Sporulation in Saccharomyces cerevisiae includes the processes of meiosis and spore formation. The genes involved in this developmental process are tightly regulated at the level of transcription to ensure that genes are expressed at the correct time and level. The co-ordinated expression of middle sporulation genes is mediated by a key timing promoter element called the middle sporulation element (MSE). While this element sets the timing of gene expression to middle sporulation, in some cases, the level of expression is mediated by cis-acting auxiliary promoter elements. This study has addressed the role that auxiliary transcription factors play in fine-tuning of timing and level of expression of the MSE-regulated middle sporulation genes SPS18 and SPS19 and the mid-late sporulation genes DIT1 and DIT2. The MSE*SPS18/19 was shown previously to set the timing of expression of SPS18 and SPS19 to middle sporulation. In order to achieve the full level of meiotic activation, a novel bipartite auxiliary promoter element called the MAE (MSE-associated element) was required (Dalton, 2004). This study has revealed that proteins bind to specific regions of the MAE motif during sporulation in vitro and has attempted to isolate the proteins by affinity chromatography and identify them by mass spectrometry. The timing of expression of DIT1 and DIT2 during sporulation was of particular interest since two MSE-like elements had been identified in the promoter of the these genes (Hepworth et al., 1995). If these MSEs were functional, it was thought that auxiliary elements may delay expression of these genes until mid-late sporulation. This study has shown that the MSE*NRE confers a normal middle sporulation pattern of expression on a reporter gene. The DRE (DIT repressor element) previously identified by Bogengruber et al. (1998) was further characterised as an element that alters the level of expression conferred by an MSE without altering the timing. Several proteins were shown to bind to specific regions of the DIT promoter surrounding the DRE motif in vitro, with a different set of proteins binding during vegetative growth and sporulation. Attempts to isolate and identify these proteins by affinity chromatography and mass spectrometry are discussed.

<p>The yeast Mediator complex is required for transcriptional regulation both in vivo and in vitro and the identification of similar complexes from metazoans indicates that its function is conserved through evolution. Mediator subunit composition and structure is well characterized both by biochemical, genetic and biophysical methods. In contrast, little is known about the mechanisms by which Mediator operates and how the complex is regulated. The aim of my thesis was to elucidate how Mediator functions at the molecular level and to investigate functional interactions within Mediator. </p><p> It is possible to recruit RNA polymerase II to a target promoter and thus to activate transcription by fusing Mediator subunits to a DNA binding domain. In order to investigate functional interactions within Mediator, we made such fusion proteins where different Mediator subunits were fused to the DNA binding domain of lexA. The expression of a reporter gene containing binding sites for lexA was subsequently measured in both a wild type strain and in strains where genes encoding specific Mediator subunits had been disrupted. We found that lexA-Med2 and lexA-Gal11 are strong activators that function independently of all Mediator subunits tested. On the other hand, lexA-Srb10 is a weak activator that depends on Srb8 and Srb11 and lexA-Med1 and lexA-Srb7 are both cryptic activators that become active in the absence of Srb8, Srb10, Srb11, or Sin4. Both lexA-Med1 and lexA-Srb7 proteins showed a stable association with the Mediator subunits Med4 and Med8 in wild type cells and in all deletion strains tested, indicating that they were functionally incorporated into the Mediator complex. We also showed that both Med4 and Med8 exist in two forms that differed in electrophoretic mobility and that these forms differed in their ability to associate with Mediator immuno-purified from the LEXA-SRB7 and LEXA-MED1 strains. Dephosphorylation assays of purified Mediator indicated that the two mobility forms of Med4 corresponded to the phosphorylated and unphosphorylated forms of the Med4 protein respectively. </p><p> Some of the data presented in this study as well as previous genetic and biochemical data obtained in our lab suggested a functional link between the Med1, Med2, Srb10 and Srb11 proteins. We extended these findings by showing that the Srb10 kinase phosphorylates the Med2 protein at residue serine 208, both in vitro and in vivo. We also showed that a point mutation of the single phosphorylation site to an alanine or to an aspartic acid residue altered the gene expression of a specific set of genes. Taken together, these data indicate that posttranslational modification of Mediator subunits is a so far uncharacterized mechanism for regulation of gene expression. </p><p> In order to study the function of the Srb7 subunit of Mediator, we isolated a temperature sensitive strain where the amino acids 2 to 8 of srb7 were deleted. The Mediator subunits Nut2 and Med7 were isolated as high copy suppressor of srb7-∆(2-8) and we were also able to show that Srb7 interacted with Nut2 and Med7 both in a 2-hybrid system and in co-immuno precipitation experiments using recombinantly expressed proteins. Interestingly, a deletion of amino acids 2 to 8 of Srb7 abolishes its interaction with both Med7 and Nut2 in vitro. Med4 also interacted with Srb7 in the 2-hybrid system and surprisingly, the first eight amino acids of Srb7 were shown to be sufficient for this interaction.</p>

The yeast Mediator complex is required for transcriptional regulation both in vivo and in vitro and the identification of similar complexes from metazoans indicates that its function is conserved through evolution. Mediator subunit composition and structure is well characterized both by biochemical, genetic and biophysical methods. In contrast, little is known about the mechanisms by which Mediator operates and how the complex is regulated. The aim of my thesis was to elucidate how Mediator functions at the molecular level and to investigate functional interactions within Mediator. It is possible to recruit RNA polymerase II to a target promoter and thus to activate transcription by fusing Mediator subunits to a DNA binding domain. In order to investigate functional interactions within Mediator, we made such fusion proteins where different Mediator subunits were fused to the DNA binding domain of lexA. The expression of a reporter gene containing binding sites for lexA was subsequently measured in both a wild type strain and in strains where genes encoding specific Mediator subunits had been disrupted. We found that lexA-Med2 and lexA-Gal11 are strong activators that function independently of all Mediator subunits tested. On the other hand, lexA-Srb10 is a weak activator that depends on Srb8 and Srb11 and lexA-Med1 and lexA-Srb7 are both cryptic activators that become active in the absence of Srb8, Srb10, Srb11, or Sin4. Both lexA-Med1 and lexA-Srb7 proteins showed a stable association with the Mediator subunits Med4 and Med8 in wild type cells and in all deletion strains tested, indicating that they were functionally incorporated into the Mediator complex. We also showed that both Med4 and Med8 exist in two forms that differed in electrophoretic mobility and that these forms differed in their ability to associate with Mediator immuno-purified from the LEXA-SRB7 and LEXA-MED1 strains. Dephosphorylation assays of purified Mediator indicated that the two mobility forms of Med4 corresponded to the phosphorylated and unphosphorylated forms of the Med4 protein respectively. Some of the data presented in this study as well as previous genetic and biochemical data obtained in our lab suggested a functional link between the Med1, Med2, Srb10 and Srb11 proteins. We extended these findings by showing that the Srb10 kinase phosphorylates the Med2 protein at residue serine 208, both in vitro and in vivo. We also showed that a point mutation of the single phosphorylation site to an alanine or to an aspartic acid residue altered the gene expression of a specific set of genes. Taken together, these data indicate that posttranslational modification of Mediator subunits is a so far uncharacterized mechanism for regulation of gene expression. In order to study the function of the Srb7 subunit of Mediator, we isolated a temperature sensitive strain where the amino acids 2 to 8 of srb7 were deleted. The Mediator subunits Nut2 and Med7 were isolated as high copy suppressor of srb7-∆(2-8) and we were also able to show that Srb7 interacted with Nut2 and Med7 both in a 2-hybrid system and in co-immuno precipitation experiments using recombinantly expressed proteins. Interestingly, a deletion of amino acids 2 to 8 of Srb7 abolishes its interaction with both Med7 and Nut2 in vitro. Med4 also interacted with Srb7 in the 2-hybrid system and surprisingly, the first eight amino acids of Srb7 were shown to be sufficient for this interaction.

<p>MicroRNAs are key post-transcriptional regulators that have been found to play critical roles in the regulation of cellular functions. There is an emerging concept that microRNAs may be just as essential for fine-tuning physiological functions and responding to changing environments and stress conditions as for viability or development. In this dissertation, two studies are presented: The first study demonstrates a role for microRNA in the regulation of oxidative stress response in erythroid cells and the functional consequences of dysregulated microRNA expression in Sickle Cell Disease (SCD) pathobiology. The second study examines a functional role for microRNA in the cellular response to changes in cellular iron concentration. Together these studies illustrate the scope of importance of microRNAs in the coordination of cellular responses to diverse stresses. </p><p>Homozygous Sickle Cell (HbSS) erythrocytes are known to have reduced tolerance for oxidative stress, yet the basis for this phenotype has remained unknown. Here we use erythrocyte microRNA expression profiles to identify a subset of HbSS patients with higher miR-144 expression and more severe anemia. We reveal that in K562 erythroid cells and primary erythroid progenitor cells, miR-144 directly regulates NRF2, a central regulator of cellular response to oxidative stress, and modulates the oxidative stress response. We further demonstrate that increased miR-144 is associated with the reduced NRF2 levels, decreased glutathione regeneration, and attenuated antioxidant capacity found in HbSS erythroid progenitors, thereby providing a mechanism for the reduced oxidative stress tolerance and increased anemia severity seen in HbSS patients. </p><p>The post-transcriptional regulation of the IRP2 regulon in the cellular response to iron deficiency is well characterized. Here we examine the potential role for microRNA-mediated regulation in the coordinated response to cellular iron deficiency.</p> / Dissertation

The mechanisms that govern the repression of 5-HT1A receptor gene expression mediated by a novel mental retardation gene, Freud-1, were examined in HEK293 and SKNSH cells. This study provides a possible mechanism of 5-HT1A receptor gene regulation by Freud-1, which, to mediate its action, recruits Swi/Snf and Sin3A/histone deacetylase (HDAC) complexes in non-neuronal HEK293 cells and Swi/Snf only in neuronal, 5-HT1A receptor-expressing SKNSH cells. Thus, Freud-1 has a dual mechanism of repression depending on cell type: HDAC dependent in HEK293 cells and HDAC independent in SKNSH cells. In addition, I present evidence that Freud-1 is not sumoylated at its consensus sumoylation sites and I present the lipid binding properties of Freud-1 and Freud-1 mutants.

How tumor cells migrate and metastasize from primary sites requires four major steps: invasion, intravasation, extravasation and proliferation from micrometastases to malignant tumor. The initiation of tumor cell invasion requires Epithelial-Mesenchymal Transition (EMT), by which tumor cells lose cell-cell interactions and gain the ability of migration. The gene expression profile during the EMT process has been extensively investigated to study the initiation of EMT. In our studies, we indicated that tyrosine phosphorylation of human p68 RNA helicase positively associated with the malignant status of tumor tissue or cells. Studying of this relationship revealed that p68 RNA helicase played a critical role in EMT progression by repression of E-cadherin as an epithelial marker and upregulation of Vimentin as a mesenchymal marker. Insight into the mechanism of how p68 RNA helicase represses E-cadherin expression indicated that p68 RNA helicase initiated EMT by transcriptional upregulation of Snail. Human p68 RNA helicase has been documented as an RNA-dependent ATPase. The protein is an essential factor in the pre-mRNA splicing procedure. Some examples show that p68 RNA helicase functions as a transcriptional coactivator in ATPase dependent or independent manner. Here we indicated that p68 RNA helicase unwound protein complexes to modulate protein-protein interactions by using protein-dependent ATPase activity. The phosphorylated p68 RNA helicase displaced HDAC1 from the chromatin remodeling MBD3:Mi2/NuRD complex at the Snail promoter. Thus, our data demonstrated an example of protein-dependent ATPase which modulates protein-protein interactions within the chromatin remodeling machine.

The mechanisms that govern the repression of 5-HT1A receptor gene expression mediated by a novel mental retardation gene, Freud-1, were examined in HEK293 and SKNSH cells. This study provides a possible mechanism of 5-HT1A receptor gene regulation by Freud-1, which, to mediate its action, recruits Swi/Snf and Sin3A/histone deacetylase (HDAC) complexes in non-neuronal HEK293 cells and Swi/Snf only in neuronal, 5-HT1A receptor-expressing SKNSH cells. Thus, Freud-1 has a dual mechanism of repression depending on cell type: HDAC dependent in HEK293 cells and HDAC independent in SKNSH cells. In addition, I present evidence that Freud-1 is not sumoylated at its consensus sumoylation sites and I present the lipid binding properties of Freud-1 and Freud-1 mutants.

<p>Post-transcriptional regulation plays an important role in governing various processes in all organisms. The development of the early embryo of <italic>Drosophila melanogaster</italic> is governed solely by post-transcriptional mechanisms; therefore, further insights into post-transcriptional regulation can be gained by studying the <italic>Drosophila </italic> embryo. This thesis addresses the actions of the translational repressor, Pumilio, in regulating two mRNAs during early embryogenesis. First, we examined the ability of Pumilio to regulate the mRNA stability of <italic>bicoid</italic>, a gene required for <italic>Drosophila </italic> head development. <italic>bicoid</italic> mRNA contains the canonical Pumilio recognition site, termed the Nanos response element (NRE), within the 3'UTR. Interestingly, we show that Pumilio binds to the NRE both in vitro and in vivo; however, no physiological significance is associated with this interaction. Furthermore, in <italic> pumilio</italic> mutant embryos <italic>bicoid</italic> mRNA stability and translation are unaltered, demonstrating that Pumilio does not regulate <italic>bicoid</italic> mRNA. Second, Pumilio has been shown to negatively regulate <italic>Cyclin B</italic>, the cyclin necessary for mitotic entry, in the somatic cytoplasm of the embryo and this repression is alleviated by the PNG Kinase complex through currently unidentified mechanisms. We further investigated the actions of Pumilio in regulating <italic>Cyclin B</italic> and discovered that the canonical partner of Pumilio, Nanos, is not involved in repressing somatic <italic>Cyclin B</italic>. Furthermore, we show that the 3'UTR of <italic>Cyclin B</italic> is not required for the regulation by Pumilio and the PNG Kinase complex. Lastly, through genetic analyses, we conclude that Pumilio may actually act upstream of the PNG Kinase complex to regulate <italic>Cyclin B</italic>.</p> / Dissertation

<p>The ability of iron (Fe) to easily transition between two valence states makes it a preferred co-factor for innumerable biochemical reactions, ranging from cellular energy production, to oxygen transport, to DNA synthesis and chromatin modification. While Fe is highly abundant on the crust of the earth, its insolubility at neutral pH limits its bioavailability. As a consequence, organisms have evolved sophisticated mechanisms of adaptation to conditions of scarce Fe availability. </p> <p>Studies in the baker's yeast Saccharomyces cerevisiae have shed light into the cellular mechanisms by which cells respond to limited Fe-availability. In response to Fe-deficiency, the transcription factors Aft1 and Aft2 activate a group of genes collectively known as the Fe-regulon. Genes in this group encode proteins involved in the high-affinity plasma membrane Fe-transport and siderophore uptake systems, as well as Fe-mobilization from intracellular stores and heme re-utilization. Concomitant with the up-regulation of the Fe-regulon, a large number of mRNAs encoding Fe-dependent proteins as well as proteins involved in many Fe-dependent processes are markedly down regulated. Thus, in response to low Fe-levels the cell activates the Fe-uptake and mobilization systems, while down-regulating mRNAs involved in highly Fe-demanding processes leading to a genome-wide remodeling of cellular metabolism that permits the funneling of the limiting Fe to essential Fe-dependent reactions. </p> <p>The Fe-regulon member Cth2 belongs to a family of mRNA-binding proteins characterized by an RNA-binding motif consisting of two tandem zinc-fingers of the CX8CX5CX3H type. Members of this family recognize and bind specific AU-rich elements (AREs) located in the 3'untranslated region (3'UTRs) of select groups of mRNAs, thereby promoting their rapid degradation. In response to Fe-limitation, Cth2 binds ARE sequences within the 3'UTRs of many mRNAs encoding proteins involved in Fe-homeostasis and Fe-dependent processes, thereby accelerating their rate of decay. </p> <p>Work described in this dissertation demonstrates that the Cth2 homolog, Cth1, is a bona fide member of the Fe-regulon, binds ARE-sequences within the 3'UTRs of select mRNAs and promotes their decay. Cth1 and Cth2 appear to be only partially redundant; Cth1 preferentially targets mRNAs encoding mitochondrial proteins, while Cth2 promotes the degradation of most of Cth1 targets in addition to other mitochondrial and non-mitochondrial Fe-requiring processes. The coordinated activity of Cth1 and Cth2 results in dramatic changes in glucose metabolism. In addition, experiments described in this dissertation indicate that the CTH1 and CTH2 transcripts are themselves subject to ARE-mediated regulation by the Cth1 and Cth2 proteins, creating an auto- and trans-regulatory circuit responsible for differences in their expression. Finally, work described here demonstrates that Cth2 is a nucleocytoplasmic shuttling protein and that shuttling is important for the early determination of cytosolic mRNA-fate.</p> / Dissertation

Galectins are a family of secreted animal lectins with a high affinity to betagalactosides commonly involved in cellular functions such as apoptosis, adhesion and migration. Galectin 15 (LGALS15), a newest member of the galectin superfamily, has a unique C-terminal RGD sequence and participates in integrin-mediated ovine trophectoderm cell attachment and migration. In the ovine uterus, LGALS15 is expressed only by the endometrial luminal (LE) and superficial glandular (sGE) epithelia, induced by progesterone between Days 10 and 12 of the cycle and pregnancy, and then stimulated by interferon tau (IFNT) from the conceptus after Day 14 of pregnancy. During early pregnancy, the canonical janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway is not active in the endometrial LE/sGE. Therefore, IFNT may utilizes a non-canonical signaling pathway to increase transcription of genes, including CST3, CTSL, HIF2A, LGALS15, and WNT7A, specifically in the endometrial LE/sGE. Alternatively, IFNT and progesterone could indirectly affect epithelial gene expression by influencing gene expression in the stroma, which then communicates with the epithelium. Although the LGALS15 gene is present in ovine, caprine and bovine species, it is only expressed in uteri of sheep and goats. Available data shows a tissue- and speciesspecific expression pattern for LGALS15, likely involving multiple layers of transcription regulation in the ruminant endometrium. Further analysis of the LGALS15 5? promoter/enhancer region revealed similar predicted transcription factor binding sites in all three species, including; PU.1, Ets-1, AP1, Sp1, and GRE or PRE sites. Interestingly, the proximal promoter region of the LGALS15 gene in all three species exhibited a conserved Sp1 binding site upstream of an AP1 binding site on both sense and antisense strands, and with similar spacing between binding sites. Sequence analysis revealed key differences in LGALS15 gene structure between ruminant species including the proximity of repetitive DNA sequences to the transcription start site (+1). Bovine LGALS15 has repetitive DNA sequences start at - 145 whereas in ovine or caprine LGALS15 it starts at about -300. The length of the repetitive DNA sequence is similar (~1.2 kb) in the 5' promoter/enhancer region of LGALS15 in all three species. Transient transfection analyses found that repetitive DNA sequences reduced basal promoter activity and responsiveness to treatments. None of the promoter construct showed responsiveness to interferon tau (IFNT). The bovine LGALS15 gene promoter showed no activity under any experimental conditions. The current studies indicate that uterine LGALS15 is expressed in ovine and caprine but not bovine species. Additionally, repetitive DNA sequences found in the promoter region may contribute to modulating the LGALS15 gene expression. Therefore, the ruminant LGALS15 gene, like other galectins, is under tight transcriptional control involving hormones, requisite transcription factors and potentially chromatin remodeling complexes working synergistically for LGALS15 promoter transactivation.