The expression level of a single gene can vary substantially within and between species, which might facilitate the emergence and fixation of novel expression patterns in the course of evolution. With rapidly accumulating data from genome-wide expression profiling, dense genotyping and individual genome re-sequencing, it is now possible to pinpoint the genetic loci that potentially give rise to gene expression variation. However, what remains elusive is how expression changes could be attributed to the differences in genetic elements, and our understanding of the phenotypic manifestation resulting from gene expression variation is far from comprehensive. In this thesis, I aim to answer these questions in budding yeast and in human. I first studied duplicated genes in budding yeast, which usually shared the identical expression patterns immediately upon duplication events. I searched for the cis-elements, whose divergence might explain the substantial expression variation between the extant paralogs, and established the role of nucleosome occupancy in driving expression differentiation between yeast duplicates. I next investigated the role of trans-factors in establishing species- or population-specific gene expression, and my study was specifically focused on primate microRNAs as a special class of regulators in trans. I first delineated the evolutionary trajectory of an X-linked primate microRNA cluster, and then proposed its function in regulating primate epididymal physiology. I extended this study to human by identifying several microRNAs with highly differentiated regulation among human populations, and such regulatory differentiation was driven by positive selection during recent human evolution. This study for the first time demonstrated high plasticity of the microRNA regulatory interactions in modulating expression variation of their target messengers. Beyond exploring the elements that control gene expression variation, I examined phenotypic manifestation of the observed expression variation in human populations, and my analysis revealed significant implication of expression variation towards differential disease susceptibility among individuals. Lastly, I examined gene expression variation at a micro scale among isogenic cell populations in budding yeast, which is termed “expression noise”. Though expression noise originates from stochasticity, my analysis demonstrated strong topological constraints on expression noise in yeast cellular networks, with which I was able to predict gene expression noise with high accuracy. These observations suggest that the seemingly stochastic gene expression may have been evolutionarily constrained. Taken together, my study presented in this thesis investigates the origin, consequence and evolutionary significance of gene expression variation in eukaryotes.

The expression level of a single gene can vary substantially within and between species, which might facilitate the emergence and fixation of novel expression patterns in the course of evolution. With rapidly accumulating data from genome-wide expression profiling, dense genotyping and individual genome re-sequencing, it is now possible to pinpoint the genetic loci that potentially give rise to gene expression variation. However, what remains elusive is how expression changes could be attributed to the differences in genetic elements, and our understanding of the phenotypic manifestation resulting from gene expression variation is far from comprehensive. In this thesis, I aim to answer these questions in budding yeast and in human. I first studied duplicated genes in budding yeast, which usually shared the identical expression patterns immediately upon duplication events. I searched for the cis-elements, whose divergence might explain the substantial expression variation between the extant paralogs, and established the role of nucleosome occupancy in driving expression differentiation between yeast duplicates. I next investigated the role of trans-factors in establishing species- or population-specific gene expression, and my study was specifically focused on primate microRNAs as a special class of regulators in trans. I first delineated the evolutionary trajectory of an X-linked primate microRNA cluster, and then proposed its function in regulating primate epididymal physiology. I extended this study to human by identifying several microRNAs with highly differentiated regulation among human populations, and such regulatory differentiation was driven by positive selection during recent human evolution. This study for the first time demonstrated high plasticity of the microRNA regulatory interactions in modulating expression variation of their target messengers. Beyond exploring the elements that control gene expression variation, I examined phenotypic manifestation of the observed expression variation in human populations, and my analysis revealed significant implication of expression variation towards differential disease susceptibility among individuals. Lastly, I examined gene expression variation at a micro scale among isogenic cell populations in budding yeast, which is termed “expression noise”. Though expression noise originates from stochasticity, my analysis demonstrated strong topological constraints on expression noise in yeast cellular networks, with which I was able to predict gene expression noise with high accuracy. These observations suggest that the seemingly stochastic gene expression may have been evolutionarily constrained. Taken together, my study presented in this thesis investigates the origin, consequence and evolutionary significance of gene expression variation in eukaryotes.

Gene expression profiles are some of the most abundant sources of data about the cellular state of a collection of cells in an organism. Comparison of the expression profiles of multiple samples allows biologists to find associations between observations at the molecular level and the phenotype of the samples. A key challenge is to distinguish variation in expression due to biological factors of interest from variation due to confounding factors that can arise for unrelated technical or biological reasons. This thesis presents models that can explicitly adjust the comparison of expression profiles to account for specific types of confounding factors. One such confounding factor arises when comparing tissue-specific expression profiles across multiple organisms to identify differences in expression that are indicative of changes in gene function. When the organisms are separated by long evolutionary distances, tissue functions may be re-distributed and introduce expression changes unrelated to changes in gene function. We developed Brownian Factor Phylogenetic Analysis, a model that can account for such re-distribution of function, and demonstrate that removing this confounding factor improves tasks such as predicting gene function. Another confounding factor arises because current protocols for expression profiling require RNA extracts from multiple cells. Often biological samples are heterogeneous mixtures of multiple cell types, so the measured expression profile is an average of the RNA levels of the constituent cells. When the biological sample contains both cells of interest and nuisance cells, the confounding expression from the nuisance cells can mask the expression of the cells of interest. We developed ISOLATE and ISOpure, two models for addressing the heterogeneity of tumor samples. We demonstrated that modeling tumor heterogeneity leads to an improvement in two tasks: identifying the site of origin of metastatic tumors, and predicting the risk of death of lung cancer patients.

Gene expression profiles are some of the most abundant sources of data about the cellular state of a collection of cells in an organism. Comparison of the expression profiles of multiple samples allows biologists to find associations between observations at the molecular level and the phenotype of the samples. A key challenge is to distinguish variation in expression due to biological factors of interest from variation due to confounding factors that can arise for unrelated technical or biological reasons. This thesis presents models that can explicitly adjust the comparison of expression profiles to account for specific types of confounding factors. One such confounding factor arises when comparing tissue-specific expression profiles across multiple organisms to identify differences in expression that are indicative of changes in gene function. When the organisms are separated by long evolutionary distances, tissue functions may be re-distributed and introduce expression changes unrelated to changes in gene function. We developed Brownian Factor Phylogenetic Analysis, a model that can account for such re-distribution of function, and demonstrate that removing this confounding factor improves tasks such as predicting gene function. Another confounding factor arises because current protocols for expression profiling require RNA extracts from multiple cells. Often biological samples are heterogeneous mixtures of multiple cell types, so the measured expression profile is an average of the RNA levels of the constituent cells. When the biological sample contains both cells of interest and nuisance cells, the confounding expression from the nuisance cells can mask the expression of the cells of interest. We developed ISOLATE and ISOpure, two models for addressing the heterogeneity of tumor samples. We demonstrated that modeling tumor heterogeneity leads to an improvement in two tasks: identifying the site of origin of metastatic tumors, and predicting the risk of death of lung cancer patients.

Les séquences non-codantes régulatrices de l’expression des gènes sont tout aussi importantes pour le phénotype d’un individu que les séquences codantes. De nombreux travaux se sont attachés à identifier les forces influençant l’évolution de ces séquences non-codantes. Ici, nous théorisons une nouvelle force sélective influençant potentiellement l’évolution de certaines séquences régulatrices. En utilisant des modèles multi-locus, nous montrons que les promoteurs génétiques les plus forts (activateurs de la transcription) gagnent un avantage à voir la copie du gène qui leur est associée (située sur le même chromosome) davantage exprimée que la copie homologue, contrôlée par un promoteur homologue moins fort. La surexpression des copies associées aboutit à une meilleure purge des mutations délétères chez ces copies, et ainsi à une association génétique entre promoteurs forts et contexte génétique favorable. Si la recombinaison entre le gène et le promoteur est suffisamment faible pour que cette association persiste, la force des promoteurs est sélectionnée pour augmenter. L’escalade des forces des promoteurs ne conduit pas forcément à une surproduction de protéines : d’autres régulateurs peuvent co-évoluer pour maintenir un niveau d’expression optimal, à condition que la sélection stabilisante tolère des niveaux d’expression transitoirement sub-optimaux. En variant les modes de reproduction, nous avons montré que ce nouveau processus sélectif ne menait pas nécessairement à une escalade de la force des promoteurs. Lorsque les chromosomes sont suffisamment isolés génétiquement (peu de recombinaison, peu de fécondation croisée), la sélection pour des associations génétiques favorables mène à une divergence des chromosomes : un chromosome accumule des promoteurs forts et possède des copies viables du gène, tandis que le chromosome homologue accumule des promoteurs faibles et des mutations délétères sur le gène. Dans le cas de lignées clonales peu ou pas recombinantes, on s’attend ainsi à observer une haploïdisation de l’expression des gènes : une copie de chaque gène concerné est éteinte et dégénère. Cette divergence s’applique aussi à des chromosomes sexuels ayant cessé de recombiner : on a pu montrer que la divergence des chromosomes menait à une extinction et une dégénérescence des gènes situés sur les chromosomes Y, et à une surexpression des gènes situés sur le chromosome X. En utilisant notre modèle, on propose ainsi une nouvelle théorie pour expliquer l’évolution des chromosomes sexuels non-recombinants. Enfin, on a utilisé des données de divergence entre Mus musculus et Rattus norvegicus pour isoler un signal ne pouvant être expliqué que par une sélection positive pour des promoteurs proximaux plus forts. Ce signal est faible, mais détectable, nous permettant d’apporter une première confirmation empirique du processus d’escalade des forces des promoteurs. / Non-coding sequences, that regulate gene expression, are as important as coding sequences to determine phenotypes. Many studies have identified the main forces affecting regulatory sequence evolution. Here, we theoretically identify a new selective force that may also play a role in this matter. Using multi-locus models, we show that stronger (activating more transcription) enhancers gain some benefit in having its associated gene copy more expressed than the homolog gene copy, controlled by a weaker homolog enhancer. Overexpressed gene copies are better purged from deleterious mutations, such that stronger enhancers get associated with a better genetic background. If recombination between the gene and the enhancer is low enough for this association to persist, enhancer strength selectively increases. Enhancer strength escalation does not necessarily lead to protein overproduction. Other regulators may indeed co-evolve to maintain optimal expression levels, provided that stabilizing selection allows for transitory sub-optimal expression levels. Implementing in the models different reproductive systems, we show that this new selective process does not necessarily lead to an enhancer strength escalation. When chromosomes are genetically isolated enough (little recombination, little outcrossing, selection for favorable genetic associations leads to chromosome divergence: one accumulates stronger enhancers and viable gene alleles, while the other accumulates weaker enhancers and deleterious gene mutations. For non-recombining clonal lineages, we expect gene expression to become haploid: for each gene, one copy is shut down and degenerates. Such divergence also applies to non-recombining sex chromosomes. We show that in such case, chromosome divergence leads to a shut down and degeneration of Y chromosome genes, and to an overexpression of genes located on X chromosomes. With our model, we propose a new theory to explain sex chromosome evolution after they stop recombining. Finally, we used divergence data between Mus musculus and Rattus norvegicus to find a signal that can only be explained by positive selection for stronger proximal enhancers. This signal is weak, but significant: this is the first empirical confirmation of enhancer strength escalation process we studied here.

Orientadores: Ana Maria Lima de Azeredo Espin, Tatiana Teixeira Torres / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T02:29:01Z (GMT). No. of bitstreams: 1 Cardoso_GiseleAntoniazzi_M.pdf: 5837869 bytes, checksum: 23826467e3b82c7f5176f5bfdcecb022 (MD5) Previous issue date: 2012 / Resumo: Os estudos da base molecular do comportamento são difíceis de serem realizados uma vez que um comportamento pode ser moldado por diversos fatores (incluindo fatores genéticos). Vários trabalhos ligaram genes a comportamentos específicos. Com bases nesses estudos, nós usamos espécies da família Calliphoridae como modelo para o estudo da evolução do hábito de parasitismo. Espécies muito próximas de califorídeos exibem comportamentos alimentares diferentes como o hábito necrófago ou parasita. Ainda não se sabe como o hábito de parasitismo surgiu em Calliphoridae, no entanto, existem diversas estratégias para tentarmos entender a evolução do hábito de parasitismo. Uma delas envolve a análise da expressão de genes candidatos relacionando as diferenças de expressão observadas com os diferentes hábitos alimentares. Assim, nós utilizamos a técnica de PCR em tempo real, que mede a expressão do gene de interesse em relação a um gene de referência. O uso do gene de referência tem como objetivo retirar uma parte da variação experimental. Portanto, esse gene deve não deve variar sua expressão nas diferentes espécies que foram estudadas. Então, primeiramente selecionamos e validamos genes de referência para obtermos uma quantificação mais precisa dos níveis de expressão dos genes candidatos. Após essa etapa, selecionamos genes candidatos e os separamos em quatro categorias: a) genes diretamente relacionados ao comportamento alimentar, b) genes relacionados ao metabolismo de substâncias tóxicas, c) genes relacionados a respostas imunológicas e; d) genes diretamente ligados ao hábito de parasitismo. A expressão de oito genes candidatos foi analisada em espécies dos gêneros Chrysomya e Cochliomyia. Além disso, foi possível inferir como a expressão desses genes evolui dentro da família Calliphoridae. Nós observamos uma grande conservação nos níveis de expressão gênica em larvas e em adultos evidenciamos diferenças de expressão correlacionadas com a divergência entre as espécies. O gene que se destacou em nossas análises por sua possível relação com o hábito alimentar deve ser estudado detalhadamente para dar continuidade ao projeto / Abstract: Studies involving the molecular basis of behavior are difficult to perform because behavior is shaped by several factors (including genetic factors). Several studies have linked genes to specific behaviors. Based in these studies, we used species of the family Calliphoridae to study the evolution of parasitism. Closely related species of this family exhibit different feeding behaviors (obligate parasites and saprophagous species). It is unclear how parasitism arose in Calliphoridae, however, it is possible to observe in their evolutionary history that this habit appears in three separate occasions. One approach to initiate this study is to examine the expression of candidate genes. For this purpose, we used real time PCR, but gene expression is measured relative to a reference gene. The use of a reference gene is to remove a part of the experimental variation. Therefore, this gene is expected not to vary its expression in the different species studied. Thus, we first selected and validated reference genes to obtain a more accurate quantification of gene expression levels. After this step, we selected candidate genes and separated them into four categories: a) genes directly related to feeding behavior, b) genes related to metabolism of toxic substances, c) genes related to immune responses and d) genes directly linked to parasitism. We analyzed the expression of eight candidate genes in species of Chrysomya and Cochliomyia genera. Moreover, it was possible to infer how the expression of these genes is evolving within family Calliphoridae. We observed a wide conservation in gene expression levels in larvae and in adults there was evidence of neutral evolution (genes differentially expressed among species). The gene Mvl may be involved in the different feeding habits and paved the way to continue the study of the evolution of parasitism / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular