Pluripotent stem cells have the capacity to develop into different cell lineages and can be manipulated into certain cell types through the use of small molecule inducers. Retinoic acid (RA) signaling through retinoic acid receptors (RAR) and retinoid X receptors (RXR) has the ability to direct lineage determination but has yielded disappointing results in promoting skeletal myogenesis in embryonic stem (ES) cells. RXR is crucial in embryonic development although it is generally considered to act as a silent partner for other nuclear receptors such as RAR. Our findings demonstrate that rexinoid specific signaling enhances skeletal myogenesis and requires β-catenin but not RAR. Moreover, RXR signalling in mouse ES cells can efficiently enhance skeletal myogenesis and closely recapitulates sequential events observed in vivo. Since ES cells closely represent the properties of the developing embryo, efficiently generating skeletal muscle provides a means to further scrutinize signaling pathways in myogenic development in view of developing therapies for muscle related diseases.

Macrophages are involved in many aspects of both the innate and acquired immunity, and participate in tissue homeostasis, bone remodelling, wound healing, and tissue repair. When their function becomes dysregulated, they contribute to the initiation and progression of inflammatory diseases. Genes expressed in activated macrophages are likely to play an important role in inflammation and/or immunity. One family of genes that is highly expressed in activated macrophages is the Schlafen (Slfn) gene family. Given that very little is known about the function of this family, particularly in macrophages, this study focused on the regulation and function of one family member, namely Slfn-4. The transcriptional regulation of Slfn-4 was characterised in murine macrophages. The expression of Slfn-4 was transiently down regulated during macrophage differentiation and dramatically up regulated in response to activation signals including lipopolysaccharide (LPS) and dsRNA Poly(I:C). A potential association with inflammation was further suggested by the enhanced expression of Slfn-4 in a mouse model of rheumatoid arthritis. Further investigations into transcriptional regulation of Slfn-4 revealed that it belongs to the subset of genes that are type I interferon (IFN)-inducible. This finding is consistent with the predicted transcription factor binding sites in the putative promoter of Slfn-4, and suggests a role for Slfn-4 in the antiviral response. To gain further insight into the function of Slfn-4 in macrophage biology, in vitro over-expression approaches were undertaken and its cellular localisation in macrophages was characterised. In resting and activated macrophages, Slfn-4 exhibited a cytoplasmic and strong perinuclear localisation. Additional studies were carried out to investigate the in vivo biology of Slfn-4 in macrophages. For this purpose, the Csf1r-GAL4VP16/UAS-ECFP (referred to as “MacBlue”) transgenic mouse line was first characterised. Enhanced cyan fluorescent (ECFP) reporter expression in the MacBlue transgenic mice was specifically detected in cells of the mononuclear phagocyte system during embryonic development and adulthood. The MacBlue transgenic mouse line was next used to drive expression of Slfn-4 in cells of the myeloid lineage in vivo, and to examine the phenotype of this line. Specific over-expression of Slfn-4 in cells of the myeloid lineage in vivo altered the percentage of peripheral blood monocytes and caused extramedullary hematopoiesis. In summary, this thesis demonstrated that Slfn-4 expression is dynamically regulated during macrophage differentiation and activation, and the enforced Slfn-4 over-expression in cells of the myeloid lineage perturbs normal monocyte/macrophage development.

Haemopoietic stem and progenitor cells from bone marrow and cord blood are well characterised with respect to their phenotype, growth in clonal assays, responsiveness to cytokine stimulation, receptor profile and their ability to sustain multilineage engraftment of receptive hosts in animal models of transplantation and of course, clinically in the treatment of some haemopoietic and immunological disorders. It is generally accepted that cells bearing the CD34+ phenotype are enriched for the most primitive of haemopoietic stem cells that possess the cardinal features of self-renewal and multipotency. However, the molecular mechanisms, the spectrum of expressed genes that give rise to the physical characteristics of haemopoietic progenitor cells are not well understood. Furthermore, although CD34+ cells from different sources (bone marrow, cord blood, mobilised peripheral blood) share many common features, there are also significant differences. (For complete abstract open document)

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Hypoxia regulates many hundreds of genes that play important roles in numerous physiological and pathophysiological processes. The hypoxia inducible transcription factors (HIFs) are central to the transcriptional activation of these hypoxia-regulated genes. However, to date, little is known about the determinants of HIF-1 and HIF-2 binding site selection. Both HIF-1 and HIF-2 appear to bind as HIF-α/HIF-1β heterodimers, and recognise the same core consensus DNA motif, the hypoxia response element (HRE). However, each has its own distinct but partially overlapping set of binding sites that accounts for only a subset of the total accessible HRE motifs. Here, I have utilised ChIP-seq to systematically compare pan-genomic HIF-1α, HIF-2α and HIF-1β DNA binding in multiple cell lines and have related this to RNA-seq analyses and other publically-accessible next-generation sequencing datasets. I show that endogenous HIF-Iα subunits exhibit a high-degree of binding site concordance with HIF-1β, consistent with largely canonical binding of intact heterodimers. Despite cell-type specific differences in HIF-1 and HIF-2 binding site occupancy, each isoform exhibits a remarkable rigidity in its preference to bind either promoter-proximal or promoter-distal sites, respectively. These specific distribution patterns are unaffected by the absence of the other HIF-Iα subunit, suggesting that they do not result from exclusion of one isoform by competition with the other, but rather are discrete properties of each. Furthermore, hypoxia regulated genes neighbouring sites that are shared by both HIF-1 and HIF-2 are more likely to be regulated by HIF-1 when the site is closer to the gene and by HIF-2 when further away, indicating that post-binding mechanisms of transcriptional regulation also follow a similar pattern. Comparison of sites preferentially bound by HIF-1 and HIF-2, respectively, revealed associations with distinct histone environments, distinct accessory transcription factor binding motifs and distinct patterns of transcription factor binding site occupancy, suggesting that each may be influencing specific HIF-1 and HIF-2 binding site selection. In particular, both the AP-1 motif and AP-1 binding site occupancy were enriched within HIF-2 binding sites compared to HIF-1 sites. Intervention on AP-1 DNA-binding using the dominant-negative protein, AFos, attenuated HIF binding, specifically at sites co-occupied by AP-1 and HIF. This indicates that a cooperative relationship exists between the two transcription factors. However, binding of both HIF-1α and HIF-2α were affected suggesting that while AP-1 binding may account for the ability of HIF to bind some HRE motifs but not others, it is not a determinant of differential binding between the two isoforms. Overall, this work reveals remarkably distinct and functionally relevant patterns of HIF-1 and HIF-2 binding across the genome, and provides insight into underlying mechanisms of binding.

This thesis characterizes RNA footprints of several RNA-binding proteins (RBPs) thatare involved in U-insertion/deletion, A-to-I, and C-to-U RNA editing in Trypanosoma brucei. Relying on iCLIP data and biochemical methods it shows that two paralogs proteins from the MRB1 complex regulate distinct editing fates of the mitochondrial transcripts. Further, this thesis provides evidence where the combinatorial interplay of RBPs might fine-tune the levels of edited mRNA. Finally, the presented thesis adds to the growing evidence of the importance of RBPs in post-transcriptional gene regulation.

The advent of high-throughput DNA and RNA sequencing has made possible the assay of millions of nucleic acid molecules in parallel. This allows functional genomic elements to be identified from background in single-tube experiments. This dissertation discusses the development of two such functional screens as well as work implementing a third that was previously developed in my thesis laboratory. Restriction-Associated DNA sequencing (RAD-Seq) is a complexity reduction sequencing method that allows the same subset of genomic sequence to be read across multiple samples. Differences in sample collection and data analysis allow manifold applications of RAD-Seq. Here we use RAD-Seq to identify mutant genes responsible for altered phenotypes in Caenorhabditis elegans and to identify hyper-invasive alleles in trout population admixtures. Apart from acquiring genomic sequence data, massively-parallel sequencing can be used for counting applications that quantify activity across a large number of test molecules. This dissertation describes the development of a technique for simultaneously quantifying the activity of a restriction enzyme across all possible DNA substrates by linking digest of a sequenced genome to Illumina-sequencing in an unbiased fashion. Finally, a powerful approach to analyze transcriptional activation is described. This method quantifies output from millions of potential DNA transcriptional enhancers via RNA amplicon sequencing of covalently-linked randomer tags and is used in conjunction with RNA-Seq to provide a mechanistic view of hypoxic gene regulation in Drosophila. This dissertation includes previously published, co-authored material

DNA methylation is an epigenetic mark that is deposited throughout the genome of mammals and plays an important role in the maintenance of transcriptionally repressive states across cell divisions. There are two major mechanisms by which DNA methylation has been proposed to act: one involves the recognition of the mark by protein complexes containing histone deacetylases (HDACs) that can remodel the local chromatin. Alternatively, methylation has been suggested to directly affect the interaction between transcription factors and their cognate binding sequence. The aim of this research was to determine the contributions of these two mechanisms in cells. The importance of HDAC activity in mediating DNA methylation-dependent transcriptional repression was assessed by comparing the genes and retrotransposons that are upregulated in response to DNA methylation loss or the disruption of HDAC activity. To this purpose, we performed whole-genome transcriptional analysis in wild type and DNA methylation-deficient mouse embryonic stem cells (DNMT.TKO mESCs) in the presence and absence of the HDAC inhibitor trichostatin A. Our data suggests that there are few genes whose repression is solely dependent on the recruitment of HDACs by DNA methylation in mESCs. Rather it appears that DNA methylation and HDAC-mediated silencing represent two independent layers of repression that converge at certain transcriptional elements. To investigate the contribution of DNA methylation on the genome-wide occupancy of transcription factors, we compared the global chromatin accessibility landscape and the binding profile of candidate transcription factors in the absence or presence of DNA methylation. We found that loss of DNA methylation associates with localised gains in accessibility, some of which can be linked to the novel binding of transcription factors such as GABPA, MAX, NRF1 and YY1. Altogether, our results present new insights into the interplay between DNA methylation and histone deacetylation and their impact on the localisation of transcription factors from different families.

Eukaryotic genomes are pervasively transcribed and frequently generate long noncoding RNAs (lncRNAs). However, most lncRNAs remain uncharacterized. In this work, a set of positionally conserved intergenic lncRNAs in the fission yeast Schizosaccharomyces pombe genome are selected for further analysis. Deleting one of these lncRNA genes (ncRNA.1343) exhibited a clear phenotype: increased drug sensitivity. Further analyses revealed that deleting ncRNA.1343 also disrupted a previously unannotated lncRNA, termed nc-tgp1, transcribed in the opposite orientation of the predicted ncRNA.1343 gene and into the promoter of the phosphate-responsive permease gene tgp1+. Detailed analyses revealed that the act of transcribing nc-tgp1 into the tgp1+ promoter increases nucleosome density and prevents transcription factor access. Decreased nc-tgp1 transcription permits tgp1+ expression upon phosphate starvation, while nc-tgp1 loss induces tgp1+ in repressive phosphate-rich conditions. Notably, drug sensitivity results directly from tgp1+ expression in the absence of nc-tgp1 transcription. Similarly, lncRNA transcription upstream of pho1+, another phosphate-regulated gene, increases nucleosome density and prevents transcription factor binding to repress pho1+ in phosphate-replete cells. Importantly, the regulation of tgp1+ and pho1+ by upstream lncRNA transcription occurs in the absence of RNAi and heterochromatin components. Instead, the regulation of tgp1+ and pho1+ by upstream lncRNA transcription resembles examples of transcriptional interference reported in other organisms. Thus, tgp1+ and pho1+ are the first documented examples of genes regulated by transcriptional interference in S. pombe.

Os genes de choque térmico groES e groEL de Caulobacter crescentus foram isolados utilizando-se os genes homólogos de E.coli como sonda e por sequenciamento demonstrou-se que estes genes estão organizados na forma de um operon em um fragmento de DNA de aproximadamente 2,5 kb, contendo também sua região regulatória. \"Northern blots\" de RNA total de células crescidas a 300C ou submetidas a choque térmico mostraram a presença de um único RNA de tamanho aproximado de 2,3kb, altamente induzido por choque térmico, permanecendo em altos níveis mesmo após longos períodos de choque térmico. Amostras de RNA total de células sincronizadas, de diferentes estágios do ciclo celular de Caulobacter, foram também analisadas mostrando que os níveis do mRNA groESL variam durante o ciclo, apresentando um máximo na célula prédivisional. Análises através de \"Western blot\" mostraram uma pequena variação nos níveis da proteína GroEL ao longo do ciclo celular, sendo os tempos 60 e 120 minutos, respectivamente, os pontos de mínimo e máximo acúmulo da proteína concordando com os resultados obtidos em \"N orthern blots\". O mesmo tipo de análise foi feito com extratos totais obtidos a partir uma população mista de células crescidas a 300C e submetidas a choque térmico, observando-se o acúmulo da proteína até 60 minutos depois do choque térmico, com aumento da ordem de 5 vezes nos níveis de GroEL, níveis estes que diminuem lentamente a partir deste ponto. Os inícios de transcrição foram determinados em experimentos de \"primer extension\" utilizando-se RNA total de células incubadas 300C e de células submetidas a diferentes condições de choque térmico. Dois possíveis sítios de início de transcrição foram determinados nas posições -119 e -88 do ATG da metionina iniciadora de groES, sendo as regiões -10 e -35 dos promotores correspondentes (P 1 e P2) identificadas. Somente a transcrição iniciando a partir de P2, que apresenta características de um promotor transcrito pelo σ32, aumenta durante o choque térmico. Fusões de transcrição com o vetor repórte placZ/290 e a região 5\' regulatória do operon groESL foram construídas para identificar as sequências responsáveis pelo controle por choque térmico e pelo controle temporal. Fusões de transcrição contendo deleções na região 5\' do operon mostraram que sequências a montante do promotor P2 não são necessárias para a indução por choque térmico ou para o controle temporal. Fusões de transcrição contendo mutações sítio-dirigidas na repetição invertida, encontrada a 3\' do promotor P2, antes do gene groES, revelaram que este elemento, conhecido como CIRCE, regula negativamente a expressão de groESL a 300C e mutações neste elemento levam à perda do controle temporal deste operon. / The heat shock genes groES and groEL of Caulobacter crescentus were isolated using the homologous genes of E.colí as a probe. DNA sequence analysis has shown that these genes are organized as an operan in a fragment of about 2.5kb, which includes the 5\' regulatory region. Northern blot analysis of total RNA from cells grown at 300C or heat shocked treated has shown the presence of a single mRNA species for groESL, of approximately 2.3kb in size, which presented increased leveis even after long periods of heat shock. Samples of total RNA from synchronized cells, corresponding to different stages of the Caulobaaer cell cycle, were also analysed, showing that the amount of groESL mRNA varies during the cycle, with maximum leveis in predivisional cells. Western blot analysis of GroEL leveis in Caulobaaer has shown that the amount of the protein decreases during the first 60 minutes of C.crescentus cell cycle and then starts to increase again. These results corroborate the data obtained with Northern blot analysis. A similar experiment was performed after exposing a mixed population of C.crescentus cells to different times of heat shock at 400C. Western blot of extracts of these cells showed a fivefold increase in the leveis of GroEL after 60 minutes of heat shock, which then begins to decrease. Primer extension experiments were performed using total RNA from cells incubated at normal growth temperature or after heat shock treatment. Two possible transcription start sites were determined at positions -119 and -88 from the ATG of the groES initiator methionine and the -10 and -35 regions of the corresponding promoters (P 1 and P2) were identified. Only trancription initiating from the P2 promoter, which has caracteristics of a σ32 promoter, I ncreases during heat shock .Transcription fusions with the reporter vector placZ/290 and the 5\' regulatory region of the groESL operan were contructed in order to identify the sequences responsible for heat shock and cell cycle contral. Deletion analysis in the 5\' region of the operon showed that no sequences upstream of the P2 promoter are necessary for heat shock induction or for temporal contral. Site-directed mutagenesis in the inverted repeat found 3\' of the P2 promoter, in front of the groES gene, revealed that this element, also known as CIRCE, negatively regulates groESL expression at 300C and mutations in it lead to loss of temporal control of this heat shock operon.