Indiana University-Purdue University Indianapolis (IUPUI) / Several experimental methods has been developed for the study of the central dogma since late 20th century. Protein mass spectrometry and next generation sequencing (including DNA-Seq and RNA-Seq) forms a triangle of experimental methods, corresponding to the three vertices of the central dogma, i.e., DNA, RNA and protein. Numerous RNA sequencing and protein mass spectrometry experiments has been carried out in attempt to understand how the expression change of known genes affect biological functions in various of organisms, however, it has been once overlooked that the result data of these experiments are in fact holograms which also reveals other delicate biological mechanisms, such as RNA splicing and the expression of long non-coding RNAs. In this dissertation, we carried out five studies based on high-throughput sequencing data, in an attempt to understand how RNA splicing and differential expression of long non-coding RNAs is associated biological functions. In the first two studies, we identified and characterized 197 stimulant induced and 477 developmentally regulated alternative splicing events from RNA sequencing data. In the third study, we introduced a method for identifying novel alternative splicing events that were never documented. In the fourth study, we introduced a method for identifying known and novel RNA splicing junctions from protein mass spectrometry data. In the fifth study, we introduced a method for identifying long non-coding RNAs from poly-A selected RNA sequencing data. Taking advantage of these methods, we turned RNA sequencing and protein mass spectrometry data into an information gold mine of splicing and long non-coding RNA activities. / 2019-05-06

Myc deregulation critically contributes to many cancer etiologies. Recent work suggests that Myc and its direct interactors can confer a distinct epigenetic state. Our goal is to better understand the Myc-conferred epigenetic status of cells. We have previously identified the long non-coding RNA (lncRNA), H19, as a target of Myc regulation and shown it to be important for transformation in lung and breast cells. These results prompted further analysis to identify similarly important Myc-regulated lncRNAs. Myc-regulated lncRNAs associated with the cell cycle and transformation have been identified by microarray analysis. A small number of candidate lncRNAs that were differentially expressed in both the cell cycle and transformation have been validated. Given the increasing importance of lncRNAs and epigenetics to cancer biology, the discovery of Myc-induced, growth associated lncRNAs could provide insight into the mechanisms behind Myc-related epigenetic signatures in both normal and disease states.

Myoclonus-Dystonia (MD) is an inherited, rare, autosomal dominant movement disorder characterized by quick, involuntary muscle jerking or twitching (myoclonus) and involuntary muscle contractions that cause twisting and pulling movements, resulting in abnormal postures (dystonia). The first MD locus was mapped to 7q21-q31 and called DYT11; this locus corresponds to the SGCE gene. Our group previously identified a second MD locus (DYT15) which maps to a 3.18 Mb region on 18p11. Two patients were chosen to undergo next-generation sequencing, which identified 2,292 shared novel variants within the critical region. Analysis of these variants revealed a 3 bp duplication in a transcript referred to as CD108131, which is believed to be a long non-coding RNA. Characterization of this transcript determined that it is 863 bp in size, it is ubiquitously expressed, with high expression in the cerebellum, and it accounts for ~3% of MD cases.

Variations in drug metabolism may alter drug efficacy and cause toxicity; better understanding of the mechanisms and risks shall help to practice precision medicine. At the 21st International Symposium on Microsomes and Drug Oxidations held in Davis, California, USA, in October 2-6, 2016, a number of speakers reported some new findings and ongoing studies on the regulation mechanisms behind variable drug metabolism and toxicity, and discussed potential implications to personalized medications. A considerably insightful overview was provided on genetic and epigenetic regulation of gene expression involved in drug absorption, distribution, metabolism, and excretion (ADME) and drug response. Altered drug metabolism and disposition as well as molecular mechanisms among diseased and special populations were presented. In addition, the roles of gut microbiota in drug metabolism and toxicology as well as long non-coding RNAs in liver functions and diseases were discussed. These findings may offer new insights into improved understanding of ADME regulatory mechanisms and advance drug metabolism research. (C) 2017 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.

The mammalian heart is a remarkable organ in that it must provide for the cardiovascular needs of the organism throughout life, without pausing. Yet, through developmental growth to adulthood and into ageing, the mammalian heart undergoes extensive physiological, morphological and biochemical remodelling. Pivotal to the age-associated alterations in cardiac phenotype is a decline in the proliferative capacity of cardiac myocytes (CMs), which is insufficient to compensate for the basal rate of CM death over time. The terminally differentiated nature of adult CMs also underlies the inability of the heart to repair itself after myocardial damage, such as infarction. As a consequence, existing CMs mount a compensatory hypertrophic response to sustain cardiac output. In parallel, the proliferation rate of resident cardiac fibroblasts, which comprise approximately 60% of total cardiac cells, increases, replacing healthy myocardium with fibrotic scar tissue. Together, CM hypertrophy and fibroblast hyperplasia progressively reduces cardiac function and the ability of the heart to adapt to environmental stressors or damage. Under continued stress or through natural ageing, the heart progresses to a failing state in which cardiac output can no longer meet the demands of the body. The societal impact of ageing-associated decline in cardiac function is great, with heart failure affecting around 8% of over 65s and consuming approximately 2% of the NHS budget. These statistics are set to rise with an ageing population. The substantial phenotypic alterations characteristic of ageing and disease-associated cardiac remodelling requires a wholesale reprogramming of the CM transcriptome. In many biological systems, although yet to be established in adult myocytes, epigenetic mechanisms underlie the transcriptome changes that arise. I hypothesised that alterations in the epigenetic landscape of CMs mediate the transcriptome remodelling that determines the phenotypic transformations that occur in cardiac ageing, hypertrophy and disease. To test this hypothesis, I examined CM-specific changes in DNA cytosine modifications, long non-coding RNA (lncRNA) expression and histone tail lysine methylation marks – epigenetic marks with central roles in transcriptional regulation in many biological systems. I examined how these changes correlate with alterations in the CM transcriptome during disease and ageing. Understanding how alterations in the transcriptome and epigenome contribute to phenotypic changes using whole tissue data is confounded by the heterogeneous nature of the heart, coupled with ageing and disease-associated changes in relative cellular composition. To overcome this, I validated a method to isolate CM nuclei specifically from post-mortem heart tissue. This method also has the advantage that it could be applied to frozen tissue, allowing access to archived material. LncRNAs are functional RNA transcripts longer than 200 bases are emerging as important regulators of gene expression. Common mechanisms of gene expression regulation by lncRNAs include by antisense suppression, as guide/co-factor molecules to direct chromatin modifying components or splicing factors to locations in the genome. Transcriptome profiling in healthy and failing human CMs identified an increase in expression of the lncRNA MALAT-1, which was consistently observed in rodent models of pathology and in ageing. Loss-of-function investigations revealed a potential anti-hypertrophic function for this lncRNA. Specifically, MALAT-1 knock down in vitro in CMs incited spontaneous hypertrophy with features reflecting pathological remodelling in the heart and hypertrophy induced by pro-hypertrophic mediators in vitro. ix In addition, novel uncharacterised transcripts were identified as differentially expressed in cardiovascular disease, including a lncRNA at 4q35.2, which was found significantly downregulated in CMs from human failing hearts. DNA methylation is a stable epigenetic modification and is generally associated with transcriptional repression. It is established by de novo DNA methyltransferases (DNMTs) in early development to determine and maintain differentiated cell states and is ‘copied’ to daughter strands in DNA synthesis by the maintenance DNMT1. Methylcytosine (MeC) can be subject to further processing to hydroxymethylcytosine (hMeC) through a TET protein-mediated oxidation reaction. This serves as a means to actively remove methylation marks as well as hMeC being a novel epigenetic modification in its own right. For the first time, I identified the cardiac myocyte genome as having a high global level of hMeC, comparable with that in neurones. I also discovered an age-associated increase in gene body hMeC that coincided with the loss of proliferative capacity and plasticity of CMs. In parallel, gene body DNA MeC levels decrease in CM ageing. Both these phenomena in gene bodies corresponded with a non-canonical upregulation in expression of genes particularly relevant to cardiac function. This relationship between gene body methylation and transcription rate is strengthened with age in CMs. Recent work in the laboratory had identified the pervasive loss of euchromatic lysine 9 dimethylation on histone 3 (H3K9me2) as a conserved feature of pathological hypertrophy and associated with re-expression of foetal genes. Concurrently, expression and activity of the enzymes responsible for depositing H3K9me2, euchromatic histone lysine methyltransferases 1 and 2 (EHMT1/GLP and EHMT2/G9a) were reduced. Consistently, microRNA-217-induced genetic or pharmacological inactivation of Ehmts was sufficient to promote pathological hypertrophy and foetal gene re-expression, while suppression of this pathway protected from pathological hypertrophy both in vitro and in mice. In summary, I provide new insight into CM-specific epigenetic changes and suggest the epigenome as an important mediator in the loss of plasticity and cardiac health in ageing and disease. Epigenetic mediators and pathways identified as responsible for this remodelling of the CM epigenome suggests opportunities for novel therapy approaches.

Les ARNs long non codants (ARNlnc) jouent un rôle clé dans les processus cellulaires vitaux, notamment le remodelage de la chromatine, la réparation de l'ADN et la traduction. Cependant, la taille et la complexité des ARNlnc présentent des défis sans précédent pour les études moléculaires mécanistiques, de sorte qu'il s'est avéré difficile jusqu'à présent de relier l'information structurelle à la fonction biologique pour les ARNlnc.Le gène 3 humain exprimé maternellement (de l’anglais "maternally expressed gene 3", MEG3), est un ARNlnc abondant, soumis à empreinte parentale et épissé alternativement. Pendant l'embryogenèse, MEG3 contrôle les protéines Polycomb, régulant la différenciation cellulaire, et dans les cellules adultes, MEG3 contrôle p53, régulant la réponse cellulaire aux stress environnementaux. Dans les cellules cancéreuses, MEG3 est régulé négativement, mais la surexpression ectopique de MEG3 réduit la prolifération incontrôlée, ce qui prouve que MEG3 agit comme un suppresseur de tumeur. Les données suggèrent que les fonctions de MEG3 pourraient être régulées par la structure de MEG3. Par exemple, on pense que MEG3 se lie directement aux protéines p53 et Polycomb. De plus, les différents variants d'épissage de MEG3, qui comprennent différents exons et possèdent ainsi des structures potentiellement différentes, présentent des fonctions différentes. Enfin, la mutagenèse par délétion, basée sur une structure de MEG3 prédit in silico, a permis d’identifier un motif MEG3 supposé structuré impliqué dans l'activation de p53. Cependant, au début de mes travaux, la structure expérimentale de MEG3 était inconnue.Pour comprendre la structure et la fonction de MEG3, j'ai utilisé des sondes chimiques in vitro et in vivo pour déterminer la structure secondaire de deux variants humains de MEG3 qui diffèrent par leurs niveaux d'activation de p53. À l'aide d'essais fonctionnels dans les cellules et de mutagenèse, j'ai systématiquement analysé la structure de MEG3 et identifié le noyau activant p53 dans deux domaines (D2 et D3) qui sont conservés structuralement dans les variants humains et conservés dans l’évolution chez les mammifères. Dans D2-D3, les régions structurales les plus importantes sont les hélices H11 et H27, car dans ces régions, j’ai pu supprimer l'activation de p53 grâce à des mutations ponctuelles, un degré de précision jamais atteint pour les autres ARNlnc jusqu’ici. J'ai découvert de manière surprenante que H11 et H27 sont reliés par des boucles connectées l’une à l’autre (de l’anglais "kissing loops") et j'ai confirmé l'importance fonctionnelle de ces interactions de structure tertiaire à longue distance par mutagenèse compensatoire. Allant au-delà de l’état de l’art, j'ai donc essayé de visualiser la structure 3D d’une isoforme de MEG3 longue de 1595 nucléotides, par diffusion de rayons X à petit angle (SAXS), microscopie électronique (EM) et microscopie à force atomique (AFM). Alors que le SAXS et l’EM sont limités par des défis techniques actuellement insurmontables, l’imagerie par AFM m’a permis d’obtenir la première structure 3D à basse résolution de MEG3 et de révéler son échafaudage tertiaire compact et globulaire. Plus remarquable encore, les mêmes mutations qui perturbent la connexion entre les «boucles» H11-H27 et qui inhibent la fonction de MEG3, perturbent aussi la structure 3D de cet ARNlnc, fournissant ainsi le premier lien direct entre la structure 3D et la fonction biologique pour un ARNlnc.Sur la base de mes découvertes, je peux donc proposer un mécanisme de l’activation de p53 basé sur la structure de MEG3, avec des implications importantes pour la compréhension de la cancérogenèse. Plus généralement, mes travaux prouvent que les relations structure-fonction des ARNlnc peuvent être disséquées avec une grande précision et ouvrent la voie à des études analogues visant à obtenir des informations mécanistes pour de nombreux autres ARNlnc d’importance médicale. / Long non-coding RNAs (lncRNAs) are key players in vital cellular processes, including chromatin remodelling, DNA repair and translation. However, the size and complexity of lncRNAs present unprecedented challenges for mechanistic molecular studies, so that connecting structural information with biological function for lncRNAs has proven difficult so far.Human maternally expressed gene 3 (MEG3) is an abundant, imprinted, alternatively-spliced lncRNA. During embryogenesis MEG3 controls Polycomb proteins, regulating cell differentiation, and in adult cells MEG3 controls p53, regulating the cellular response to environmental stresses. In cancerous cells, MEG3 is downregulated, but ectopic overexpression of MEG3 reduces uncontrolled proliferation, proving that MEG3 acts as a tumour suppressor. Evidence suggests that MEG3 functions may be regulated by the MEG3 structure. For instance, MEG3 is thought to bind p53 and Polycomb proteins directly. Moreover, different MEG3 splice variants, which comprise different exons and thus possess potentially different structures, display different functions. Finally, deletion mutagenesis based on a MEG3 structure predicted in silico identified a putatively-structured MEG3 motif involved in p53 activation. However, at the beginning of my work, the experimental structure of MEG3 was unknown.To understand the MEG3 structure and function, I used chemical probing in vitro and in vivo to determine the secondary structure maps of two human MEG3 variants that differ in their p53 activation levels. Using functional assays in cells and mutagenesis, I systematically scanned the MEG3 structure and identified the p53-activating core in two domains (D2 and D3) that are structurally conserved across human variants and evolutionarily conserved across mammals. In D2-D3, the most important structural regions are helices H11 and H27, because in these regions I could tune p53 activation even by point mutations, a degree of precision never achieved for any other lncRNA to date. I surprisingly discovered that H11 and H27 are connected by “kissing loops”, and I confirmed the functional importance of these long-range tertiary structure interactions by compensatory mutagenesis. Going beyond state-of-the-art, I thus attempted to visualize the 3D structure of a 1595-nucleotide long MEG3 isoform by small angle X-ray scattering (SAXS), electron microscopy (EM), and atomic force microscopy (AFM). While SAXS and EM are limited by currently-insurmountable technical challenges, single particle imaging by AFM allowed me to obtain the first low resolution 3D structure of MEG3 and reveal its compact, globular tertiary scaffold. Most remarkably, functionally-disrupting mutations that break the H11-H27 “kissing loops” disrupt such MEG3 scaffold, providing the first direct connection between 3D structure and biological function for an lncRNA.Based on my discoveries, I can therefore propose a structure-based mechanism for p53 activation by human MEG3, with important implications in understanding carcinogenesis. More broadly, my work serves as proof-of-concept that lncRNA structure-function relationships can be dissected with high precision and opens the field to analogous studies aimed to gain mechanistic insights into many other medically-relevant lncRNAs.

Orientador: Guilherme Targino Valente / Resumo: A levedura Saccharomyces cerevisiae é o microrganismo mais utilizado para a produção de etanol devido a sua alta capacidade fermentativa e resistência aos estresses oriundos desse processo. Entretanto, a própria concentração de etanol é um dos fatores mais limitantes no processo de produção desse combustível. Os aspectos da genômica funcional relacionada à tolerância ao etanol são ainda pouco esclarecidos, e nem mesmo se sabe se os lncRNAs tem papel nesse processo. Poucos lncRNAs foram identificados em S. cerevisiae, e nem mesmo se conhece as redes lncRNAs-proteínas nessa espécie e nem se podem codificar micropeptídeos. Nesse contexto, este trabalho visa identificar lncRNAs em linhagens de S. cerevisiae com diferentes níveis de tolerância ao etanol. Para isso, foi realizado a montagem dos lncRNAs, predição de ligações lncRNA-proteínas, buscas de micropepetídeos, análises de conservação genômica, estrutural e funcional dos lncRNAs, avaliação da influência do lncRNAs em regular as expressões de seus vizinhos e comparação dos resultados entre linhagens mais e menos tolerantes ao etanol. As análises de enriquecimento ontológico apontam para uma relação próxima entre os lncRNAs e a tolerância ao etanol e uma conservação funcional, embora os dados não reportem nenhuma conservação nem genômica nem estrutural. Além disso, variados tipos de prováveis regulações foram sugeridas, sendo a regulação em trans majoritariamente inversa entre os lncRNAs e seus genes-alvo, diferentemente da ma... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The yeast Saccharomyces cerevisiae is the most used microorganism for ethanol production due to its high fermentative capacity and resistance to different stressors along this process. However, the ethanol concentration is one of the most limiting factors of fuel production. The functional genomics aspects related to the ethanol tolerance are still unclear, and it is not clear if the lncRNAs really have a role in this process. Few lncRNAs were identified in S. cerevisiae, lncRNA-protein networks of this species are still unknown and also if they can code micropeptides. In this context, this thesis aims to identify lncRNAs and evaluate their roles in S. cerevisiae ethanol tolerance. Then, it was performed the assembling of lncRNAs, predictions of lncRNA-protein interactions, searches for potential micropeptides coding-lncRNAs, analysis of genomic, structural and functional conservation of lncRNAs, evaluation of the lncRNAs influence in regulating the expressions of their neighbors, and comparison between strains that are more and less tolerant to the ethanol. Moreover, many putative regulatory pathways were here suggested, being that most trans regulations act on an inversely manner between the expression of the lncRNAs and their target-genes, unlike observed in most of cis regulations. The current literature confirms the lncRNAs functional conservation here observed, and the role of these non-coding molecules as regulators. Finally, here we suggest that lncRNAs are acting to ... (Complete abstract click electronic access below) / Mestre

L’autisme est un syndrome neuro-développemental hétérogène à l’étiologie génétique complexe. Afin d’identifier les dérèglements initiaux responsables de ce mal-développement cérébral, des travaux antérieurs au sein de notre équipe se sont basés sur des cellules représentatives des stades précoces de l’ontogenèse : les cellules souches olfactives. Le gène MOCOS, codant pour la sulfurase du cofacteur à molybdène, a été trouvée sous-exprimé chez la majorité des patients autistes comparés à des sujets contrôles de même âge et du même sexe.Nous avons ensuite postulé que la dissection minutieuse des mécanismes moléculaires pouvant rendre compte de cette dérégulation aiderait à trouver des mécanismes sous-jacents contribuant aux troubles du spectre autistique (TSA). Ceci a conduit à l'identification de COSMOC, un long ARN non codant généré à partir d'une transcription divergente dans la région promotrice de MOCOS, dont l'expression est diminuée chez 10 des 11 patients autistes de notre cohorte. A l’aide de diverses techniques de biologie moléculaire, nous avons montré que la déplétion de COSMOC induit : (1) une sous-expression de MOCOS, (2) une déstabilisation de l'organisation de la chromatine, ce qui suggère une fonction de régulateur transcriptionnel, et (3) une altération du métabolisme des lipides et de l’homéostasie redox de la cellule, deux voies dérégulés dans les TSA. Par ailleurs, COSMOC régule de l’expression de la PTBP2 (polypirimidine track biding protein 2), un facteur d’épissage contrôlant l’expression de nombreuses protéines synaptiques. En conclusion, la dérégulation de COSMOC pourrait expliquer certains des dysfonctionnements observés dans les TSA. / Autism is a heterogeneous neuro-developmental syndrome with a complex genetic etiology. In order to unveil the initial disturbances responsible for this brain maldevelopment, previous works in our team relied on cells representative of the early stages of ontogenesis: olfactory stem cells. The MOCOS gene, coding for molybdenum cofactor sulfurase, was found under-expressed in most of autistic patients of our cohort when compared with age- and gender-matched control adults without any neuropsychiatric disorders. We postulated that the meticulous dissection of the molecular mechanisms involved this deregulation would help to unveil pathogenic mechanisms underlying autism spectrum disorders (ASD). This led to the identification of COSMOC, a long non-coding RNA, generated from a divergent transcription in the promoter region of MOCOS, whose expression is decreased in 10 out of 11 autistic patients in our cohort. Using various molecular biological techniques (interference RNA, DNA microarray, qPCR...), we showed that COSMOC depletion induces: (1) an under-expression of MOCOS, (2) a destabilization of chromatin organization, suggesting a transcriptional regulatory function, and (3) an alteration of cellular lipid metabolism and redox homeostasis, two deregulated pathways in ASD. In addition, COSMOC regulates the expression of PTBP2 (polypirimidine track biding protein 2), a splicing factor that controls the expression of many synaptic proteins, including PSD95. In conclusion, the deregulation of COSMOC may explain some of the dysfunctions observed in ASDs.

Myoclonus-Dystonia (MD) is an inherited, rare, autosomal dominant movement disorder characterized by quick, involuntary muscle jerking or twitching (myoclonus) and involuntary muscle contractions that cause twisting and pulling movements, resulting in abnormal postures (dystonia). The first MD locus was mapped to 7q21-q31 and called DYT11; this locus corresponds to the SGCE gene. Our group previously identified a second MD locus (DYT15) which maps to a 3.18 Mb region on 18p11. Two patients were chosen to undergo next-generation sequencing, which identified 2,292 shared novel variants within the critical region. Analysis of these variants revealed a 3 bp duplication in a transcript referred to as CD108131, which is believed to be a long non-coding RNA. Characterization of this transcript determined that it is 863 bp in size, it is ubiquitously expressed, with high expression in the cerebellum, and it accounts for ~3% of MD cases.

Myc deregulation critically contributes to many cancer etiologies. Recent work suggests that Myc and its direct interactors can confer a distinct epigenetic state. Our goal is to better understand the Myc-conferred epigenetic status of cells. We have previously identified the long non-coding RNA (lncRNA), H19, as a target of Myc regulation and shown it to be important for transformation in lung and breast cells. These results prompted further analysis to identify similarly important Myc-regulated lncRNAs. Myc-regulated lncRNAs associated with the cell cycle and transformation have been identified by microarray analysis. A small number of candidate lncRNAs that were differentially expressed in both the cell cycle and transformation have been validated. Given the increasing importance of lncRNAs and epigenetics to cancer biology, the discovery of Myc-induced, growth associated lncRNAs could provide insight into the mechanisms behind Myc-related epigenetic signatures in both normal and disease states.