Histone acetylation is an epigenetic post-translational modification recognised by the bromodomain, a protein module that forms part of multi-component complexes affecting transcription. This interaction plays fundamental cellular roles, and shows association with particular diseases including inflammation and cancer. The biological roles of bromodomains and the progress of ligands developed so far has been summarised in introductory Chapter 1. Work within the group has led to the development of a nanomolar ligand for BRD4, a BET bromodomain implicated in cancer and numerous diseases. Evaluation in an NCI-60 cancer cell screen indicated antiproliferative activity in a variety of cancer types. However, metabolic predictions indicate that this compound is unoptimised for use in vivo. Chapter 2 describes synthesis of a collection of analogues to improve the physical and pharmacokinetic properties of this series of compounds. This work identified compounds with equivalent affinity but greater predicted metabolic stability, as well as more potent derivatives. This research will direct the design of potent and metabolically stable derivatives that can be used in animal models. Chapter 3 describes work carried out towards the development of small molecules to target bromodomains for which there are no known ligands, using the FALZ bromodomain as an initial target. A fragment-based approach has identified a number of compounds that bind to different non-BET bromodomains. These fragments will be a useful starting point for the development of more potent and selective non-BET bromodomain ligands. As well as acetylated lysines, a number of other acylation post-translational modifications occur on lysine residues. Chapter 4 describes work carried out to investigate the interaction of other acylated lysine residues with bromodomains. This work highlighted that other acylated lysines can interact with bromodomains, and selectivity for particular bromodomains can also be achieved. These modified lysines could be incorporated into cognate peptides to improve in vitro peptide displacement assays, aiding the development of small molecular bromodomain probes.

Mechanical ventilation, a lifesaving intervention for patients with acute respiratory distress syndrome (ARDS), also unfortunately contributes to excessive mechanical stress and impaired lung physiological and structural integrity. We have elsewhere established the pivotal role of increased nicotinamide phosphoribosyltransferase (NAMPT) transcription and secretion as well as its direct binding to the toll-like receptor 4 (TLR4) in the progression of this devastating syndrome; however, regulation of this critical gene in ventilator-induced lung injury (VILI) is not well characterized. On the basis of an emerging role for epigenetics in enrichment of VILI and CpG sites within the NAMPT promoter and 5'UTR, we hypothesized that NAMPT expression and downstream transcriptional events are influenced by epigenetic mechanisms. Concomitantly, excessive mechanical stress of human pulmonary artery endothelial cells or lipopolysaccharide (LPS) treatment led to both reduced DNA methylation levels in the NAMPT promoter and increased gene transcription. Histone deacetylase inhibition by trichostatin A or Sirt-1-silencing RNA attenuates LPS-induced NAMPT expression. Furthermore, recombinant NAMPT administration induced TLR4-dependent global H3K9 hypoacetylation. These studies suggest a complex epigenetic regulatory network of NAMPT in VILI and ARDS and open novel strategies for combating VILI and ARDS.

The chicken embryonic β-type globin gene, ρ, is silenced on day five of embryogenesis. Concomitant with this silencing is methylation of cytosine residues in the promoter and proximal transcribed region of the gene, which is first detected on day seven and is complete in adult cells. Once methylated, expression of the gene cannot be induced unless the methylation is removed by treatment of cells with Sazacytidine. Therefore ρ-globin is a member of a small group of genes whose normal developmentally regulated expression is mediated at least in part by DNA methylation.A methyl-DNA binding complex, termed the MeCPC (Erythroid Methyl Cytosine-binding Protein Complex), has been found to bind to the methylated, but not unmethylated, ρ-globin promoter and proximal transcribed region in nuclear extracts from definitive erythrocytes. This complex has a stronger binding affinity for its cognate binding sequence, the methylated ρ-globin proximal transcribed region (M-ρ248), than for an artificial 5-methylcytosine-rich sequence (M-CG11).To define the components of the MeCPC, we developed two chromatographic procedures to purify the complex from adult chicken red blood cell nuclear extracts (Purification Strategies I and II). Mass spectrometry was performed on the MeCPC obtained by Purification Strategy I and proteins were identified by a novel application of peptide mass fingerprint data fitting. Four components of the previously-purified MeCPl transcriptional repression complex were identified in the sample: MBD2, RbAp48, HDAC2 and MTA1. Another identified protein, MENT, is a factor expressed only in chicken hematopoietic cells. These five proteins, as well as the MeCPl component Mi2, were found to tightly coelute by Western blotting of gel-filtration fractions from Purification Strategy II. Therefore, we conclude that these five proteins are components of the MeCPC.To confirm that MBD2 is associated with the ρ-globin gene in vivo, we perfomed the chromatin immunoprecipitation assay using anti-MBD2 antibodies. In adult erythrocytes, significant enrichment for MBD2 is seen at the transcriptionally inactive ρ-globin gene, but no enrichment is observed at the transcriptionally active βA globin gene. These experiments confirm that MBD2 binds to the methylated p-globin gene in adult chicken erythroid cells.

L’étude du contrôle de l’expression des gènes a été, au cours de ces dernières années, révolutionnée par la découverte des régulations épigénétiques. Parmi les différents acteurs participant à ces régulations se trouvent les protéines du groupe Polycomb (PcG). Ces protéines, initialement découvertes chez la drosophile, sont responsables de la mise en place et du maintien de "marques épigénétiques" au niveau de gènes cibles, qui sont alors réprimés. Les protéines PcG agissent sous forment de trois complexes dinstincts chez les animaux nommés PRC1 (Polycomb Repressive Complex 1), PRC2 (Polycomb Repressive Complex 2) et PhoRC (Pleiohomeotic Repressive Complex); le PRC2 possédant une activité histone méthyltransférase de type H3 K9/27. Chez les plantes, seules trois classes de protéines PcG sont retrouvées: la classe des Enhancer of zeste E(z), des Extra Sex Combs (ESC) et des Supressor of zeste 12 (Su(z)12), formant le complexe PRC2. Leur rôle dans le développement des plantes a été mis en évidence chez Arabidopsis, au niveau du gamétophyte femelle et de la graine, du maintien de l’état végétatif, de l’identité florale et de la vernalisation. Cependant leur implication dans le développement du fruit reste inconnue. Mon travail a permis d'identifier et de caractériser deux gènes PcG de la classe des E(z) de tomate exprimés dans le fruit, nommés SlEZ1 et SlEZ2. Les proteines SlEZ1 et SlEZ2 présentent l’ensemble des domaines caractéristiques des protéines de cette classe et sont localisées dans les noyaux. Les expériences de double hybride révèlent que les protéines SlEZ1 et SlEZ2 sont capables de former des complexes de type PRC2 avec certaines autres protéines PcG de tomate (de type ESC et Su(z)12). Ceci suggère que SlEZ1 et SlEZ2 sont effectivement des protéines fonctionnelles. L’analyse de des profils d’expression des gènes SlEZ1 et SlEZ2 révèle une expression ubiquitaire dans la plante au niveau de l’appareil végétatif, de la fleur et dans le fruit. Cependant, dans la fleur, seul SlEZ1 présente une expression dans les étamines tandis que les ARNm de SlEZ2 sont présent de façon spécifique dans le tissu de transmission du style. Dans le fruit, SlEZ1 est exprimé de façon constante, tandis que SlEZ2 semble faiblement exprimé dans les fruits en cours de mûrissement. Afin d’identifier la fonction de SlEZ1 dans le développement du fruit, des plantes transgéniques sous-exprimant SlEZ1 de façon constitutive ont été générées. Elles présentent une morphologie altérée de la fleur: les étamines sont torsadées et ne forment pas de cône staminal fermé. De plus, une augmentation du nombre moyen de carpelles par fruit est observée. / The control of gene expression has been challenged by the discovery of epigenetic regulation. Among the different factors involved in epigenetic regulations, the Polycomb (PcG) proteins are known to repress gene expression by setting epigenetic marks. The PcG protein, initially discovered in drosophila, act together in three distinct complexes named PRC1 (Polycomb Repressive Complex 1), PRC2 (Polycomb Repressive Complex 2) and PhoRC (Pleiohomeotic Repressive Complex). PRC2 complexes methylate histone H3 on lysines K9/27. In plants, only three classes of PcG protein has been found: the Enhancer of zeste (E(z)) class, the Extra Sex Combs (ESC) class and the Supressor of zeste 12 (Su(z)12) class, which belong to the PRC2. Their function in plant development has been brought to light in Arabidopsis thaliana. They control female gametophyte and seed development, maintain the vegetative development, and are involved in floral identity and vernalization. However, their function in fruit development is still unknown. My work was aimed to identify and characterize two PcG genes, named SlEZ1 and SlEZ2, encoding tomato E(z) class proteins. SlEZ1 and SlEZ2 proteins contain all the five E(z) characteristic domains and are both localized in the nucleus. Furthermore, as double-hybrid experiments reveal that both SlEZ1 and SlEZ2 proteins are able to form PRC2 complexes and interact with PcG proteins of other classes (ESC and Su(z)12 classes), it seems that these proteins are functional. Their expression profiles reveal ubiquitous expression during vegetative development (leaves, buds, stems) and reproductive development (flowers and fruits). However SlEZ1 is specifically expressed in the stamens whereas SlEZ2 shows specific expression in the transmitting tissue of the style. Moreover, their expression during fruit development shows some differences: if SlEZ1 expression is almost constant, SlEZ2 expression decreases during fruit development. In order to indentify SlEZ1 functions in fruit development, transgenic plants underexpressing constitutively SlEZ1 have been generated. These plants present altered flower morphology with twisted stamens and increased carpel number fruits.

Leptosphaeria maculans ‘brassicae’ (Lmb) est un ascomycète de la classe des Dothideomycètes faisant partie d’un complexe d’espèces présentant différents niveaux d’adaptation au colza. Lmb est responsable d’une des maladies les plus dommageables sur colza : la nécrose du collet. Lmb présente un cycle de vie complexe au cours duquel il alterne différents modes de vie, traduisant l’existence de mécanismes de régulation fine de l’expression des gènes lui permettant de s’adapter rapidement à de nouvelles conditions. Le séquençage de son génome a révélé une structure originale, avec l’alternance de deux types de régions : les isochores GC et les isochores AT. Alors que les isochores GC sont riches en gènes, les isochores AT sont pauvres en gènes et présentent des caractéristiques de l’hétérochromatine (régions génomiques riches en éléments transposables et présentant un faible taux de recombinaison). Bien que pauvres en gènes, les isochores AT représentent une « niche écologique » pour les gènes codant des effecteurs puisque 20 % des gènes des isochores AT codent des effecteurs putatifs contre seulement 4 % des gènes localisés en isochores GC. Les gènes codant des effecteurs situés en isochores AT présentent un comportement transcriptionnel différent de ceux localisés en isochores GC : une faible expression pendant la croissance mycélienne et une forte induction d’expression pendant l’infection primaire du colza. Sur la base de ces observations, l’objectif de ma thèse était de caractériser le déterminisme de la co-expression des effecteurs situés dans les isochores AT et en particulier d’évaluer si la régulation de l’expression de ces gènes se fait par un contrôle épigénétique lié à leur localisation particulière et/ou par l’intervention de régulateurs communs. Afin de déterminer le rôle de la structure des isochores AT, l’analyse fonctionnelle de protéines impliquées dans le remodelage de la chromatine (i.e. HP1, DIM-5 et DMM-1) a été réalisée et leur implication dans la régulation de l’ensemble des gènes prédits dans le génome de L. maculans a été évaluée. Cette étude a permis de démontrer l’implication de la structure hétérochromatinienne des isochores AT dans la répression de l’expression pendant la croissance mycélienne des gènes situés dans cet environnement génomique, en particulier les gènes codant des effecteurs. Parmi les gènes sous contrôle épigénétique, nous avons pu observer qu’en plus des gènes localisés en isochores AT, des zones en isochores GC étaient aussi affectées et pouvaient constituer des « hot-spots » de contrôle épigénétique. Afin d’identifier des régulateurs candidats pouvant être impliqués dans le contrôle de l’expression des effecteurs pendant l’infection, le répertoire des gènes codant des facteurs de transcription (FTs) chez Lmb a été établi et l’analyse de la conservation de ce répertoire parmi les autres espèces du complexe d’espèces Leptosphaeria a permis d’identifier les FTs spécifiques, ou spécifiquement sur-exprimés pendant l’infection du colza, chez Lmb. Des candidats ont été sélectionnés pour réaliser leur analyse fonctionnelle : des gènes codant des FTs sur-exprimés pendant l’infection (9 FTs) ainsi que les orthologues de FTs qui avaient été décrits chez d’autres espèces comme régulateurs majeurs de la pathogénie (StuA, Sge1 et Fox1). L’analyse fonctionnelle de FTs candidats a permis d’établir que StuA, comme chez d’autres champignons phytopathogènes, joue un rôle important dans la mise en place de l’infection et l’expression des effecteurs chez L. maculans. Le « silencing » d’un FT de type AT-Hook, famille de FTs se fixant préférentiellement au niveau de séquences riches en AT, a un fort effet sur la pathogénie du champignon et entraîne une diminution d’expression de 2 effecteurs. Cette thèse a permis d’apporter de nouveaux éléments concernant la régulation des gènes codant des effecteurs chez un champignon phytopathogène impliquant, pour la première fois, un mécanisme épigénétique. / Leptosphaeria maculans is an ascomycete belonging to the Dothideomycete class and is part of a species complex showing different level of adaptation toward oilseed rape. Within this species complex, Lmb is responsible for the most damaging disease of this crop: “stem canker”. Lmb presents a complex life cycle during which it alternates between different life styles and nutritional strategies underlying the involvement of precise regulatory networks for gene expression to rapidly adapt to new conditions. The sequencing of the Lmb genome has revealed an unusual structure, alternating two types of regions, GC- and AT-isochores. While GC-isochores are gene-rich, AT-isochores are gene-poor and have several characteristics of heterochromatin (they are rich in transposable elements and present a lower rate of recombination compared to GC-isochores). Although gene-poor, AT-isochores are “ecological niches” for effector genes as 20% of the genes in these regions encode for putative effectors against only 4% of the genes in GC-isochores. Effector-encoding genes located in AT-isochores present a different transcriptional behavior compared to those located in GC-isochores: a very low expression in axenic culture and a drastic increase in expression during primary leaf infection. On these bases, the aim of my thesis was to characterize the determinism of the concerted effector gene expression. Are AT-isochores targets of reversible epigenetic modifications that affect the regulation of effector genes? and/or are one or several common regulators involved in the control of the concerted expression of effector genes? To assess the role of the structure of AT-isochores, functional analysis of three key players involved in chromatin remodeling (i.e. HP1, DIM-5 and DMM-1) was performed and their role in global gene expression was assessed. This study validated that heterochromatic structure of AT-isochores represses expression of genes located in such a genomic environment, notably effector genes. Among genes under an epigenetic control, we also identified genes located in GC-isochores that were similarly influenced and may represent “hot spots” for epigenetic control. To identify putative regulators of effector gene expression, we established the complete repertoire of transcription factors (TFs) of Lmb and by analyzing the conservation of this repertoire among species of the Leptosphaeria species complex, we identified TFs specific of Lmb, or specifically induced during infection. Functional analysis of 12 TFs was set up: nine TF-encoding genes induced during infection and three orthologs of TFs described as required for pathogenesis in other phytopathogenic fungi (StuA, Sge1, Fox1). This functional analysis showed that StuA, as in other phytopathogenic fungi, plays a major role in infection and expression of effector genes in Lmb. The silencing of an AT-Hook type TF, family of TFs that specifically interact with AT-rich sequences, was associated with a reduction of the expression of two effector genes during infection and with pathogenicity defects. This study brought new insights into the regulation of effector genes in a phytopathogenic fungus involving, for the first time, an epigenetic mechanism.

La tomate (Solanum lycopsersicum), qui forme un clade monophylétique restreint au sein de la large famille des Solanacées, est utilisée comme modèle pour l’analyse du génome, et le développement du fruit. A ce jour, de nombreux efforts ont été consacrés à l'analyse de la diversité génétique des espèces de tomate. Cependant peu de travaux ont porté sur l'analyse de la diversité épigénétique, alors qu’il est aujourd’hui admis que les processus épigénétiques jouent un rôle essentiel dans la diversité phénotypique. Dans un premier temps, le niveau de méthylation de l'ADN a été comparé dans les feuilles et les fruits de différentes variétés de tomates sauvages et cultivées. Puis la famille des gènes Enhancer of zeste (E (z)) a été analysée. Chez la tomate, cette famille comprend deux gènes fonctionnels ainsi qu’un pseudogène. Finalement la stabilité épigénétique reste un facteur majeur pouvant avoir un impact essentiel sur les stratégies de sélection végétales. En outre nous avons fait une caractérisation fine des différents aspects du développement du fruit et de la maturation. / Tomato (Solanum lycopsersicum) which forms a small monophyletic clade within the large Solanaceae family has been chosen as a model system for studying the Solanaceae genome, fruit development and ripening. At that time, many efforts have been devoted to the analysis of the genetic diversity of tomato species, little work has focused on the analysis epigenetic diversity in this clade, although there is a general agreement that epigenetic processes play essential role in the phenotypic diversity in animal and plant system. As first step, DNA methylation level was analyzed in leaves and fruits of various wild and cultivated tomato species.Additionally, the Enhancer of zest (E(z)) gene family has been analyzed. In tomato, the E(z) family consists in two functional genes (SlEZ1, SlEZ2) and in a pseudogene (SlEZ3). In addition, the epigenetic stability is an important consideration that could have a significant on strategies for crop breading. Finally, we made a fine characterization of the different aspects of fruit development and ripening. / All’interno della grande famiglia delle Solanacee è stato scelto il pomodoro (Solanum lycopsersicum) come sistema modello per studio dello sviluppo e maturazione del frutto. Molti sforzi sono stati fatti per analizzare la diversità genetica delle specie di pomodoro, pochi lavori invece riguardano l’analisi della diversità epigenetica, sebbene ci sia accordo sul fatto che processi epigenetici giochino un ruolo essenziale nella diversità fenotipica dei sistemi animali e vegetali. Inizialmente è stato analizzato il livello di metilazione del DNA in foglie e frutti delle diverse specie di pomodoro selvatico e coltivato. Inoltre, è stata analizzata la famiglia genica Enhancer of Zeste (E (z)). In pomodoro la famiglia E(z) consiste di 2 geni funzionali SlEZ1, SlEZ2 e di uno pseudogene SlEZ3. Inoltre la stabilità epigenetica è importante in quanto può avere un impatto sulle strategie di miglioramento genetico delle specie coltivate. Infine è stata condotta una attenta caratterizzazione dei meccanismi cellulari dello sviluppo del frutto e della sua maturazione.

HIV-1 infections can be treated but not cured by the current antiretroviral therapy regimens. One of the major barriers to HIV-1 eradication is the persistence of the virus in treated HIV+ individuals under the form of reservoirs. A continuum of molecular mechanisms, at the epigenetic, transcriptional and post-transcriptional levels maintains HIV-1 gene expression silent in its reservoirs. A better understanding of HIV-1 molecular mechanisms of persistence thus allows to devise novel therapeutic approaches to eradicate the virus. In this context, our thesis aimed at characterizing the molecular mechanisms of HIV-1 persistence in its T-lymphoid and myeloid reservoirs. More specifically, we have studied the epigenetic and transcriptional mechanisms of HIV-1 persistence at three different levels. First, we have investigated the DNA methylation-mediated mechanisms underlying the heterogeneity of the DNA methylation inhibitor 5-aza-2’-deoxycytidine potency in the reactivation of HIV-1 gene expression from latently-infected CD4+ T cells. Second, we have studied the contribution of the intragenic binding sites for the cellular PU.1 transcription factor in the specific regulation of HIV-1 gene expression in myeloid lineages. Finally, in a third study, we have designed a new tool to study the transcriptional landscape of HIV-1 in LTRs-suppressed proviruses. Collectively, our work has offered individual insights into the molecular mechanisms underlying the heterogeneity of HIV-1 T-lymphoid and myeloid reservoirs, with the ultimate goal of developing new HIV-1 curative strategies and improving the quality of life of HIV+ individuals. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Introduction: Preterm birth is increasing worldwide and is a major cause of neonatal death. Survivors are at increased risk of neurodisability, cognitive, social and psychiatric disorders in later life. Alterations to the white matter can be assessed using diffusion tensor imaging (DTI) MRI and are associated with poor neurodevelopmental outcome. The pathogenesis of white matter injury is multifactorial and several clinical risk and resilience factors have been identified. DNA methylation (DNAm) is an epigenetic process which links stressful early life experience to later life disease and is associated with normal brain development, neuronal processes and neurological disease. Several studies have shown DNAm is altered by the perinatal environment, however its role in preterm white mater injury is yet to be investigated. Aims: 1. To examine the relationship between preterm birth and white matter integrity 2. To investigate the effect of neuroprotective treatments and deleterious clinical states on white matter integrity in preterm infants 3. To assess the best DTI method of quantifying white matter integrity in a neonatal population 4. To investigate the effect of preterm birth on DNA methylation and 5. To determine the clinical and imaging factors that contribute to the variance in DNA Methylation caused by preterm birth Methods: DTI data was acquired from preterm infants (< 32 weeks' gestation or < 1500 grams at birth) at term equivalent age (TEA) and term controls (> 37 weeks' gestation at birth). Region-of-interests (ROI) and tract-averaged methods of DTI analysis were performed to obtain measurements of fractional anisotropy (FA) and mean diffusivity (MD) in the genu of corpus callosum, posterior limb of internal capsule and centrum semiovale. Clinical data was collected for all infants and the effect of prematurity, neuroprotective agents and clinical risk factors on white matter integrity were analysed. 8 major white matter tracts were segmented using probabilistic neighbourhood tractography (PNT), a tract-averaged technique which also allowed the calculation of tract shape. The two DTI techniques were compared to evaluate agreement between results. DNA was collected from preterm infants and term controls at TEA, and a genome-wide analysis of DNAm was performed. DTI parameters from probabilistic neighborhood tractography (PNT) methodology and clinical risk and resilience factors were used to inform a principal components analysis to investigate the contribution of white matter integrity and clinical variables to variance in DNAm. Results: FA and MD were significantly affected by preterm birth on ROI analysis. In addition, DTI parameters were affected by clinical factors that included antenatal magnesium sulphate, histological chorioamnionitis and bronchopulmonary dysplasia. Evaluation of DTI methodology revealed good accuracy in repeated ROI measurements but limited agreement with tract-averaged values. Differential methylation was found within 25 gene bodies and 58 promoters of protein-coding genes in preterm infants, compared with controls. 10 of these genes have a documented association with neural function or neurological disease. Differences detected in the array were validated with pyrosequencing which captured additional differentially methylated CpGs. Ninety-five percent of the variance in DNAm in preterm infants was explained by 23 principal components (PC); corticospinal tract shape associated with 6th PC, and gender and early nutritional exposure associated with the 7th PC. Conclusions: Preterm birth is associated with alterations in white matter integrity which is modifiable by clinical risk factors and neuroprotective agents. ROI analysis may not provide sufficient representation of white matter tracts in their entirety. Prematurity is related to alterations in the methylome at sites that influence neural development and function. Differential methylation analysis has identified several promising candidate genes for future work and contributed to the understanding of the pathogenesis of preterm brain injury.

Friedreich ataxia (FRDA) is a neurodegenerative disorder that is inherited in an autosomal recessive pattern. The most common FRDA mutation is hyperexpansion of a GAA triplet repeat sequence in the first intron of the affected gene, frataxin (FXN), resulting in decreased frataxin protein expression. The hyperexpanded GAA repeats can adopt unusual DNA structures and induce aberrant epigenetic changes leading to heterochromatin mediated gene silencing. Several epigenetic changes, including increased levels of DNA methylation, histone modifications, repressive chromatin formation and elevated levels of non-coding RNA have been reported in FRDA. It has been reported that a novel FXN antisense transcript (FAST-1), is present at higher levels in FRDA patient-derived fibroblasts and its overexpression is associated with the depletion of CTCF, a chromatin insulator protein, and heterochromatin formation involving the critical +1 nucleosome. Previously, characteristics of FAST-1 were investigated in our lab and a full-length FAST-1 transcript containing a poly (A) tail was identified. To investigate any possible effects of FAST-1 on FXN expression, I first overexpressed this FAST-1 transcript in three different non-FRDA cell lines and a consistent decrease of FXN expression was observed in each cell type compared to control cells. I also identified that FAST-1 copy number is positively correlated with increased FAST-1 expression, which in turn is negatively correlated with FXN expression in FAST-1 overexpressing cells. Additionally, we found that FAST-1 overexpression is associated with increased levels of DNA methylation at CpG sites U6 and U11 of the FXN upstream GAA repeat region, together with CTCF depletion and heterochromatin formation at the 5'UTR of the FXN gene. To further investigate the role of FAST-1 in FXN gene silencing, I used a small hairpin RNA (shRNA) strategy to knock down FAST-1 expression in FRDA fibroblast cells. I found that knocking down FAST-1 increases FXN expression, but not to the level of control cells. Lastly, I investigated the pattern of FAST-1 expression and histone modifications at the FXN transgene in our new FRDA mouse model, designated YG8LR. The YG8LR mice showed decreased levels of FXN expression and H3K9ac and increased levels of FAST-1 expression and H3K9me3. Our data suggest that since FAST-1 is associated with FXN gene silencing, inhibition of FAST-1 may be an approach for FRDA therapy.

In higher eukaryotes, the DNA base Cytosine can exist in a variety of modified forms when in the dinucleotide CpG. Although a methylated form tends to dominate within the genome, approximately 1% of all CpG dinucleotides are found unmodified at high densities spanning around 1Kb and tend to co-localise to the 5’ ends of around 60% of annotated gene promoters. These unique DNA sequences are known as CpG islands (CGIs) and their role within the genome to date is largely unknown. Methylation of CGIs in cancers however has been linked to silencing of associated genes implying a role in gene regulation. Furthermore these sites are also interesting as they remain specifically nonmodified within a genome rich in methylated CpG. We set out to better understand the roles for CGIs through the characterisation of any specific CGI binding proteins. Digestion of nuclei with methyl sensitive restriction enzymes facilitates the purification of CGI fragments. Subsequent immunohistochemistry on the CGI chromatin fragments along with ChIP-PCR over several CGIs revealed an enrichment of the “active” histone modifications including H3K4me3, a depletion of the “silencing” marks such as H3K27me3, as well as a group of CGI specific binding factors. These latter proteins contained a domain previously shown to bind to non-methylated CpG dinucleotides (the CXXC domain) and as such were ideal candidates for CGI specific factors, in particular a protein called Cfp1. Genome wide sequencing revealed a striking correlation between Cfp1 and H3K4me3 which were both seen at around 80% of islands. Furthermore, the presence of Cfp1/H3K4me3 at islands tended to have a negative correlation with the presence of chromatin rich in the silencing histone modification H3K27me3. Closer investigation of the Cfp1 protein reveals it to be a true non-methyl CGI binding factor in vivo and shRNA reduction of Cfp1 levels to around 10% of wild type resulted in a precipitous drop in H3K4me3 levels over CGIs without a dramatic reduction in global H3K4me3 levels. As Cfp1 has been shown to be part of the Set1 histone H3K4 methyltransferase complex responsible for this modification, this CXXC protein may be attracting this histone modifying complex and as such represents a method whereby the underlying DNA sequence (CpG) can drive the overlying epigenetic state. This study may go some way to understanding the functional significance of CGIs within the genome.