Discovering Conserved cis-Regulatory Elements That Regulate Expression in Caenorhabditis elegans

The aim of this dissertation is two-fold:: 1) To catalog all cis-regulatory elements within the intergenic and intronic regions surrounding every gene in C.elegans: i.e. the regulome) and: 2) to determine which cis-regulatory elements are associated with expression under specific conditions. We initially use PhyloNet to predict conserved motifs with instances in about half of the protein-coding genes. This initial first step was valuable as it recovered some known elements and cis-regulatory modules. Yet the results had a lot of redundant motifs and sites, and the approach was not efficiently scalable to the entire regulome of C. elegans or other higher-order eukaryotes. Magma: Multiple Aligner of Genomic Multiple Alignments) overcomes these shortcomings by using efficient clustering and memory management algorithms. Additionally, it implements a fast greedy set-cover solution to significantly reduce redundant motifs. These differences make Magma ~70 times faster than PhyloNet and Magma-based predictions occur near ~99% of all C. elegans protein-coding genes. Furthermore, we show tractable scaling for higher-order eukaryotes with larger regulomes. Finally, we demonstrate that a Magma-predicted motif, which represents the binding specificity for HLH-30, plays a critical role in the host-defense to pathogenic infections. This novel finding shows that hlh-30(-) animals are more susceptible to S. aureus and P. aeruginosa than their wild type counterparts

High-throughput genomic/proteomic studies : finding structure and meaning by similarity

The post-genomic challenge was to develop high-throughput technologies for measuring genome scale mRNA expression levels. Analyses of these data rely on computers in an unprecedented way to make the results accessible to researchers. My research in this area enabled the first compendium of microarray experiments for a multi-cellular eukaryote, Caenorhabditis elegans. Prior to this research approximately 6% of the C. elegans genome had been studied, and little was known about global expression patterns in this organism. Here I cluster data from 553 different microarray experiments and show that the results are stable, statistically significant and highly enriched for specific biological functions. These enrichments allow identification of gene function for the majority of C. elegans genes. Tissue specific expression patterns are discovered suggesting the role of particular proteins in digestion, tumor suppression, protection from bacteria and from heavy metals. I report evidence that genome instability in males involves transposons, and find co-expression patterns between sperm proteins, protein kinases and phosphatases suggesting that sperm, that are transcriptionally inactive cells, commonly use phosphorylation to regulate protein activities. My subsequent research addresses protein concentrations and interactions, beginning with a simultaneous comparison of multiple data sets to analyze Saccharomyces cerevisiae gene-expression (cell cycle and exit from stationary phase/G0) and protein-interaction studies. Here, I find that G1-regulated genes are not co-regulated during exit from stationary phase, indicating that the cells are not synchronized. The tight clustering of other genes during exit from stationary-phase does indicate that the physiological responses during G0 exit are separable from cell-cycle events. Subsequently, I report in vivo proteomic research investigating population phenotypes in stationary phase cultures using the yeast Green Fluorescent Protein-fusion library (4156 strains) together with flow cytometry. Stationary phase cultures consist of dense quiescent (Q) and less dense non-quiescent (NQ) fractions. The Q-cell fraction is generally composed of daughter cells with high concentrations of proteins involved in the citric acid cycle and the electron transport chain, for example Cit1p. The NQ fraction has subpopulations of cells that can be separated by the low and high concentrations of these mitochondrial proteins, i.e., NQ cells often have double intensity peaks: a bright fraction and a much dimmer fraction, which is the case for Cit1p. The Q fraction uses oxygen 6 times as rapidly as the NQ fraction, and 1.6 times as rapidly as exponentially growing cells. NQ cells are less reproductively capable than Q cells, and show evidence of reactive oxygen species stress. These phenotypes develop as early as 20-24 hours after the diauxic shift, which is as early as we can make a differentiating measurement using fluorescence intensities. Finally, I propose a new way to analyze multidimensional flow cytometry data, which may lead to better understanding of Q/NQ cell differentiation

Identifying modifiers of age‐dependent protein aggregation in C. elegans

The misfolding of specific proteins and their accumulation in insoluble aggregates has long been recognized as a pathological hallmark of several neurodegenerative diseases. In recent years, widespread protein aggregation occurring during healthy aging has become a hot topic of research. However, to this date little is known about the regulation of this aggregation, the tissue‐specificity and the consequences in a disease context. This thesis answers several questions about different aspects of protein aggregation with aging and in disease. Notably, we analysed the solubility of RNA‐binding proteins that are important for the formation of stress granules (sgRBPs) in the nematode Caenorhabditis elegans (C. elegans). We showed the impact of sgRBP insolubility on organismal health and the importance of maintaining their solubility in long‐lived animals. We identified regulators of sgRBP aggregation. In addition, we showed that aggregation‐prone sgRBPs are highly prone to interact with other proteins and that this co‐localization can influence aggregation patterns or protein localization. Furthermore, we analysed the tissue‐specificity of the regulation of age‐related protein aggregation. Disruption of the protein‐quality control network has contrasting effects on protein aggregation in different tissues, surprisingly reducing age‐related protein aggregation in the pharyngeal muscle of C. elegans. Specifically, we showed that impaired proteinquality control prevented the accumulation of newly synthesized aggregation‐prone proteins. Additionally we demonstrated how screening approaches identifying mutations that influence disease‐associated phenotypes, like protein aggregation in C. elegans, can help prioritise variants found by whole exome sequencing in large cohorts of patients with Parkinson’s disease. To validate promising candidates found to be influencing protein aggregation in C. elegans, we have established a cell culture model of age‐related protein aggregation. In conclusion, these findings give important insights into mechanism and regulation of age‐related protein insolubility and highlight the importance of age‐related protein aggregation for neurodegenerative diseases

Neural Circuit Dependence of Acute and Subacute Nociception in C. Elegans

Nociception, the detection and avoidance of harmful cues, is a crucial system in all organisms. Animals use nociceptive systems to escape from substances that decrease survival, and can also modulate the threshold for avoidance behaviors to weigh the attractive features of an environment against its harmful features. To allow regulation, the nociception system of mammals incorporates multiple feedback and feedforward loops in its central and peripheral pathways. The nociception system of the roundworm Caenorhabditis elegans shares many features of the mammalian circuit. Both neural circuits feature a direct path from sensory neurons to motor neurons that is connected by a single class of interneuron, bypassing the higher processing centers. Both neural circuits also feature higher processing pathways that receive information from sensory neurons and provide further input onto the direct pathway. While the anatomical wiring of the C. elegans nervous system has been known for decades, how sensory neurons access different downstream paths in the circuit is less clear. One possible route of differential access of sensory input to downstream neurons is through different dynamics of activation. The temporal dimension of neural circuits cannot be deduced by anatomical wiring, but must be measured directly. In my thesis, I have characterized and manipulated the dynamic properties of a classical nociceptor in C. elegans, the polymodal sensory neuron ASH, and asked how these properties instruct downstream circuits and behavior. I thus first elucidated ASH calcium activation dynamics using simple step responses and using a newly developed systems identification approach for C. elegans. Using both long pulses and rapidly fluctuating “white noise” sequences of different nociceptive stimuli, I deduced their ASH activation profiles and linear temporal filters describing how the neuron summates the history of stimulus encounter. This analysis demonstrated that ASH calcium responses to natural stimuli include both linear features and multiple nonlinear components. Mutations in G protein-coupled sensory signaling disrupt both fast linear filtering and sustained responses to nociceptive stimuli. Mutations in a voltage gated calcium channel alter the temporal qualities of the ASH response in a pattern suggesting a role of this channel in sensory adaptation. In the course of these studies, I discovered several additional classes of sensory neurons that respond to nociceptive stimuli with robust calcium responses, even though past studies did not demonstrate a role for these neurons in nociceptive behavior. To gain experimental control over the dynamic activity that initiates nociceptive signaling, I ectopically expressed the pheromone receptors SRG-34 and SRG-36 in ASH and activated this system with their endogenous ligand, the ascaroside C3. ASH does not normally detect C3, but when it expresses either of these receptors it generates robust calcium responses to C3. These calcium signals have distinct temporal dynamics: SRG-34 mediated calcium signals are fast rising and fast adapting, while SRG-36 mediated calcium signals increase slowly during stimulation with little adaptation. Expression of SRG-34 or SRG-36 in ASH caused animals to avoid C3. Remarkably, time-aligned histograms of C3-induced avoidance behavior during stimulus onset, presence, and removal closely followed the dynamics of ASH calcium activity at these same time points, with a fast onset and adaptation for SRG-34 and a slow, sustained avoidance of SRG-36. ASH can directly activated the backward command motor neuron AVA or indirectly activate AVA through other neuronal pathways, including the intermediate interneuron AIB. Selectively silencing the AIB interneuron with the a chemical genetics system using the histamine-gated chloride channel resulted in complete loss of nociceptive avoidance behaviors induced by slow-ramping SRG-36 receptor in ASH, but had less of an effect on SRG-34 avoidance. Selectively silencing the AVA backward command interneuron reduced reversals, but spared or increased other avoidance behaviors for both SRG-34 and SRG-36. These results indicate that downstream interneurons are engaged in different ways, and to different degrees, depending on the mechanism of ASH activation. I next monitored the activity of AIB and AVA neurons in freely-moving ASH:srg-34 or ASH:srg-36 animals responding to C3. In ASH:srg-34 animals, AIB and AVA begin increasing activity upon C3 onset. In ASH:srg-36 worms, AIB increased activity before AVA. Together with my AIB silencing results, these observations suggest that AIB accumulates signals from ASH over time to promote AVA activity. Using a coherent type-1 feed forward loop with a calcium slope-determined AND or OR logic, I modeled features of AIB contribution to nociceptive behaviors in response to different ASH temporal dynamics. These findings suggest that feedforward excitation loops, a motif seen in C. elegans and mammalian nervous systems, can result in behaviorally-salient consequences in response to different sensory neuron calcium dynamics

Integrative statistical methods for decoding molecular responses to insect herbivory in Nicotiana attenuata

This work focuses on the development of statistical methods to select features (genes and metabolites) exhibiting induced local and systemic defense responses to insect attack in Nicotiana attenuata along with the extraction of additional information regarding their timing of action. To characterize the dynamics of activation in time and space of herbivory-induced responses, I designed a framework by combining methods previously developed for feature selection and extraction to identify activated network motifs. These motifs are the set of features that are differentially perturbed in local and systemic tissues in response to herbivory. The extraction of multifactorial statistical information in terms of time response variable simultaneously captured the dynamic response of a gene/metabolite in more than one tissue and therefore helped in identifying tissue-specific activation of biochemical pathways during herbivory, their transition points and shared patterns of regulation with other physiological processes and gene-metabolite interactions at the level of isolated motifs. I utilized this framework to evaluate the transcriptional and metabolic dynamics in the roots to investigate their role in aboveground stress responses. I discovered an emergent property of an inversion in root-specific semidiurnal (12h) rhythms in response to simulated leaf herbivory. In addition, I illustrated the benefits of our statistical framework, used for generating spatio-temporally resolved transcriptional/metabolic maps, by visualizing the chronology of the activation of pathways central to signaling, tolerance and defense in N. attenuata. The research described in this thesis, in addition to being valuable in deciphering dynamic responses to insect attack in a whole plant context, lays the foundation for future analyses in which statistical modeling of these networks assisted with experimental data could predict the logical rules governing these dynamic interactions

Identification of transcription factor targets, gene expression profiles and accessible chromatin regions in the Caenorhabditis elegans epidermis using targeted DamID

Development is an exceptionally complex process that is performed with exquisite control. A series of developmental programmes allow the orchestrated and tightly-regulated deployment of the genomic information, governing events like cell division, cell fate maintenance and differentiation. Understanding the complete regulatory states that instruct a selective decoding of the genome capable of bringing about morphogenetic events is central to developmental biology. Among all cells, stem cells maintain the potential to produce cells that undergo transitions in developmental trajectories and thus are particularly interesting. In this study, I have used the postembryonic development of the Caenorhabditis elegans epidermis driven by the stem cell-like seam cells, to begin exploring the gene regulatory network, transcriptional states and epigenomic regulation involved in cell fate patterning. To that end, I have adapted and present here the first application of the targeted DamID (TaDa) methodology in C. elegans, for assaying protein-DNA interactions, to use as a single technique in approaching all of the above objectives. I show that TaDa requires little starting material, is reproducible and tissue-specific. Using TaDa I identify targets for the transcription factors LIN-22 and NHR-25 that propose new biological functions for these regulators in epidermal development. I acquire gene expression profiles for the seam cells and hypodermis that lead to the discovery of novel transcription and chromatin factors, as well as new miRNAs. Finally, I produce the first cell-type-specific chromatin accessibility maps in C. elegans for the seam cells and hypodermis and use them to identify tissue-specific enhancers. These findings expand our knowledge of the mechanisms underlying fate decisions in epidermal patterning and provide a proof-of-concept for the application of TaDa in C. elegans.Open Acces