<p>Changes in gene transcription driven by the activation of intracellular calcium signaling pathways play an important role in neural development and plasticity. A growing body of evidence suggests that stimulus-driven modulation of histone modifications play an important role in the regulation of neuronal activity-regulated gene transcription. However, the histone modifying enzymes that are targets of activity-regulated signaling cascades in neurons remain to be identified. The histone demethylases (HDMs) are a large family of enzymes that have selective catalytic activity against specific sites of histone methylation. To identify HDMs that may be important for activity-regulated gene transcription in neurons, we induced seizures in mice and screened for HDMs whose expression is induced in the hippocampus. Among the few HDMs that changed expression, Kdm6b showed the highest induction. Kdm6B is a histone H3K27-specific HDM whose enzymatic activity leads to transcriptionally permissive chromatin environments. In situ hybridization analysis revealed that Kdm6b is highly induced in post-mitotic neurons of the dentate gyrus region of the hippocampus. We can recapitulate the activity-dependent induction of Kdm6b expression in cultured hippocampal neurons by application of Bicuculline, a GABAA receptor antagonist that leads to synaptic NMDA receptor activation and calcium influx. Kdm6b expression is also induced following application of BDNF, a neurotrophic factor that is upregulated in the seized hippocampus. To investigate possible functions of Kdm6b in neuronal development, we performed in situ hybridization analysis that allows for the identification of regions with high Kdm6b expression that could be sites of potential function in the developing mouse brain. We found high levels of Kdm6b expression in the inner layer of the external granule layer of the cerebellum, a region where pre-migratory immature neurons reside and a site of significant apoptosis. On the basis of this data and the fact that intracellular calcium signaling arising from synaptic firing supports neuronal survival, we explored the necessity for Kdm6b in the survival of cultured cerebellar granule cells. Knock down of Kdm6b by RNAi increases cell death, demonstrating that Kdm6b contributes to neuronal survival. Ongoing experiments are addressing the role of Kdm6b in neuronal differentiation. Overall these data raise the possibility that stimulus-dependent regulation of Kdm6b, and perhaps regulation of H3K27 methylation mediated by Kdm6b, may contribute to the regulation of gene expression in neurons and thus to their proper development and plasticity.</p> / Dissertation

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.

No description available.

Les cellules souches hématopoïétiques (CSH) sont rares, mais indispensables pour soutenir la production des cellules matures du sang, un tissu en constant renouvellement. Deux caractéristiques principales les définissent; la propriété d’auto-renouvellement (AR), ou la capacité de préserver leur identité cellulaire suivant une division, et la multipotence, ce potentiel de différentiation leur permettant de générer toutes les lignée hématopoïétiques. De par leurs attributs, les CSH sont utilisée en thérapie cellulaire dans le domaine de la transplantation. Une organisation tissulaire hiérarchique est aussi préservée dans la leucémie, ou cancer du sang, une masse tumorale hétérogène devant être maintenue par une fraction de cellules au potentiel prolifératif illimité, les cellules souches leucémiques (CSL). Les travaux présentés dans ce manuscrit visent à explorer les bases moléculaires de l’AR, encore mal définies. Certains membres de la famille des facteurs de transcription à homéodomaine HOX sont impliqués dans la régulation de l’hématopoïèse normale, et leur dérégulation peut contribuer à la transformation leucémique. En particulier, la surexpression du gène Hoxb4 dans les CSH influence leur destin cellulaire, favorisant des divisions d’auto-renouvellement et leur expansion en culture et in vivo. En général, les CSH s’épuisent rapidement lorsque maintenue hors de leur niche ex vivo. Différents facteurs interagissent avec les HOX et modulent leur liaison à l’ADN, dont la famille des protéines TALE (Three Amino acid Loop Extension), comme MEIS1 et PBX1. En utilisant une stratégie de surexpression combinée de Hoxb4 et d’un anti-sens de Pbx1 dans les CSH, générant ainsi des cellules Hoxb4hiPbx1lo, il est possible de majorer encore d’avantage leur potentiel d’AR et leur expansion in vitro. Les CSH Hoxb4hiPbx1lo demeurent fonctionnellement intactes malgré une modulation extrême de leur destin cellulaire en culture. Les niveaux d’expressions de facteurs nucléaires, seules ou en combinaison, peuvent donc s’avérer des déterminants majeurs du destin des CSH. Afin d’identifier d’autres facteurs nucléaires potentiellement impliqués dans le processus d’AR des CSH, une stratégie permettant d’évaluer simultanément plusieurs gènes candidats a été élaborée. Les progrès réalisés en termes de purification des CSH et de leur culture en micro-puits ont facilité la mise au point d’un crible en RNAi (interférence de l’ARN), mesurant l’impact fonctionnel d’une diminution des niveaux de transcrits d’un gène cible sur l’activité des CSH. Les candidats sélectionnés pour cette étude font partie du grand groupe des modificateurs de la chromatine, plus précisément la famille des histones déméthylases (HDM) contenant un domaine catalytique Jumonji. Ce choix repose sur la fonction régulatrice de plusieurs membres de complexes méthyl-transférases sur l’AR des CSH, dont l’histone méthyl-transférases MLL (Mixed Lineage Leukemia). Cette stratégie a aussi été utilisée dans le laboratoire pour étudier le rôle de facteurs d’asymétrie sur le destin des CSH, en collaboration. Ces études ont permis d’identifier à la fois des régulateurs positifs et négatifs de l’activité des CSH. Entre autre, une diminution de l’expression du gène codant pour JARID1B, une HDM de la lysine 4 de l’histone H3 (H3K4), augmente l’activité des CSH et s’accompagne d’une activation des gènes Hox. En conclusion, divers déterminants nucléaires, dont les facteurs de transcription et les modificateurs de la chromatine peuvent influencer le destin des CSH. Les mécanismes sous-jacents et l’identification d’autres modulateurs de l’AR demeurent des voies à explorer, pouvant contribuer éventuellement aux stratégies d’expansion des CSH ex vivo, et l’identification de cibles thérapeutiques contre les CSL. Mots-clés : cellules souches hématopoïétiques, Hoxb4, Pbx1, auto-renouvellement, histone déméthylases, RNAi / Hematopoietic stem cells (HSC) are rare, but essential to sustain the constant production of all mature blood cells, a constantly renewing tissue. They are defined by two main characteristics; namely self-renewal (SR), or the capacity to preserve cell identity following division, and multipotency, the differentiation potential that allows them to generate all hematopoietic lineages. Given their attributes, HSC are used for cellular therapy in the transplantation field. A hierarchy in tissue organisation is also preserved in leukemia, or blood cancer, a heterogeneous tumor mass that is sustained by a subset of cells with unlimited SR potential, the leukemia stem cells (LSC). Studies presented in this manuscript aim to explore the molecular basis underlying SR, which are still poorly defined. Certain members of the HOX family of homeodomain transcription factors are involved in the regulation of normal hematopoiesis, and their deregulation can contribute to leukemia development. In particular, Hoxb4 overexpression in HSC influences cells fate, favouring SR divisions and their subsequent expansion in culture and in vivo. In general, HSC exhaust rapidly when maintained ex vivo, outside of their niche. Several factors interact with HOX and modulate their binding to DNA, including members of the TALE (Three Amino acid Loop Extension) protein family, such as MEIS1 and PBX1. Using a strategy of combined overexpression of Hoxb4 and an anti-sense to Pbx1in HSC, generating Hoxb4hiPbx1lo cells, it is possible to further impact on their SR potential and expansion in vitro. These Hoxb4hiPbx1lo cells remain functionally intact despite extreme modulation of their cell fate in culture. Levels of expression of nuclear factors, alone or in combination, can thus impact significantly on HSC fate. In order to identify other nuclear factors potentially involved in the process of HSC self-renewal, a strategy enabling simultaneous assessment several gene candidates was elaborated. To this end, progress made in terms of HSC purification and their culture in micro-wells facilitated the setup of an RNAi (RNA interference) screen, measuring the functional impact of lowering gene candidate transcript levels on HSC activity. Gene candidates selected for this study belong to the greater group of chromatin modifiers, more specifically the family of histone demethylases (HDM) containing a Jumonji catalytic domain. This choice stems from the regulatory function of several members of histone methyl-transferase complexes on HSC self-renewal, including the histone methyl-transferase MLL (Mixed Lineage Leukemia). This strategy was also used in the laboratory to study the role of asymmetry factors on HSC fate, in a collaborative study. These studies enabled identification of both positive and negative regulators of HSC activity. Among these, reduced expression of the gene coding for JARID1B, a histone 3 lysine 4 (H3K4) HDM, increased HSC activity was associated with Hox genes activation. In conclusion, several nuclear determinants, including transcription factors and chromatin modifiers, can influence HSC fate. Underlying mechanisms and identification of additional modulators of SR remain areas to explore, which could eventually contribute to HSC expansion strategies ex vivo, and identification of therapeutic targets against LSC. Keywords: hematopoietic stem cells, Hoxb4, Pbx1, self-renewal, histone demethylases, RNAi

Xiao, Zhangang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 118-131). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Les cellules souches hématopoïétiques (CSH) sont rares, mais indispensables pour soutenir la production des cellules matures du sang, un tissu en constant renouvellement. Deux caractéristiques principales les définissent; la propriété d’auto-renouvellement (AR), ou la capacité de préserver leur identité cellulaire suivant une division, et la multipotence, ce potentiel de différentiation leur permettant de générer toutes les lignée hématopoïétiques. De par leurs attributs, les CSH sont utilisée en thérapie cellulaire dans le domaine de la transplantation. Une organisation tissulaire hiérarchique est aussi préservée dans la leucémie, ou cancer du sang, une masse tumorale hétérogène devant être maintenue par une fraction de cellules au potentiel prolifératif illimité, les cellules souches leucémiques (CSL). Les travaux présentés dans ce manuscrit visent à explorer les bases moléculaires de l’AR, encore mal définies. Certains membres de la famille des facteurs de transcription à homéodomaine HOX sont impliqués dans la régulation de l’hématopoïèse normale, et leur dérégulation peut contribuer à la transformation leucémique. En particulier, la surexpression du gène Hoxb4 dans les CSH influence leur destin cellulaire, favorisant des divisions d’auto-renouvellement et leur expansion en culture et in vivo. En général, les CSH s’épuisent rapidement lorsque maintenue hors de leur niche ex vivo. Différents facteurs interagissent avec les HOX et modulent leur liaison à l’ADN, dont la famille des protéines TALE (Three Amino acid Loop Extension), comme MEIS1 et PBX1. En utilisant une stratégie de surexpression combinée de Hoxb4 et d’un anti-sens de Pbx1 dans les CSH, générant ainsi des cellules Hoxb4hiPbx1lo, il est possible de majorer encore d’avantage leur potentiel d’AR et leur expansion in vitro. Les CSH Hoxb4hiPbx1lo demeurent fonctionnellement intactes malgré une modulation extrême de leur destin cellulaire en culture. Les niveaux d’expressions de facteurs nucléaires, seules ou en combinaison, peuvent donc s’avérer des déterminants majeurs du destin des CSH. Afin d’identifier d’autres facteurs nucléaires potentiellement impliqués dans le processus d’AR des CSH, une stratégie permettant d’évaluer simultanément plusieurs gènes candidats a été élaborée. Les progrès réalisés en termes de purification des CSH et de leur culture en micro-puits ont facilité la mise au point d’un crible en RNAi (interférence de l’ARN), mesurant l’impact fonctionnel d’une diminution des niveaux de transcrits d’un gène cible sur l’activité des CSH. Les candidats sélectionnés pour cette étude font partie du grand groupe des modificateurs de la chromatine, plus précisément la famille des histones déméthylases (HDM) contenant un domaine catalytique Jumonji. Ce choix repose sur la fonction régulatrice de plusieurs membres de complexes méthyl-transférases sur l’AR des CSH, dont l’histone méthyl-transférases MLL (Mixed Lineage Leukemia). Cette stratégie a aussi été utilisée dans le laboratoire pour étudier le rôle de facteurs d’asymétrie sur le destin des CSH, en collaboration. Ces études ont permis d’identifier à la fois des régulateurs positifs et négatifs de l’activité des CSH. Entre autre, une diminution de l’expression du gène codant pour JARID1B, une HDM de la lysine 4 de l’histone H3 (H3K4), augmente l’activité des CSH et s’accompagne d’une activation des gènes Hox. En conclusion, divers déterminants nucléaires, dont les facteurs de transcription et les modificateurs de la chromatine peuvent influencer le destin des CSH. Les mécanismes sous-jacents et l’identification d’autres modulateurs de l’AR demeurent des voies à explorer, pouvant contribuer éventuellement aux stratégies d’expansion des CSH ex vivo, et l’identification de cibles thérapeutiques contre les CSL. Mots-clés : cellules souches hématopoïétiques, Hoxb4, Pbx1, auto-renouvellement, histone déméthylases, RNAi / Hematopoietic stem cells (HSC) are rare, but essential to sustain the constant production of all mature blood cells, a constantly renewing tissue. They are defined by two main characteristics; namely self-renewal (SR), or the capacity to preserve cell identity following division, and multipotency, the differentiation potential that allows them to generate all hematopoietic lineages. Given their attributes, HSC are used for cellular therapy in the transplantation field. A hierarchy in tissue organisation is also preserved in leukemia, or blood cancer, a heterogeneous tumor mass that is sustained by a subset of cells with unlimited SR potential, the leukemia stem cells (LSC). Studies presented in this manuscript aim to explore the molecular basis underlying SR, which are still poorly defined. Certain members of the HOX family of homeodomain transcription factors are involved in the regulation of normal hematopoiesis, and their deregulation can contribute to leukemia development. In particular, Hoxb4 overexpression in HSC influences cells fate, favouring SR divisions and their subsequent expansion in culture and in vivo. In general, HSC exhaust rapidly when maintained ex vivo, outside of their niche. Several factors interact with HOX and modulate their binding to DNA, including members of the TALE (Three Amino acid Loop Extension) protein family, such as MEIS1 and PBX1. Using a strategy of combined overexpression of Hoxb4 and an anti-sense to Pbx1in HSC, generating Hoxb4hiPbx1lo cells, it is possible to further impact on their SR potential and expansion in vitro. These Hoxb4hiPbx1lo cells remain functionally intact despite extreme modulation of their cell fate in culture. Levels of expression of nuclear factors, alone or in combination, can thus impact significantly on HSC fate. In order to identify other nuclear factors potentially involved in the process of HSC self-renewal, a strategy enabling simultaneous assessment several gene candidates was elaborated. To this end, progress made in terms of HSC purification and their culture in micro-wells facilitated the setup of an RNAi (RNA interference) screen, measuring the functional impact of lowering gene candidate transcript levels on HSC activity. Gene candidates selected for this study belong to the greater group of chromatin modifiers, more specifically the family of histone demethylases (HDM) containing a Jumonji catalytic domain. This choice stems from the regulatory function of several members of histone methyl-transferase complexes on HSC self-renewal, including the histone methyl-transferase MLL (Mixed Lineage Leukemia). This strategy was also used in the laboratory to study the role of asymmetry factors on HSC fate, in a collaborative study. These studies enabled identification of both positive and negative regulators of HSC activity. Among these, reduced expression of the gene coding for JARID1B, a histone 3 lysine 4 (H3K4) HDM, increased HSC activity was associated with Hox genes activation. In conclusion, several nuclear determinants, including transcription factors and chromatin modifiers, can influence HSC fate. Underlying mechanisms and identification of additional modulators of SR remain areas to explore, which could eventually contribute to HSC expansion strategies ex vivo, and identification of therapeutic targets against LSC. Keywords: hematopoietic stem cells, Hoxb4, Pbx1, self-renewal, histone demethylases, RNAi

Regulation of gene expression comprises a wide range of mechanisms that control the abundance of gene products in response to environmental and developmental changes. These biological processes can be modulated by posttranslational modifications including arginine methylation. Among the enzymes that catalyze the methylation, protein arginine methyltransferase 7 (PRMT7) is known to modify histones to repress gene expression. Jumonji domain-containing protein 6 (JMJD6) is a putative arginine demethylase that potentially antagonize PRMT7. However, the biological significance of these enzymes is not well understood. This thesis summarizes the investigation of both PRMT7 and JMJD6 in cell culture models for adipocyte differentiation. The results suggest that PRMT7 is not required for the differentiation, whereas JMJD6 is necessary for the differentiation by promoting the expression of the lineage determining transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancerbinding proteins (C/EBPs). The underlying mechanisms by which JMJD6 regulate differentiation involve transcriptional and post-transcriptional control of gene expression. Unexpectedly, the adipogenic function of JMJD6 is independent of its enzymatic activity. Collectively, the present research reveals a novel role of JMJD6 in gene regulation during the differentiation of adipocytes.

L’importance des modificateurs de la chromatine dans la régulation de l’hématopoïèse et des hémopathies malignes est illustrée par l’histone méthyltransférase Mixed-Lineage Leukemia (MLL) qui est essentielle au maintien des cellules souches hématopoïétiques (CSH) et dont le gène correspondant, MLL, est réarrangé dans plus de 70% des leucémies du nourrisson. Les histones déméthylases (HDM), récemment découvertes, sont aussi impliquées dans le destin des CSH et des hémopathies malignes. Le but de ce projet est d’étudier l’expression des HDM dans les cellules hématopoïétiques normales et leucémiques afin d’identifier de potentiels régulateurs de leur destin. Nous avons réalisé un profil d'expression génique des HDM par qRT-PCR et par séquençage du transcriptome (RNA-seq) dans des cellules de sang de cordon (cellules CD34+ enrichies en CSH et cellules différenciées) et des cellules de leucémie aiguë myéloïde (LAM) avec réarrangement MLL. Les deux techniques montrent une expression différentielle des HDM entre les populations cellulaires. KDM5B et KDM1A sont surexprimés dans les cellules CD34+ par rapport aux cellules différenciées. De plus, KDM4A et PADI2 sont surexprimés dans les cellules leucémiques par rapport aux cellules normales. Des études fonctionnelles permettront de déterminer si la modulation de ces candidats peut être utilisée dans des stratégies d’expansion des CSH, ou comme cible thérapeutique anti-leucémique. Nous avons aussi développé et validé un nouveau test diagnostique pour détecter les mutations de GATA2 qui code pour un facteur de transcription clé de l’hématopoïèse impliqué dans les LAM. Ces travaux soulignent l’importance des facteurs nucléaires dans la régulation de l’hématopoïèse normale et leucémique. / The importance of chromatin modifiers in regulation of hematopoiesis and hematologic malignancies is illustrated by the Mixed-Lineage Leukemia (MLL) histone methyltransferase, which is essential to maintain hematopoietic stem cells (HSC) and whose corresponding gene, MLL, is rearranged in over 70% of infant leukemia. The recently discovered histone demethylases (HDM) are also involved in HSC fate and in hematologic malignancies. The purpose of this project is to study the expression of HDM in normal and leukemic hematopoietic cells to identify potential regulators of their fate. We performed a comprehensive gene expression profile of HDM by qRTPCR and transcriptome sequencing (RNA-seq) in cord blood cells (CD34+ cells enriched in HSC and differentiated cells) and in acute myeloid leukemia (AML) cells with MLL rearrangement. Both techniques revealed differential expression of HDM between these cell populations. KDM5B and KDM1A are overexpressed in CD34+ cells compared to differentiated cells. Moreover, KDM4A and PADI2 are overexpressed in leukemic cells compared to normal cells. Functional studies will determine whether modulation of these candidates can be used in HSC expansion strategies or as anti-leukemic drug target. We have also developed and validated a new diagnostic test to detect mutations of GATA2, a gene encoding a key transcription factor involved in hematopoiesis and in AML. This work highlights the importance of nuclear factors in the regulation of normal and leukemic hematopoiesis.

Myeloid-derived suppressor cells (MDSCs) prolong sepsis by promoting immunosuppression. We reported that sepsis MDSC development requires long non-coding RNA Hotairm1 interactions with S100A9. Using a mouse model that simulates the immunobiology of sepsis, we find that histone demethylase KDM6A promotes Hotairm1 transcription by demethylating transcription repression H3K27me3 histone mark. We show that chemical targeting of KDM6A by GSK-J4 represses Hotairm1 transcription, which coincides with decreases in transcription activation H3K4me3 histone mark and transcription factor PU.1 binding to the Hotairm1 promoter. We further show that immunosuppressive IL-10 cytokine promotes KDM6A binding at the Hotairm1 promoter. IL-10 knockdown repletes H3K27me3 and reduces Hotairm1 transcription. GSK-J4 treatment also relocalizes nuclear S100A9 protein to the cytosol. To support translation to human sepsis, we demonstrate that inhibiting H3K27me3 demethylation by KDM6A ex vivo in MDSCs from patients with protracted sepsis decreases Hotairm1 transcription. These findings suggest that epigenetic targeting of MDSCs in human sepsis might resolve post-sepsis immunosuppression and improve sepsis survival.