Eukaryotic cytosolic ribosomes are composed of two distinct subunits consisting of four individual ribosomal RNAs and, in Arabidopsis thaliana, 81 ribosomal proteins. Functional subunit assembly is dependent on the production of each ribosomal component. Arabidopsis thaliana r-protein genes exist in multi-gene families ranging in size from two to seven transcriptionally active members. The cytosolic RPS15a gene family consists of four members (RPS15aA, -C, -D and -F) that, at the amino acid level, share 87-100% identity. Using semi-quantitative RT-PCR I have shown that RPS15aC is not expressed and that transcript abundance differs both spatially and temporally among the remaining RPS15a genes in non-treated Arabidopsis tissues and in seedlings following a variety of abiotic stresses. A comprehensive analysis of the RPS15a 5' regulatory regions (RRs) using a series of deletion constructs was used to determine the minimal region required for gene expression and identify putative cis-regulatory elements. Transcription start site mapping using 5' RACE indicated multiple sites of initiation for RPS15aA and -F and only a single site for RPS15aD while all three genes contain a leader intron upstream of the start codon. Analysis of reporter gene activity in transgenic Arabidopsis containing a series of 5' RR deletion::GUS fusions showed that, similar to previous RT-PCR results, there was a trend for mitotically active tissues to stain for GUS activity. Putative cis-elements including the TELO box, PCNA Site II motif and pollen specific elements were identified. However, there was not always a clear correlation between the presence of a putative element and RPS15a transcript abundance or GUS activity. Although variation in transcriptional activity of each RPS15a gene has been observed, subcellular localization of both RPS15aA and -D in the nucleolus has been confirmed in planta by confocal microscopy. The results of this thesis research suggest while all three active RPS15a genes are transcriptionally regulated, additional post-transcriptional and/or translational regulation may be responsible for final RPS15a levels while differential isoform incorporation into ribosomal subunits may be the final point of r-protein regulation.

Ribosomal proteins (RPs), synthesized in the cytoplasm, need to be transported from the cytoplasm to the nucleolus (a nuclear compartment), where a single molecule of each RP assembles with rRNAs to form the large and small ribosomal subunits. The objectives of this research were to identify nuclear/nucleolar localization signals (NLSs/NoLSs; generally basic motifs) that mediate the transport of Arabidopsis RPL23aA, RPL15A and RPS8A into the nucleus and nucleolus, and to study transcriptional regulation and subcellular localization of RPs. While all previous research has shown that nucleolar localization of proteins is mediated by specific basic motifs, in this study, I showed that a specific number of basic motifs mediated nucleolar localization of RPL23aA, rather than any specific motifs. In this protein, single mutations of any of its eight putative NLSs (pNLSs) had no effect on nucleolar localization, however, the simultaneous mutation of all eight completely disrupted nucleolar localization, but had no effect on nuclear localization. Furthermore, mutation of any four of these pNLSs had no effect on localization, while mutation of more than four increasingly disrupted nucleolar localization, suggesting that any combination of four of the eight pNLSs is able to mediate nucleolar localization. These results support a charge-based system for the nucleolar localization of RPL23aA. While none of the eight pNLSs of RPL23aA were required for nuclear localization, in RPS8A and RPL15A, of the 10 pNLSs in each, the N-terminal two and three NLSs, respectively, were absolutely required for nuclear/nucleolar localization. Considering the presence of only a single molecule of each RP in any given ribosome, which obligates the presence of each RP in the nucleolus in equal quantities, I studied transcriptional regulation of Arabidopsis RP genes and the subcellular localization of five RP families to determine the extent of coordinated regulation of these processes. Variation of up to 300-fold was observed in the expression levels of RP genes. However, this variation was drastically reduced when the expression level was considered at the RP gene family level, indicating that coordinate regulation of expression of RP genes, coding for individual RP isoforms, is more stringent at the family level. Subcellular localization also showed differential targeting of RPs to the cytoplasm, nucleus and nucleolus, together with a significant difference in the nucleolar import rates of RPS8A and RPL15A. Although one could expect coordinated regulation of the processes preceding ribosomal subunit assembly in the nucleolus, my results suggest differential regulation of these processes.

Eukaryotic cytosolic ribosomes are composed of two distinct subunits consisting of four individual ribosomal RNAs and, in Arabidopsis thaliana, 81 ribosomal proteins. Functional subunit assembly is dependent on the production of each ribosomal component. Arabidopsis thaliana r-protein genes exist in multi-gene families ranging in size from two to seven transcriptionally active members. The cytosolic RPS15a gene family consists of four members (RPS15aA, -C, -D and -F) that, at the amino acid level, share 87-100% identity. Using semi-quantitative RT-PCR I have shown that RPS15aC is not expressed and that transcript abundance differs both spatially and temporally among the remaining RPS15a genes in non-treated Arabidopsis tissues and in seedlings following a variety of abiotic stresses. A comprehensive analysis of the RPS15a 5' regulatory regions (RRs) using a series of deletion constructs was used to determine the minimal region required for gene expression and identify putative cis-regulatory elements. Transcription start site mapping using 5' RACE indicated multiple sites of initiation for RPS15aA and -F and only a single site for RPS15aD while all three genes contain a leader intron upstream of the start codon. Analysis of reporter gene activity in transgenic Arabidopsis containing a series of 5' RR deletion::GUS fusions showed that, similar to previous RT-PCR results, there was a trend for mitotically active tissues to stain for GUS activity. Putative cis-elements including the TELO box, PCNA Site II motif and pollen specific elements were identified. However, there was not always a clear correlation between the presence of a putative element and RPS15a transcript abundance or GUS activity. Although variation in transcriptional activity of each RPS15a gene has been observed, subcellular localization of both RPS15aA and -D in the nucleolus has been confirmed in planta by confocal microscopy. The results of this thesis research suggest while all three active RPS15a genes are transcriptionally regulated, additional post-transcriptional and/or translational regulation may be responsible for final RPS15a levels while differential isoform incorporation into ribosomal subunits may be the final point of r-protein regulation.

Ribosomal proteins (RPs), synthesized in the cytoplasm, need to be transported from the cytoplasm to the nucleolus (a nuclear compartment), where a single molecule of each RP assembles with rRNAs to form the large and small ribosomal subunits. The objectives of this research were to identify nuclear/nucleolar localization signals (NLSs/NoLSs; generally basic motifs) that mediate the transport of Arabidopsis RPL23aA, RPL15A and RPS8A into the nucleus and nucleolus, and to study transcriptional regulation and subcellular localization of RPs. While all previous research has shown that nucleolar localization of proteins is mediated by specific basic motifs, in this study, I showed that a specific number of basic motifs mediated nucleolar localization of RPL23aA, rather than any specific motifs. In this protein, single mutations of any of its eight putative NLSs (pNLSs) had no effect on nucleolar localization, however, the simultaneous mutation of all eight completely disrupted nucleolar localization, but had no effect on nuclear localization. Furthermore, mutation of any four of these pNLSs had no effect on localization, while mutation of more than four increasingly disrupted nucleolar localization, suggesting that any combination of four of the eight pNLSs is able to mediate nucleolar localization. These results support a charge-based system for the nucleolar localization of RPL23aA. While none of the eight pNLSs of RPL23aA were required for nuclear localization, in RPS8A and RPL15A, of the 10 pNLSs in each, the N-terminal two and three NLSs, respectively, were absolutely required for nuclear/nucleolar localization. Considering the presence of only a single molecule of each RP in any given ribosome, which obligates the presence of each RP in the nucleolus in equal quantities, I studied transcriptional regulation of Arabidopsis RP genes and the subcellular localization of five RP families to determine the extent of coordinated regulation of these processes. Variation of up to 300-fold was observed in the expression levels of RP genes. However, this variation was drastically reduced when the expression level was considered at the RP gene family level, indicating that coordinate regulation of expression of RP genes, coding for individual RP isoforms, is more stringent at the family level. Subcellular localization also showed differential targeting of RPs to the cytoplasm, nucleus and nucleolus, together with a significant difference in the nucleolar import rates of RPS8A and RPL15A. Although one could expect coordinated regulation of the processes preceding ribosomal subunit assembly in the nucleolus, my results suggest differential regulation of these processes.

We previously described BT2, a BTB/POZ domain containing protein, as an activator of telomerase in Arabidopsis thaliana. In the current study, I present evidence of its interesting roles in mediating multiple hormone, stress and metabolic responses in plants. Steady-state expression of BT2 mRNA was regulated diurnally and was under the control of circadian clock, with a maximum expression in the dark. BT2 mRNA was responsive to nutrient status and to multiple biotic and abiotic stress signals. Using bt2 loss-of-function and BT2 over-expressing lines, I show that BT2 suppresses sugar and ABA-mediated responses during germination. BT2 is also essential for transcriptional gene activation mediated by CaMV 35S enhancers in Arabidopsis. Loss of BT2 in several well-characterized 35S enhancer activation-tagged lines such as yucca1d, pap1d, jaw1d etc., resulted in suppression of the activation phenotypes. The suppression of the phenotypes was due to decreased transcription of the activation-tagged genes. I further demonstrate that BT2 genetically interacts with CULLIN3. I propose that BT2 and CULLIN3 are components of a ubiquitin ligase complex. Together with associated proteins BET9 and BET10, the BT2 complex is required for CaMV 35S enhancer-mediated activation of gene expression and may regulate expression of target genes involved in multiple responses to fluctuating biotic and abiotic conditions. I also found that BT2 protein levels are tightly regulated in plants. BT2 protein was primarily localized in the nucleus and was developmentally regulated. BT2 turn-over was regulated in part by the 26S-proteosome, and rare codons present in its open reading frame affected BT2 protein accumulation. In addition to BT2, its orthologs, BT1, BT3, BT4 and BT5, also responded to light, clock and nutrients, with some differences. Moreover, BT1, BT3 and BT4 were also required for 35S enhancer-mediated activation of gene expression. I propose that BT family proteins assemble into multi-protein complexes to mediate multiple responses to changing environmental and nutritional conditions.

Receptors that transport vitellogenin into oocytes are of vital importance to egg-laying species because they promote oocyte development. In this study, we describe the cloning of the first hymenopteran vitellogenin receptor (VgR) cDNA. Using reverse transcription polymerase chain reaction (RT-PCR) and both 5’- and 3’- rapid amplification of cDNA ends (RACE), cDNA fragments encompassing the entire coding region of a putative VgR from fire ant (= SiVgR) were cloned and sequenced. The complete SiVgR cDNA has a length of 5764 bp encoding a 1782-residue protein with a predicted molecular mass of 201.3 kDa. The deduced amino acid sequence of the SiVgR revealed that it encoded a protein belonging to the low-density lipoprotein receptor superfamily. The number and arrangement of modular domains of SiVgR are the same as those of mosquito and fruit fly VgRs, except there are only four Class A cysteine-rich repeats in the first ligand binding domain of SiVgR compared to five in the mosquito and fruit fly. The deduced amino acid sequence of the SiVgR exhibited 35% and 31% identity to those of the mosquito and fruit fly VgRs, respectively. Northern blot analysis demonstrated that the 7.4-kb SiVgR mRNA was present only in Northern blot analysis demonstrated that the 7.4-kb SiVgR mRNA was present only in ovaries of reproductive females − both alates (virgins) and queens (mated) and was more abundant in alates. The developmental profile of transcriptional expression was determined by semiquantitative RT-PCR. It showed that the SiVgR transcript increased 6-fold from 0- to 10-days after mating, then remained constant through 30 days. It also showed that the SiVgR transcripts increased with age in alate virgin females. The transcriptional expression of the SiVgR was up-regulated more than two-fold by methoprene, a juvenile hormone analog, as determined by using an in vitro system. This suggested the SiVgR gene is JH regulated.

The cyclic nucleotide monophosphates (CNs), cAMP and cGMP, are second messengers that participate in the regulation of development, metabolism and adaptive responses. In plants, CNs are associated with the control of pathogen responses, pollen tube orientation, abiotic stress response, membrane transport regulation, stomatal movement and light perception. In this study, we hypothesize that cAMP and cGMP promote changes in the transcription level of genes related to photosynthesis, high light and membrane transport in Arabidopsis thaliana leaves and, that these changes at the molecular level can have functional biological consequences. For this reason we tested if CNs modulate the photosynthetic rate, responses to high light and root ion transport. Real time quantitative PCR was used to assess transcription levels of selected genes and infrared gas analyzers coupled to fluorescence sensors were used to measure the photosynthetic parameters. We present evidence that both cAMP and cGMP modulate foliar mRNA levels early after stimulation. The two CNs trigger different responses indicating that the signals have specificity. A comparison of proteomic and transcriptional changes suggest that both transcriptional and post-transcriptional mechanisms are modulated by CNs. cGMP up-regulates the mRNA levels of components of the photosynthesis and carbon metabolism. However, neither cAMP nor cGMP trigger differences in the rate of carbon assimilation, maximum efficiency of the photosystem II (PSII), or PSII operating efficiency. It was also demonstrated that CN regulate the expression of its own targets, the cyclic nucleotide gated channels - CNGC. Further studies are needed to identify the components of the signaling transduction pathway that mediate cellular changes and their respective regulatory and/or signaling roles.

For cancer cells to initiate cell migration and progress to metastasize, epithelial genes must be silenced and the expression of mesenchymal genes must be upregulated. During prostate carcinogenesis, E-cadherin expression is downregulated through multiple mechanisms, the majority of which combine to silence E-cadherin expression through transcriptional regulation at the level of the E-cadherin promoter. Recently it has been discovered that there is transcriptional upregulation of the mesenchymal cadherin, N-cadherin during prostate cancer metastasis. Although N-cadherin expression can be detected in human prostate cancer and in prostate carcinoma cell lines, the mechanisms controlling the transcriptional regulation of N-cadherin in cancer are uncharacterized. This body of work offers the first evidence for the mechanisms controlling the transcriptional upregulation of N-cadherin expression in prostate carcinoma. We utilized anchorage independent culture to induce downregulation of N-cadherin expression, and then analyzed the necessary events for N-cadherin upregulation when cells attached to Fibronetin (FN). In order to determine the functional regions of the N-cadherin proximal promoter that were involved in the upregulation of N-cadherin expression, we cloned regions of the human N-cadherin 5’ proximal promoter, and regions of the first intron of the N-cadherin gene into a luciferase reporter vector. It was determined that the bHLH transcription factor Twist1 controlled the upregulation of N-cadherin transcription in PC-3 cells, through β1 integrin dependent nuclear localization of Twist1. A cis-element located in the first intron of the N-cadherin gene was shown to be necessary for Twist1 mediated effects on the N-cadherin promoter. We then determined the requirements for cell-type specific expression of the N-cadherin promoter. It was determined that an additional cis-element located in the first intron of the N-cadherin gene was necessary to repress N-cadherin promoter activity in cells lacking N-cadherin. Through deletion analysis of the N-cadherin promoter luciferase construct, a DNA binding site for the transcription factor FoxP1 was discovered. FoxP1 binds to the repressive cis-element in vitro, and mutation of the FoxP1 DNA binding site eliminated cell-type specific activity of the N-cadherin promoter. Therefore, we have documented that the aberrant expression of N-cadherin in prostate carcinoma involves alterations in both positive and negative transcriptional regulators.

<p><italic>Cryptococcus neoformans </italic>is a human fungal pathogen that is also ubiquitous in the environment. To cause disease inside a human host, <italic>C. neoformans</italic> must be able to sense and respond to a multitude of stresses. One of the major responses to the host is the induction of a polysaccharide capsule, which allows the fungus to resist damage and evade the host immune response. This capsule is regulated by a number of signal transduction cascades, but a major contributor is the conserved cAMP/PKA pathway. </p><p> Using genetic and molecular biology techniques, I identified Gcn5 and Rim101 as key transcriptional regulators of capsule within the host. I determined that <italic>C. neoformans</italic> Rim101 is activated by a combination of the canonical pH sensing pathway and the cAMP/PKA pathway. This novel connection potentially gives the pathogen greater flexibility in responding to environmental stimuli, thus allowing for a greater capacity for disease. </p><p> I determined that the Rim101 transcription factor regulates cell wall remodeling in the context of the host by deep mRNA sequencing, electron microscopy, and biochemical assays. Using chromatin immunoprecipitation, I confirmed that these cell wall changes are under direct control of Rim101. I then confirmed the importance of cell wall changes in the host by nanoString profiling of fungal RNA in the context of a murine lung infection. I also examined the lungs of infected mice for cytokine and immune cell infiltrate and determined that <italic>C. neoformans</italic> cell wall changes are important in avoiding triggering an aberrant host response. I hypothesize that this cell wall remodeling via Rim101 activation is required for full capsule attachment and for masking immunogenic molecules from the host immune system.</p> / Dissertation

The Drosophila hindsight (hnt) gene encodes a C2H2-type zinc-finger (ZNF) protein crucial for epithelial morphogenesis. The human HNT homologue, RREB1, functions as a transcriptional modulator and regulates several tumor suppressor genes. HNT’s functional motifs, target genes and its regulatory abilities have not been elucidated. Here I showed that the C-terminal region of HNT containing the last five of 14 ZNFs (ZNF 10-14) binds in vitro to DNA-elements similar to those identified for RREB-1. I then mapped HNT’s endogenous binding sites on polytene chromosomes and focus on two, at 4C and 60C, which are associated with the hnt and nervy (nvy) loci, respectively. Sequence analysis of the bound fragments shows conservation of motifs similar to those bound by HNT in vitro. Data from both hnt loss- and gain-of-function experiments show that HNT attenuates the transcription of the hnt and nvy genes in several tissues and developmental stages. I show that the identified HNT C-terminal DNA binding domain ZNF 10-14 is not required for these regulatory functions. I further delineate the minimal functional motifs of HNT in transcriptional regulation and show that its ninth ZNF in isolation has a repressive activity and is sufficient to confer many regulatory functions of HNT. On the other hand, mutation of ZNF 9 in the context of the full-length protein indicates that it is not necessary for HNT functions. Interestingly, ZNF 9 has been lost from HNT vertebrate homologues. I propose two redundant mechanisms of transcriptional regulation by HNT: one is mediated by the potential protein-interaction abilities of ZNF 9; another is through cooperation of other ZNF motifs of HNT; the DNA binding abilities conferred by the C-terminal five fingers may be essential for the latter mechanism.