Although advances have been made toward understanding the complex mechanisms that regulate the process of DNA transcription, the specific mechanisms of activation for many individual genes remain unknown. In this study, we focus on the role the transcription factor SOX9 plays in activating cartilage-specific genes, specifically Col9a1 and Cartilage Link Protein (CRTL1). Previously, enhancers of these genes containing single SOX9 binding sites were shown to be activated through SOX9 binding. However, the hypothesis was made that in cartilage-specific genes dimeric SOX9, as opposed to monomeric SOX9, is necessary for activation. We identified a putative binding site adjacent to each of the known single SOX9 binding sites in the Col9a1 D and E enhancers and in the CRTL1 enhancer. Electrophoretic Mobility Shift Assays (EMSAs) were performed to determine whether SOX9 bound to these putative sites. Transient transfections were then performed using wild-type and mutant enhancer- reporter plasmids to determine whether these putative SOX9 binding sites were important for activation in vivo. Although dimeric SOX9 bound to each of the enhancers in vitro, several different effects were seen in vivo. In the presence of the wild-type Col9a1 D enhancer, no activation was seen. However, when the enhancer was extended to include an additional pair of newly found SOX9 binding sites, expression was increased 10-fold. When any of the four SOX9 binding sites within this enhancer were mutated, expression was completely eliminated, suggesting that interdependent dimers or a tetramer of SOX9 is necessary for the activation of transcription. The weaker Col9a1 enhancer E was found to increase gene expression minimally through binding of either dimeric or monomeric SOX9. However, dimeric SOX9 was required for the activation of gene expression by the CTRL1 enhancer. Through this study we validate the importance of not just monomeric but of dimeric and possibly tetremeric SOX9 as an activator of cartilage-specific gene expression.

No description available.

Controlling when genes are expressed is critical for the growth of an organism. Studying gene expression regulation in Streptomyces presents an opportunity to better understand how these complex bacteria develop and how they control their impressive biosynthetic capabilities. In this work we investigated the potential role of a G-quadruplex binding protein, and two ribonucleases (RNases) in regulating gene expression in Streptomyces venezuelae. G-quadruplexes are structures that form in DNA or RNA molecules. Depending on their location in DNA, G-quadruplexes can increase or decrease the expression of nearby genes and the stability of a G-quadruplex structure can be affected by G-quadruplex binding proteins. We probed the ability of a G-quadruplex-associated protein from S. venezuelae, TrmB (a tRNA-methyltransferase), to bind and methylate G-quadruplexes and prevent the formation of these structures. We were unable to conclude that TrmB bound or methylated G-quadruplex structures or motifs. RNases are enzymes that cleave RNA molecules and have important roles in controlling cellular RNA levels, and thus gene expression. We investigated the roles of RNase J and RNase III in S. venezuelae. Both of these RNases impact development and specialized metabolism in Streptomyces. We found that the RNase J mutant was unable to grow properly on classical medium containing glycerol. We also documented small RNA fragments that were unique to the RNase J mutant and sought to identify them. To better understand the RNase J and RNase III strains, we conducted RNA-sequencing of wild type S. venezuelae and mutant strains lacking RNase III or RNase J. Comparisons between each mutant and the wild type strain revealed significant changes in genes related to nitrogen assimilation, phosphate uptake, and specialized metabolite production in both the RNase III and RNase J mutant. Together these results contribute to our understanding of the diverse regulatory features that exist in S. venezuelae. / Thesis / Master of Science (MSc) / Studying how gene expression is regulated in the Gram-positive, soil-dwelling bacteria Streptomyces presents an opportunity to better understand how these complex organisms develop and how they control their impressive biosynthetic capabilities. This study investigated the potential role of a G-quadruplex binding protein, and two ribonucleases (RNases) in regulating gene expression in Streptomyces venezuelae. We probed the ability of a G-quadruplex associated protein from S. venezuelae, TrmB, to bind, methylate, and prevent the formation of G-quadruplex structures in DNA. We also investigated the roles of RNase J and RNase III in S. venezuelae growth and development. In RNase J and RNase III mutants, RNA-sequencing revealed dramatic changes in the transcript levels of genes related to phosphate uptake, nitrogen assimilation, and specialized metabolite production. Together these results contribute to our understanding of the diverse and complex regulatory features that exist in S. venezuelae.

Cells generally manage to maintain stable phenotypes in the face of widely varying environmental conditions. This fact is particularly surprising since the key step of gene expression is fundamentally a stochastic process. Many hypotheses have been suggested to explain this robustness. First, the special topology of gene regulatory networks (GRNs) seems to be an important factor as they possess feedforward loops and certain other topological features much more frequently than expected. Second, genes often regulate each other in a canalizing fashion: there exists a dominance order amidst the regulators of a gene, which in silico leads to very robust phenotypes. Lastly, an entirely novel gene regulatory mechanism, discovered and studied during the last two decades, which is believed to play an important role in cancer, is shedding some light on how canalization may in fact take place as part of a cell’s gene regulatory program. Short segments of single-stranded RNA, so-called microRNAs, which are embedded in several different types of feedforward loops, help smooth out noise and generate canalizing effects in gene regulation by overriding the effect of certain genes on others. Boolean networks and their multi-state extensions have been successfully used to model GRNs for many years. In this dissertation, GRNs are represented in the time- and statediscrete framework of Stochastic Discrete Dynamical Systems (SDDS), which captures the cell-inherent stochasticity. Each gene has finitely many different concentration levels and its concentration at the next time step is determined by a gene-specific update rule that depends on the current concentration of the gene’s regulators. The update rules in published gene regulatory networks are often nested canalizing functions. In Chapter 2, this class of functions is introduced, generalized and analyzed with respect to its potential to confer robustness. Chapter 3 describes a simulation study, which supports the hypothesis that microRNA-mediated feedforward loops have a stabilizing effect on GRNs. Chapter 4 focuses on the cellular DNA mismatch repair machinery. A first regulatory network for this machinery is introduced, partly validated and analyzed with regard to the role of microRNAs and certain genes in conferring robustness to this particular network. Due to steady exposure to mutations, GRNs have evolved over time into their current form. In Chapter 5, a new framework for modeling the evolution of GRNs is developed and then used to identify topological features that seem to stabilize GRNs on an evolutionary time-scale. Chapter 6 addresses a completely separate project in Bioinformatics. A novel functional enrichment method is developed and compared to various popular methods. Funding for this work was provided by NSF grant CMMI-0908201 and NSF grant 1062878. / Ph. D.

The cessation of egg-laying during the incubation period of the turkey hen is a source of major economic loss to the turkey industry. In August of 2000 there were approximately 2.7 million turkey breeder hens in the United States. Since the value of one fertile turkey egg is $0.62, the loss of only one egg per hen per year would cost the industry $1.7 million. A number of management procedures have been implemented to control egg production and prevent incubation. However, these methods are labor intensive. The anterior pituitary hormone prolactin (PRL) is involved in the onset of incubation in the turkey hen. Levels of circulating PRL and PRL mRNA are 10X greater in photostimulated hens than in photorefractory hens, 20X greater in laying hens, and 100X greater in incubating hens. It would be useful to determine the molecular mechanisms controlling regulation of the turkey (t) PRL gene. This information could be used to modulate the release of PRL and thereby prevent the induction of the incubation period in turkey hens. Approximately 2 kilobases (kb) of the tPRL 5'-flanking region were examined by the electrophoretic mobility shift assay (EMSA) using nuclear extracts from turkey pituitaries and liver. Within this 2 kb fragment, only three regions of the tPRL gene were identified that participate in tissue- and sequence-specific DNA-protein interactions with nuclear extracts from turkey pituitaries. These are the regions from nucleotides (nt) -41 to -73, -105 to -137, and -175 to -199, named tprl-1, tprl-2 and tprl-3, respectively. Three shifted bands were observed using tprl-1 and tprl-2 while two shifted bands were seen using tprl-3. Competition EMSAs done on these three regions showed that in the presence of unlabeled, excess, specific competitor DNA, the proteins bound to competitor DNA and no shifted bands were observed. If the competitor was a nonspecific DNA sequence, then there was no effect on the shifted bands. When using labeled tprl-2 and unlabeled tprl-1 as competitor DNA, no shifted bands were observed. However, when using labeled tprl-1 and unlabeled tprl-2 as competitor DNA, only one of three shifted bands was eliminated. These data indicate that tprl-1 and tprl-2 bind both common and specific pituitary nuclear proteins and have different affinities for pituitary nuclear proteins. A supershift EMSA involving the addition of rabbit-anti-rat Pit-1 indicated that tPit-1 is a common pituitary nuclear protein that is bound to tprl-1 and tprl-2. However, this interaction may not occur in the turkey in vivo. The mapping of transcription factor binding sites in the tPRL 5'-flanking region is the first step toward the identification and isolation of factors that bind to and regulate transcription of the PRL gene. / Master of Science

AreA, a 96 kDa gene regulatory protein involved in nitrogen metabolite repression in Aspergillus nidulans, is a member of the GATA family of zinc finger DNA binding proteins, and regulates the expression of around 100 genes. This project was designed to examine the domain structure of AreA in this region of the protein, and to characterise the DNA binding domain Limited proteolysis has been employed to identify structural domains in the Cterminal region of AreA, which has been cloned and over-produced in E.coli. A variety of proteases have been used, and each reveals a dominant stable fragment of approximately 17-22 kDa. N-terminal sequencing and mass spectroscopy have been used to identify a number of these fragments. The major product following limited proteolysis by Glu-C is composed of two closely related species, a 164 residue fragment (17,489 Da) and a 157 residue fragment (16,857 Da). Both fragments encompass the Zn-finger motif, and share the same Cterminus, differing at the N-terminus by only 7 amino acids. The DNA sequence coding for the 157 residue fragment (16,857 Da) has been cloned and over-produced as a His-tag fusion protein. Further studies on this domain have shown that this putative domain has a relatively strong DNA binding constant with values in the nanomolar range. Structural analysis using Circular Dichroism, NMR and fluorescence suggests that the domain contains some irregular or unstructured regions. The regions that are structured are likely to be from the zinc-finger region, since DNA binding is maintained.

Chromatin structure and epigenetic mechanisms play an important role in initiating and maintaining the intricate patterns of gene expression required for embryonic development. One such mechanism, DNA methylation (5mC), involves the chemical modification of cytosine bases in DNA and is implicated in maintaining patterns of transcription. However, many fundamental aspects of DNA methylation are not fully understood, including the mechanisms by which it influences transcriptional states. Recent data suggest functional links between DNA methylation and a second epigenetic mechanism that has important roles in transcriptional repression, the polycomb group (PcG) repressor system. Here, I suggest that an intact DNA methylation system is required for the repression of many PcG target genes by influencing the genomic targeting of the polycomb repressor 2 complex (PRC2) and its signature histone modification, H3K27me3 (K27me3). I demonstrate differential genomic localisation of K27me3 at gene promoter regions in hypomethylated mouse embryonic fibroblast (MEF) cells deficient for the major maintenance DNA methyltransferase, Dnmt1. Globally, Dnmt1-/- MEFs have a higher level of the K27me3 mark than controls, as assessed by western blot and immunofluorescence. I observe increased K27me3 at a relatively small number of gene promoters in Dnmt1-/- MEFs that often are associated with high levels of DNA methylation in wildtype MEFs, consistent with the notion that DNA methylation is capable of antagonising PRC2 binding at certain loci. Conversely, I show that a large number of developmentally important genes that are normally repressed and highly bound by K27me3, including classic polycomb targets, the Hox genes, display dramatically reduced association with K27me3 in Dnmt1-/- MEFs. Many of these genes, but not all, show reciprocal increases in promoter H3K4me3 modification and are transcriptionally de-repressed in Dnmt1-/- MEFs. I suggest that these genes are mostly associated with CpG-rich promoters with low levels of DNA methylation in wildtype cells, implying that their silencing is not dependent on the canonical role of DNA methylation. Consistent with the findings of recently published work, I suggest a working model where PRC2 binding in wildtype cells is restricted by CpG methylation. According to this model, the differential genomic location of K27me3 in hypomethylated Dnmt1-/- MEFs is explained by a redistribution of PRC2 to normally DNA methylated, unbound loci, resulting in a titration effect and coincident loss of K27me3 from normal targets. It was also apparent that certain PRC2-target genes, including the developmentally important Hox gene clusters, are strongly affected in Dnmt1-/- MEFs, displaying striking loss of K27me3. As intergenic transcription has been implicated in relief from polycomb silencing and abundant intergenic transcription has been reported within Hox clusters, I measured RNA expression at Hox clusters and a small number of other PcG target genes in Dnmt1-/- MEFs using highdensity tiling arrays. In Dnmt1-deficient MEFs, widespread increases in intergenic transcription were observed within Hox clusters. In addition, mapping of the elongatingpolymerase- associated H3K36me3 histone modification showed widespread increases in this mark at intergenic and promoter regions in Dnmt1-/- MEFs. Increased local intergenic RNA and H3K36me3 were found to correlate with K27me3 loss for this cohort of genes. I suggest a working model where increased intergenic transcription and H3K36me3 in Dnmt1-/- MEFs leads to accelerated loss of K27me3 at certain loci, including Hox clusters. Taken together with recently published data, this work suggests that a major role of DNA methylation is in shaping the PRC2/K27me3 landscape. The potential implications of this putative role for DNA methylation are widespread, including our knowledge of how DNA methylation influences transcriptional regulation, and the consequence of rearranged DNA methylation patterns that are observed in many diseases including cancers.

No description available.

Transposons were first discovered as "jumping genes" by Barbara McClintock, who continued to study them in maize through the 1940's and 1950's. Since then, transposons have been shown to make up a large percentage of eukaryotic genomes, including close to half of the human genome, but have been dismissed as simply "junk DNA." Recently, the importance of keeping transposons tightly regulated within the cellular environment has begun to be appreciated; the mechanisms to accomplish this have been studied and the current understanding of pathways governing transposon regulation is discussed within this dissertation. However, recent work presented within the scope of this dissertation in Drosophila melanogaster revealed a previously unknown function for condensin complexes in transposable element regulation. These studies provide a link between pathways governing chromosome pairing and transposon regulation. The potential interplay between these two pathways is intriguing and until now, largely unexplored.Aside from how transposons themselves are regulated, studies into potential roles they may play in the regulation of other protein coding genes within the cell may provide clues into the functionality of these elements within our genome. As a specific example, BRCA1 has a high density of retrotransposon sequences within its primary transcript, and studies of BRCA1 regulation presented within this dissertation has led to the development of a model for a novel gene regulatory mechanism occurring in human cells involving retrotransposons. This mechanism may provide direct relevance to cancer etiology, as retrotransposons have long been known to be misregulated in cancer.As a sum, the work presented within this dissertation extends our knowledge of how transposons are regulated and provides some of the first evidence for their functionality in gene regulatory pathways within human cells.

No description available.