Gastrulation is the process by which the three germ layers are generated during vertebrate development. Nodal ligands, which form a subgroup of the Transforming Growth Factor β (TGFβ) superfamily, regulate the expression several transcription factors implicated in gastrulation. Among these are the paired-like homeodomain transcription factors Goosecoid (Gsc) and Mixl1. At the molecular level, Gsc has been described to function as a transcriptional repressor by directly binding to paired homedomain binding sites on target promoters. Here, I describe a novel mechanism of transcriptional repression by Gsc. Using a molecular and embryological approach, I demonstrate that the forkhead transcription factor Foxh1, a major transducer of Nodal signaling, associates with Gsc which in turn recruits histone deacetylases to negatively regulate Mixl1 expression during early mouse development. Post-translational modification of transcription factors by SUMO proteins represents an important mechanism through which their activity is controlled. Here, I also demonstrate that Gsc is sumoylated in mammalian cells by members of the PIAS family of proteins and this modification potentiates the repressive activity of Gsc on direct targets such as the Xbra and Gsc promoters, but not on indirect targets such as Mixl1. Taken together, work presented in this thesis describes two novel mechanisms of transcriptional repression by Gsc.

Early region 1A (E1A) protein is the first protein expressed following viral infection. E1A proteins initiate the cell cycle in infected cell by altering cellular gene expression and also activate expression of other viral genes enabling viral replication. The C-terminus of E1A is the least-characterized region of the protein, with few known binding partners, however DREF has been identified as a novel binding partner. My studies have determined that DREF directly binds to E1A and has a role in the virus life cycle. DREF is a restriction factor for virus growth and is a component of viral replication centres. DREF is SUMOylated and SUMOylation appears to affect localization to viral replication centres. DREF co-localizes with PML bodies and subcellular distribution of DREF is altered by the presence of E1A. This work provides a platform to study the role of DREF in uninfected cells, and in HAdV biology and its possible role in antiviral response. / October 2015

The aim of this diploma thesis was to study the role of posttranslational modifications of phosducin and their role in the interaction with the 14-3-3 protein as well as the influence of the complex formation on these modifications. Phosducin is a 33kDa protein commonly present in photoreceptor cells of the retina as well as other tissues. Despite many experiments, its physiological functions are still elusive. It has been speculated that fosducin plays an important regulatory role in visual phototransduction pathway, regulation of blood pressure and expression of G-proteins. The phosducin function is regulated through binding to the 14-3-3 protein, a regulatory protein involved in many biochemical processes. Phosducins binding to 14-3-3 protein requires phosphorylation of two serine residues Ser-54 and Ser-73 within the N-terminal domain of phosducin. However, the role of the 14-3-3 protein binding in the regulation of phosducin function is still unclear. In this diploma thesis proteins 14-3-3ζ∆C and phosducin (mutation Q52K) were successfully expressed and purified. The effect of the complex formation on phosducin posttranslational modifications was investigated using limited proteolysis and dephosphorylation. These experiments revealed that the complex formation significantly slowed down both...

Huntington's disease (HD) is a neuropsychiatric disorder characterized by choreiform movement of the limbs, cognitive disability, psychosis and dementia. It is untreatable, incurable, and ultimately fatal. HD is invariably associated with an abnormally long CAG expansion within the IT15 gene on human chromosome 4. Although the mutant huntingtin protein (mHtt) is ubiquitously expressed in HD patients, cellular degeneration occurs only in neurons within the striatum and cerebral cortex. The Ras homolog Rhes is expressed very selectively in the precise brain areas affected by HD. Recent work using cultured cells suggests that Rhes may be a co-factor with mHtt in cell death. However, there is controversy as to whether cell death underlies the symptoms of HD. We used a validated transgenic mouse model of HD crossed with Rhes knockout mice to show that the behavioral symptoms of HD are regulated by Rhes. HD/Rhes-/- mice showed greatly delayed expression of HD-like symptoms in this in vivo model. Drugs that block or inhibit the actions of Rhes may be useful as the first treatments for HD.

Meiosis is a highly regulated process, partly due to the need to break and then repair DNA as part of the meiotic program. In this thesis, mechanisms of meiotic regulation are investigated, including the post-translational modification termed SUMOylation, and a potential novel negative regulator of error-prone DNA repair pathways. Post-translational modifications are widely used during meiotic events to regulate steps such as protein complex formation, checkpoint activation, and protein attenuation. In this thesis, we investigate how proteins that are obligatory components of the SUMO pathway, one such post-translational modification, affect the C. elegans germline. We show that UBC-9, the E2 conjugation enzyme, and the C. elegans homolog of SUMO, SMO-1, localized to germline nuclei throughout prophase I. Mutant analysis of smo-1 and ubc-9 revealed increased recombination intermediates throughout the germline, originating during the mitotic divisions. SUMOylation mutants also showed late meiotic defects including defects in the restructuring of oocyte bivalents and endomitotic oocytes (EMO). Increased rates of non-interfering crossovers (COs) were observed in ubc-9 heterozygotes, even though interfering COs were unaffected. We have also identified a physical interaction between UBC-9 and DNA repair protein MRE-11. ubc-9 and mre-11 null mutants exhibited similar phenotypes at germline mitotic nuclei and were synthetically sick. These phenotypes and genetic interactions were specific to MRE-11 null mutants as opposed to RAD-50 or resection-defective MRE-11. We propose that the SUMOylation pathway acts redundantly with MRE-11, and in this process MRE-11 likely plays a structural role. We also found a candidate negative regulator of non-homologous end joining through an RNAi screen using the mre-11(iow1) resection-defective mutant. Mutant analysis did not rescue the resection-defective phenotype when multiple CRISPR-generated nhr-2 deletion alleles were generated. Therefore, nhr-2 does not appear to be directly involved in DNA repair.

SUMO is a small ubiquitin-like modifier. The fluorescent fused SUMO (active for sumoylation) localized in the nucleus, while C-terminal truncated SUMO (inactive for sumoylation) diffused in the cytoplasm. Daxx is a SUMO target protein, locates predominantly in the nucleus. It has been identified as a component of the PODs. During extracellular stimulation, Daxx could be recruited to the cytoplasm with the existence of Ask1. Therefore, it is a shuttle protein. Daxx should contain nuclear localization signal (NLS) and nuclear export signal (NES) motifs. To identify the NES and NLS motifs on Daxx, Daxx were truncated into four segments. Several amino acids on the predicted NES and NLS motifs were mutated. Our results showed that the truncated Daxx fragments D1 (containing NES) and D4 (containing NLS2) could be translocated into nucleus independently. However, either NES or NLS2 mutants disrupted their translocation into nucleus. It indicated that both NES and NLS2 motif of Daxx were involved in the nuclear transport. Nevertheless the co-transfection of SUMOs and Daxx showed that the interactions between SUMO active form and Daxx mutants and between inactive SUMO and Daxx wild type rescued the nuclear transport function of Daxx mutants and inactive SUMO. Therefore, SUMO may play a role in the nuclear transport of Daxx by either sumoylation or interaction with Daxx in cytoplasm, and Daxx may recruit inactive SUMOs into nucleus by interaction.

No description available.

Gastrulation is the process by which the three germ layers are generated during vertebrate development. Nodal ligands, which form a subgroup of the Transforming Growth Factor β (TGFβ) superfamily, regulate the expression several transcription factors implicated in gastrulation. Among these are the paired-like homeodomain transcription factors Goosecoid (Gsc) and Mixl1. At the molecular level, Gsc has been described to function as a transcriptional repressor by directly binding to paired homedomain binding sites on target promoters. Here, I describe a novel mechanism of transcriptional repression by Gsc. Using a molecular and embryological approach, I demonstrate that the forkhead transcription factor Foxh1, a major transducer of Nodal signaling, associates with Gsc which in turn recruits histone deacetylases to negatively regulate Mixl1 expression during early mouse development. Post-translational modification of transcription factors by SUMO proteins represents an important mechanism through which their activity is controlled. Here, I also demonstrate that Gsc is sumoylated in mammalian cells by members of the PIAS family of proteins and this modification potentiates the repressive activity of Gsc on direct targets such as the Xbra and Gsc promoters, but not on indirect targets such as Mixl1. Taken together, work presented in this thesis describes two novel mechanisms of transcriptional repression by Gsc.

yes / Multiple pathways participate in the AMPA receptor trafficking that underlies long-term potentiation (LTP) of synaptic transmission. Here we demonstrate that protein SUMOylation is required for insertion of the GluA1 AMPAR subunit following transient glycine-evoked increase in AMPA receptor surface expression (ChemLTP) in dispersed neuronal cultures. ChemLTP increases co-localisation of SUMO-1 and the SUMO conjugating enzyme Ubc9 and with PSD95 consistent with the recruitment of SUMOylated proteins to dendritic spines. In addition, we show that ChemLTP increases dendritic levels of SUMO-1 and Ubc9 mRNA. Consistent with activity dependent translocation of these mRNAs to sites near synapses, levels of the mRNA binding and dendritic transport protein CPEB are also increased by ChemLTP. Importantly, reducing the extent of substrate protein SUMOylation by overexpressing the deSUMOylating enzyme SENP-1 or inhibiting SUMOylation by expressing dominant negative Ubc9 prevent the ChemLTP-induced increase in both AMPAR surface expression and dendritic SUMO-1 mRNA. Taken together these data demonstrate that SUMOylation of synaptic protein(s) involved in AMPA receptor trafficking is necessary for activity-dependent increases in AMPAR surface expression. / Medical Research Council, the European Research Council and the Wellcome Trust

No description available.