For a cell to exert a specialized function certain genes have to be expressed, others repressed. Transcription factors, regulating this expression, do not function alone, but are often part of multi-protein complexes. Regulating a single gene with more than one transcription factor is an efficient way to integrate responses to a variety of signals using a limited number of proteins. DNA binding proteins often interact with each other and with non-DNA binding proteins in a specific arrangement. The assembly of these complexes is often highly cooperative and promotes high levels of transcriptional synergy. The center of my thesis is the family of POU transcription factors. Specifically, I elaborate the interaction within the POU protein family, with members of other transcription factor families and with cofactors. In all cases, the assembly of the correct array of polypeptides on the DNA requires specific protein-protein and protein-DNA interactions. As an example of POU factors interacting with each other and with a cofactor I investigated the properties of a protein-DNA complex with the B-cell-specific cofactor OBF! and the Octl dimer. Depending on the DNA sequence they bind to, Octl dimers are arranged in configurations that are either accessible (PORE sequence) or inaccessible (MORE sequence) to OBF!. In Chapter 3 I show that the expression of Osteopontin, which contains a PORE sequence in its enhancer region, depends on the presence of OBFI in B-cells. OBFI alleviates DNA sequence requirements of the Octl dimer on PORE-related sequences in vitro. Furthermore, OBFI enhances POU dimer-DNA interactions and overrides Oct! interface mutations, which abolish PORE-mediated dimerization without OBFl. Based on the biochemical data, I propose a novel Oct! dimer arrangement when OBF 1 is bound. As an example of Oct factors interacting with members of another transcription factor family I studied the interactions of Sox2 with Octl and Oct4, respectively. POU and Sox transcription factors exemplify partnerships established between various transcriptional regulators during early embryonic development. The combination of Oct4 and Sox2 on DNA is considered to direct the establishment of the first three lineages in the mammalian embryo. Although functional cooperativity between key regulator proteins is pivotal for milestone decisions in mammalian development, little is known about the underlying molecular mechanisms. The data in Chapter 4 validate experimental highresolution structure determination, followed by model building. The study shows that Oct4 and Sox2 are able to dimerize on DNA in distinct conformational arrangements. The binding site characteristics of their target genes are responsible for the correct spatial alignment of the Velcro-like interaction domains on their surface. Interestingly, these surfaces frequently have redundant functions and are instrumental in recruiting various interacting protein partners. In Chapter 5 I investigated how Sox2 and Oct4 regulate transcription of a target gene. The first intron of Osteopontin contains a Sox-binding site and a unique PORE to which Oct4 can either bind as a monomer or a dimer. The study reveals that Sox2-specific repression depends on an upstream Sox site and an intact PORE, although neither the Sox nor the PORE sites are negative elements on their own. A mechanism is being proposed how Sox2 represses Oct4-mediated activation of Osteopontin.

Genes are the basic units of heredity, containing the DNA code for RNA molecules. Transcription is the process that converts the genetic code of genes into RNA molecules, which then perform a range of functions, including encoding proteins in the case of mRNA. Transcription is commonly regulated by proteins known as transcription factors, which can act in either a positive or negative manner.

The biosynthesis of fungal highly reduced polyketides was investigated by domainain shuffling, heterologous expression and gene mactivation expenments. In order to produce novel polyketides and to study chain length control of fungal iterative type I PKS, the ketosynthase (KS) and acyltransferase (AT) domains of squalestatin tetraketide synthase (SQTKS) were replaced by the corresponding domains from tenellin 1 and fusarin C synthetases that catalyse the synthesis of a pentaketide and a hexaketide, respectively. However, the expression of the chimeric genes encoding the hybrid PKS enzymes in Aspergillus oryzae did not result in production of any polyketides in detectable amount.

Transcription by RNA polymerase 1 (Pol-1) is the main driving force behind ribosome biogensis, a fundamental cellular process that requires the coordinated transcription of all three nuclear polymerases. Increased Pol-1 transcription, and the concurrent increase in ribosome biogensis has been linked to the high rates of proliferation in many cancers. The ellipticine family contains a number of potent anticancer therapeutic agents with some having progressed to stage 1 and II clinical trial; however the mechanism by which these compounds works remain unclear.

BCL11A (also known as CTIPl and Evi9) is a Kruppel zinc finger protein that functions as a developmental regulator in haematopoietic and neuronal tissues. It was originally identified as an oncoprotein whose overexpression is associated with acute leukaemia or B-cell lymphoma. Recent genetic studies have linked inactivating mutations in the BCL11A gene to sickle cell disease and B-thalassaemia, and it was subsequently shown that BCL11A protein is a master regulator of foetal haemoglobin switching. A major role of BCL 11 A is its function as a corepressor for DNA binding transcription factors such as NR2F2/COUP-TFII, GATAl and SOX6 transcription factors, and BCL11A appears to act in conjunction with other coregulators such as BCL6, SIRTl, FOG2 and the NuRD complex. In this study, the molecular basis of interaction of BCL11A with the orphan nuclear receptor COUP-TFII (NR2F2) was investigated. Using yeast two hybrid mapping, as well as in vitro and in vivo protein interactions assays, it has been established that BCL11A contains two motifs that are necessary and sufficient for binding to the ligand binding domain of COUP-TFII. These motifs (termed receptor interacting domains RIDl and RID2) are novel variants of the LXXLL and CoRNR box motifs found in other nuclear receptor cofactors. RID 1 and RID2 share the consensus FNSXXLXXLN, but unlike other motifs display highly selective binding to the NR2E/F subfamily of orphan nuclear receptors. I present evidence for functional interactions of RIDl and RID2 with the LBDs ofNR2F2/COUP-TFII, NR2El/TLX and NR2E3/PNR, whereas PNR shows an additional selectivity for RID1. No detectable interactions were observed with more than twenty other LBDs.

G-protein coupled receptors (GPCRs) are a diverse superfamily of membrane proteins. They have a wide range of physiological roles and include many successful drug targets. Sequencing of the human genome has revealed a distinct subfamily of GPCRs known as Adhesion GPCRs. These receptors possess unusually large extracellular N-terminal domains which are believed to be involved in cell-cell adhesion. Few data are available which demonstrate that these receptors are able to couple to G-proteins, their classification as GPCRs is primarily based on homology and predicted topology. These receptors are also mainly orphans. This investigation aims to demonstrate G-protein coupling of Adhesion GPCR members and use this coupling to aid de-orphanisation and pharmacological targeting. In this study, a selection of Adhesion GPCRs are expressed in a range of yeast (S. cerevisiae) strains each harbouring different mammalian-yeast chimeric G-proteins. Constitutive coupling of four different Adhesion GPCRs to the chimeric G-proteins is observed via a reporter gene assay. The chimeric G-proteins used represent the human complement, allowing prediction of the G-protein specificities of these receptors. This yeast assay is then used for high throughput screening to identify both potentially native ligands and inhibitors/potential therapeutic compounds. Following analysis in yeast, the Adhesion GPCRs CD97 and EMR2 were expressed in mammalian HEK293 cells where they also displayed constitutive activity when co-expressed with the appropriate Go. subunits (GaJ6).This constitutive activity is strong and mirrors the G-protein specificities seen in yeast. Using this assay, the effects of candidate CD97 and EMR2 ligands were assessed, their putative binding sites

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