Glucocorticoids (GC) are powerful metabolic hormones with anti-inflammatory actions. Despite major side effects they remain widely prescribed therapies. GC regulates gene expression through an intracellular receptor (GR), which is a ligand activated transcription factor. Macrophages are innate immune cells and major targets of GC. Traditionally repression of pro-inflammatory genes in the context of an inflammatory stimulus has been considered the primary mode of action of GC in macrophages. The work described in this thesis has demonstrated that GC act primarily as inducers of gene expression in primary macrophages from both mouse and man, but the set of induced genes is very different between the two species. Chromatin immunoprecipitation and sequencing (ChIP-seq) in each species using anti-GR antibodies revealed candidate enhancers in the vicinity of inducible genes that were generally not shared between mouse and man. The differences in binding were correlated with DNA sequence changes at the enhancer sites between the two species, that caused gain or loss of predicted GR receptor-binding motifs. The mechanism of action of GC was investigated by imaging several different target loci using fluorescence in situ hybridisation in macrophage nuclei. Chromatin at specific GC responsive loci was found to decondense within minutes of exposure of macrophages to the ligand. The apparent decondensation was effect was maintained for at least 24 hours and was not prevented by inhibitors of transcription. The general principles of the GC response were shared between species. However the divergence found underlines the caution that must be used when translating specific findings from mouse to man. Additionally, the data support a role for GR driven changes to chromatin structure in gene regulation in macrophages.

Chromatin remodelers are ATP-dependent multisubunit assemblies that regulate transcription and other processes by altering DNA-histone contacts. The mechanism of action is based on the transduction of energy released by ATP hydrolysis to translocation on DNA and ultimately the movement of histones in cis or trans. Though the critical ATP burning and translocation activities are fulfilled by a conserved ATPase domain in the catalytic subunit, there are accessory domains and subunits that are speculated to regulate these activities. Important questions in the field center around the identification of these domains and subunits, whether they affect complex formation, substrate affinity or a critical step in remodeling. If they do affect remodeling, what is the structural basis of the regulatory activity. In this study, these questions have been addressed using the prototype remodeler SWI/SNF from budding yeast. ySWI/SNF is a 12 subunit complex that includes the catalytic subunit Swi2/Snf2. It affects 6% of the yeast genome being primarily involved in gene activation. We employed a systematic protein or domain deletion strategy and characterized the mutant complexes in vitro and in vivo. A key finding was that SWI/SNF is organized in distinct structural modules and that the Snf2 module regulates most of its activities. Snf2 is a central subunit in this module and the function of conserved regions within Snf2 were studied. The N terminus preceding the HSA and ATPase domain has three major roles - complex assembly, recruitment and regulation of catalytic activity. A novel SnAC domain located C terminal to ATPase domain was identified to play critical role in coupling ATP hydrolysis to nucleosome movement by acting as a histone anchor. Finally the tandem AT-hooks between SnAC and bromodomain serve as DNA binding domains but also affect ATPase activity and nucleosome mobilization independent of its binding activity. Taken together, this study provides a comprehensive overview of the function of regulatory domains in SWI/SNF.

In eukaryotes, the SET1 family of methyltransferases carry out the methylation of Lysine 4 on Histone H3. Alone, these enzymes exhibit low enzymatic activity and require the presence of additional regulatory proteins, which include RbBP5, Ash2L, WDR5 and DPY-30, to stimulate their catalytic activity. While previous structural studies established the structural basis underlying the interaction between RbBP5, Ash2L and WDR5, the formation of the Ash2L/DPY-30 complex remains elusive. Here we report the crystal structure of the Ash2L/DPY-30 complex solved at 2.2Å. Our results show that a Cterminal amphipathic α-helix on Ash2L makes several hydrophobic interactions with the DPY-30 homodimer. Moreover, the structure reveals that a tryptophan residue on Ash2L, which directly precedes its C-terminal amphipathic α-helix, makes key interactions with one of DPY-30 α-helix. Finally, biochemical studies of Ash2L revealed a hitherto unknown ability of this protein to bind anionic lipids.

Folliculogenesis is a complex process which describes the maturation of the ovarian follicle, from the primordial stage all the way to the ovulation of the antral follicle, and its sequela, the formation of the corpus luteum (CL). Imitation switch (ISWI) proteins are a class of ATP-dependent chromatin remodelers which mobilize nucleosomes to regulate a number of cellular processes including transcription, replication, and DNA repair. The pattern of expression of the mammalian ISWI proteins SNF2H and SNF2L in the mouse ovary suggests a role in the coordination of the proliferation and differentiation of granulosa cells during folliculogenesis. Here, we report that SNF2H is associated with proliferating granulosa cells, while SNF2L expression is induced following the LH surge which triggers their terminal differentiation into luteal cells. Knockdown of Snf2l by siRNA is sufficient to downregulate the expression of StAR, an important steroidogenic enzyme, and marker of the CL. Furthermore, SNF2L is thought to directly regulate StAR expression by physically binding to its promoter as indicated by chromatin immunoprecipitation (ChIP). In order to identify additional targets regulated by SNF2L, an unbiased microarray screen was developed to look for genes induced by LH in a SNF2L-dependent manner. One of the candidates, Fgl2 is strongly induced at 8h post hCG only in granulosa cells with intact SNF2L activity. Furthermore overexpression of SNF2L is sufficient to induce FGL2, and SNF2L is present on its promoter in the SIGC rat granulosa cell line. Some of the SNF2L binding partners that may be important in this regulation are PR-A and FLI-I, which have been found to interact with SNF2L by IP. Finally we describe here the phenotype of a Snf2l KO mouse which includes multiple reproductive defects, including resistance to superovulation, low secondary follicle counts, and a high incidence of abnormal antral follicles. Taken together these data suggest an important role of ISWI proteins in folliculogenesis, particularly SNF2L, which may regulate multiple genes important for the terminal differentiation of granulosa cells into luteal cells following the LH surge.

The work presented here included the development of a highly sensitive method to estimate the size and complexity of poly(ADP-ribose). This involved radiolabeling of the precursor pools, purification of polymers using a boronate resin, polymer fractionation according to size by molecular sieve chromatography and analysis of polymer complexity by enzymatic digestion to nucleotides which were quantified by strong anion exchange chromatography.

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Distal enhancer elements regulate the tissue-specific expression of their target genes via the establishment of physical interactions with the gene promoter. In mice, a cluster of five enhancers, jointly classified as a super-enhancer, specifically upregulate α-globin gene expression during erythroid differentiation. Aside from the Nprl3 gene, whose promoter is located inside this enhancer region, expression-levels of other genes within a short distance (&lt,50kb) of the enhancer region are not affected by the activation of the enhancer in erythroid cells, despite being located within the same sub-TAD in erythroid cells. The CCCTC-binding factor (CTCF) is implicated in the organisation of chromosome topology through the formation of interactions between its binding sites in an orientation-dependent manner. In this thesis, I demonstrate that CTCF functions in vivo as a boundary to maintain α-globin enhancer-promoter specificity in erythroid cells. The study of the local chromatin architecture by next-generation Capture-C reveals that α-globin enhancer and promoter interactions are constrained to a compartment of roughly 70kb. The unidirectional interaction profiles of the α-globin enhancers are delimited by the interactions between two genomic domains flanking the α-globin cluster. Further investigation shows that each of these domains contains several CTCF binding sites orientated in tandem, such that CTCF binding orientation between domains is convergent. Although CTCF binding across the α-globin locus is identical between mouse embryonic stem (ES) cells and erythroid cells, interaction between these domains occurs only in erythroid cells suggesting it is dependent on the formation of tissue-specific α-globin enhancer-promoter interactions. By generating a series of mouse models, deleting CTCF binding sites at the α-globin enhancers singly and in combination, I show that the deletion of two CTCF binding sites directly flanking the enhancer cluster results in a shift in interactions between flanking domains, away from the enhancer region. This leads to an expansion of enhancer interactions to include two genes directly upstream of the α-globin enhancers: Rhbdf1 and Mpg. Despite the Rhbdf1 gene being subject to polycomb group protein-mediated gene repression in erythroid cells, ablation of CTCF binding results in increased interactions between both the Rhbdf1 and Mpg gene promoters and the α-globin enhancers and concurrent strong transcriptional upregulation of both genes. The Rhbdf1 gene promoter acquires the active histone mark H3K4me3, but doesn't lose Polycomb Repressive Complex 2 (PRC2) mark H3K27me3 or binding of its catalytic component Ezh2. Despite the presence of this repressive mark, robust levels of Rhbdf1 expression are detected at levels higher than those in ES cells where this gene is actively expressed under the influence of its own enhancer. I conclude that regulation of the direction of enhancer interactions by CTCF is required for the promoter specificity of enhancers and the maintenance of transcriptional states of nearby genes.