Browsing by Subject "Transcriptional regulation"
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Item Open Access COMPARATIVE ANALYSIS OF TRANSCRIPTIONAL RESPONSE TO STRESS AND CARBOHYDRATE AVAILABILITY IN HALOARCHAEA(2023) Hackley, Rylee KHypersaline-adapted archaea, or haloarchaea, inhabit extreme environments where changes in near-saturated salinity, oxygen, and nutrients require rapid regulation of essential cellular processes. Transcriptional regulatory networks govern the dynamical responses enabling these organisms to sense and respond to rapidly changing conditions. Additionally, timely regulation of carbon metabolic pathways is essential to respond to intermittent nutrient availability and prevent futile cycling of intracellular metabolites. In Halobacterium salinarum, a hypersaline-adapted archaeon that does not rely on carbohydrates for carbon or energy, over 1,500 transcriptome profiling experiments have yielded a genome-scale model of regulatory interactions and revealed a general transcriptional response to diverse stressors (Chapter 2). Among regulators in Hbt. salinarum, a sugar-sensing TrmB family protein has previously been shown to control gluconeogenesis and other biosynthetic pathways. This characterization expanded the set of regulatory functions known for TrmB-family proteins in archaea, which regulate carbohydrate metabolism in hyperthermophilic archaea. TrmB regulators are particularly interesting in Haloarchaea because an expansion of the protein family is presumed to have occurred alongside a diversification of carbohydrate catabolic pathways (Chapter 3).
To investigate whether the expanded set of TrmB functions is shared among haloarchaea with different metabolic capabilities and better understand how regulatory variation arises in extremophiles, we characterized the role of TrmB in two haloarchaeal model species that catabolize carbohydrates: Haloarcula hispanica and Haloferax volcanii (Chapters 4 and 5, respectively). We hypothesized that TrmB would maintain a role in the regulation of gluconeogenesis through homologous targets but would acquire targets involved in the concordant catabolic processes of these saccharolytic models. To characterize the role of TrmB homologs, we conducted high-throughput growth assays, microscopy and other microbiological phenotyping techniques, gene expression profiling via RNA-seq, promoter activity analysis, and protein-DNA binding assays of TrmB homologs in Har. hispanica and Hfx. volcanii. Our results show that TrmB homologs indeed activate gluconeogenesis through the recognition of conserved cis-regulatory motifs. However, contrary to its role in Hbt. salinarum, TrmB does not act as a global regulator in Har. hispanica or Hfx. volcanii: it does not directly repress the expression of peripheral pathways such as cofactor biosynthesis or catabolism of other carbon sources. A key bidirectional control point, activation of ppsA and repression of pyk, is lost in Har. hispanica.
Our results indicate substantial rewiring of the TrmB regulon in Hfx. volcanii. A novel transcriptional regulator, TbsP, is responsible for repressing gluconeogenic genes when glucose is available. TrmB and TbsP appear to compete for partially overlapping binding sites in the promoter of gapII, which encodes the gluconeogenic-specific glyceraldehyde-3-phosphate dehydrogenase. Loss-of-function mutations in tbsP are sufficient to recover partial gapII expression and gluconeogenic activity when trmB is deleted. In Hfx. volcanii, TrmB is predicted to activate ppsA and repress the gene encoding bacterial phosphoenopyruvate carboxylase, perhaps preserving the dynamical and functional behavior of TrmB in Hbt salinarum, but reflecting species-specific anaplerotic strategies. Moreover, TrmB is predicted to repress the expression of bacterial type I GAPDH: gapI and gapII may comprise an additional bidirectional control point in Hfx. volcanii, although this hypothesis requires additional testing.
Cumulatively, this dissertation outlines specific examples of TRN rewiring highlighting the incorporation of metabolic enzymes gained through inter-domain horizontal gene transfer, suggesting rewiring of the TrmB regulon via gain and loss of binding sites alongside metabolic network evolution in Haloarchaea.
Item Open Access Computational Inference of Genome-Wide Protein-DNA Interactions Using High-Throughput Genomic Data(2015) Zhong, JianlingTranscriptional regulation has been studied intensively in recent decades. One important aspect of this regulation is the interaction between regulatory proteins, such as transcription factors (TF) and nucleosomes, and the genome. Different high-throughput techniques have been invented to map these interactions genome-wide, including ChIP-based methods (ChIP-chip, ChIP-seq, etc.), nuclease digestion methods (DNase-seq, MNase-seq, etc.), and others. However, a single experimental technique often only provides partial and noisy information about the whole picture of protein-DNA interactions. Therefore, the overarching goal of this dissertation is to provide computational developments for jointly modeling different experimental datasets to achieve a holistic inference on the protein-DNA interaction landscape.
We first present a computational framework that can incorporate the protein binding information in MNase-seq data into a thermodynamic model of protein-DNA interaction. We use a correlation-based objective function to model the MNase-seq data and a Markov chain Monte Carlo method to maximize the function. Our results show that the inferred protein-DNA interaction landscape is concordant with the MNase-seq data and provides a mechanistic explanation for the experimentally collected MNase-seq fragments. Our framework is flexible and can easily incorporate other data sources. To demonstrate this flexibility, we use prior distributions to integrate experimentally measured protein concentrations.
We also study the ability of DNase-seq data to position nucleosomes. Traditionally, DNase-seq has only been widely used to identify DNase hypersensitive sites, which tend to be open chromatin regulatory regions devoid of nucleosomes. We reveal for the first time that DNase-seq datasets also contain substantial information about nucleosome translational positioning, and that existing DNase-seq data can be used to infer nucleosome positions with high accuracy. We develop a Bayes-factor-based nucleosome scoring method to position nucleosomes using DNase-seq data. Our approach utilizes several effective strategies to extract nucleosome positioning signals from the noisy DNase-seq data, including jointly modeling data points across the nucleosome body and explicitly modeling the quadratic and oscillatory DNase I digestion pattern on nucleosomes. We show that our DNase-seq-based nucleosome map is highly consistent with previous high-resolution maps. We also show that the oscillatory DNase I digestion pattern is useful in revealing the nucleosome rotational context around TF binding sites.
Finally, we present a state-space model (SSM) for jointly modeling different kinds of genomic data to provide an accurate view of the protein-DNA interaction landscape. We also provide an efficient expectation-maximization algorithm to learn model parameters from data. We first show in simulation studies that the SSM can effectively recover underlying true protein binding configurations. We then apply the SSM to model real genomic data (both DNase-seq and MNase-seq data). Through incrementally increasing the types of genomic data in the SSM, we show that different data types can contribute complementary information for the inference of protein binding landscape and that the most accurate inference comes from modeling all available datasets.
This dissertation provides a foundation for future research by taking a step toward the genome-wide inference of protein-DNA interaction landscape through data integration.
Item Open Access Cryptococcus neoformans transcriptional regulation of the host-pathogen interface(2013) O'Meara, Teresa RodgersCryptococcus neoformans is a human fungal pathogen that is also ubiquitous in the environment. To cause disease inside a human host, C. neoformans 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.
Using genetic and molecular biology techniques, I identified Gcn5 and Rim101 as key transcriptional regulators of capsule within the host. I determined that C. neoformans 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.
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 C. neoformans 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.
Item Open Access Novel Methods to Identify Chromatin Accessibility Differences Across Primates(2019) Edsall, Lee ElizabethOne of the aims of evolutionary biology is to identify gene regulatory regions (and the resulting level of expression) that evolved between species. The conventional method of analysis for this is to perform pairwise comparisons on data generated for each species. Software programs for this approach are mature and work well when there are only two species of interest. These same programs can be used when there are three species of interest. However, the analysis becomes more cumbersome and the statistical significance (p-value) difficult to calculate. Performing pairwise comparisons when there are more than three species have significant limitations. One is the exponential increase in the number of tests performed, greatly reducing the sensitivity after false discovery rate correction. For n species, (n-1) tests are performed on each region. Another limitation is the lack of a principled way to identify and classify genes (or regulatory regions) containing changes in multiple species.
To address these limitations, we developed a novel method of jointly modelling the data from all of the species using a negative binomial generalized linear model. In addition to providing a principled way of identifying and classifying sites with multiple changes, our method is more sensitive largely due to a substantial decrease in the number of tests performed. Our method jointly models all of the data in a single test, regardless of the number of species. As a result, the correction for number of independent tests performed is (n-1) times larger for the multiple pairwise method than for the joint modelling approach.
We applied this joint modelling approach to DNase-seq data generated from skin fibroblast cells from five primate species; human, chimpanzee, gorilla, orangutan, and rhesus macaque. We identified 89,744 DNase I Hypersensitive sites (DHS sites) that were comparable across all species, of which 41% (36,666) were classified as differential in one or more species. 30% of the differential sites (11,095) are likely due to a single change in chromatin accessibility in one species. Changes that likely occurred on the internal human-chimpanzee branch or human-chimpanzee-gorilla branch account for 15% (5,385) of the differential sites. 16% (6,034) of the differential sites contain changes that happened on either the human-chimpanzee-gorilla-orangutan internal branch or the rhesus macaque species branch. 32% (11,698) of the differential sites are due to multiple changes in chromatin accessibility (e.g., independent changes on the human and orangutan species branches).
The accuracy of this new approach was demonstrated by a high degree of concordance with an earlier study from our laboratory that analyzed data from human, chimpanzee, and rhesus macaque. Additionally, we performed a conventional pairwise analysis of the DHS sites from the five species and classified only 33% as differential, indicating decreased sensitivity compared to the joint modelling approach. Together, these results indicate that this novel joint modelling approach provides an improved method for comparative analysis of DNase-seq data.
Although we developed this method for DNase-seq data, we expect that it can be applied to other count-based data types such as ChIP-seq, ATAC-seq, and RNA-seq. We also expect that it can be applied to other experimental designs such as time-series, multi-tissue comparisons, and multiple developmental stage comparisons. The R script for performing the joint modelling analysis and instructions for modifying the script for use by other investigators are available in a GitHub repository (http://github.com/ledsall/2019primate).
Item Open Access Pumilio-mediated Repression of mRNAs in the Early Drosophila Melanogaster Embryo(2009) Nomie, Krystle JoliPost-transcriptional regulation plays an important role in governing various processes in all organisms. The development of the early embryo of Drosophila melanogaster is governed solely by post-transcriptional mechanisms; therefore, further insights into post-transcriptional regulation can be gained by studying the Drosophila 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 bicoid, a gene required for Drosophila head development. bicoid 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 pumilio mutant embryos bicoid mRNA stability and translation are unaltered, demonstrating that Pumilio does not regulate bicoid mRNA. Second, Pumilio has been shown to negatively regulate Cyclin B, 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 Cyclin B and discovered that the canonical partner of Pumilio, Nanos, is not involved in repressing somatic Cyclin B. Furthermore, we show that the 3'UTR of Cyclin B 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 Cyclin B.
Item Open Access The Transcriptional Regulation of the Central Plant Defense Signal, Salicylic Acid(2014) Zheng, XiaoyuSalicylic acid (SA) is a central plant defense signal. It is not only required for closing the stomata upon infection to prevent pathogens from entering into the plant apoplast, but also mediates defense responses activated by pathogen-originated microbe-associated molecular patterns (MAMPs) and effectors in the infected tissues. In addition, SA is a necessary and sufficient signal for systemic acquired resistance (SAR). In Arabidopsis thaliana, SA level increases in response to pathogen attack, which is essential for activating defense responses. This SA accumulation involves transcriptional activation of several genes including ICS1 (ISOCHORISMATE SYNTHASE 1), EDS5 (ENHANCED DISEASE SUSCEPTIBILITY 5), EDS1 (ENHANCED DISEASE SUSCEPTIBILITY 1), PAD4 (PHYTOALEXIN-DEFICIENT 4) and PBS3 (avrPphB SUSCEPTIBLE 3). However, it is not well understood how pathogenic signals induce these SA accumulation genes. Interestingly, our time-course transcriptome analysis showed that these five genes share a similar pathogen-induced expression pattern, suggesting the existence of common transcription factors (TFs). Through yeast-one-hybrid screening, a TF NTL9 was identified for its interactions with the promoters of the SA accumulation genes. Preferentially expressed in guard cells, NTL9 activates the expression of SA accumulation genes in guard cells. The ntl9 mutant is defective in pathogen-induced stomatal closure mediated by a well-characterized MAMP, flg22. Consistent with the stomatal closure defect, the ntl9 mutant exhibits elevated susceptibility to surface-inoculated pathogens. The stomatal closure defect of the ntl9 mutant can be rescued by exogenous application of SA, demonstrating that NTL9 acts upstream of SA in stomatal closure response. These results suggest that NTL9-mediated activation of SA accumulation genes is essential for MAMP-triggered stomatal closure.
While plants induce SA to activate defense responses, pathogens can also produce virulence factors to counteract the effects of SA. Coronatine is one such virulence factor produced by Pseudomonas syringae. Coronatine is known to promote opening of stomata for bacterial entry, bacterial growth in the apoplast, systemic susceptibility and development of disease symptoms such as chlorosis. In the process of examining the mechanisms underlying coronatine-mediated virulence, three homologous TFs, ANAC019, ANAC055 and ANAC072, were found to be activated by coronatine directly through the TF, MYC2. Genetic characterization of these three TF mutants revealed that these TFs mediate multiple virulence effects of coronatine by inhibiting SA accumulation. To exert this inhibitory effect, these TFs repress ICS1 and activate BSMT1, genes involved in SA biosynthesis and inactivation modification, respectively. Thus, a signaling cascade downstream of coronatine was illustrated to dampen SA-mediated defense responses through differential transcriptional regulation of genes related to SA level.
Taken together, my dissertation studies revealed novel transcriptional regulation of SA production and demonstrated that this transcriptional regulation is a vital point not only for plant defense activation but also for pathogen manipulation to counteract defense responses. Further studies on the interplay of this transcriptional regulation by different TFs would broaden our understanding about the dynamics of plant-pathogen interaction.