The JmjC-histone lysine demethylases (JmjC-KDMs) are epigenetic regulators responsible for the demethylation of methylated lysine residues on the N-terminal histone tails. As Fe²⁺ and 2-oxoglutarate dependent oxygenases (2OG oxygenases), the JmjC-KDMs possess an absolute requirement for molecular oxygen and are related to the cellular oxygen sensing HIF hydroxylases, PHD2 and FIH. Several JmjC-KDMs are known HIF target genes, hence are upregulated in hypoxia. Moreover, a number of JmjC-KDMs have been shown to have differential oxygen dependences, while aberrant histone methylation has been observed in both hypoxic cells and disease states such as cancer and cardiovascular disease. The work described in this thesis aimed to investigate the impact of hypoxia on the JmjC-KDM, KDM4A. In vitro kinetic analyses revealed a K_m^app(O₂) for recombinant KDM4A of 173 ± 23 μM, which is higher than reported values for the 2OG oxygenases C-P4H, mPAHX and even FIH, and approaching those evaluated for the key oxygen sensor PHD2 (230-1746 μM). These results indicate that KDM4A activity is highly sensitive to oxygen availability, and has the biochemical potential to act as an oxygen sensor in the context of epigenetic regulation. Subsequent investigation of the cellular oxygen dependence of KDM4A, and found that the activity of ectopically expressed KDM4A in U2OS cells demonstrates a graded response to oxygen. Importantly, this trend correlates with the in vitro results, providing further evidence that hypoxia may impact upon epigenetic regulation by the JmjC-KDMs. The various factors that may contribute to the hypoxic inhibition of KDM4A were investigated both in vitro and in cells. The results of these studies suggested that altered concentrations of TCA cycle intermediates, comprising reduced levels of the 2OG oxygenase co-substrate 2OG and increased concentrations of the reported inhibitor 2HG, are likely to only minimally affect the activity of KDM4A in hypoxia. Interestingly, the 2OG oxygenase inhibitor IOX1 possessed increased inhibitory potency against KDM4A under conditions of low oxygen, implying that the use of mixed-mode inhibitors against KDM4A may be of therapeutic benefit in hypoxic disease states. This may be of particular pertinence to cardiac hypertrophy (CH), in which KDM4A activity is reported to have pathophysiological consequences. In a collaboration with Dr Tim McKinsey (University of Colorado, Denver), the KDM4 inhibitor CCT1 was tested in a phenotypic screen of cardiomyocyte hypertrophy, the results of which further support a role for KDM4A in this disease, and suggest that the use of small-molecule inhibitors of KDM4A may be a viable therapeutic strategy in CH. Finally, the effect of reactive oxygen species, levels of which may be increased in hypoxia, on KDM4A activity was explored. Recombinant KDM4A was found to be acutely sensitive to inhibition by hydrogen peroxide (H₂O₂) when compared to the HIF hydroxylases PHD2 and FIH. These results imply that KDM4A may act as a sensor of oxidative stress at the chromatin level, and further investigation in a more biologically relevant context is proposed. Overall, the work described herein demonstrates that the activity of KDM4A is sensitive to oxygen availability, a phenomenon that is likely to have significant implications for epigenetic regulation in hypoxia and the expression of KDM4A-regulated genes in ischaemic disease states.

Lysine methylation is an important post-translational modification found on histones that is added and removed by histone lysine methyltransferases and demethylases, respectively. Lysine methylation occurs in a specific and well-regulated manner, and plays key roles in regulating important biological processes such as transcription, DNA damage and cell cycle. Regulation of the protein abundance of these methylation enzymes particularly by the ubiquitin-proteasome system has emerged as a key mechanism by which the histone methylation status of the cell can be regulated, allowing cells to respond rapidly to specific developmental and environmental cues. In my thesis, I focus on two histone lysine demethylases, KDM4A and PHF8, both of which appear to be regulated by E3 ligases; this regulation impacts their function in the cell. Chapter 2 shows that KDM4A is targeted for proteasomal degradation by the SCFFBXO22, and mis-regulation of KDM4A results in changes in global histone 3 lysine 9 and 36 (H3K9 and H3K36) methylation levels and impacts the transcription of a KDM4A target gene, ASCL2. Chapter 3 shows how PHF8 is targeted for proteasomal degradation by the APCCDC20 via a novel, previously unreported LxPKxLF motif on PHF8. I also found that similar to other APCCDC20 substrates like Cyclin B, PHF8 is an important G2-M regulator, loss of which results in cell cycle defects such as prolonged G2 and defective M phases. To further interrogate PHF8 biology, Chapter 4 describes the generation of a PHF8 conditional knockout mouse. PHF8 biology is interesting and relevant to human disease, as mutations are found in X-linked intellectual disability and autism. Complete loss of PHF8 by full body knockout in the mouse appears to be embryonically lethal, underscoring its key role in early development. This mouse model would allow us to extensively study the biochemistry and biology of PHF8 in the context of development and especially in brain function, where it is anticipated to play key roles. Overall, my dissertation work provides mechanistic and biological insights into how histone demethylases are dynamically regulated by the ubiquitin-proteasome system, providing an extra dimension to our understanding of how chromatin marks can be regulated.

Developmental processes are carefully controlled at the level of transcription to ensure that the fertilized egg develops into an adult organism. The mechanisms that controls transcription of protein-coding genes ultimately ensure that the Pol II machine synthesizes mRNA from the correct set of genes in every cell type. Transcriptional control involves Pol II recruitment as well as transcriptional elongation. Recent genome-wide studies shows that recruitment of Pol II is often followed by an intermediate step where Pol II is halted in a promoter-proximal paused configuration. The release of Pol II from promoter-proximal pausing is thus an additional and commonly occurring mechanism in metazoan gene regulation. The serine kinase P-TEFb is part of the Super Elongation Complex that regulates the release of paused Pol II into productive elongation. However, little is known about the role of P-TEFb mediated gene expression in development. We have investigated the function of P-TEFb in early Drosophila embryogenesis and find that P-TEFb and other Super Elongation Complex subunits are critical for activation of the most early expressed genes. We demonstrate an unexpected function for Super Elongation Complex in activation of genes with non-paused Pol II. Furthermore, the Super Elongation Complex shares phenotypes with subunits of the Mediator complex to control the activation of essential developmental genes. This raises the possibility that the Super Elongation Complex has an unappreciated role in the recruitment of Pol II to promoters. The unique chromatin landscape of each cell type is comprised of post-translational chromatin modifications such as histone methylations and acetylations. To study the function of histone modifications during development, we depleted the histone demethylase KDM4A in Drosophila to evaluate the role of KDM4A and histone H3 lysine 36 trimethylation (H3K36me3) in gene regulation. We find that KDM4A has a male-specific function and regulates gene expression both by catalytic-dependent and independent mechanisms. Furthermore, we used histone replacement to investigate the direct role of H3K14 acetylation in a multicellular organism. We show that H3K14 acetylation is essential for development, but is not cell lethal, suggesting that H3K14 acetylation has a critical role in developmental gene regulation. This work expands our knowledge of the mechanisms that precisely controls gene regulation and transcription, and in addition highlights the complexity of metazoan development. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 3: Manuscript.</p>

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Cette étude a été conçue pour identifier les protéines qui interagissent potentiellement avec Déméthylase 4A spécifique de la lysine (KDM4A) dans le contexte du cancer en utilisant l’essai d'identification de la biotine dépendante de la proximité 2 (BioID2). KDM4A est une lysine déméthylase et un régulateur épigénétique qui joue un rôle dans la carcinogenèse en favorisant la prolifération. Nous avons cherché à identifier l'interactome protéique de KDM4A dans la lignée cellulaire du cancer du col de l'utérus HeLa. Ces interactions protéiques ont été caractérisées en fonction de leur dépendance à l’activité catalytique de KDM4A et / ou au domaine Tandem Tudor. De nouveaux interactants de KDM4A ont été détectés, tout en observant des partenaires protéiques précédemment identifiés, comme FBXO22. KDM4A semble interagir avec certains membres du complexe de remodelage de la chromatine pBAF, en particulier, ARID2, BRD7 et SMARCA2. Le complexe pBAF facilite ou empêche l'accessibilité à l'ADN en restructurant le nucléosome. Une analyse plus approfondie est nécessaire pour valider si l'interaction complexe KDM4A-pBAF est directe ou indirecte. Cette étude suggère également l’importance du domaine Tandem Tudor dans le rôle de KDM4A dans la réparation de bris double brin. Enfin, nous proposons également une implication potentielle de KDM4A dans l'épissage de l'ARNm et le transport d'anions organiques. Cette étude fournit de nouvelles informations sur le rôle de KDM4A dans le développement du cancer. / This study was designed to identify potential interacting proteins of Lysine-specific demethylase 4A (KDM4A) in the context of cancer using proximity-dependent biotin identification 2 (BioID2) assay. KDM4A is a lysine demethylase and an epigenetic regulator that plays a role in carcinogenesis by promoting proliferation. Herein, we sought out to identify the protein interactome of KDM4A in cervical cancer cell line HeLa. These protein interactions were characterized by their dependency on KDM4A’s catalytic activity and/or Tandem Tudor domain. It succeeded at detecting novel interactors of KDM4A as well as previously studied interactions, such as FBXO22. KDM4A seems to be interacting with some members of the pBAF chromatin remodeling complex, specifically, ARID2, BRD7, and SMARCA2. The pBAF complex facilitates or prevents accessibility to DNA by restructuring the nucleosome. Further analysis is required to validate whether the KDM4A-pBAF complex interaction is direct or indirect. This study also implied the importance of the Tandem Tudor domain in KDM4A’s role in the double stranded break repair. Finally, we also propose the potential involvement of KDM4A in mRNA splicing and organic anion transport. This study provides new insights into KDM4A’s role in cancer development.

La sénescence cellulaire et l'apoptose ont évolué comme des puissantes barrières protectrices contre la transformation néoplasique. La sénescence est un état d'arrêt permanent de la prolifération dans lequel les cellules restent métaboliquement actives. La sénescence cellulaire est déclenchée par différentes sources de stress, notamment les oncogènes activés, le dysfonctionnement des télomères, les dommages à l'ADN et des défauts dans la réplication provoqués par des agents génotoxiques, des espèces réactives de l'oxygène, etc. Ce processus complexe engage deux voies différentes de suppresseurs de tumeurs, les voies p53/p21 et p16INK4a/pRb, et les deux voies doivent être compromises dans les cellules humaines afin de contourner la sénescence. Par conséquent, décrire la relation entre l'activation des oncogènes, l'arrêt de la prolifération induite par la sénescence et l'échappement à l'état de sénescence est essentiel pour comprendre le processus de tumorigenèse. KDM4A est un membre de la sous-famille KDM4 des Jumonji lysine déméthylases ciblant les variantes di- et triméthylées de l'histone H3 lysine 9 (H3K9) et l'histone H3 lysine 36 (H3K36). Les trois premiers membres de la sous-famille KDM4A, KDM4B et KDM4C sont également capables de lier l'histone 4 lysine 20 di-méthyl/tri-méthyl (H4K20me2/3) et l'histone 3 lysine 4 tri-méthyl (H3K4me3), via leurs domaines Tudor consécutifs. KDM4A module négativement l'activité de la voie p53, en ciblant directement le suppresseur de tumeur CHD5, et est également un régulateur négatif de la réponse aux dommages de l'ADN. Les niveaux d'expression de KDM4A sont souvent élevés dans les cellules cancéreuses et diminués pendant la sénescence cellulaire. La motivation principale de cette thèse est d'élargir nos connaissances actuelles sur la façon dont la réorganisation de la chromatine influence la stabilité du phénotype de sénescence. Dans la première partie de ce travail, nous abordons la fonction de méthylation de H4K20 et H3K9, dans le contexte des foyers d'hétérochromatine associés à la sénescence (SAHF: senescence-associated heterochromatin foci). Nous démontrons que l'intégration de H4K20me3 dans les SAHF dépend de l'incorporation précédente de H3K9me3 et révélons les méthyltransférases H4K20 impliquées dans ce processus. Nous proposons un mécanisme moléculaire par lequel H4K20me3 et H3K9me3 coopèrent avec p53 dans la répression stable des gènes cibles de E2F au cours de la sénescence induite par l'oncogène Ras. Dans la deuxième partie de la thèse, nous présentons une voie de dégradation lysosomale (c'est-à-dire l'autophagie médiée par des chaperons) en tant que nouveau mécanisme potentiel par lequel les cellules modulent les niveaux de KDM4A pendant la sénescence. Nos résultats suggèrent que la méthylation dans les lysines des histones régule la stabilité de sénescence en réponse à l'oncogène Ras et révèlent le potentiel d'induction de la sénescence par inhibition ciblée de KDM4A dans le traitement du cancer. / Cellular senescence and apoptosis have evolved as potent protective barriers against neoplastic transformation. Senescence is a state of stable arrest of proliferation in which cells remain metabolically active. Cellular senescence is triggered by different sources of stress, including activated oncogenes, telomere dysfunction, DNA damage and replication defects elicited by genotoxic agents, reactive oxygen species, etc. This complex process engages two different tumor suppressor pathways, the p53/p21 and p16INK4a/pRb pathways that need to be compromised in human cells in order to circumvent the senescence-associated growth halt. Hence, describing the relationship between oncogene activation, senescence-induced proliferation arrest and escape from the senescence state remains essential to understand tumorigenesis. KDM4A is a member of the KDM4 sub-family of Jumonji lysine demethylases targeting di- and tri-methylated histone H3 lysine 9 (H3K9) and histone H3 lysine 36 (H3K36). The first three sub-family members KDM4A, KDM4B and KDM4C are also able to bind histone 4 lysine 20 di-methyl/tri-methyl (H4K20me2/3) and histone 3 lysine 4 tri-methyl (H3K4me3), via their tandem Tudor domain. KDM4A negatively modulates the activity of the p53 pathway, by directly targeting the tumor suppressor CHD5, and is also a negative regulator of the DNA damage response. KDM4A expression levels are often elevated in cancer cells and decreased during cellular senescence. The principal motivation for this thesis is to expand our current knowledge on how chromatin reorganization influences the stability of the senescence phenotype. In the first part of this work we address the function of H4K20 and H3K9 methylation, in the context of the senescence-associated heterochromatin foci (SAHF). We demonstrate that integration of H4K20me3 into the SAHF depends on the previous incorporation of H3K9me3 and reveal the H4K20 methyltransferases involved in this process. We propose a molecular mechanism by which H4K20me3 and H3K9me3 cooperate with p53 in the stable repression of E2F target genes during oncogenic Ras-induced senescence. In the second part of the thesis we present a lysosomal-degradation pathway (i.e. chaperone-mediated autophagy) as a novel potential mechanism by which cells modulate KDM4A protein levels during senescence. Our results strongly suggest that histone lysine methylation contributes to the stability of the senescence response to the Ras oncogene and reveal the potential of senescence induction by targeted inhibition of KDM4A in the treatment of cancer.