expresión de sus genes diana.

It is evident that components of the 26S proteasome function beyond protein degradation in the regulation of transcription. Studies in yeast implicate the 26S proteasome, specifically the 19S cap, in the epigenetic regulation of transcription. Saccharomyces cerevisiae 19S ATPases remodel chromatin by facilitating histone acetylation and methylation. However, it is unclear if the 19S ATPases play similar roles in mammalian cells. We previously found that the 19S ATPase Sug1 positively regulates transcription of the critical inflammatory gene MHC-II and that the MHC-II promoter fails to efficiently bind transcription factors upon Sug1 knockdown. MHC-II transcription is regulated by the critical coactivator CIITA. We now find that Sug1 is crucial for regulating histone H3 acetylation at the cytokine inducible MHC-II and CIITA promoters. Histone H3 acetylation is dramatically decreased upon Sug1 knockdown with a preferential loss occurring at lysine 18. Research in yeast indicates that the ortholog of Sug1, Rpt6, acts as a mediator between the activating modifications of histone H2B ubiquitination and H3 methylation. Therefore, we characterized the role the 19S proteasome plays in regulating additional activating modifications. As with acetylation, Sug1 is necessary for proper histone H3K4 and H3R17 methylation at cytokine inducible promoters. In the absence of Sug1, histone H3K4me3 and H3R17me2 are substantially inhibited. Our observation that the loss of Sug1 has no significant effect on H3K36me3 implies that Sug1’s regulation of histone modifications is localized to promoter regions as H3K4me3 but not H3K36me3 is clustered around gene promoters. Here we show that multiple H3K4 histone methyltransferase subunits bind constitutively to the inducible MHC-II and CIITA promoters and that over-expressing one subunit significantly enhances promoter activity. Furthermore, we identified a critical subunit of the H3K4 methyltransferase complex that binds multiple histone modifying enzymes, but fails to bind the CIITA promoter in the absence of Sug1, implicating Sug1 in recruiting multi-enzyme complexes responsible for initiating transcription. Finally, Sug1 knockdown maintains gene silencing as elevated levels of H3K27 trimethylation are observed upon Sug1 knockdown. Together these studies strongly implicate the 19S proteasome in mediating the initial reorganization events to relax the repressive chromatin structure surrounding inducible genes.

DNA damage can cause genome instability, which may lead to human cancer. The most common form of DNA damage is DNA base damage, which is efficiently repaired by DNA base excision repair (BER). Thus BER is the major DNA repair pathway that maintains the stability of the genome. On the other hand, BER mediates DNA demethylation that can occur on the promoter region of important tumor suppressor genes such as Breast Cancer 1 (BRCA1) gene that is also involved in prevention and development of cancer. In this study, employing cell-based and in vitro biochemical approaches along with bisulfite DNA sequencing, we initially discovered that an oxidized nucleotide, 5’,2-cyclo-2-deoxyadenosine in DNA duplex can either cause misinsertion by DNA polymerase β (pol β) during pol β-mediated BER or inhibit lesion bypass of pol β resulting in DNA strand breaks. We then explored how a T/G mismatch resulting from active DNA demethylation can affect genome integrity during BER and found that pol β can extend the mismatched T to cause mutation. We found that AP endonuclease 1 (APE1) can use its 3'-5' exonuclease to remove the mismatched T before pol β can extend the nucleotide preventing a C to T mutation. The results demonstrate that the 3'-5' exonuclease activity of APE1 can serve as a proofreader for pol β to prevent mutation. We further explored the effects of exposure of environmental toxicants, bromate and chromate on the DNA methylation pattern on the promoter region of BRCA1 gene with bisulfite DNA sequencing. We found that bromate and chromate induced demethylation of 5-methylcytosines (5mC) at the CpG sites as well as created additional methylation at several unmethylated CpG sites at BRCA1 gene in human embryonic kidney (HEK) 293 cells. We further demonstrated that the demethylation was mediated by pol β nucleotide misinsertion and an interaction between pol β and DNA methyltransferase 1 (DNMT1) suggesting a cross-talk between BER and DNA methyltransferases. We suggest that DNA base damage and BER govern the interactions among the environment, the genome and epigenome, modulating the stability of the genome and epigenome and disease development.

Abstract 2-oxoglutarate-dependent dioxygenases (2-OGDDs) are an enzyme family that contains many enzymes that modify chromatin in extensive ways. These enzymes include several histone lysine demethylases (KDMs) and TET enzymes that convert methylated cytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) ultimately leading to DNA demethylation. Disturbed DNA and histone methylation are found in many cancers. However, the role of KDMs and TETs behind these oncogenic changes has so far not been fully investigated. This study focused on the role of these chromatin-modifying enzymes in cancers with special emphasis on enzyme kinetic studies. Cancers with inactivating fumarate hydratase (FH), succinate dehydrogenase (SDH) and isocitrate dehydrogenase (IDH) mutations accumulate fumarate, succinate and R-2-hydroxyglutarate, respectively. In this study we showed how these cancer-associated 2-oxoglutarate (2-OG) analogues can inhibit the TET enzymes and many of the KDMs leading to lower 5-hmC levels and increased H3K27 and H3K9 methylation on chromatin, respectively. We also characterized kinetic properties of acute myeloid leukaemia (AML)-associated TET2 mutants and found that their ability to bind 2-OG or iron was impaired leading to diminished catalytic activity. Tumours are often hypoxic due to inadequate vasculature and blood supply. The TET enzymes and KDMs require oxygen for the reactions they catalyse. We determined the oxygen affinity of TETs and many KDMs and found that a H3K27 demethylase KDM6A has a remarkably low affinity for oxygen indicating that it is inactivated in hypoxic tumours and tissues. H3K27 methylation was found to be increased in hypoxic cells and this blocked cell differentiation. Altogether, these studies shed light on the mechanisms behind the altered DNA and histone methylation found in several cancers with hypoxic conditions or FH, SDH and IDH mutations. Altered DNA and histone methylation has previously been associated with progression of cancer, such as epithelial-to-mesenchymal transition (EMT). We now linked catalytic inhibition of 2-OGDDs to disturbed DNA and histone methylation that can account for altered cell differentiation, EMT and increased aggressiveness and invasiveness of cancers
Tiivistelmä 2-oksoglutaraatista riippuvaiset dioksygenaasit ovat entsyymiperhe, johon kuuluu useita entsyymejä, jotka muokkaavat kromatiinin epigeneettisiä merkkejä monin tavoin. Näitä entsyymejä ovat mm. DNA:n demetylaatioon vaikuttavat TET-entsyymit sekä useat histonidemetylaasit. Vaikka muutoksia DNA:n ja histonien metylaatiotasoissa on havaittu useissa syövissä, ei näiden entsyymien roolia muutosten taustalla ole vielä tutkittu. Tämä tutkimus kohdistui näiden epigenetiikkaan vaikuttavien entsyymien roolin ymmärtämiseen syövissä keskittyen erityisesti kyseisten entsyymien kinetiikkaan. Useissa syövissä on havaittu fumaraattihydrataasin, sukkinaattidehydrogenaasin ja isositraattidehydrogenaasien aktiivisuuteen vaikuttavia mutaatioita, jotka johtavat fumaraatin, sukkinaatin ja R-2-hydroksiglutaraatin kertymiseen syöpäsoluihin. Tässä tutkimuksessa osoitimme, kuinka nämä karsinogeeniset 2-oksoglutaraattianalogit voivat inhiboida TET-entsyymejä ja histonidemetylaaseja, mikä alentaa 5-hydroksimetyylisytosiinitasoja ja lisää histonien metylaatiota. Näytämme myös, kuinka tietyillä akuutissa myelooisessa leukemiassa esiintyvillä TET2-mutanteilla on heikentynyt kyky sitoa 2-oksoglutaraattia tai rautaa, mikä johtaa entsyymien aktiivisuuden laskuun. Kasvainkudoksissa happipitoisuudet ovat usein matalia nopean kasvun ja puutteellisen verisuonituksen vuoksi. TET-entsyymit ja histonidemetylaasit vaativat happea katalysoimissaan reaktioissa. Määritimme TET-entsyymien ja monien histonidemetylaasien riippuvuutta hapesta ja osoitimme, että H3K27-histonidemetylaasi KDM6A on erittäin riippuvainen hapesta, mikä osoittaa, ettei se pysty toimimaan kasvaimissa ja kudoksissa, joissa happipitoisuudet ovat matalia. Huomasimme, että vähähappisissa olosuhteissa solujen H3K27 metylaatio on lisääntynyt, mikä johti erilaistumisen estymiseen soluissa. Tämä tutkimus paljasti uusia mekanismeja useista syövistä löytyneiden muuntuneiden DNA:n ja histonien metylaatiotasojen taustalla. Häiriintynyt DNA:n ja histonien metylaatio on aiemmin yhdistetty syöpien etenemiseen, erityisesti solujen erilaistumisen häiriintymisen kannalta. Tässä tutkimuksessa yhdistimme 2-oksoglutaraatista riippuvaisten entsyymien inhibition häiriintyneeseen DNA:n ja histonien metylaatioon, joka voi johtaa muuntuneeseen solujen erilaistumiseen ja lopulta lisääntyneeseen syöpien aggressiivisuuteen ja invasiivisuuteen

A major therapeutic challenge in musculoskeletal regenerative medicine is how to effectively replenish bone tissue lost due to pathological conditions such as fracture, osteoporosis, or rheumatoid arthritis. Mesenchymal stem cells are currently investigated for applications in bone-tissue engineering and human bone marrow-derived mesenchymal stem cells (hMSCs) could be a promising source for generation of tissue-engineered bone. However, the therapeutic potential of MSCs has not been fully exploited due to a lack of knowledge regarding the identity, nature, and differentiation of hMSCs. Epigenetic mechanisms regulating the chromatin structure as well as specific gene transcription are crucial in determination of stem cell differentiation. With the aim to systematically identify epigenetic factors that modulate MSC differentiation, the work in this thesis encompasses an approach to identify epigenetic mechanisms underlying, initiating, and promoting osteoblast differentiation, and the investigation of individual epigenetic modulators. Various osteogenic inducers were validated for differentiation of MSCs and an assay allowing assessment of differentiation outcome was developed. This assay was subsequently employed in knockdown experiments with lentiviral short hairpin RNAs and inhibitor screens with small molecules targeting putative druggable epigenetic modulator classes. This approach identified around 100 epigenetic modulator candidates involved in osteoblast differentiation, of these candidates approximately 2/3 downregulated and 1/3 upregulated alkaline phosphatase (ALP) activity. Serving as a proof-of-concept, orthogonal validation experiments employing locked nucleic acid (LNA) knockdown were performed to validate a subset of candidates. Two identified target genes were selected for further investigation. Bromodomain-containing protein 4 (BRD4) was identified as one component of epigenetic regulation; its inhibition led to a decrease in ALP expression, downregulation of key osteoblast transcription factors Runx2 and Osterix, as well as impaired bone matrix formation. Knockdown of lysine (K)-specific demethylase 1A (KDM1A/LSD1) upregulated ALP activity and treatment with a small molecule inhibitor targeting KDM1A led to an increase in ALP, RUNX2, and bone sialoprotein expression. Intriguingly, in a transgenic mouse model overexpressing Kdm1a a decrease in bone volume and bone mineral density was observed, thus supporting the hypothesis that KDM1A is a central regulator of osteoblast differentiation.

Mon travail de thèse porte sur l’étude du rôle de l’histone acétyl-transférase MOZ et de l’histone méthyle-transférase MLL dans l’hématopoïèse. Elles contrôlent l’expression de nombreux gènes, nottament des gènes HOX, des facteurs de transcription connus pour leur rôle dans l’hématopoïèse normale et pathologique. Les deux protéines ont des gènes cibles communs tel qu'HOXA9. Ces observations nous ont conduit à rechercher une coopération fonctionnelle entre MOZ et MLL. Nous avons montré que MOZ était associée avec MLL dans les cellules souches/progénitrices humaines CD34+ afin d’activer la transcription des gènes HOXA5, HOXA7 et HOXA9. En effet, les deux protéines interagissent et sont recrutées au niveau de leur promoteur. Nous avons mis en évidence une interférence fonctionnelle entre ces deux facteurs épigénétiques, puisque MOZ est nécessaire au recrutement et à l’activité enzymatique de MLL au niveau des gènes HOXA5, HOXA7 et HOXA9 et réciproquement.Afin de caractériser le mécanisme d’action impliquant la coopération entre MOZ et MLL, nous avons recherché d’autres partenaires associés à ce duo. Nous avons identifié la Symplekin, un membre de la machinerie de polyadénylation. Nous avons mis en évidence l’interaction de la Symplekin avec MOZ et MLL dans les cellules de la lignée hématopoïétique humaine KG1. Les trois protéines sont co-recrutées sur le promoteur du gène HOXA9. Nous avons démontré le rôle ambivalent de la Symplekin. Bien qu’elle soit importante pour la polyadénylation et par conséquent pour la stabilité de l’ARN Hoxa9, la Symplekin empêche le recrutement de MOZ et de MLL au niveau du gène HOXA9, conduisant ainsi à une diminution de sa transcription
My thesis project has consisted of the study of MOZ, and MLL. They are epigenetic regulators. MOZ and MLL activate transcription of HOX genes, which are transcription factors essential during haematopoiesis. MOZ and MLL have some target genes in common. In our study, we characterised a cooperation between MOZ and MLL in human haematopoietic stem/progenitor cells CD34+. They are both recruited onto HOX promoters. MOZ is essential for MLL recruitment, and this is reciprocal. In conclusion, we provided an example of a mechanism involving a direct cross-talk between two histone modifying enzymes.In order to dissect the mechanism of action of this complex, we decided to identify novel proteins interacting with both MOZ and MLL. A member of the RNA polyadenylation machinery has been isolated: Symplekin. We confirmed the interaction between MOZ, MLL and Symplekin in the human haematopoietic immature cell line KG1. We showed that Symplekin is co-recruited to HOXA9 promoter along with MOZ and MLL. We demonstrated the dual role of this member of the polyadenylation machinery. Indeed, besides the fact that Symplekin is important for Hoxa9 polyadenylation, thus its stability, it prevents MOZ and MLL recruitment onto HOXA9 promoter, leading to a decrease of HOXA9 transcription.Our work improved the understanding of the mechanism of action of MOZ and MLL in HOX control

La résistance aux thérapies anti-cancéreuses demeure un enjeu majeur, en particulier dans le cancer du sein triple négatif (TNBC), dont le traitement repose principalement sur la chimiothérapie. L'acquisition de la résistance est un processus en plusieurs étapes qui commence par la survie d'une sous-population rare de cellules cancéreuses. Ces cellules, appelées cellules persistantes, constituent un réservoir à partir duquel les cellules résistantes finiront par émerger. L'état de persistance est transitoire et réversible, ouvrant des perspectives d'intervention thérapeutique. Cependant, comprendre les mécanismes récurrents qui conduisent à l'émergence des cellules persistantes au cours du traitement reste un défi, notamment en raison de leur accessibilité limitée chez les patientes. L'objectif de cette thèse était d'améliorer notre compréhension de la persistance et de proposer de nouvelles cibles pharmacologiques afin d'améliorer la réponse aux chimiothérapies dans les cancers TNBC. Dans cette perspective, nous avons étudié les caractéristiques épigénétiques, telles que le remodelage des paysages chromatiniens, et transcriptomiques des cellules persistantes en utilisant de nombreux modèles de TNBC in vitro et in vivo. Ces modèles reproduisent l'émergence de cellules persistantes lors d'un traitement avec un panel de chimiothérapies. Ils permettent de décortiquer l'évolution phénotypique des cellules tout au long du traitement et d'identifier au niveau transcriptomique et épigénomique les régulateurs moléculaires conduisant la transition d'un état chimio-naïf à un état chimio-persistant. Nous avons ainsi précédemment montré un rôle clé de la marque d'histone répressive H3K27me3 agissant comme une barrière qui, une fois levée au niveau de gènes spécifiques après traitement, permet aux cellules cancéreuses de tolérer le stress thérapeutique. À travers ce travail, nous avons affiné la définition des cellules persistantes dans le TNBC en identifiant les points communs entre les programmes de persistance de plusieurs patientes en réponse à des traitements de chimiothérapie aux modes d'actions variés. L'état de persistance est partagé et caractérisé par l'activation de voies de signalisation telles que la réponse au stress et l'inflammation, représentant des cibles potentielles pour prévenir la persistance avant même la résistance. Nous avons également obtenu des clés de compréhension sur les mécanismes d'action des acteurs moléculaires conduisant à l'expression du programme de persistance. Grâce à des prédictions utilisant des réseaux de régulation des gènes, nous avons observé que certains facteurs de transcription, tels que les protéines de la famille AP-1, sont des régulateurs “maîtres” pouvant conduire à l'activation des gènes impliqués dans cet état de persistance aux chimiothérapies. Parmi les facteurs AP-1, nous avons montré que FOSL1 se lie aux enhancers et reprogramme le transcriptome des cellules cancéreuses pour leur permettre de persister sous chimiothérapie. En parallèle, en testant comment les enzymes épigénétiques contrôlent le programme de persistance, nous avons démontré que la méthyltransférase EZH2, responsable de la triméthylation de H3K27, est un régulateur maître du programme de persistance qui pourrait lui-même être régulé par d'autres partenaires. En résumé, les régulateurs maîtres que nous avons identifiés orchestrent au niveau génomique, épigénomique et transcriptomique l'activation de gènes spécifiques et sont nécessaires et/ou suffisants pour conduire à l'état de persistance. A travers ces découvertes, nous proposons des stratégies prometteuses afin de surmonter la persistance et améliorer la réponse aux traitements dans le TNBC : l'inhibition de régulateurs clés tels que le facteur de transcription FOSL1 et les déméthylases KDM6A/B responsables de la déméthylation de H3K27 ; ainsi que le ciblage des voies spécifiques du programme de persistance
Resistance to anti-cancer therapies remains a major challenge, particularly in triple-negative breast cancer (TNBC), which primarily relies on chemotherapy for treatment. The acquisition of resistance is a multi-step process that begins with the survival of a rare subpopulation of cancer cells. These cells, known as persister cells, form a reservoir from which resistant cells will emerge. The persister state being transient and reversible, it offers opportunities for therapeutic intervention. However, understanding the recurrent mechanisms leading to the emergence of persister cells during treatment remains a challenge, particularly due to their limited accessibility in patients. The goal of this thesis was to improve our understanding of persistence and propose new pharmacological targets to enhance chemotherapy responses in TNBC. To this end, we studied the epigenetic characteristics, such as chromatin landscape remodeling, and transcriptomic profiles of persister cells using numerous in vitro and in vivo TNBC models. These models reproduce the emergence of persister cells during treatment with a panel of chemotherapies. They allow us to dissect the phenotypic evolution of cells during cancer treatment and identify at the transcriptomic and epigenomic levels the molecular regulators driving the transition from a chemo-naive state to a chemo-persister state. This way, we have previously shown a key role for the repressive histone mark H3K27me3, acting as a barrier that, once removed at specific genes after treatment, enables cancer cells to tolerate therapeutic stress. Through this work, we refined the definition of persister cells in TNBC by identifying commonalities between the persister programs of several patients in response to chemotherapies with varying modes of action. The persister state is shared and characterized by the activation of signaling pathways such as stress response and inflammation, representing potential targets to prevent persistence before resistance even develops.We also gained insights into the mechanisms of action of the molecular players driving the expression of the persister program. Using predictions from gene regulatory networks, we observed that key transcription factors, such as the AP-1 family proteins, are master regulators capable of activating genes involved in chemotherapy persistence. Among the AP-1 factors, we showed that FOSL1 binds enhancers and reprograms the transcriptome of cancer cells to confer them the ability to persist under chemotherapy. In parallel, by testing how epigenetic enzymes control the persister program, we demonstrated that the methyltransferase EZH2, responsible for H3K27 trimethylation, is a master regulator of the persister program, which itself may be regulated by other partners. In summary, the master regulators that we have identified orchestrate the activation of specific genes at the genomic, epigenomic, and transcriptomic levels and are necessary and/or sufficient to drive the persister state. Through these discoveries, we propose promising strategies to overcome persistence and improve treatment responses in TNBC: inhibiting key regulators such as the transcription factor FOSL1 and the demethylases KDM6A/B responsible for H3K27 demethylation; as well as targeting specific pathways of the persistence program

Epigenetic regulation is a complex process involving the interplay of multiple different cellular factors. Work described in this thesis concerned the characterisation of proteins involved in the binding to, and demethylation of, histone 3 (H3) tails modified by N-methylation. Initial work focussed on the biophysical characterisation of the tandem plant homeodomains (PHD) of the chromatin remodeller CHD4. NMR spectroscopy was used to investigate the solution structure of the tandem PHDs. Studies on a more native-like construct including the C terminal tandem chromodomains are also presented. Binding studies of the PHDs with H3 peptides reveal that the individual PHD fingers can independently bind a histone peptide. The remainder of the work involved characterisation of JmjC histone demethylases (KDMs), enzymes that catalyse removal of Nε-methyl groups from histone lysyl-residues. Initially, two members of the KDM7 subfamily, PHF8 and KIAA1718, were studied; a high throughput screening assay for them was developed, which enabled identification of a selective inhibitor of the KDM2/7 subfamilies of KDMs, the plant growth regulator Daminozide. A disease relevant mutation in PHF8 was studied and shown to cause mis-localisation of the enzyme to the cytoplasm, providing a potential explanation for the clinically observed phenotype. Subsequent chapters describe unprecedented activities for the JmjC KDMs. 2OG oxygenases catalyse a wide range of oxidative reactions, predominantly mediated by initial substrate hydroxylation. The activity of PHF8 with lysine analogous was tested; the results demonstrated that PHF8, and other KDMs, can oxidatively remove Nε-alkyl groups other than methyl groups, such as ethyl and isopropyl groups. The substrate scope of the JmjC KDMs thus has the potential to be wider than previously thought. Observation of β-hydroxylation of the Nε-isopropyl group of a histone peptide including Nε methylisopropyllysine by JMJD2A/E supports the presumed mechanism of histone lysine demethylation as proceeding via initial hydroxylation. This work led to the discovery that JmjC KDMs can catalyse arginine demethylation. This novel arginine demethylase activity by JmjC KDMs was characterised and the work extended to encompass potential arginine demethylase activity in cells. Biochemical characterisation of UTY, a homologue of the H3 K27 demethylases JMJD3 and UTX, which is reported to be inactive, was carried out; UTY was shown to catalyse demethylation at H3 trimethylated at K27 on peptidic substrates, albeit it at substantially lower rates than the other family members. To investigate the reason for this reduced activity, two variants were made, S1142G and P1214I; the latter variant was shown to be considerably more active than wildtype UTY, likely due to an increased peptide-binding interaction. Preliminary experiments in cells did not conclusively demonstrate histone demethylation, but a luciferase assay suggested that UTY may have catalytic activity in cells. Overall the findings in the thesis suggest that the process of cellular epigenetic regulation is likely even more complex than previously thought, with the potential that JmjC KDMs carry out multiple, context dependent functions.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, the 5-year survival rate being less than 5%. At the time of diagnosis, 90% of PDACs extend beyond the pancreas and distant metastases are often present. Due to aggressive growth, local expansion and early appearance of metastasis, primary PDAC tumours are local enough for curative surgical resection in only 10–20% of the cases. Adjuvant chemotherapy is indicated in these curative-treated cases, with slight improvement in survival. PDAC is considered to represent a heterogeneous group of biologically and prognostically different malignancies. Characterization of these subgroups is essential and there is an urgent need for more accurate biomarkers and targeted treatments in PDAC. In the current work, we immunohistochemically investigated the expression levels and prognostic values of oxidative stress markers (8-OHdG, Keap1, Prx I, II, III, V and VI), epigenetic histone modifiers (KDM4A, KDM4B, KDM4D and SIRT1–4), and cell-cycle regulators (p16, Rb, CDK4) and DNA-repair enzymes (FEN1 and MGMT) in the cohort of surgically treated PDAC patients. We found that Keap1 expression was associated with better pancreatic cancer-specific survival. Expression of antioxidative peroxiredoxins I, III, V and VI was also connected with a more favourable tumour characteristics and Prx I and VI showed prognostic value. When considering the biology of PDAC, we noticed that pivotal epigenetic regulation also occurred in exocrine pancreatic tissue adjacent to resection margins. Overexpression of the cell-cycle regulator CDK4 and the DNA-repair enzyme FEN1 in the whole population, and elevated expression level of MGMT in the most high-risk patients were connected with worse prognosis. The results of the study can be utilized in the future when individualized therapies are being designed for PDAC patients. Due to occurrence of the epigenetic regulation also in exocrine pancreatic tissue adjacent to resection margins, it could be evaluated in future for routine diagnostics and treatment optimization. The potential role of MGMT in the development of PDAC chemoresistance should be studied in the future
Tiivistelmä Haiman duktaalinen adenokarsinooma (PDAC) on yksi aggressiivisimmista syöpäsairauksista. Viiden vuoden elossaoloennuste on vain lähellä 5 prosenttia. Diagnoosihetkellä 90% haiman adenokarsinoomista yltää haiman ulkopuolelle ja usein kasvain on jo lähettänyt etäpesäkkeitä. Kasvutaipumuksen sekä metastasoinnin takia kuratiivinen kirurginen hoito on mahdollista vain 10–20% tapauksista. Liitännäissolunsalpaajahoito on aiheellista näissä kuratiivistavoitteisesti hoidetuissa tapauksissa. Kuitenkin vaikutus kokonaiselossaoloaikaan on melko vähäinen. Uusimman tutkimustiedon valossa PDAC:aa pidetäänkin heterogeenisenä ryhmänä biologisesti ja ennusteellisesti erilaisia tautiryhmiä. Näiden tautiryhmien tunteminen ja tunnistaminen riittävän tarkkojen merkkiaineiden avulla olisi ensiarvoisen tärkeää, jotta hoitoja voitaisiin kohdentaa niistä hyötyville potilaille. Väitöskirjatutkimuksessa selvitimme immunohistokemiallisin menetelmin oksidatiivisen stressin merkkiaineiden (8-OHdG, Keap1, Prx I, II, III, V ja VI), epigeneettisten histonimodifikaattorien (KDM4A, KDM4B, KDM4D ja SIRT1–4) sekä solusyklin säätelijöiden (p16, Rb, CDK4) ja DNA-korjausentsyymien (FEN1 ja MGMT) ilmentymistä ja ennusteellista arvoa kirurgisesti hoidetuilla PDAC-potilailla. Tutkimuksessamme totesimme, että kasvainkudoksen Keap1-ilmentymä yhdistyi parempiennusteiseen taudinkuvaan. Antioksidatiivisten peroksiredoksiinien I, III, V ja VI ilmentyminen yhdistyi niin ikään suotuisampaan kasvaimen fenotyyppiin ja Prx I ja VI osoittivat ennusteellista arvoa. Havaitsimme lisäksi, että PDAC:n biologiaan keskeistesti vaikuttavaa epigeneettistä säätelyä tapahtuu myös malignin haimakudoksen viereisessä eksokriinisessä haimakudoksessa. Solusyklin säätelijä CDK4:n ja DNA-korjausentsyymi FEN1:n voimakas ilmentyminen koko tutkimuspopulaatiossa sekä kohonnut MGMT:n ilmentyminen korkeimman riskin potilailla yhdistyivät huonompaan taudin ennusteeseen. Väitöskirjatyön tutkimustuloksia voidaan tulevaisuudessa hyödyntää, kun tutkitaan yksilöllisiä hoitomuotoja PDAC-potilailla. Koska epigeneettistä säätelyä tapahtuu myös syövän viereisessä eksokriinisessa haimakudoksessa, voidaan tulevaisuudessa tämän kudoksen arviointia mahdollisesti käyttää rutiinisti diagnostiikassa sekä hoidon optimoinnissa. MGMT:n mahdollinen rooli PDAC:n kemoresistenssin kehittymisessä tulisi tulevaisuudessa selvittää

Fine motor skills are learned through repetitive practice and once learned, last for a long time. Skilled reaching is linked to structural and functional changes in multiple brain regions including, in particular, the primary motor cortex. Previous studies demonstrated that fine motor skill learning is associated with cortical synaptogenesis and motor map reorganization. At present, studies have implicated an indispensable role of epigenetic alterations in both hippocampal- and striatal-dependent memory formations, while examinations into the epigenetic changes in the primary motor cortex are lacking. The current study was aimed to identify epigenetic changes in motor cortex as a result of extensive motor skill learning using the single pellet skilled reaching task. Male Wistar rats were trained in the single pellet skilled reaching task (n = 6) for 10 consecutive days or were, under similar conditions, given access to pellets that did not require skilled reaches (n = 6). Skilled motor trained rats exhibited a rapid increase in successful reaches during the first four days of motor training before reaching a plateau, indicative of the acquisition and consolidation of the learned task, respectively. Expression profiles of chromatin modifying enzymes were screened using epigenetic-targeted PCR arrays. Results suggest that gene expression levels of multiple chromatin regulatory enzymes were down-regulated in the motor cortex of trained animals compared to controls following 10 days of motor training in the skilled reaching task. Among the chromatin modifying enzymes, the transcription level of Smyd1 (SET and MYND domain containing 1; NM_001106595) was lower (-2.17 fold-change) in motor cortex after 10 days of training compared to controls. Our results point to an epigenetic regulation of chromatin modification markers in the primary motor cortex that possibly underlie the mechanisms of synaptic plasticity, synaptogenesis and the formation of procedural memory.

La cromatina és una estructura nucleoproteica, dinàmica i jeràrquicament organitzada dins del nucli cel•Iular. La seva estructura, organització i funció està controlada al llarg del cicle cel.lular, per processos epigenètics que produeixen canvis heretables que no afecten la seqüència de DNA. Entre aquests canvis es troben la metilació del DNA, les modificacions postraduccionals de les histones i desplegament de la cromatina orquestrat pels complexes remodeladors i proteïnes estructurals. Les modificacions postraduccionals de les histones es donen principalment de forma dinàmica i regulada a l'extrem N-terminal, canviant la seva càrrega i funció. Entre les diferents modificacions descrites, l'acetilació i la metilació de les histones H3 i H4, semblen tenir una importància especial sobre l'estructura i estat de la cromatina. En particular, l'acetilació de la lisina 16 de la histona H4 (H4K16Ac) per se impedeix la formació d'estructures compactes de cromatina. D'aquesta manera, s'associa l'estat desacetilat d'aquest residu a zones d'heterocromatina i transcripcionalment inactives, mentre que la hiperacetilació d'H4K16 està estretament vinculada a zones eucromàtiques i transcripcionalmentactives. La dinàmica d'acetilació-desacetiIació d' H 4K 16 està controlada principalment per l'equilibri de les activitats acetiltransferasa de MOF i desacetilasa de Si rT1 i Si rT2. Per tant, aquests dos grups d'enzims són essencials per el control de l'expressió gènica i de l'arquitectura de la cromatina dins del nucli, regulant la transició entre l'estat transcripcionalment actiu i inactiu de la cromatina. MOF és crucial per el desenvolupament embrionari , I a reparació del DNA i la progressió del cicle cel .lular, i la seva pèrdua està associada a l'augment de les aberracions cromosòmiques, a l'aturada del cicle cel.lular en G2/M i a l'augment de la inestabilitat genòmica. SirT1 i Si rT2 pertanyen a la classe III de desacetilases d'histones (HDACs), conegudes com sirtuïnes, i són crucials per el manteniment de la integritat genòmica, l'adaptació a l'entorn i l'envelliment, entre altres funcions. No obstant, dels set membres de sirtuïnes presents a mamífers, només Si rT2 i en menor mesura SirT1, han estat vinculades amb el control del cicle cel.lular. Concretament, Si rT2, tot i ser principalment citoplasmàtica, és transportada al nucli durant I a transició G2/M , moment en el que a través de la desacetilació d'H4K16 permet la monometilació d'H4K20 per PR-SET7 (la monometiI transferasa d'H4K20), determinant els nivells d'H4K2Ome2,3 en la resta del cicle cel.lular. Com a conseqüència, SirT2 està implicat en processos estrictament associat a aquestes modificacions, com la compactació dels cromosomes metafàsics, Ia progressió a través de mitosi, la replicació i reparació del DNA o la formació de l'heterocromatina. Fins al moment el paper de Si rT2 en el control de I a progressió a través de la mitosi ha estat atribuït a la regulació de diferents substrats. En particular, el control d'H4K2Ome1 dependent de SirT2 ha estat relacionat amb l'activació del checkpoint de G2/M en resposta a estrès, tot i que els mecanismes involucrats en l'aturada del cicle cel.lular eren del tot desconeguts. En aquest treball hem volgut indagar sobre com Si rT2 regula la transició G2/M i la seva coordinació amb la maquinària regulatória del checkpoint. Les nostres dades semblen indicar que SirT2 condueix a l'activació del checkpoint de G2/M a través de la regulació d' H4K 1 6Ac, H4K2Ome1 i el control de I a transcripció de gens relacionats amb el cicle cel.lular. Es descriu per primera vegada, com SirT2 controla H4K16Ac a través de la regulació de l'activitat i de l'estabilitat proteica de MOF durant G2/M. Concretament, SirT2 desacetila MOF durant G2/M i en promou la sortida de la cromatina, la inactivació i degradació, afavorint l'estat desacetilat d'H4K16 i la monometilació d'H4K20. En concordança, hem vist que MOF controla negativament la presència de PR-SET7 a la cromatina, mantenint així uns nivells adequats d'H4K2Omel abans d'entrar a mitosi i evitant la condensació prematura dels cromosomes. El nostre estudi suggereix que la interconnexió entre SirT2 i MOF està directament implicada en el control epigenètic del cicle cel.lular, contribuint al manteniment de l'estabilitat genòmica.
Chromatin is a dynamical structure hierarchically organized to fit inside the nucleus. The structure, organization and function of chromatin are tightly controlled throughout the cell cycle by different epigenetic mechanisms, including DNA methylation and histone modifications. The histone post-translational modifications occur mainly in their N-terminal tail, and give rise to changes in the charge and function of the protein. Among the different histone modifications, lysine acetylation (K) is one of the best characterized. Acetylation of lysine 16 of histone H4 is the most frequently acetylated residue in eukaryotes and is a key regulator of high orders of chromatin structure. Thus, the deacetylated state of this residue is associated with heterochromatic and transcriptional inactive regions, whereas the acetylated form is found in euchromatic and transcriptional active regions. The dynamics of this histone mark is mainly governed by the acetyltransferase MOF and the NAD±-dependent deacetylases SirT1 and SirT2, which makes both groups of enzymes essential for the regulation of the gene expression and the control of chromatin organization. MOF is crucial in embryogenesis, DNA repair and the cell cycle progression. In fact, loss of MOF has been shown to induce cell cycle arrest during G2/M transition, increased chromosomal aberrations and genome instability. SirT 1 and SirT2 belong to Class III of histone deacetylases (HDACs), commonly referred as sirtuins. They play a key role in stress response, and in particular in protecting genome integrity. Among the seven mammalian sirtuins (SirT1-7), only SirT2 and to a lesser extend SirT1, have been linked with cell cycle regulation. In particular, SirT2, which mainly localizes to the cytoplasm during most of the cell cycle, shuttles to the nucleus in G2/M transition, where deacetylates H4K16Ac driving, among other things, H4K2Omel deposition by the histone methyltransferase PR-SETT. The control of H4K2Omel deposition determines the levels of H4K2Ome2,3 in the next cell cycle, which links SirT2 to the regulation of DNA replication and repair, as well as heterochromatin formation. Work from our group and others have shown that SirT2 plays a role in the control of mitosis progression. In particular, SirT2 is required for the cell cycle arrest in the G2/M checkpoint during stress response, process that has been related to SirT2-dependent regulation of H4K2Omel. However, the mechanisms behind the cell cycle arrest are still undefined. In the present work we aimed to elucidate the function of SirT2 in G2/M transition and its coordination with the checkpoint regulatory machinery. Our results seem indicate that SirT2 drives the G2/M checkpoint activation through the regulation of H4K16Ac, H4K2Omel and the control of the expression of cell cycle related genes. We also describe for the first time, a complementary mechanism whereby SirT2 regulates the levels of H4K16Ac during mitosis. We observe that SirT2 not only deacetylates MOF during G2/M, suppressing its acetyltransferase activity, but also induces both chromatin eviction and degradation of MOF. This in turn, results in H4K16 hypoacetylation and subsequent monomethylation of H4K20. Additionally, we show that MOF inhibits PR-SETT chromatin localization, maintaining the appropriate levels of H4K2Omel before entering mitosis and avoiding premature chromosome condensation. Our study suggests that the crosstalk between MOF and SirT2 is directly involved in the epigenetic control of the cell cycle, contributing to the maintenance of genome stability.

L’ontogenèse des lymphocytes B (LB) comporte une première phase de différenciation, dans la moelle osseuse en l'absence de toute stimulation antigénique spécifique, aboutissant au LB immature. La seconde phase, d’activation et de maturation finale, est dépendante des antigènes et se déroule dans les organes lymphoïdes secondaires, au sein de structures transitoires appelées centres germinatifs (CG). Elle génère des plasmocytes et des cellules B mémoires spécifiques d’un antigène.Ce travail de thèse s’intéresse à différents acteurs impliqués dans la régulation épigénétique et transcriptionnelle de la différenciation lymphoïde B terminale : les enzymes TET2 et TET3 et le facteur de transcription (FT) SPI1/PU.1. Des mutations affectant les gènes codant pour ces protéines sont trouvées dans les hémopathies chez l’Homme et nous avons cherché à déterminer leurs conséquences fonctionnelles en utilisant des modélisations in vivo et in vitro.J’ai d’une part analysé l’impact de la perte de fonction de TET2 sur la différenciation et la maturation des cellules B. Les résultats montrent un blocage de la différenciation plasmocytaire associé à une hyperplasie des CG et une augmentation du pourcentage et du nombre absolu des LB du CG (BGC). L’analyse par PCR quantitative de l’expression des FT importants pour la différenciation des BGC et des plasmocytes a montré que les cellules déficientes pour TET2 présentent une répression du gène Prdm1 codant pour BLIMP1, un régulateur essentiel de la différenciation plasmocytaire. Je me suis ensuite intéressée à TET3, autre protéine de la famille TET exprimée dans la lignée B. Les modèles Tet3-déficients in vivo et in vitro n’ont pas montré d’altération marquée de la différenciation B terminale.J’ai par ailleurs étudié une mutation somatique de SPI1/PU.1, identifiée par notre équipe chez des patients atteints de maladie de Waldenström (MW). Dans plus de 95% des cas, la mutation activatrice L265P du gène MYD88 est également présente. Nous avons montré que la mutation de SPI1, bien que n'empêchant pas sa liaison à l’ADN, modifie son affinité de liaison sur les sites normalement reconnus par la forme sauvage. La mutation semble faire adopter à cette protéine ETS de classe III un comportement qui ressemble à celui d'une classe I/IIa. J’ai ensuite cherché à documenter les bases de la coopération oncogénique entre SPI1 et MYD88 de deux façons. La première, en étudiant la prolifération et la différenciation de lymphocytes B naïfs issus d’un modèle murin knock-in pour la mutation SPI1 développé dans l’équipe, transduits avec un rétrovirus apportant la mutation de MYD88. Les résultats montrent une augmentation de la prolifération dans la condition double mutante ainsi qu’une augmentation de la différenciation terminale. La seconde approche consiste à modifier la lignée humaine BCWM1 de MW par CRISPR/Cas9 afin d’y introduire la mutation de SPI1 en même temps que l’expression de la GFP. Ce modèle sera notamment utilisé pour réaliser des expériences de ChIP-seq afin d’identifier les cibles de la protéine mutante dans un contexte MW-like.En conclusion, le respect des programmes transcriptionnels est essentiel pour le bon déroulement de la différenciation B terminale et peut être impacté soit directement, par des mutations affectant des FT comme SPI1, soit indirectement lorsque le profil de méthylation de gènes codant pour des FT (PRDM1) est altéré suite à des mutations affectant des enzymes comme TET2
B-cell development involves a first phase of differentiation in the bone marrow, in the absence of any specific antigenic stimulation, leading to immature B-cells. The second phase, staging activation and final maturation, is antigen-dependent and takes place in the secondary lymphoid organs, within transient structures called germinal centers (GC). It generates antigen-specific plasma cells and memory B cells.This thesis work focuses on different actors involved in the epigenetic and transcriptional regulation of B-cell differentiation: the enzymes TET2 and TET3 and the transcription factor (TF) SPI1/PU.1. Mutations in genes encoding these proteins are found in human neoplasms, we used in vivo and in vitro models to determine their functional consequences.I analyzed the impact of TET2 loss of function on the differentiation and maturation of B-cells. The results show an impaired plasma cell differentiation associated with GC hyperplasia and an increase in the percentage and absolute number of GC B-cells (BGC). Quantitative PCR analysis of the expression of key BGC and plasma cell TF showed that Tet2-deficient cells exhibit repression of the Prdm1 gene encoding BLIMP1, a master regulator of plasma cell differentiation. I then turned my attention to TET3, another TET family protein expressed in the B-cell lineage. In vivo and in vitro Tet3-deficient models show that the loss of TET3 does not significantly affect terminal B differentiation.In addition, I studied a somatic mutation of SPI1/PU.1, identified by our team in patients with Waldenström's disease (WM). In more than 95% of cases, the L265P activating mutation of MYD88 gene is also present. We have shown that SPI1 mutation, although not preventing its binding to DNA, alters its binding affinity at sites normally recognized by the wild-type form. The mutation appears to cause this class III ETS protein to behave in a manner similar to a class I/IIa ETS protein. I then sought to document the basis for oncogenic cooperation between SPI1 and MYD88 in two ways. First, by studying the proliferation and differentiation of naïve B-cells from a locally developped mouse model knock-in for the SPI1 mutation, transduced with a retrovirus carrying the MYD88 mutation. The results show an increase in proliferation in the double mutant condition as well as an increase of the terminal differentiation. Second, by modifying the human BCWM1 WM cell line by CRISPR/Cas9 in order to introduce the SPI1 mutation at the same time as the expression of the GFP. This model will be used in particular to perform ChIP-seq experiments to identify the targets of the mutant protein in a MW-like context.In conclusion, compliance to transcriptional programs is essential for the smooth progress of B-terminal differentiation and can be impacted either directly, by mutations affecting TF such as SPI1, or indirectly when the methylation profile of key TF-encoding genes (PRDM1) is altered following mutations in enzymes such as TET2

Covalent modifications of histone tails play essential roles in mediating chromatin structure and epigenetic regulation. JmjD3 is a JumonjiC domain containing histone demethylase, belongs to the KDM6 subfamily, and catalyses the removal of methyl groups on methylated lysine 27 on histone 3 (H3K27), a critical mark to promote polycomb mediated repression and gene silencing. The importance of JmjD3 has been implicated in development, cancer biology and immunology. In this thesis, I report the recombinant production of active human JmjD3, development of two in vitro screening assays, a cell-based assay, and structural determination of JmjD3 in complex with the inhibitor 8-hydroxy-5-carboxyquinoline (8HQ). A highly selective and potent small molecule inhibitor GSK-J1 was subsequently identified. The inhibitor is active in HeLa cells and promotes a dose-dependent increase of global H3K27 methylation. The inhibitor GSK-J1 was used in two different cell assay systems related to inflammation and differentiation, to understand how H3K27 demethylation controls cellular functions. By inhibiting H3K27me3 demethylation, it is demonstrated that tumor necrosis factor (TNF) and other pro-inflammatory cytokines are regulated by H3K27 demethylase inhibition in M1- type macrophages derived from healthy volunteers and rheumatoid arthritis patients. It is also shown that inhibition of H3K27me3 demethylation abrogates cellular fusion of M2- type macrophages. During RANKL induced osteoclast differentiation, JmjD3 is up-regulated and promotes the expression of the key transcription factor NFATc1. By inhibiting JmjD3, NFATc1 expression is reduced and osteoclastogenesis is inhibited. This mechanism demonstrates a novel anti-resorptive principle of potential utility in conditions of excess bone resorption such as osteoporosis, bone erosion in inflammatory arthritis or cancer of the bone. These experiments further resolve the ambiguity between scaffold and catalytic function associ- ated with the H3K27 demethylase in these biological systems, and demonstrate that its enzymatic activity is crucial for epigenetic regulation of macrophage and osteoclast function.

As depicted in Chapter I, 2-oxoglutarate- (2OG) dependent oxygenases are ubiquitous in living systems and display a wide range of cellular functions, spanning metabolism, transcription, and translation. Although functionally diverse, the 2OG oxygenases share a high degree of structural similarities between their catalytic sites. From a medicinal chemistry point of view, the combination of biological diversity and structural similarity presents a rather challenging task for the development of selective small molecules for functional studies in vivo. The non-selective metal chelator 8-hydroxyquinoline (8HQ) was used as a template for the generation of tool compound I for the KDM4 subfamily of histone demethylases via application of the Betti reaction. Structural analogue II was used as the corresponding negative control (Figure A). These compounds were characterised in vitro against a range of 2OG oxygenases and subsequently used for studies in cells. I displays selectivity for KDM4 and increases the level of the H3K9me3 histone mark in cells. It has an effect on the post-translational modification pattern of histone H3, but not other histones, and reduces the viability of lung cancer cells, but not normal lung cells, derived from the same patient. I also stabilises hypoxia-inducable factor HIF in cells via a mechanism which seems to be independent from prolyl hydroxylase inhibition. This work is described in Chapters II and III. The chemical biology research in epigenetics is complemented by qualitative analysis conducted in the social sciences at Said Business School. With a global view on how innovation occurs and may actively be fostered, Chapter IV focuses on the potential of epigenetics in drug discovery and how this process may actively be promoted within the framework of open innovation. Areas of focus include considerations of incremental and disruptive technology; how to claim, demarcate, and control the market; how knowledge brokering occurs; and insights about process, management, organisation, and culture of open innovation. In contrast to the open-skies approach adopted for the development of a tool compound in Chapters II and III, a focused-library approach was taken for the generation of a tool compound for the OGFOD1 ribosomal prolyl hydroxylase. The development of a suitable in vitro activity assay for OGFOD1 in Chapter V enabled the development of lead compound III in Chapter VI. III is selective for OGFOD1 against the structurally closely related prolyl hydroxylase PHD2.

Indiana University-Purdue University Indianapolis (IUPUI)
Epigenetic alterations have been shown to control cell type specification and differentiation leading to the changes in chromatin structure and organization of many genes. HDACs have been well documented to play an important role in both neurogenesis and gliogenesis in ganglionic eminence and cortex-derived cultures. However, the role of HDACs in glial cell type specification and differentiation in the optic nerve has not been well described. As a first step towards understanding their role in glial cell type specification, we have examined histone acetylation and methylation levels as well as the expression levels and patterns of the classical HDACs in both murine and chick optic nerve. Analysis of mRNA and protein levels in the developing optic nerve indicated that all 11 members of the classical HDAC family were expressed, with a majority declining in expression as development proceeded. Based on the localization pattern in both chick and murine optic nerve glial cells, we were able to group the classical HDACs: predominantly nuclear, nuclear and cytoplasmic, predominantly cytoplasmic. Nuclear expression of HDACs during different stages of development studied in this project in both murine and chick optic nerve glial cells suggests that HDACs play a role in stage-dependent changes in gene expression that accompany differentiation of astrocytes and oligodendrocytes. Examination of localization pattern of the HDACs is the first step towards identifying the specific HDACs involved directly in specification and differentiation of glia in optic nerve.

Current therapies for human parasite infections rely on a few drugs, most of which have severe side effects, and their usefulness is being seriously threatened by the drug resistance problem. Globally, this is pushing anti-parasitic drug discovery research towards new agents endowed with novel mechanisms of action. Epigenetic processes and the histone modifying enzymes perform vital functions in parasite growth and survival and are validated therapeutic targets. A wealth of current literature indicates that by the use of the so called “drug repurposing” approach, small molecule epigenetic modulators, which were originally developed to treat other human disease conditions, are being investigated for treatment of parasitic diseases, including P. falciparum and S. mansoni infections. The current project focuses on the development and synthesis of novel epigenetic modulators as potential antimalarial and antischistosomal agents. The first part of the project deals with the target-based drugs approach using a small library of HDAC inhibitors to identify potential antimalarial agents. Antiplasmodial activity revealed that MC1742, a uracil-based hydroxamide HDAC inhibitor, as a potent compound with an IC50 value of 4 nM and 6 nM against Pf3D7 sensitive and W2 multidrug resistant strains, respectively. Another project deals with development and synthesis of smSirt2 inhibitors, where several compounds showed modest activity against S. mansoni and good selectivity over hSirt2 that could be potentially used as hits/leads for further medicinal chemistry optimization. Finally, as far as the LSD1 inhibitors as potential antischistosomal agents is considered, preliminary data indicated that most of the tested compounds were relatively toxic to the juvenile stage of schistosomula and to a lesser extent to adult worms. Among these tested compounds, MC3935 was found to be extremely toxic to both schistosomula and adults. Taking into account the preliminary effects of these compounds, it has been demonstrated, as expected, that histone demethylase inhibitors are essential and attractive targets for development of new antimalarial and antischistosomal agents.

DNA methylation is an epigenetic modification that is nearly ubiquitous. Eukaryotic DNA methylation contributes to the regulation of gene expression and maintaining genome integrity. In mammals, DNA methylation occurs primarily on the C5 carbon of cytosine in a CpG dinucleotide context and is catalyzed by the DNA methyltransferases, DNMT1, DNMT3A and DNMT3B. While dnmt3a and dnmt3b genes are highly homologous, the enzymes have distinct functions. Some previous reports suggested differences in the enzymatic behavior of DNMT3A and 3B, which could affect their biological roles. The goal of my thesis work was to characterize kinetics mechanisms of DNMT3A and 3B, and to identify the similarities and differences in their catalytic properties that contribute to their distinct biological functions. Given the sequence similarity between the enzymes, we asked whether DNMT3B was kinetically similar to DNMT3A. In a series of experiments designed to distinguish between various kinetics mechanisms, we reported that unlike DNMT3A, DNMT3B methylated tandem CpG on DNA in a processive manner. We also reported that the disruption of the R-D interface, critical for the cooperativity of DNMT3A, had no effect on DNMT3B activity, supporting the non-cooperative mechanism of this enzyme.

DNMT3A is frequently mutated in numerous cancers. Acute Myeloid Leukemia (AML) is a malignancy of hematopoietic stem cells in which numerous patients exhibit a high frequency of the heterozygous somatic mutation Arg882His in DNMT3A. Through thorough consensus motif building, we discovered a strong similarity in CpG flanking sequence preference between DNMT3A Arg882His variant and DNMT3B enzyme. Moreover, we found that the variant enzyme has the same kinetics mechanism as DNMT3B, indicating a gain-of-function effect caused by the mutation. This change is significant because the variant enzyme can aberrantly methylate DNMT3B targets in AML cells and effect global gene expression. In particular, given that DNMT3B has been shown to have oncogenic properties, this suggests that the Arg882His variant can acquire similar oncogenic properties and drive AML development.

Taken together, my thesis work provides novel insights into the relationship between the biochemical properties and the biological functions of DNMT3A and 3B.

Indiana University-Purdue University Indianapolis (IUPUI)
Multiple sclerosis (MS) is a disabling disease that afflicts an estimated two million people worldwide. The disease is characterized by degradation of the myelin sheath that insulates neurons of the central nervous system manifesting as a heterogeneous collection of symptoms. Two enzymes, protein arginine deaminases type 2 and 4 (PAD2 and PAD4) have been implicated to play an etiologic role in demyelination and neurodegeneration by catalyzing a post-translational modification of arginine peptide residues to citrulline. The pathogenesis of MS is poorly understood, though vitamin D deficiency is a well-associated risk factor for developing the disorder. Using the experimental autoimmune encephalomyelitis (EAE) model of MS we demonstrate vitamin D treatment to attenuate over-expression of PAD 2 and 4 in the brain and spine during EAE. In addition, we identify two molecules produced by peripheral immune cells, IFNɣ and IL-6, as candidate signaling molecules that induce PAD expression in the brain. We demonstrate vitamin D treatment to inhibit IFNɣ mediated up regulation of PAD2 and PAD4 both directly within the brain and by modulating PAD-inducing cytokine production by infiltrating immune cells. These results provide neuroprotective rational for the supplementation of vitamin D in MS patients. More importantly, these results imply an epigenetic link between vitamin D deficiency and the pathogenesis of MS that merits further investigation.