The p53 protein is a primary mediator of apoptosis and growth arrest after exposure to DNA-damaging agents. Previous work has categorised a wild type p53 gene in the majority of childhood acute lymphoblastic leukaemia (ALL) cases, in which instance the p53 protein functions as a modulator of chemotherapy-induced cell death. In contrast, certain p53-induced proteins, such as p21WAF1, can act in an anti-apoptotic manner, and bestow resistance to chemotherapy. Previous studies of the p53 pathway in ALL have utilised cell lines and primary material. In this study a model of ALL was utilised that had previously been developed from a heterogeneous panel of patient biopsies established as xenografts in immune-deficient mice, and are adaptable for short term in vitro culture. A wild-type p53 protein response to etoposide and nutlin-3 exposure was a feature of the whole ALL xenograft panel, irrespective of clinical characteristics and disease biology. While a range of p53 target genes were induced in B-cell precursor (BCP)-ALL and T-ALL xenografts after etoposide exposure, there was negligible induction of p21WAF1 in T- ALL samples. Further work with the histone deacetylase inhibitor vorinostat facilitated p53-independent induction of p21WAF1 in BCP-ALL samples, yet failed to induce p21WAF1 in T- ALL. An association was observed between reduced p21WAF1 expression in the T-ALL samples and decreased histone H3 acetylation in the p21WAF1 promoter together with increased cytosine methylation in the first exon/intron of the p21WAF1 gene. These results suggest that p21WAF1 in T-ALL cells is subject to epigenetic modifications that cause transcriptional silencing. Defective induction of p21WAF1 in T-ALL xenografts was associated with increased sensitivity to the death-inducing effects of drugs, phosphatidylserine (PS) externalisation and caspase-3/-7 activity after drug exposure, indicating that p21WAF1 may exert an anti-apoptotic activity. As proof of principle, p21WAF1 was silenced in Nalm-6 cells by micro-RNA transduction and these cells exhibited increased sensitivity and rapid PS externalisation after drug exposure. A combination of a p21WAF1 inhibitory agent and vorinostat gave some pharmacological evidence to suggest that p21WAF1 inhibition could enhance drug efficacy. Overall, these investigations provide insight into the epigenetic regulation of p21WAF1 and demonstrate an anti-apoptotic role for p21WAF1 in childhood ALL cells.

This thesis proposes the relevance of epigenetic research to feminist studies and gender studies, and vice versa, and asks how epigenetics speaks to the so-called sex-gender distinction. It also discusses what epigenetics could potentially tell us about ourselves, and our place in a world where we are all creatures of both nature and nurture. The author proposes that with its promise of insight into the relationship of the body to environment and experience over time, epigenetics could be an inextricable link between nature and nurture. Combining a modified version of diffractive analysis, and gender/sex as an analytical device, the author engages with epigenetic research and its representation in popular science and in the public imaginary. After discussing the striations of feminist discourse on permeable bodies, the author proposes epigenetics as another layer in the strata, placing epigenetics within feminist and gender studies literature and discourse. Noting that as research gains ground the way in which the public imagines and describes epigenetics gives shape to its materialization and development, this thesis asserts the urgent need for social sciences, and in particular feminist and gender studies, to engage in critical discourse

This thesis proposes the relevance of epigenetic research to feminist studies and gender studies, and vice versa, and asks how epigenetics speaks to the so-called sex-gender distinction. It also discusses what epigenetics could potentially tell us about ourselves, and our place in a world where we are all creatures of both nature and nurture. The author proposes that with its promise of insight into the relationship of the body to environment and experience over time, epigenetics could be an inextricable link between nature and nurture. Combining a modified version of diffractive analysis, and gender/sex as an analytical device, the author engages with epigenetic research and its representation in popular science and in the public imaginary. After discussing the striations of feminist discourse on permeable bodies, the author proposes epigenetics as another layer in the strata, placing epigenetics within feminist and gender studies literature and discourse. Noting that as research gains ground the way in which the public imagines and describes epigenetics gives shape to its materialization and development, this thesis asserts the urgent need for social sciences, and in particular feminist and gender studies, to engage in critical discourse with epigenetic research as it is carried out and as it is translated to the wider public.

Rheumatoid arthritis (RA) is a debilitating chronic inflammatory disease causing bone and cartilage degradation. Macrophages are known to play a role in RA pathology by producing pro-inflammatory cytokines and chemokines, which activates immune cells, drives inflammation and facilitates the degradation of bone and cartilage. Alterations in epigenetic mechanisms, processes that regulate gene expression, have been implicated in the regulation of pro-inflammatory cytokines in RA. Therefore, the aim of this thesis was to determine specific epigenetic variation between RA patient blood and synovial fluid (SF)-derived macrophages (SF MLS). Granulocyte and macrophages colony stimulating factor (GM-CSF) was used to differentiate healthy donor and RA patient blood monocytes into macrophages. Lipopolysaccharide (LPS) was used to stimulate blood and SF-derived macrophages to initiate inflammatory cytokine production. A library of small molecule inhibitors was used to identify key epigenetic regulators of pro-inflammatory cytokine production. Bromodomain and extra-terminal (BET) protein inhibitors (JQ1, I-BET151, PFI-1) were the only class of inhibitor to show consistent down regulation of pro-inflammatory cytokines in both healthy and RA patient-derived macrophages. However, only JQ1 was shown to reduce TNFα production significantly in SF MLS. Transcriptional profiling of RA patient SF MLS indicated a preference for a pro-inflammatory phenotype, and a resistance to steroids (a trait found in 30% of RA patients); SF MLS production of chemokines and cytokines were not downregulated by glucocorticoids in comparison to corresponding blood-derived macrophages. However, JQ1 treatment successfully suppressed these genes. In addition, silencing of BRD4 in blood-derived macrophages from healthy donors reduced pro-inflammatory cytokine production. Chromatin immunoprecipitation studies showed BRD4 was localised to pro-inflammatory promoter regions upon LPS stimulation and displaced in the presence of JQ1. These studies identified BET proteins BRD2, 3 and 4, as essential epigenetic regulators of pro-inflammatory cytokine and chemokine production in both healthy donors and RA patient macrophages. Furthermore, the observation that BET inhibitors can regulate genes that are steroid resistant in RA patient SF MLS, highlights their therapeutic potential in RA.

Nature has evolved an additional level of genetic regulation by-passing direct changes in genetic code through the means of posttranslational modifications (PTMs) of nucleobases and histone proteins. Acetylation, methylation, phosphorylation, O-GlcNAcylation, ubiquitination, sumoylation, and ADP ribosylation are few common examples of various histone modifications. Identification of these modifications and subsequent access to homogeneously modified histone proteins are key for understanding the functional consequence of these PTMs. In this doctoral thesis, the role of PTMs of histone proteins in epigenetics was investigated with emphasis on understanding the role of O-GlcNAcylation in particular. In the second chapter, the functional consequence of O-GlcNAcylation at histone protein, H2B-Ser112 was explored. Homogeneously GlcNAcylated histones and nucleosomes were synthesized using protein chemical reactions. Mass Spectrometry (MS) based quantitative interaction proteomics revealed a direct interaction between GlcNAcylated nucleosomes and the Facilitates Chromatin Transcription (FACT) complex. Preferential binding of FACT to GlcNAcylated nucleosomes provides a molecular mechanism for FACT-driven transcriptional control. In the third chapter, the physical effect of O-GlcNAcylation on the nucleosome structure is described. Homogeneously GlcNAcylated histone protein, H2A-Thr101 was synthesized. The modified protein was used to reconstitute histone sub-complexes and nucleosomes. Various biophysical studies involving circular dichroism and native mass spectrometry revealed that H2A-T101 GlcNAcylation regulates the stability of the nucleosome structure, suggesting a role in transcriptional activation. In the fourth chapter, we discuss an interesting scenario where two PTMs - O-GlcNAcylation and phosphorylation - can compete for the same modification site of histone protein, H2B-Ser36. The resulting outcome is possibly a competitive antagonism or cross-talk, which can modulate the overall control of chromatin regulation. Using a "Tag-and-modify" approach, modified histone proteins bearing both modifications was synthesized, and was later used for nucleosome reconstitution. Quantitative interaction proteomics experiments with the modified nucleosome revealed key interacting protein partners for both the modifications.

Pendant les derniers stades de la spermatogenèse, les cellules germinales mâles post-méiotiques subissent une réorganisation dramatique de l'architecture de leur chromatine, impliquant notamment le remplacement presque total des histones par les protamines, créant des noyaux fortement condensés que l'on trouve dans le sperme mature. Au cours de ce processus, un événement précoce clé est la vague d'hyperacetylation des histones, qui précède leur remplacement. Notre équipe a précédemment identifié le facteur d'expression testiculaire de la famille BET, Brdt (BRomoDomain Testis), qui se lie aux histones acétylées via ses deux bromodomaines, comme essentiel au cours de ce processus. Cependant, les mécanismes aboutissant à l'hyperacétylation des histones à l'échelle génomique sont encore inconnus, ce qui reste l'une des questions majeures dans le domaine. La protéine NUclear in Testis (NUT) est un facteur spécifique testiculaire dont la fonction physiologique dans les cellules germinales mâles était inconnue. Cette protéine se trouve exprimée de manière ectopique dans un cancer rare mais très agressif, le carcinome de la ligne médiane (NUT Midline Carcinoma), en fusion avec BRD4, produisant ainsi une protéine de fusion hautement oncogène. Dans les cellules cancéreuses NUT est capable de recruter et d'activer l'histone acétyltransférase p300, contribuant ainsi à l'activité oncogénique de la protéine de fusion BRD4-NUT. Mon projet de doctorat est d'explorer la fonction physiologique de NUT, en étudiant des souris knock-out pour NUT qui ont été générées par notre équipe en collaboration avec Mathieu Gérard (Saclay). L'absence de NUT provoque une stérilité mâle associée à un arrêt de la spermatogenèse lors de l'allongement et de la condensation des spermatides, au stade où normalement les histones sont remplacées. D'autres expériences suggèrent que NUT pourrait agir sur la régulation de marques épigénétiques, y compris l'hyperacétylation des histones. Les mécanismes par lesquels NUT interfère avec la vague d'acétylation et les facteurs en interaction, y compris Brdt, sont explorées. Au total, cette étude démontre la contribution essentielle du NUT à la régulation épigénétique et au remplacement des histones au cours de la maturation post-méiotique des cellules germinales mâles. / During the late stages of spermatogenesis, post-meiotic male germ cells undergo a dramatic reorganization of their chromatin architecture involving the almost genome wide replacement of histones by protamines, creating highly condensed nuclei that are found in the mature sperm. During this process a key early event is known to be the wave of histone hyperacetylation, which precedes their replacement. Our team previously reported that the testis specific BET factor BRDT (BRomoDomain Testis specific), which binds acetylated histones, is essential during this process. However, how this genome wide hyperacetylation occurs has remained one of the major questions in the field. NUclear protein in Testis (NUT) is a testis specific factor whose physiological function in male germ cells was unknown. It has been found ectopically expressed in NUT Midline Carcinoma, a rare but highly aggressive cancer, in fusion with BRD4, resulting in a highly oncogenic fusion protein. In cancer cells, NUT is able to recruit and activate the histone acetyltransferase p300, hence contributing to the oncogenic activity of the BRD4-NUT fusion protein. My Ph.D. project investigates the original function of NUT by using NUT knockout mice that were generated by our team in collaboration with Mathieu Gerard (Saclay). The absence of NUT causes male sterility associated with a spermatogenic arrest during spermatids elongation/condensation, at a stage when histone replacement normally takes place. Additional experiments suggest that NUT could act through the regulation of epigenetic marks, including histone hyperacetylation. The mechanisms by which NUT interferes with the hyperacetylation wave and interacting factors, including Brdt, are explored. Altogether this study demonstrates the essential contribution of NUT to the epigenetic regulation and histone replacement during the post-meiotic maturation of male germ cells.

L'épigénétique joue un rôle majeur dans le développement érythrocytaire de Plasmodium falciparum, tels que variation antigénique, pathogenèse, différenciation sexuée. Jusqu'à présent, ces éléments n'ont jamais été décrits chez les sporozoïtes. Pour caractériser la régulation épigénétique au niveau des sporozoïtes de P. falciparum, nous avons étudié les principaux régulateurs épigénétiques PfHP1 (P. falciparum hétérochromatine Protein 1) ainsi que PfSET6 et PfSET7 (méthyltransférases histone lysine). J'ai établi une cartographie génomique des marques épigénétiques répressives associées à l'hétérochromatine, et actives associées à l'euchromatine. J'ai identifié un nouveau mécanisme stade-spécifique de contrôle de l'expression génique, qui réprimés plusieurs gènes codant pour des protéines exportées. Ce mécanisme repose sur une expansion d'hétérochromatine. De plus, je démontre qu'un membre de la famille des gènes var, qui code pour le facteur de virulence PfEMP1 des stades sanguins, est exprimé à la surface des sporozoïtes. Cette localisation contraste avec les stades sanguins, où PfEMP1 est transporté à la surface des érythrocytes et participe à cytoadhérence. L'ensemble de ces résultats ouvre de nouvelles questions biologiques: quels sont les facteurs qui régulent la formation d'hétérochromatine chez les sporozoïtes? Quelle est la fonction de PfEMP1 sur la surface d'un sporozoïte? Mes conclusions indiquent un rôle putatif de PfEMP1 lors de la migration des sporozoïtes. En outre, l'expression, à la surface du sporozoïte, d'un antigène polymorphique et spécifique de souche pourrait expliquer la réponse immunitaire souche-spécifique, induite par les sporozoïtes atténués. / Epigenetic mechanisms control key processes during Plasmodium falciparum blood stage development such as antigenic variation, malaria pathogenesis and sexual commitment. However, the epigenetic landscape has not been reported for the sporozoites stage. To characterize epigenetic regulation in sporozoites, we tested the major epigenetic regulators P. falciparum Heterochromatin Protein 1 (PfHP1) and the histone lysine methyltransferases (PfSET6 and PfSET7) in P. falciparum sporozoites. I obtained a reliable genome-wide occupancy data for repressive heterochromatin and active euchromatin marks. Notably, I discovered an unprecedented stage specific mechanism of silencing, which represses several hundreds of genes, encoding parasite surface exported proteins. This is based on an expansion of facultative heterochromatin boundaries in sporozoites. Moreover, I demonstrate that a single member of the polymorphic var gene family, encoding the blood stage virulence factor PfEMP1, is expressed at the surface of sporozoites. This is in contrast to blood stages where PfEMP1 is transported to the erythrocyte surface participating in cytoadhesion. Overall, my findings rise new biological questions including what are the factors that regulate heterochromatin boundaries and what is the function of a virulence-associated surface antigen in sporozoites stage. My findings point to a putative function of this adhesion molecule in sporozoites migration. Moreover, the expression of a highly polymporphic and strain-specific antigen on the surface of sporozoites might provide a molecular explanation for the strain-specific protective immune response induced by attenuated sporozoites.

No description available.

Epigenetics is the study of heritable biological variation not related to changes in DNA sequence. Epigenetic processes are responsible for establishing and maintaining transcriptional programs that define cell identity. Defects to epigenetic processes have been linked to a host of disorders, including mental retardation, aging, cancer and neurodegenerative diseases. The ability to control and engineer epigenetic systems would be valuable both for the basic study of these critical cellular processes as well as for synthetic biology. Indeed, while synthetic biology has made progress using bottom-up approaches to engineer transcriptional and signaling circuitry, epigenetic systems have remained largely underutilized. The predictive engineering of epigenetic systems could enable new functions to be implemented in synthetic organisms, including programmed phenotypic diversity, memory, reversibility, inheritance, and hysteresis. This thesis broadly focuses on the development of foundational tools and intellectual frameworks for applying synthetic biology to epigenetic regulation in the model eukaryote, Saccharomyces cerevisiae. Epigenetic regulation is mediated by diverse molecular mechanisms: e.g. self-sustaining feedback loops, protein structural templating, modifications to chromatin, and RNA silencing. Here we develop synthetic tools and circuits for controlling epigenetic states through (1) modifications to chromatin and (2) self-templating protein conformations. On the former, the synthetic tools we develop make it possible to study and direct how chromatin regulators operate to produce distinct gene expression programs. On the latter, we focus our studies on yeast prions, which are self-templating protein conformations that act as elements of inheritance, developing synthetic tools for detecting and controlling prion states in yeast cells. This thesis explores the application of synthetic biology to these epigenetic systems through four aims: Aim 1. Development of inducible expression systems for precise temporal expression of epigenetic regulators Aim 2. Construction of a library of chromatin regulators to study and program chromatin-based epigenetic regulation. Aim 3. Development of a genetic tool for quantifying protein aggregation and prion states in high-throughput Aim 4. Dynamics and control of prion switching Our tools and studies enable a deeper functional understanding of epigenetic regulation in cells, and the repurposing of these systems for synthetic biology toward addressing industrial and medical applications. / 2019-10-08T00:00:00Z

Human pluripotent stem cells (hPSCs) are an invaluable model for cellular and developmental biology, and hold great potential for translational applications. While great progress has been made in elucidating the signalling pathways regulating pluripotency and differentiation, our mechanistic understanding of the downstream regulations is still incomplete. Moreover, studies aimed at clarifying these aspects are severely impeded by the lack of efficient methods to conditionally modulate gene expression in hPSCs and hPSC-derived cells. In this dissertation I provide new insights into the molecular mechanisms controlled by the Activin/Nodal-SMAD2/3 signalling pathway, whose activity dictates the balance between hPSC pluripotency and differentiation. First, I show that SMAD2/3 modulates the chromatin epigenetic landscape of hPSCs by cooperating with the pluripotency factor NANOG to recruit the DPY30-COMPASS complex and promote histone 3 lysine 4 trimethylation (H3K4me3). This regulation promotes expression of pluripotency genes, while poising developmental regulators for activation during differentiation. Secondly, I describe a novel efficient approach for inducible gene knockdown in hPSCs and hPSC-derived cells. By taking advantage of this technology, I demonstrate that DPY30 is required for early differentiation of hPSCs into certain mesoderm and endoderm derivatives. Finally, I report the first large-scale proteomic identification of SMAD2/3 interacting proteins in both undifferentiated and differentiating hPSCs. This analysis not only confirms that SMAD2/3 interacts with multiple epigenetic modifiers involved in hPSC fate choices, but also implicates SMAD2/3 in several functions other than transcriptional regulation. In particular, I describe how SMAD2/3 physically and functionally interacts with the METTL3-METTL14-WTAP complex to promote the formation of N6-methyladenosine (m6A). This epitranscriptional modification antagonizes the expression of selected mRNAs, including pluripotency factors whose transcription is promoted by SMAD2/3. Therefore, this provides a negative feedback that facilitates rapid exit from pluripotency upon inhibition of Activin/Nodal signalling. Overall, the work presented in this dissertation advances the stem cell field in two ways. First, it demonstrates that the Activin/Nodal-SMAD2/3 pathway finely orchestrates the balance between pluripotency and differentiation by shaping both the epigenome and the epitranscriptome of hPSCs. Secondly, it provides a novel powerful technology to facilitate further studies of the mechanisms that regulate cell fate decisions.