The classical cell cycle regulatory pathway is well appreciated as a key regulator of cell fate determination during neurogenesis; however, the extent of pRB/E2F function in neural stem and progenitor cells is not fully understood, and insight into the mechanisms underlying its connection with cell fate regulation are lacking. The E2F3 transcription factor has emerged as an important regulator of neural precursor cell (NPC) proliferation in the embryonic and adult forebrain, and we demonstrate here that it also influences the self-renewal potential of NPCs. Using knockout mouse models of individual E2F3 isoforms, we demonstrate the surprising result that the classical transcriptional activator E2F3a represses NPC self-renewal and promotes neuronal differentiation, while E2F3b promotes the expansion of the NPC pool and inhibits differentiation. We attribute these opposing activities to a unique mechanism of transcriptional regulation at the Sox2 locus, a key regulator of stem cell pluripotency, whereby E2F3a recruits transcriptional repressors to this site, and E2F3b promotes Sox2 activation. Importantly, E2F3a-mediated Sox2 regulation is necessary for cognitive function in the adult. Additionally, through the determination of genome-wide promoter binding sites for E2f3 isoforms as well as E2F4, another key regulator of NPC self-renewal, we determined that E2Fs are poised to regulate an extensive set of target genes with key roles in regulating diverse cell fate choices in NPCs, including self-renewal, cell death, progenitor expansion, maintenance of the precursor state, and differentiation. Together, these results reveal a diversity of function for E2Fs in the control of neural precursor cell fate, and identify E2F3 isoforms as important regulators of the pluripotency and stem cell maintenance gene Sox2.

Developmental processes are carefully controlled at the level of transcription to ensure that the fertilized egg develops into an adult organism. The mechanisms that controls transcription of protein-coding genes ultimately ensure that the Pol II machine synthesizes mRNA from the correct set of genes in every cell type. Transcriptional control involves Pol II recruitment as well as transcriptional elongation. Recent genome-wide studies shows that recruitment of Pol II is often followed by an intermediate step where Pol II is halted in a promoter-proximal paused configuration. The release of Pol II from promoter-proximal pausing is thus an additional and commonly occurring mechanism in metazoan gene regulation. The serine kinase P-TEFb is part of the Super Elongation Complex that regulates the release of paused Pol II into productive elongation. However, little is known about the role of P-TEFb mediated gene expression in development. We have investigated the function of P-TEFb in early Drosophila embryogenesis and find that P-TEFb and other Super Elongation Complex subunits are critical for activation of the most early expressed genes. We demonstrate an unexpected function for Super Elongation Complex in activation of genes with non-paused Pol II. Furthermore, the Super Elongation Complex shares phenotypes with subunits of the Mediator complex to control the activation of essential developmental genes. This raises the possibility that the Super Elongation Complex has an unappreciated role in the recruitment of Pol II to promoters. The unique chromatin landscape of each cell type is comprised of post-translational chromatin modifications such as histone methylations and acetylations. To study the function of histone modifications during development, we depleted the histone demethylase KDM4A in Drosophila to evaluate the role of KDM4A and histone H3 lysine 36 trimethylation (H3K36me3) in gene regulation. We find that KDM4A has a male-specific function and regulates gene expression both by catalytic-dependent and independent mechanisms. Furthermore, we used histone replacement to investigate the direct role of H3K14 acetylation in a multicellular organism. We show that H3K14 acetylation is essential for development, but is not cell lethal, suggesting that H3K14 acetylation has a critical role in developmental gene regulation. This work expands our knowledge of the mechanisms that precisely controls gene regulation and transcription, and in addition highlights the complexity of metazoan development. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 3: Manuscript.</p>

Bacterial cells reproduce by doubling in size and dividing. The molecular control systems which regulate the cell cycle must do so in a manner which maintains a similar cell size over many generations. A cell can under conditions of fast growth conclude cell cycles in shorter time than the time required to replicate its chromosome. Under such conditions several rounds of replication are maintained in parallel and a cell will inherit replication processes which were initiated by an ancestor. To accomplish this the cell has to initiate and terminate one round of replication during each cell cycle. To investigate the effects of the cell cycle on gene-regulation in the gut bacterium Escherichia coli, an experimental method combining microfluidics, single molecule fluorescence microscopy and automated analysis capable of acquiring an arbitrary number of complete cell cycles per experiment was developed. The method allowed for the rapid exchange of the chemical environment surrounding the cells. Using this method it was possible to measure the dissociation time of the transcription factor molecule, LacI-Venus, from the native lactose operator sequence, lacO1, and an artificially strong operator, lacOsym, in vivo. The results indicated that regulation of gene-expression from the lactose operon does not occur at equilibrium in living cells. Furthermore, by studying the intracellular location of non-specifically interacting transcription factor molecules it was possible to determine that these do not form long-lived gradients inside the cell as was previously proposed. By studying the replication machinery and the origin of replication it was found that replication is initiated according to a cell volume per origin which did not vary over different growth conditions. Further, division timing was found to be determined by the initiation event to occur after a fixed time-delay. A consequence of this mode of regulation is an uncertainty relation between the size at birth and the cell cycle time, in which cells will vary more in in the cycle time during conditions of slow growth as compared to fast growth and vary more in birth length during conditions of fast growth as compared to slow growth.

<p>During embryogenesis, a single zygote gives rise to a multicellular embryo with distinct spatial territories marked by differential gene expression. How is this patterning process organized? How robust is this function to perturbations? Experiments that examine normal and regulative development will provide direct evidence for reasoning out the answers to these fundamental questions. Recent advances in technology have led to experimental determinations of increasingly complex gene regulatory networks (GRNs) underlying embryonic development. These GRNs offer a window into systems level properties of the developmental process, but at the same time present the challenge of characterizing their behavior. A suitable modeling framework for developmental systems is needed to help gain insights into embryonic development. Such models should contain enough detail to capture features of interest to developmental biologists, while staying simple enough to be computationally tractable and amenable to conceptual analysis. Combining experiments with the complementary modeling framework, we can grasp a systems level understanding of the regulatory program not readily visible by focusing on individual genes or pathways. </p><p>This dissertation addresses both modeling and experimental challenges. First, we present the autonomous Boolean network modeling framework and show that it is a suitable approach for developmental regulatory systems. We show that important timing information associated with the regulatory interactions can be faithfully represented in autonomous Boolean models in which binary variables representing expression levels are updated in continuous time, and that such models can provide direct insight into features that are difficult to extract from ordinary differential equation (ODE) models. As an application, we model the experimentally well-studied network controlling fly body segmentation. The Boolean model successfully generates the patterns formed in normal and genetically perturbed fly embryos, permits the derivation of constraints on the time delay parameters, clarifies the logic associated with different ODE parameter sets, and provides a platform for studying connectivity and robustness in parameter space. By elucidating the role of regulatory time delays in pattern formation, the results suggest new types of experimental measurements in early embryonic development. We then use this framework to model the much more complicated sea urchin endomesoderm specification system and describe our recent progress on this long term effort. </p><p>Second, we present experimental results on developmental plasticity of the sea urchin embryo. The sea urchin embryo has the remarkable ability to replace surgically removed tissues by reprogramming the presumptive fate of remaining tissues, a process known as transfating, which in turn is a form of regulative development. We show that regulative development requires cellular competence, and that competence is lost early on but can be regained after further differentiation. We demonstrate that regulative replacement of missing tissues can induce distal germ layers to participate in reprogramming, leading to a complete re-patterning in the remainder of the embryo. To understand the molecular mechanism of cell fate reprogramming, we examined micromere depletion induced non-skeletogenic mesoderm (NSM) transfating. We found that the skeletogenic program was greatly temporally compressed in this case, and that akin to another NSM transfating case, the transfating cells went through a hybrid regulatory state where NSM and skeletogenic marker genes were co-expressed.</p> / Dissertation

Adenylyl cyclase (Adcy) is an important early effector of adrenocorticotrophin (ACTH) on the adrenal cortex; however, this enzyme consists of ten isozymes in mammalian cells and the factors governing the expression of different Adcy isozymes have not been well defined. The aim of this study is to investigate the regulation of mouse Adcy4, one of ten isozymes, in Y1 adrenocortical tumor cells and in mutant subclones derived from the Y1 cells. Adcy4 is expressed at a high level in brain but at lower levels in many other tissues including the Y1 cells. Moreover, this isozyme is specifically deficient in Y1 mutants with impaired steroidogenic factor 1 (SF1) activity. These observations support a hypothesis that Adcy4 expression is influenced by both ubiquitously expressed and tissue-specific transcription factors. My sequencing results indicate that mouse Adcy4 is highly homologous to the human and rat counterparts; its gene is located less than 1 kb downstream of Ripk3 and contains 26 exons. Primer extension and in silico analyses suggest that Adcy4 contains a TATA-less promoter and initiates transcription from multiple sites. Luciferase reporter gene assays indicate that Adcy4 promoter activity is mainly stimulated by the proximal GC-rich region but is inhibited by the first intron. This 124 bp GC-rich region is well conserved among several mammalian species and exhibits strong promoter activity in Y1 cells, which is functionally compromised in the Adcy4-deficient mutant. Within this region, three Sp1/Sp3- and one SF1-binding sites have been identified which bind the corresponding proteins Sp1 and Sp3 or SF1 in electrophoretic mobility shift assays (EMSAs). Site-directed mutagenesis reveals that the 5’-most Sp1/Sp3 site enhances Adcy4 promoter activity, whereas the middle Sp1/Sp3 and SF1 sites each repress this activity. In Y1 mutant cells, mutating the SF1 site restores Adcy4 promoter activity and knocking down SF1 with shRNA increases Adcy4 expression. All these data demonstrate that Adcy4 expression is under the control of the ubiquitous factors Sp1 and Sp3 and the tissue-specific factor SF1 and establish that SF1 is a repressor for Adcy4 promoter activity. This study is the first to demonstrate a repressor function for SF1 in certain promoter contexts.

Fundamental aspects of biologically-inspired regulatory mechanisms are considered in a robotics context, using artificial neural-network control systems . Regulatory mechanisms are used to control expression of genes, adaptation of form and behavior in organisms. Traditional neural network control architectures assume networks of neurons are fixed and are interconnected by wires. However, these architectures tend to be specified by a designer and are faced with several limitations that reduce scalability and tractability for tasks with larger search spaces. Traditional methods used to overcome these limitations with fixed network topologies are to provide more supervision by a designer. More supervision as shown does not guarantee improvement during training particularly when making incorrect assumptions for little known task domains. Biological organisms often do not require such external intervention (more supervision) and have self-organized through adaptation. Artificial neural tissues (ANT) addresses limitations with current neural-network architectures by modeling both wired interactions between neurons and wireless interactions through use of chemical diffusion fields. An evolutionary (Darwinian) selection process is used to ‘breed’ ANT controllers for a task at hand and the framework facilitates emergence of creative solutions since only a system goal function and a generic set of basis behaviours need be defined. Regulatory mechanisms are formed dynamically within ANT through superpositioning of chemical diffusion fields from multiple sources and are used to select neuronal groups. Regulation drives competition and cooperation among neuronal groups and results in areas of specialization forming within the tissue. These regulatory mechanisms are also shown to increase tractability without requiring more supervision using a new statistical theory developed to predict performance characteristics of fixed network topologies. Simulations also confirm the significance of regulatory mechanisms in solving certain tasks found intractable for fixed network topologies. The framework also shows general improvement in training performance against existing fixed-topology neural network controllers for several robotic and control tasks. ANT controllers evolved in a low-fidelity simulation environment have been demonstrated for a number of tasks on hardware using groups of mobile robots and have given insight into self-organizing system. Evidence of sparse activity and use of decentralized, distributed functionality within ANT controller solutions are found consistent with observations from neurobiology.

This thesis mainly concerns modeling, approximation and inference of gene regulatory dynamics on the basis of gene expression patterns. The dynamical behavior of gene expressions is represented by a system of ordinary dierential equations. We introduce a gene-interaction matrix with some nonlinear entries, in particular, quadratic polynomials of the expression levels to keep the system solvable. The model parameters are determined by using optimization. Then, we provide the time-discrete approximation of our time-continuous model. We analyze the approximating model under the aspect of stability. Finally, from the considered models we derive gene regulatory networks, discuss their qualitative features of the networks and provide a basis for analyzing networks with nonlinear connections.

DNA methylation is an epigenetic modification in which a methyl group is covalently added to the DNA. In vertebrate genomes methylation occurs almost exclusively at cytosines immediately followed by a guanine (CpG dinucleotides). Two important aspects of DNA methylation have inspired several recent scientific investigations including those in this dissertation. First, methylated cytosines are hotspots of point mutation due to a methylation-dependent mutation mechanism, which has caused a deficiency of CpGs in vertebrate genomes. Second, DNA methylation in promoters is linked with transcriptional silencing of the associated genes. This dissertation presents the results of four studies in which I investigated the impacts of DNA methylation on the neutral and functional evolution of vertebrate genomes. The results of the first two studies demonstrate that DNA methylation has profound impacts on both inter- and intra-genomic neutral substitution rate variation. The third and fourth studies demonstrate that DNA methylation has played critical roles in shaping the evolution of vertebrate promoters and gene regulation.

The human genome contains tens of thousands of gene loci which code for an even greater number of protein and RNA products. The highly complex temporal and spatial expression of these genes makes possible all the biological processes of life. Altered gene expression by mutation or deregulation is fundamental for the development of many human diseases. The ultimate aim of this thesis was to identify gene expression changes relevant to cancer. The advent of genome-wide expression profiling techniques, such as microarrays, has provided powerful new tools to identify such changes and researchers are now faced with an explosion of gene expression data. Processing, comparing and integrating these data present major challenges. I approached these challenges by developing and assessing novel methods for cross-platform analysis of expression data, scalable subspace clustering, and curation of experimental gene regulation data from the published literature. I found that combining results from different expression platforms increases reliability of coexpression predictions. However, I also observed that global correlation between platforms was generally low, and few gene pairs reached reasonable thresholds for high-confidence coexpression. Therefore, I developed a novel subspace clustering algorithm, able to identify coexpressed genes in experimental subsets of very large gene expression datasets. Biological assessment against several metrics indicates that this algorithm performs well. I also developed a novel meta-analysis method to identify consistently reported genes from differential expression studies when raw data are unavailable. This method was applied to thyroid cancer, producing a ranked list of significantly over-represented genes. Tissue microarray analysis of some of these candidates and others identified a number of promising biomarkers for diagnostic and prognostic classification of thyroid cancer. Finally, I present ORegAnno (www.oreganno.org), a resource for the community-driven curation of experimentally verified regulatory sequences. This resource has proven a great success with ~30,000 sequences entered from over 900 publications by ~50 contributing users. These data, methods and resources contribute to our overall understanding of gene regulation, gene expression, and the changes that occur in cancer. Such an understanding should help identify new cancer mechanisms, potential treatment targets, and have significant diagnostic and prognostic implications.

Floral stripes are a common pigmentation pattern in plants. Defining the molecular mechanisms of the striped pattern formation will aid understanding of how a gene can be differentially regulated across a population of similar cells. In the venation phenotype of Antirrhinum majus, the anthocyanin pigment is typically confined to the adaxial epidermal cells overlaying the petal veins. To explore how this pattern forms this study focused on the expression and regulation of Venosa, a Myb regulator of anthocyanin biosynthesis. Pigment complementation experiments demonstrated that the lack of a MYB factor caused the lack of pigment in the cells outside the venation pigmentation domain. An allele of Venosa was isolated and identified. It was a mutant version of functional Venosa due to the central part being replaced by a transposon. Phenotype / genotype analysis indicated that the venation pigmentation patterning was due to the functional Venosa. In situ mRNA hybridisation showed that Venosa was expressed from the xylem to the adaxial epidermis, and was controlled spatially and quantitatively by a signal associated with the petal veins. Venosa expression provided the longitudinal axis for venation pigmentation stripes, and determined the location and intensity of the pigmented cells. Because another factor required for pigmentation, a bHLH factor, is specifically expressed in epidermal cells and it provides the transverse axis. The pigmented stripes are the cross expression domain of these two kinds of factors. The transcriptional controlling property of a 2.4 kb (relative to the ATG) promoter region of the Venosa gene was analysed. The -900 bp fragment was characterised in detail using 5'-end deletion mutagenesis. A heterologous host, tobacco, was used for analysis in stable transgenics. The homologous host, Antirrhinum, was used for transient assays. The efficacy and efficiency of different reporter genes (intron-containing GUS, GFP, Venosa cDNA and genomic Venosa) and enhancement systems (transcriptional enhancer, translational enhancer, inhibitor of post transcriptional gene silencing and a two-step signaling amplification system) for the detection of low-level reporter gene expression were also tested. The strength of expression correlated to the length of the promoter fragment, and expression was detected using deletions down to -500 bp, although only weak expression was found. This expression was flower specific but not vein related in both plant hosts. No expression was detected in petals of either host with fragments shorter than -500 bp. The results suggest that the fragment from -380 bp to -900 bp positively affected Venosa expression at the transcriptional level, but might not be sufficient to define venation. A possibility is that the venation controlling property is negatively controlled at the epigenetic level, such as DNA methylation status and / or chromatin structure. The role of gibberellin and sugar in the pigment and venation patterning formation of Antirrhinum was studied. The results suggest that gibberellin is not required for pigmentation or venation patterning. Convincing evidence on the role of sugar signaling could not be obtained from the experiments, due to the difficulty in separating the impact on pigmentation from other functions of sugars in petal development. In addition, the in situ analysis detected the expression of a gene probably related to aurone biosynthesis that may be a regulatory gene of this biosynthetic pathway.