Sickle cell disease and β-thalassemia affect millions of people worldwide. γ-globin is the fetal counterpart to the adult β-globin. Research has shown that affected patients with higher than normal γ-globin show less severe symptoms. Therefore, reversing or preventing the hemoglobin switch from γ- to β- globin is a promising avenue of research for treating these diseases. KLF1 is an erythroid transcription factor involved in hemoglobin switching. Herein, we show that KLF1 directly regulates the γ-globin repressor gene LRF in both the mouse and human systems. KLF1 may also directly activate γ-globin expression by binding the promoter. In human HUDEP-2 cells, an increase in γ-globin expression is seen upon modest knockdown (~50%) of KLF1, whereas normal amounts of KLF1 are observed upon robust knockdown (>75%) of KLF1. The data suggest that KLF1 plays both a positive and negative role in γ-globin expression.

Genome-wide binding and expression studies in Drosophila melanogaster have revealed widespread roles for Dichaete and SoxNeuro, two group B Sox proteins, during fly development. Although they have distinct target genes, these two transcription factors bind in very similar patterns across the genome and can partially compensate for each other's loss, both phenotypically and at the level of DNA binding. However, the inherent noise in genome-wide binding studies as well as the high affinity of transcription factors for DNA and the potential for non-specific binding makes it difficult to identify true functional binding events. Additionally, externalfactors such as chromatin accessibility are known to play a role in determining binding patterns in Drosophila. A comparative approach to transcription factor binding facilitates the use of evolutionary conservation to identify functional features of binding patterns. In order to discover highly conserved features of group B Sox binding, I performed DamID-seq for SoxNeuro and Dichaete in four species of Drosophila, D. melanogaster, D. simulans, D. yakuba and D. pseudoobscura. I also performed FAIRE-seq in D. pseudoobscura embryos to compare the chromatin accessibility landscape between two fly species and to examine the relationship between open chromatin and group B Sox binding. I found that, although the sequences, expression patterns and overall transcriptional regulatory targets of Dichaete and SoxNeuro are highly conserved across the drosophilids, both binding site turnover and rates of quantitative binding divergence between species increase with phylogenetic distance. Elevated rates of binding conservation can be found at bound genomic intervals overlapping functional sites, including known enhancers, direct targets of Dichaete and SoxNeuro, and core binding intervals identified in previous genome-wide studies. Sox motifs identified in intervals that show binding conservation are also more highly conserved than those in intervals that are only bound in one species. Notably, regions that are bound in common by SoxNeuro and Dichaete are more likely to be conserved between species than those bound by one protein alone. However, by examining binding intervals that are uniquely bound by one protein and conserved, I was able to identify distinctive features of the targets of each transcription factor that point to unique aspects of their functions. My comparative analysis of group B Sox binding suggests that sites that are commonly bound by Dichaete and SoxNeuro, primarily at targets in the developing nervous system, are highlyconstrained by natural selection. Uniquely bound targets have different tissue expression profiles, leading me to propose a model whereby the unique functions of Dichaete and SoxNeuro may arise from a combination of differences in their own expression patterns and the broader nuclear environment, including tissue-specific cofactors and patterns of accessible chromatin. These results shed light on the evolutionary forces that have maintained conservation of the complex functional relationships between group B Sox proteins from insects to mammals.

The idea of morphogen gradients encoding positional information for a developing organism has long been discussed in the field of developmental biology, but only recently have quantitative models been proposed that relate measured transcription factor concentrations to enhancer activity. However, successful models are typically computationally time-consuming, thus limiting full exploration and interpretation of the data. This thesis addresses these problems using standard statistical techniques applied to a comprehensive data set with the even skipped (eve) locus as a test case. The first part of the thesis introduces the data set. This is the precellular Virtual Embryo from the Berkeley Drosophila Transcription Network project. It comprises expression measurements of almost 100 genes in more than 6,000 individual nuclei at six time points. Different modelling approaches are evaluated in the context of this data set leading to a justification of logistic regression and the methods used to prepare the data set for further analysis. The second part applies logistic regression to describe the response of the eve enhancers to known regulating transcription factors such as Hunchback. Predictions of behaviour under regulator perturbation are consistent with experimental results and the functional form is shown not to be arbitrarily flexible, both in terms of the regulators and regions of the embryo included. The third part uses the framework developed above to find minimal explanatory models in the context of statistical model selection. It is found that the best scoring models depend on well-known regulators. The model selection techniques are then extended by directing the process using previous biological observations to analyse the eve 2 and eve 3+7 enhancers. The results are consistent with published research, but suggest specific additional hypotheses for the enhancers' regulation. Finally, the thesis concludes by proposing a general model of positional information and discussing the biological implications of the results. Overall, the results show how transcriptional control can be allocated to discrete enhancers and that characterising their activity in relatively simple terms is sufficient to explain their precise spatially-defined response to transcription factor concentrations.

No description available.

Salamanders have an extraordinary ability to regenerate appendages after loss or amputation, irrespective of age. My dissertation research explored the possibility that regenerative ability is associated with the evolution of novel, salamander-specific genes. I utilized transcriptional and genomic databases for the axolotl to discover previously unidentified genes, to the exclusion of other vertebrate taxa. Among the genes identified were multiple mmps (Matrix metalloproteases) and a jnk1/mapk8 (c-jun-N-terminal kinase) paralog. MMPs function in extracellular matrix remodeling (ECM) and tissue histolysis, processes that are essential for successful regeneration. Jjnk1/mapk8 plays a pivotal role in regulating transcription in response to cellular stress stimuli, including ROS (reactive oxygen species). Discovery of these novel genes motivated further bioinformatic studies of mmps and wet-lab experiments to characterize JNK and ROS signaling. The paralogy of the newly discovered mmps and orthology of 15 additional mmps was established by analyses of predicted, protein secondary structures and gene phylogeny. A microarray-analysis identified target genes downstream of JNK signaling that are predicted to function in cell proliferation, cellular stress response, and ROS production. These inferences were validated by additional experiments that showed a requirement for NOX (NADPH oxidase) activity, and thus presumably ROS production for successful tail regeneration. In summary, my dissertation identified novel, salamander-specific genes. The functions of these genes suggest that regenerative ability is associated with a diverse extracellular matrix remodeling and/or tissue histolysis response, and also stress-associated signaling pathways. The bioinformatic findings and functional assays that were developed to quantify ROS, cell proliferation, and mitosis will greatly empower the axolotl embryo model for tail regeneration research.

Activation of the NLRP3 inflammasome has been shown in response to numerous activators; here we show that the oxazolidinone antibiotic linezolid results in both the NLRP3-dependent in vitro release of the proinflammatory cytokine IL-1 Α; and in vivo neutrophilic influx following its intraperitoneal administration. Clinical use of linezolid is commonly limited by hematologic side effects; herein we also show NLRP3-deficiency protected animals against linezolid-induced effects on the bone marrow. Importantly, all previously described activators of the NLRP3 inflammasome have required the generation of reactive oxygen species (ROS). Linezolid is however unique amongst NLRP3 agonists in that its ability to activate the NLRP3 inflammasome in a ROS-independent manner. The pathways for ROS-dependent and ROS-independent NLRP3 activation converge upon mitochondrial dysfunction and specifically the mitochondrial lipid cardiolipin. We demonstrated that interference with cardiolipin synthesis specifically inhibits NLRP3 inflammasome activation. These findings firstly suggests that ROS generation is not the canonical activator of NLRP3 but rather an intermediary step leading to the mitochondrial perturbation that is tied to NLRP3 inflammasome activation and also implicate the involvement of mitochondrial lipid cardiolipin in this process; secondarily, linezolid-induced NLRP3 activation may account for thetoxicity associated with prolonged usage of this antibiotic.

<p>The relationship between gene copy number and protein expression levels has not thoroughly been examined in humans or mouse models of Down syndrome (DS) in relationship to developmental changes in the trisomic brain. Found on human chromosome 21 (Hsa21) and triplicated in DS, Dual-specificity tyrosine-phosphorylated regulated kinase 1A (<i>DYRK1A)</i> has been linked in DS to neurological deficits by restricting cell growth and proliferation. Little information exists regarding DYRK1A during perinatal development and how its expression may lead to cognitive deficits, and none exists that explores the gene-to-protein relationship during these critical time periods. This study aims to 1) Quantify variable DYRK1A expression across development as a function of age, sex, and brain region in trisomic Ts65Dn mice compared to euploid counterparts and 2) establish that the spatiotemporal pattern of developmental DYRK1A in the brain is not influenced solely by gene copy number, and that reduction of <i>Dyrk1a</i> in euploid and trisomic mice does not result in a corresponding global reduction of DYRK1A expression. DYRK1A was quantified in three areas of the postnatal brain at seven ages using the Ts65Dn mouse, the most studied model of DS, and found that trisomic expression is significantly increased on postnatal day ([P]6), declining by the third week to near euploid levels. We also uncovered a sexual dimorphic expression of DYRK1A when comparing animals of different sexes within the same genotype. Data from <i>Dyrk1a</i> knockdown mice indicated that reducing only <i>Dyrk1a</i> in euploid and in otherwise trisomic animals yields highly variable levels of DYRK1A, dependent on sex and tissue type, supporting the non-intuitive relationship between gene dosage and protein expression. These data emphasize the need to understand the age-dependent regulation of antecedent conditions that are causing changes in <i>Dyrk1a</i> expression in the brain.</p>

Previous research has shown variation of thermal tolerance in clones of the aquatic crustacean Daphnia magna. In general, genotypes from regions with warmer climate possess a greater tolerance to high temperatures than those from colder climates. However, the mechanism by which they achieve this thermal tolerance is unclear. One possible explanation may lie in how the animals utilize polyunsaturated fatty acids (PUFAs), which are macromolecules essential for maintaining cell membrane fluidity and to prevent transition to gel-like phase. The need for PUFAs is exacerbated further in poikilothermic animals at colder temperatures. Therefore, it can be hypothesized that D. magna originating from colder climates would more readily uptake and incorporate PUFAs into their cell membranes when in a cold environment. This study examined twenty-one D. magna clones known to differ in thermal tolerance. A life-table experiment was conducted in order to see which clones could produce viable eggs at 10°C, when given a diet low in PUFAs (green alga Scenedesmus obliquus). Since egg-laying requires a significant investment of PUFAs, fecundity was used as the measurement of cold tolerance. Separately, eggs and their mothers from select clones were collected for lipid analysis. No D. magna clone showed success in every egg clutch during the experiment, although a few showed failures in every clutch. Clones from cold climate regions were not more likely to have more successful clutches when compared to those from warm climates. Lipid analysis via gas chromatography is ongoing and will compare lipid content in eggs and mothers at 25°C and 10°C with different diets—either low in PUFAs (S. obliquus) or high in PUFAs (heterokont alga Nannochloropsis limnetica). The lipid analysis will reveal if there is a lack of PUFA investment in eggs at cold temperatures by comparing unsuccessful cold-temperature eggs to successful cold-temperature eggs and warm-temperature eggs. Adult uptake of PUFAs can also be discerned by comparing adults with a low-PUFA diet at cold temperatures to those with a high-PUFA diet at cold temperatures and those at warm temperatures. This analysis will help understand trade-offs between high performance at different temperatures and mechanisms of plastic response to temperature.

The mammalian lung is an exquisitely designed organ with two structurally distinct compartments, one that comprises multiple generations of branched tubules to conduct and clean the air (airways) and another that consists of a vast network of thin-walled alveolar structures to allow gas exchange (alveoli). In the embryo these compartments arise from highly dynamic patterning events during branching morphogenesis that define two major domains, a proximal (Sox2+) and a distal (Sox9+), which ultimately form the airways and alveoli, respectively. Although the signaling pathways controlling branching morphogenesis are increasingly understood, the mechanisms that regulate the transition zone (TZ) between the proximal and distal domains are still elusive. The goals of this thesis are to identify markers and molecular regulators of the TZ, to examine the role of Hippo-Yap signaling in the establishment of the TZ and to investigate the evolutionary conservation of this process in the lung of the snake Pantherophis guttata, which lacks a branched airway tree. Using a combination of mouse genetics, single cell RNAseq, computational approaches and immunofluorescence-confocal analyses I show that Yap transcriptional activity and nucleocytoplasmic shuttling are essential for patterning of the lung by being pivotal for initiation of the events that give rise to the TZ, as well as for subsequent lineage differentiation of compartment-specific progenitors. I show that cytoplasmic sequestration of Yap in Sox2+ epithelial progenitors is a crucial mechanism to prevent the deleterious effects of maintaining nuclear Yap once airway progenitors are specified. Moreover, PISCES-inferred protein activity profiling identified Hspa8, Krt19, Btg2, Anxa2, Cldn10 and Icam1 in the TZ. Notably, analyses of Yap loss and gain function in mice revealed Icam1 as a key marker of the TZ and a downstream target of Yap. Lastly, I show that Sox2 and Sox9 are conserved markers of proximal (bronchiolar) and distal (respiratory) cell fate in the respiratory tract. However, in the snake Pantherophis guttata, the early proximal-distal event that specifies the Sox9+ compartment in the mouse appears delayed. I speculate that proximal-distal patterning in murine lung development actually represents a precocious specification event of respiratory identity, as well as that this ultimately enabled the incorporation of a program of branching morphogenesis in the ancestral program of lung development. Considering that in humans the primordial lungs are double Sox2+ Sox9+, this suggests an unsuspected heterogeneity in the early lung developmental events of human, mice, and reptiles. Altogether, the findings revealed by this work open new avenues of research to further understand the molecular mechanisms that drive lung development.

The Caenorhabditis elegans somatic gonad is patterned by the activity of regulatory cell types, which govern its morphology, serve as the germline niche, and pattern its connection to the outside. All regulatory cell types are specified by activity of the basic helix-loop-helix gene hlh-2/E/Daughterless, and differences in how functions are assigned between the regulatory cells in males and hermaphrodites lead directly to their sexually-dimorphic gonads. Here, I present evidence that a code of bHLH genes function together with hlh-2 to promote the specification and function of each regulatory cell type except for the hermaphrodite anchor cell, which is specified by HLH-2 activity alone. Each regulatory cell type expresses an overlapping but distinct set of bHLH genes, which we find are required for its specification and associated functions. Notably, ectopic expression of regulatory cell bHLH complements are sufficient to transform cells with anchor cell potential into the expected regulatory cell, albeit transiently, suggesting that they are master regulators of regulatory cell fate. As all nematode species pattern their gonads through cognate regulatory cells and bHLH genes are highly conserved, we hypothesized that a similar bHLH code might function in specifying the regulatory cells of other species. In some nematode species the anchor cell, which remains stationary in C. elegans, is able to migrate. In C. elegans, the bHLH gene hlh-12 is necessary for proper migration of hermaphrodite distal tip cells and male linker cell, the two migrating regulatory cell types; addition of hlh-12 to the C. elegans anchor cell causes it to become displaced in a manner dependent on the endogenous hermaphrodite distal tip cell and male linker cell machinery, suggesting that the anchor cell gains the ability to migrate with the addition of hlh-12. We thus hypothesized that ectopic expression of an hlh-12 ortholog in these species might have led them to evolve migrating anchor cells. However, phylogenetic analysis of the bHLH genes of several other species, including the ones with migratory anchor cells, suggests that hlh-12 may be novel to the Caenorhabditis genus and does not have orthologs in the species with migrating anchor cells, raising the possibility that either these species use another bHLH gene for migration or that their regulatory cells are specified in a bHLH-independent manner.