There are about 20,000 genes in the human genome. The lowly nematode worm, C. elegans, has about the same number of genes. How could two organisms that are so different arise from a similar number of genes? The answer is epigenetics, or the factors that help control when and where genes are expressed. There are many layers that comprise the epigenetic control of genes. One of which is the structure or architecture of chromatin. Chromatin is a complex of DNA and proteins. Histone proteins with DNA wrapped around them form the fundamental component of chromatin, the nucleosome. Chromatin exists in two forms, euchromatin and heterochromatin. Euchromatin is made of loosely packed nucleosomes while in heterochromatin nucleosomes are tightly packed. Genome elements are not accessible in heterochromatin but are in euchromatin. In this way chromatin architecture provides a layer of control of genetic expression. Where nucleosome form is a function of several factors including the underlying DNA sequence, and binding competition between histones and other DNA binding proteins. Here we test the ability of various DNA sequences to position and repel histone proteins in C. elegans worms. We find that the 601sequence can position nucleosomes and that the PRS-322 sequence does repel nucleosomes in vivo. Assessing chromatin architecture requires sequences to be aligned to a reference genome, however, there are numerous programs with which to do this. Each program performs this task in a different way. These differences can have a large impact on the downstream analysis of the results. To this end, we have tested various alignment programs to assess how well they align reads to a reference genome. Here we have found that Bowtie2, BWA, and Chromap perform alignments accurately and we suggest using them. As an organism develops its genetic expression changes. This change in expression is often the result of temporally specific genomic elements such as enhancers. Understanding when enhancers are accessible during development can lead to a better understanding of the genetic control needed for development. Here we utilize data gathered at specific developmental stages in C. elegans to elucidate enhancer accessibility. In this work we have furthered the understanding of epigenetic control of expression by quantifying positioning and repelling sequences, testing read mapping programs for accuracy and identifying temporally specific enhancers in developing worms.

No description available.

No description available.

<p> Quantum dots are semiconductor nanocrystals with unique optical properties that give them the potential to be excellent probes for bio-imaging applications. However, before quantum dots can be employed for such applications, their toxicity and cellular interactions need to be thoroughly assessed. The Caenorhabditis elegans (C. elegans) embryo was chosen as a test environment to study both the toxicity and dynamics of carboxyl terminated CdSe/ZnS quantum dots. Using confocal imaging, it was found that the C. elegans embryo is not morphologically affected by the introduction of quantum dots up to a concentration of about 1 OOnM. However, the embryo was observed to respond to the nanomaterial by packaging it into aggregates during development in a dose and time dependant manner. Image analysis and fluorescence correlation spectroscopy revealed that this packaging process happens from the nm scale to the J.Ull scale and that it reduces quantum dot mobility over development. This work shows that the dynamics of the quantum dots are highly influenced by the cellular environment in the embryo, as they appear to aggregate and possibly also interact with cellular structures and organelles in the embryo. </p> / Thesis / Master of Applied Science (MASc)

Caenorhabditis elegans es un nematodo de vida libre utilizado como organismo modelo en diferentes disciplinas de la ciencia. Su tamaño reducido, plan corporal anatómicamente simple, ciclo de vida corto y amplio repertorio de comportamientos, lo han transformado en un organismo muy útil en investigación. Además, emerge como un modelo de interés en la industria farmacéutica para realizar ensayos in vivo rápidos y económicos, y para la detección de compuestos con actividad biológica. C. elegans comparte características fisiológicas y farmacológicas con nematodos parásitos y además es sensible a la mayoría de las drogas antiparasitarias que se utilizan en el hombre y en los animales. Dado que es difícil trabajar con nematodos parásitos en el laboratorio, C. elegans ha emergido como un excelente modelo de nematodo parásito y ha contribuido al conocimiento de los mecanismos de acción de diversos fármacos. C. elegans cuenta con la mayor familia de receptores Cys-loop. En sus músculos, posee tres receptores Cys-loop principales: dos receptores nicotínicos (nAChRs), el L-AChR y el N-AChR, y el receptor de GABA, UNC-49. Los nAChRs median la contracción de los músculos de la pared del cuerpo mientras que los receptores de GABA median la relajación muscular, permitiendo el movimiento sinusoidal típico del nematodo. Estos receptores son los blancos moleculares de drogas antihelmínticas. El levamisol, actuando como agonista del L-AChR, genera contracción sostenida de los músculos y finalmente la parálisis espástica del nematodo. La piperazina, actuando como agonista de los receptores de GABA, genera relajación muscular y parálisis flácida. Otros receptores Cys-loop presentes en el nematodo también son blancos de fármacos antihelmínticos. El receptor de glutamato permeable a cloruro (GluCl) presente en neuronas y células musculares es el blanco molecular de la ivermectina (IVM), uno de los antiparasitarios más utilizados a nivel mundial. En cuanto a los receptores Cys-loop, C. elegans no es más diferente a los nematodos parásitos de lo que cada especie individual de parásito lo es de otra. Esto se evidencia en la amplia diversidad de subunidades que generan receptores Cys-loop con diferente composición y propiedades farmacológicas en los nematodos y cuyas bases moleculares no se comprenden completamente. En esta Tesis se utilizó a C. elegans como modelo de nematodo parásito. Se estudiaron las propiedades antihelmínticas y los blancos de acción de diferentes compuestos químicos a través de ensayos de comportamiento. Para determinar sus mecanismos de acción se realizaron registros electrofisiológicos de corrientes unitarias y macroscópicas sobre receptores presentes en células musculares de C. elegans o expresados heterologamente en células de mamífero. En el Capítulo 1 se estudió el befenio, un antihelmíntico colinérgico cuyo modo de acción no se conocía completamente. Mediante ensayos de comportamiento se determinó que befenio genera parálisis espástica en nematodos salvajes adultos jóvenes. Utilizando cepas mutantes se determinó que el L-AChR es el blanco molecular involucrado en la actividad paralizante de befenio. Estos resultados sugieren que no existiría un receptor específico para befenio en los músculos de C. elegans. Cuando befenio fue combinado con levamisol el efecto paralizante fue aditivo. Esto es de importancia ya que la combinación de drogas es una buena estrategia para reducir la resistencia en nematodos parásitos. A nivel molecular, mediante registros de canal único, se determinó que befenio activa el L-AChR de C. elegans tanto en larvas L1 como L2, y a mayores concentraciones, actúa como un bloqueador de canal abierto de dicho receptor. Los estudios de docking molecular mostraron que befenio se une al sitio de unión ortostérico del agonista y forma las interacciones cation-π requeridas para la activación del receptor. Estos resultados podrían explicar la alta eficacia para activar el L-AChR. La selectividad de befenio por el nAChR muscular de mamífero fue estudiada mediante registros de canal único y de corrientes macroscópicas. Se determinó que befenio activa el nAChR pero actúa como un agonista muy débil y un bloqueador de canal potente. Según estudios de docking molecular, befenio generaría las interacciones necesarias para la activación solamente en uno de los dos sitios ortostéricos del receptor. Esto explicaría su baja eficacia en receptores de mamífero con respecto a los receptores de nematodos. Cepas mutantes de C. elegans que carecen de la subunidad LEV-8 podrían contener LAChRs formados por la subunidad ACR-8 en su reemplazo. Estos L-AChRs imitan un receptor de nematodo parásito, como el receptor de H. contortus, cuya subunidad ACR-8 podría mediar la actividad de befenio. Mediante ensayos de comportamiento con la cepa mutante se determinó que la subunidad ACR-8 no es requerida para el efecto paralizante de befenio en C. elegans. A nivel de canal único, los receptores que carecen de la subunidad LEV-8 también fueron activados por befenio y dicha droga, al igual que ACh, indujo una rápida desensibilización del receptor. En el Capítulo 2 se estudiaron tres terpenoides, carvacrol, timol y eugenol, presentes en plantas. Mediante ensayos de comportamiento utilizando nematodos salvajes, se determinó que los terpenoides paralizan rápidamente a C. elegans. El orden de potencia de parálisis fue: carvacrol>timol>eugenol. Las larvas fueron más sensibles que los nematodos adultos jóvenes. Además, los compuestos inhibieron irreversiblemente la eclosión de los huevos con el mismo orden de potencia. Estos hallazgos indican que los terpenoides producen efectos antihelmínticos a corto y largo plazo. Se evaluaron tres combinaciones de drogas: timol/levamisol, timol/piperazina y timol/ivermectina. El efecto paralizante de la combinación timol/levamisol fue sinérgico y dicha combinación también fue efectiva en la inhibición de la eclosión de huevos. Mediante ensayos de comportamiento con nematodos mutantes se determinó que los L-AChRs y los receptores de GABA son los blancos moleculares de los terpenoides. Los registros de corrientes macroscópicas revelaron que los compuestos no son capaces de activar los receptores, pero inhiben las corrientes evocadas por los agonistas. En registros de canal único, los terpenoides disminuyeron la actividad de L-AChRs generada por ACh y levamisol, redujeron la frecuencia de aperturas del L-AChR e indujeron un componente de estado cerrado más prolongado. Sin embargo, no afectaron las propiedades del canal como la conductancia y la duración de apertura. El análisis global indicó que los terpenoides ejercen su efecto antihelmíntico actuando como antagonistas no competitivos del L-AChR. En el Capítulo 3 se estudió la doxepinona, considerada una estructura química privilegiada. Mediante ensayos de comportamiento se demostró que la doxepinona ejerce su acción paralizante sobre nematodos salvajes adultos jóvenes actuando a través el GluCl, el blanco molecular de la IVM. Este compuesto sintético generó parálisis estacionaria en nematodos salvajes. La IVM actúa sobre GluCls presentes en la faringe del nematodo e inhibe el bombeo faríngeo. Doxepinona también redujo la velocidad de bombeo faríngeo en nematodos salvajes y el efecto fue mediado por los GluCls. Mediante registros de corrientes macroscópicas se caracterizaron las corrientes del receptor heteromérico GluCl α 1/GluClß de C. elegans evocadas por el agonista glutamato. Se determinó que la doxepinona no es un agonista de dicho receptor ya que no es capaz de activarlo. Mediante diferentes protocolos de aplicación de drogas, se determinó que la doxepinona actúa como un inhibidor alostérico de los GluCls. Se propuso a la inhibición del GluCl como un nuevo mecanismo antihelmíntico. En resumen, en esta Tesis Doctoral, utilizando a C. elegans como modelo de nematodo parásito, se identificaron los sitios y se descifraron los mecanismos de acción molecular de diferentes compuestos químicos, con actividad antihelmíntica. / Caenorhabditis elegans is a free-living nematode used as a model organism in different science disciplines. Its reduced size, anatomically simple body plan, short life cycle and broad repertoire of behaviours have turned it in a useful organism for research. It also emerges as an interesting model in the pharmaceutical industry for fast and cheap in vivo assays and for the detection of compounds with biological activity. C. elegans shares pharmacological and physiological characteristics with parasitic nematodes and is sensitive to most antiparasitic drugs used in humans and animals. Given that parasitic nematodes are difficult to work with in the laboratory, C. elegans has emerged as an excellent parasitic model and has contributed to the understanding of mechanisms of action of anthelmintic drugs. C. elegans has the largest Cys-loop receptor family. In its muscle, it has three main Cysloop receptors: two nicotinic receptors (nAChRs), L-AChR and N-AChR, and the UNC-49 GABA receptor. nAChRs mediate body wall muscle contraction while GABA receptors mediate muscle relaxation, thus allowing the typical sinusoidal movement of the nematode. These receptors are the molecular targets of anthelmintic drugs. Levamisole, acting as an L-AChR agonist, generates sustained muscle contraction which ends in spastic paralysis of the nematode. Piperazine, by acting as an agonist of GABA receptors, generates muscle relaxation and flaccid paralysis. Other Cys-loop receptors in the nematode are also targets of anthelmintic drugs. The glutamate-activated chloride channel (GluCl) present in neurons and muscle cells is the molecular target of ivermectin (IVM), which is one of the most used antiparasitic drug worldwide. Considering Cys-loop receptors, C. elegans is no more dissimilar to parasitic nematodes than each individual species of parasite is to another. This results from the wide subunit diversity that generates Cys-loop receptors with different compositions and pharmacological properties among nematodes; the molecular basis of this diversity remains not fully understood. In this Thesis, C. elegans was used as parasitic nematode model. The anthelmintic properties and molecular targets of different chemical compounds were studied through behavioural assays. To determine their mechanisms of action, electrophysiological recordings, single-channel and macroscopic current recordings, were carried out in C. elegans muscle cells or in mammalian cells heterologously expressing the receptor under study. In Chapter 1 bephenium was studied. It is a cholinergic anthelmintic drug whose mechanism of action was not fully understood. Through behavioural assays it was determined that bephenium generates spastic paralysis in young adult wild-type worms. By using different mutant strains, it was determined that L-AChR is the molecular target involved in the paralyzing activity of bephenium. The results suggested that there may not be a specific receptor for bephenium in C. elegans muscle. When bephenium was combined with levamisole, the paralyzing effects were additive; which is of significance since drug combination is a good strategy to reduce resistance in parasitic nematodes. At the molecular level, through single channel recordings, it was determined that bephenium activates L-AChR in larvae L1 and L2 C. elegans. At higher concentrations, it acted as an L-AChR open channel blocker. Molecular docking studies showed that bephenium binds to the orthostetic agonist binding site and forms the cation-π interactions required for receptor activation. This result may explain the high efficacy for L-AChR activation. Bephenium selectivity for the mammalian muscle nAChR was studied through singlechannel and macroscopic current recordings. Bephenium activated nAChRs, but it acted as a very weak agonist and a potent channel blocker. According to the molecular docking studies, bephenium would generate the necessary interactions for activation only in one of the two orthosteric sites of the receptor. This may explain the low efficacy in the mammalian receptor with respect to nematode receptors. C. elegans mutant strains that lack LEV-8 subunit may have L-AChRs containing the spare ACR-8 subunit in its replacement. These L-AChRs may mimic those in certain nematode parasites, like the H. contortus receptor, for which it was suggested that its ACR-8 subunit may mediate bephenium activity. Through behavioural assays in the mutant strain, it was determined that the ACR-8 subunit is not required for the paralyzing effects of bephenium on C. elegans. At the single channel level, the receptors that lack LEV-8 subunit were similarly activated by bephenium. Bephenium, like ACh, induced fast receptor desensitization. In the Chapter 2 terpenoids present in plants (carvacrol, thymol and eugenol) were studied. Through behavioural assays in wild-type nematodes, it was determined that terpenoids produced fast paralysis of the worms. The paralyzing potency order was: carvacrol > thymol > eugenol. The larvae were more sensitive than young adults. Also, the compounds irreversibly inhibited egg hatching with the same potency order. These findings indicate that terpenoids generate short- and long-term anthelmintic effects. Three drug combinations were evaluated: thymol/levamisole, thymol/piperazine and thymol/ivermectin. The paralyzing effect of thymol/levamisole combination was synergic, and this combination was effective in the inhibition of egg hatching too. Through behavioural assays in mutant nematodes, it was determined that L-AChRs and GABA receptors are the molecular targets of the terpenoids. The macroscopic current recordings revealed that the compounds could not activate the receptors but inhibited the currents evoked by the agonists. In single channel recordings, terpenoids reduced L-AChR activity generated by ACh and levamisole, reduced the frequency of L-AChR openings and induced a longer closed state component. However, terpenoids did not affect channel properties, such as conductance and open duration. The global analysis indicated that, terpenoids exert their anthelmintic effect, acting as L-AChR non-competitive antagonists. In the Chapter 3, doxepinone was studied. Doxepinone is considered a privileged chemical structure. Through behavioural assays, it was demonstrated that doxepinone exert the paralyzing action in wild-type young adult worms acting through GluCls, which are the molecular targets of IVM. The synthetic compound generated stationary paralysis on wild-type worms. IVM acts on nematode pharyngeal GluCls and inhibits pharyngeal pumping. Doxepinone also reduced the pharyngeal pumping rate in wild-type worms and the effect was mediated by GluCls. Through macroscopic current recordings, the responses of GluCl α1/GluClß receptors of C. elegans evoked by the agonist glutamate were characterized. It was determined that doxepinone is not a GluCl agonist because it is not capable of activating the receptor. Through different drug application protocols, it was determined that doxepinone acts as an allosteric inhibitor of GluCls. The inhibition of GluCls was proposed as a new anthelmintic mechanism. In summary, in this Doctoral Thesis, using C. elegans as a model of parasitic nematode, the target sites and mechanisms of action of different chemical compounds with anthelmintic activity were deciphered.

Yes / The application of omics to study Caenorhabditis elegans (C. elegans) in the context of spaceflight is increasing, illuminating the wide-ranging biological impacts of spaceflight on physiology. In this review, we highlight the application of omics, including transcriptomics, genomics, proteomics, multi-omics, and integrated omics in the study of spaceflown C. elegans, and discuss the impact, use, and future direction of this branch of research. We highlight the variety of molecular alterations that occur in response to spaceflight, most notably changes in metabolic and neuromuscular gene regulation. These transcriptional features are reproducible and evident across many spaceflown species (e.g., mice and astronauts), supporting the use of C. elegans as a model organism to study spaceflight physiology with translational capital. Integrating tissue-specific, spatial, and multi-omics approaches, which quantitatively link molecular responses to phenotypic adaptations, will facilitate the identification of candidate regulatory molecules for therapeutic intervention and thus represents the next frontiers in C. elegans space omics research.

Under unfavorable environmental conditions Caenorhabditis elegans larvae enter a dauer stage which is a specialized non-feeding larval stage. In the dauer stage, worms display astonishingly low metabolism, which allows them to adapt themselves to environmental stress and to dwell without food for several months. Dauer larvae can enter into the reproductive larval stage, when environmental conditions become favorable. In this study, the metabolic transition of dauers into the reproductive larval stage is analyzed in detail: a. During the exit of dauers, several metabolic traits were examined. Primarily, dauer larva initiates the metabolic transition by activating feeding, which is followed by upregulated oxygen consumption and mitochondrial remodeling, as well as enhanced protein synthesis. b. To better understand the metabolic transition, inhibitors of the dauer exit were introduced. Lithium ions were shown to inhibit the transition of dauers to reproductive larvae and prevent the upregulation of metabolic activities required for this process. c. In liquid culture, the transition from the dauer to the reproductive larva is also inhibited, presumably because of the hypoxic character of the liquid culture. Thus, hypoxia has a negative effect on the metabolic transition. d. In the course of our investigation we discovered that the dauer larva is not a closed system but indeed, it can dwell on the externally available ethanol as a carbon source by incorporating it into the energy metabolism. This allows dauers to survive for longer periods in the absence of bacteria, the preferred food of worms. These findings clarify the nature of dauers, how they utilize distinct pathways during the metabolic transition and how they take advantage of the externally available carbon source. These results may in the future enable us to elucidate the complex pathways of metabolism, as well as the ways in which it can be regulated.

Depuis une vingtaine d’années, il est possible d’allonger la durée de vie génétiquement. Nombre d’études réalisées sur des espèces allant de la levure aux primates, ont permis d’identifier des cascades de signaux intracellulaires ayant un impact sur la longévité et la qualité du vieillissement. Il est important de noter que certaines de ces interventions réduisent considérablement l’incidence de cancers et de maladies liées au vieillissement chez les mammifères. Ceci témoigne des liens existant entre vieillissement et carcinogénèse et il probable que le développement de stratégies pharmacologiques ayant pour cible le vieillissement se révèlent efficaces contre les maladies du vieillissement comme le cancer, la maladie d’Alzheimer ou de Parkinson. Nous avons criblé la banque de mutants de Saccharomyces cerevisiae pour identifier des mutations génétiques qui augmentent la durée de vie. La plupart des gènes identifiés se sont révélés conservés puisqu’ils influencent aussi la longévité chez C. elegans. La protéine de liaison à l’acyl-CoA (ACBP) est une petite protéine (10 kDa) qui se lie avec une haute affinité aux chaîne d’acyl-CoA esters (moyennes et longues) et les transporte vers les sites de consommation de l'acyl-CoA. ACBP est hautement conservée parmi les espèces eucaryotes et joue un rôle important dans la biosynthèse des lipides et le trafic vésiculaire. Chez Saccharomyces cerevisiae, la délétion d’ACBP (ACB1) entraîne une augmentation de la longévité et favorise la résistance au stress. Pour tester si l’impact d’ACBP sur la longévité s'étend aux eucaryotes supérieurs, nous avons exploré le lien entre les gènes codant pour des ACBPs chez Caenorhabditis elegans et la longévité en utilisant l’ARN interfèrent. Chez C. elegans, sept paralogues ACBP ont été identifiés, qui sont exprimés dans différents tissus. Nous avons constaté que la réduction de l'expression de maa-1 (codant une ACBP associée aux membranes) prolonge la durée de vie des vers sauvages. Nos résultats démontrent que: 1) une perte de fonction de maa-1 entraîne une résistance au superoxyde, 2) et aux événements protéotoxiques telle que l'agrégation protéique associées aux maladies neurodégénératives comme la maladie de Huntington. Enfin, nous avons montré que l'activité du facteur de transcription HIF-1 (hypoxia inducible factor-1) contribue à la longévité causée par la mutation maa-1. En effet, la délétion du gène hif-1 annule complètement l’augmentation de la longévité causée par maa-1. / Understanding the aging process, its regulation, and how to delay it has become a priority for an increasing number of scientists worldwide. The principal reason for this is that it is becoming more and more evident that anti-aging interventions may be effective against age-related diseases such as cancer, cardiovascular, and neurodegenerative diseases. Simple model organisms such as Caenorhabditis elegans and Saccharomyces cerevisiae have been instrumental to identify the principal genes implicated in aging whose role has turned out to be conserved in mammals. The project presented here has originated from a genome-wide screen performed in S. cerevisiae that has led to discover several novel life span-regulatory genes whose deletion prevents aging. One of these genes encodes for Acyl-CoA binding protein (ACBP). ACBP is a small (10 kDa) protein that binds medium- and long-chain fatty acyl-CoA esters with high affinity and transports them to acyl-CoA consuming processes. ACBP is highly conserved among eukaryotic species and plays important roles in lipid biosynthesis and vesicle trafficking. In S. cerevisiae, lack of ACBP (Acb1) extends longevity and promotes stress resistance. To test whether the life span-regulatory role of ACBP extends to higher eukaryotes, we explored the link between the C. elegans ACBP genes and longevity by RNAi screening. In C. elegans, seven ACBP paralogs have been identified, which are expressed in different tissues. We found that reducing the expression of maa-1 (encoding a membrane associated ACBP) extended the longevity of wild-type worms. Our results show that 1) a loss of function maa-1 mutant is resistant to the superoxide-generating agent paraquat and 2) reduction of maa-1 expression increases resistance to the proteotoxicity associated with the aggregation of the Huntington's disease-associated polyQ peptide. The activity of the anti-aging transcription factor HIF-1 (hypoxia inducible factor-1) contributes to the extended longevity caused by lack of maa-1. The effect of MAA-1 loss on longevity was fully reverted by the deletion of the hif-1 gene.

L’adaptation cellulaire au stress dépend en partie de changements dans l’expression de gènes de réponse au stress, souvent accompagnés par des modifications dans la structure chromatinienne. Des facteurs chromatiniens pourraient être à l’origine de ces modifications mais leurs mécanismes d’action restent mal connus au cours du développement. La réponse aux protéines malconformées (UPR) est une réponse à des conditions de stress physiologique qui ciblent le réticulum endoplasmique (RE) ; l’UPR a été impliquée dans de nombreuses maladies humaines incluant le cancer et différents composants de cette réponse pourraient être de potentielles cibles pharmaceutiques. Nous avons démontré que HPL-2, l’homologue de la protéine HP1 chez Caenorhabditis elegans, est nécessaire pour la réponse au stress du RE. L’inactivation d’HPL-2 montre une résistance accrue au stress du RE qui dépend d’une part de la voie XBP-1 de l’UPR et d’autre part d’un flux autophagique augmenté. La résistance accrue des vers dépourvu d’HPL-2 est associée avec une augmentation de l’activation d’XBP-1 et de chaperonnes du RE en conditions physiologiques. L’expression d’HPL-2 est ubiquitaire et nous avons déterminé qu’HPL-2 joue un rôle antagoniste dans les cellules neuronales et intestinales pour influencer la réponse au stress du RE. Nous avons également montré qu’une modulation de l’état de la chromatine par une inhibition chimique d’histones déacétylases donnait le même phénotype que l’absence d’HPL-2. De plus, l’augmentation ou la diminution de la méthylation de la lysine 4 de l’histone 3 (H3K4me) joue également un rôle dans la réponse au stress du RE. Ces travaux contribuent ainsi à une meilleure compréhension du lien entre l’UPR, le stress du RE et la structure chromatinienne aussi bien dans un processus normal que dans certaines pathologies. / Cellular adaptation to environmental changes and stress relies on a wide range of regulatory mechanisms which are tightly controlled at several levels, including transcription. Chromatin structure and chromatin binding proteins are important factors contributing to the transcriptional response to stress. However, it remains largely unknown to what extent specific chromatin factors influence these distinct responses in a developmental context. One of the best characterized stress response pathways is the unfolded protein response (UPR), which is activated by accumulation of misfolded proteins in the endoplasmic reticulum (ER). Here, we show that Caenorhabditis elegans HPL-2, the homologue of the HP1 chromatin associated protein, is required for the ER stress response. Inactivation of HPL-2 results in enhanced resistance to ER stress dependent on the XBP-1 branch of the UPR and the closely related process of autophagy. Increased resistance to ER stress in animals lacking HPL-2 is associated with increased basal levels of XBP-1 activation and ER chaperones under physiological conditions. Using tissue specific rescue experiments, we find that HPL-2 plays antagonistic roles in intestinal and neuronal cells to influence the ER stress response. We further show that chemical inhibition of histone deacetylase activity mimics the HPL-2 loss of function phenotype, and that increasing or decreasing histone H3 lysine 4 methylation (H3K4me) has antagonistic effects on animal survival in response to ER stress. Altogether our results point to an important function for specific chromatin factors and chromatin modifications in maintaining ER homeostasis in a developmental context.

As embryos proceed through development, they must undergo a series of cell fate decisions. At each division, potency is progressively restricted until a terminally differentiated, postmitotic cell is produced. An important part of that cell type determination is repression of alternative fate possibilities. In this thesis, I have explored the mechanisms by which a single transcription factor activates certain cell fates while inhibiting others, using the Caenorhabditis elegans ALM and BDU neurons as a model. ALM neuron identity is regulated by two interacting transcription factors: the POU homeobox gene unc-86 and the LIM homeobox gene mec-3. I investigated fate determination in BDU neurons, the sister cells of ALM. I found that BDU identity is broadly defined by a combination of unc-86 and the Zn finger transcription factor pag-3, while the neuropeptidergic subroutine of BDU is determined by the LIM homeobox gene ceh-14. In addition, I found that reciprocal homeotic transformations occur between ALM and BDU neurons upon loss of either mec-3 or pag-3. In mec-3 mutants, ALM neurons acquire the gene expression profile and morphological characteristics of BDU cells, while in pag-3 mutants, BDU neurons express genes normally found in ALM and change some aspects of their morphology to resemble ALM. While these fate switches appear to be a simple case of cross-repression, the mechanism is in fact more complicated, as pag-3 is expressed not just in BDU but also in ALM. In this thesis, I present evidence that MEC-3 inhibits execution of BDU identity in ALM by physically binding to UNC-86 and sequestering it away from the promoters of BDU genes. This work expands upon the literature examining simultaneous activation of one identity program and repression of alternate programs by introducing a novel mechanism by which a transcription factor competes to direct specific cell fates.