A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine.

No description available.

The trophoblast transcription factor glial cell missing-1 (GCM1) regulates asymmetric division of placental cytotrophoblast to form the differentiated syncytiotrophoblast. Reduced GCM1 expression is a key feature of the hypertensive disorder preeclampsia. In-silico techniques identified a novel calcium-dependent transcriptional repressor – DREAM as a regulatory candidate for GCM1. The overall objective of this thesis was to determine if DREAM regulates GCM1 expression and therefore villous trophoblast turnover. siRNA-mediated DREAM silencing in both BeWo cells and floating villous explants significantly upregulated GCM1 causing reduced cytotrophoblast proliferation. Calcium-dependency was demonstrated in both BeWo cells and floating villous explants by contrasting the effects of ionomycin and nimodipine. A direct interaction between DREAM and the GCM1 promoter was demonstrated using EMSA and ChIP assay. DREAM is a negative upstream regulator of GCM1 expression in human placenta that participates in calcium-dependent trophoblast differentiation.

The trophoblast transcription factor glial cell missing-1 (GCM1) regulates asymmetric division of placental cytotrophoblast to form the differentiated syncytiotrophoblast. Reduced GCM1 expression is a key feature of the hypertensive disorder preeclampsia. In-silico techniques identified a novel calcium-dependent transcriptional repressor – DREAM as a regulatory candidate for GCM1. The overall objective of this thesis was to determine if DREAM regulates GCM1 expression and therefore villous trophoblast turnover. siRNA-mediated DREAM silencing in both BeWo cells and floating villous explants significantly upregulated GCM1 causing reduced cytotrophoblast proliferation. Calcium-dependency was demonstrated in both BeWo cells and floating villous explants by contrasting the effects of ionomycin and nimodipine. A direct interaction between DREAM and the GCM1 promoter was demonstrated using EMSA and ChIP assay. DREAM is a negative upstream regulator of GCM1 expression in human placenta that participates in calcium-dependent trophoblast differentiation.

Vts1p is the Saccharomyces cerevisiae member of the Smaug family of post-transcriptional regulators, which is a group of sequence-specific RNA-binding proteins that regulate target mRNA expression. Vts1p is known to mediate deadenylation-dependent degradation of target transcripts through the recruitment of the Ccr4p/Pop2p/Not deadenylase complex. By conducting a functional analysis of Vts1p deletion mutants, I demonstrate that two regions within Vts1p are independently capable of downregulating the expression of an mRNA reporter. I provide both genetic and biochemical evidence that suggests residues 170-523 regulate reporter expression at the level of mRNA stability and function through a mechanism that requires the Ccr4p/Pop2p/Not deadenylase, whereas residues 1-237 repress reporter expression at the level of translation and function through a novel mechanism. In addition, I map a direct interaction between the eIF4E-binding protein, Eap1p, and the Vts1p SAM domain, which suggests a model in which residues 170-523 recruit Eap1p to mediate efficient target transcript degradation.

Vts1p is the Saccharomyces cerevisiae member of the Smaug family of post-transcriptional regulators, which is a group of sequence-specific RNA-binding proteins that regulate target mRNA expression. Vts1p is known to mediate deadenylation-dependent degradation of target transcripts through the recruitment of the Ccr4p/Pop2p/Not deadenylase complex. By conducting a functional analysis of Vts1p deletion mutants, I demonstrate that two regions within Vts1p are independently capable of downregulating the expression of an mRNA reporter. I provide both genetic and biochemical evidence that suggests residues 170-523 regulate reporter expression at the level of mRNA stability and function through a mechanism that requires the Ccr4p/Pop2p/Not deadenylase, whereas residues 1-237 repress reporter expression at the level of translation and function through a novel mechanism. In addition, I map a direct interaction between the eIF4E-binding protein, Eap1p, and the Vts1p SAM domain, which suggests a model in which residues 170-523 recruit Eap1p to mediate efficient target transcript degradation.

The localization and translation of messenger ribonucleic acids (mRNAs) play crucial roles in cellular function and diseases, and are regulated by numerous RNA-binding proteins (RBPs) and small non-coding RNAs, called trans-acting factors. Biochemical and imaging methods used to study RNA interactions with these trans-acting elements have made important discoveries in characterizing how these factors regulate gene expression and determining the RNA sequence to which they bind. However, the spatiotemporal information regarding these interactions in subcellular compartments have been difficult to determine or to quantify accurately. To image and quantify native RNA and RNA–protein interactions simultaneously in situ, we developed a proximity ligation assay that combines peptide-modified RNA imaging probes. It can detect the RNAs in live cells and the interactions at a single-interaction level. Lastly, it can produce results that are easily quantifiable. We tested the specificity and sensitivity of this technique using two models: interactions between the genomic RNA and the N protein of human respiratory syncytial virus as well as those between exogenous transcripts with or without the Human antigen R (HuR) binding site and HuR. To validate this method, its accuracy and utility have been demonstrated in three models: poly(A)+ or β-actin mRNAs binding to different cytoskeleton for localization, poly(A)+ or β-actin mRNAs interacting with HuR for stabilization, and programmed cell death 4 (PDCD4) mRNA binding to HuR or T-cell intracellular antigen (TIA1) for translational regulation.

<p>Post-transcriptional regulation of cytoplasmic mRNAs is an efficient mechanism of regulating the amounts of active protein within a eukaryotic cell. RNA sequence elements located in the untranslated regions of mRNAs can influence transcript degradation or translation through associations with RNA-binding proteins. Tristetraprolin (TTP) is the best known member of a family of CCCH zinc finger proteins that targets adenosine-uridine rich element (ARE) binding sites in the 3’ untranslated regions (UTRs) of mRNAs, promoting transcript deadenylation through the recruitment of deadenylases. More specifically, TTP has been shown to bind AREs located in the 3’-UTRs of transcripts with known roles in the inflammatory response. The mRNA-binding region of the protein is the highly conserved CCCH tandem zinc finger (TZF) domain. The synthetic TTP TZF domain has been shown to bind with high affinity to the 13-mer sequence of UUUUAUUUAUUUU. However, the binding affinities of full-length TTP family members to the same sequence and its variants are unknown. Furthermore, the distance needed between two overlapping or neighboring UUAUUUAUU 9-mers for tandem binding events of a full-length TTP family member to a target transcript has not been explored. To address these questions, we recombinantly expressed and purified the full-length C. albicans TTP family member Zfs1. Using full-length Zfs1, tagged at the N-terminus with maltose binding protein (MBP), we determined the binding affinities of the protein to the optimal TTP binding sequence, UUAUUUAUU. Fluorescence anisotropy experiments determined that the binding affinities of MBP-Zfs1 to non-canonical AREs were influenced by ionic buffer strength, suggesting that transcript selectivity may be affected by intracellular conditions. Furthermore, electrophoretic mobility shift assays (EMSAs) revealed that separation of two core AUUUA sequences by two uridines is sufficient for tandem binding of MBP-Zfs1. Finally, we found evidence for tandem binding of MBP-Zfs1 to a 27-base RNA oligonucleotide containing only a single ARE-binding site, and showed that this was concentration and RNA length dependent; this phenomenon had not been seen previously. These data suggest that the association of the TTP TZF domain and the TZF domains of other species, to ARE-binding sites is highly conserved. Domains outside of the TZF domain may mediate transcript selectivity in changing cellular conditions, and promote protein-RNA interactions not associated with the ARE-binding TZF domain. </p><p>In summary, the evidence presented here suggests that Zfs1-mediated decay of mRNA targets may require additional interactions, in addition to ARE-TZF domain associations, to promote transcript destabilization and degradation. These studies further our understanding of post-transcriptional steps in gene regulation.</p> / Dissertation

The Mediator complex was originally discovered in the yeast Saccharomyces cerevisiae and has since then been shown to be required for transcriptional regulation both in vitro and in vivo. The Mediator complex also stimulates basal, unregulated transcription and serves as a bridge by conveying signals from promoter-bound transcriptional regulatory proteins such as activators and repressors to the RNA Polymerase II general transcriptional machinery. The Mediator consists of 21 subunits and can be divided into three distinct modules head, middle and tail. Despite the tremendous progress that has been achieved so far in characterizing the Mediator complex both functionally and structurally, many aspects of the complex are not yet well understood. The objective of this work is to achieve further understanding of the Mediator complex by studying the folding of different protein subunits, their interactions and how that affects assembly of the Mediator complex. In our first study we made a temperature-sensitive med21 mutant and used it to identify genes that can suppress the mutation when present in high copy number. Among the 10 genes that we identified, the strongest suppressors were Med7 and Med10, which encode Mediator subunits, and Ash1, which encodes a repressor of the HO gene. We also used 2-hybrid experiments and immunoprecipitation to study protein-protein interactions between Med21 and the Med4, Med7 and Med10 proteins which are all essential for viability and located within the middle domain of the Mediator complex. We found that the N-terminal 2-8 amino acids of Med21 are required for interactions with Med7 and Med10. These results led us to propose a model in which the N-terminal part of Med21 functions as a molecular switchboard where competing signals from various activators, repressors and mediator subunits are integrated prior to reaching the general transcription machinery. In our second study, we extended our studies of protein-protein interactions to another part of the mediator complex by studying the folding and the assembly processes of the mediator head domain subunits Med8, Med18 and Med20. Using purified proteins and a combination of several different methods such as immunoprecipitation, far-UV circular dichroism and fluorescence, we demonstrated that the Med8, Med18 and Med20 subunits are interdependent on each other for proper folding and complex formation.

Versican, a chondroitin sulfate proteoglycan, is one of the main components of the extracellular matrix and hence plays a central role in tissue morphogenesis and a number of pathologic processes. My main goal has been to investigate the mechanisms of versican gene regulation, focusing on the signal transduction pathways, promoter regions, cis-acting elements,and trans- factors. This thesis puts forth new knowledge regarding transcriptional regulation of the human versican gene. In chapter III, I present the cloning of a 752-bp fragment of the human versican promoter (- 634/+118 bp) and nine stepwise 5' deletion fragments in the PGL3-luciferase reporter plasmid. Furthermore, I identify three potential enhancer and two repressor regions in this promoter. I also demonstrate that both cAMP and C/EBPf3 enhanced and repressed versican transcription in HeLa cells and rat aortic smooth muscle cells (SMC),respectively, suggesting that versican transcription is differentially regulated by the respective mediator and transcription factor in epithelial cells and SMC. In chapter IV, I reveal the role ofPI3K/PKB/GSK-30 signaling pathway in regulating versican promoter activity and transcription. Furthermore, I identify that the 0-catenin/TCF-4 transcription factor complex, one of the downstream targets of GSK-3[3, mediates versican promoter activity and transcription. In chapter V, I identify that variations in C-terminal regions of TCF family members determine the irrepressor or enhancer properties on Wnt target genes. Furthermore, I show that curcumin is a strong inhibitor of the P-catenin/TCF-p300 mediated gene expression. In chapter VI, I demonstrate that the androgen receptor trans-activates versican transcription in prostate cancer cells. Furthermore, I show cross-talk between the androgen receptor and 13-catenin in regulating versican transcription in prostate stromal fibroblasts. Overall, this study charts previously uncharacterized promoter elements, transcription factors, and signal transduction pathways involved in regulation of the versican gene.