<p>After finishing a human genome reference sequence in 2002, the genomics community has</p><p>turned to the task of interpreting it. A primary focus is to identify and characterize not only</p><p>protein-coding genes, but all functional elements in the genome. The effort has identified</p><p>millions of regulatory elements across species and in hundreds of human cell-types. Nearly</p><p>all identified regulatory elements are found in non-coding DNA, hypothesizing a function</p><p>for previously unannotated sequence. The ability to identify regulatory DNA genome-wide</p><p>provides a new opportunity to understand gene regulation and to ask fundamental questions</p><p>in diverse areas of biology.</p><p>One such area is the aim to understand the molecular basis for phenotypic differences</p><p>between humans and other primates. These phenotypic differences are partially driven</p><p>by mutations in non-coding regulatory DNA that alter gene expression. This hypothesis</p><p>has been supported by differential gene expression analyses in general, but we have not</p><p>yet identified specific regulatory variants responsible for differences in transcription and</p><p>phenotype. I have worked to identify regulatory differences in the same cell-type isolated</p><p>from human, chimpanzee, and macaque. Most regulatory elements were conserved among</p><p>all three species, as expected based on their central role in regulating transcription. How-</p><p>ever, several hundred regulatory elements were gained or lost on the lineages leading to</p><p>modern human and chimpanzee. Species-specific regulatory elements are enriched near</p><p>differentially expressed genes, are positively correlated with increased transcription, show</p><p>evidence of branch-specific positive selection, and overlap with active chromatin marks.</p><p>ivSpecies-specific sequence differences in transcription factor motifs found within this regu-</p><p>latory DNA are linked with species-specific changes in chromatin accessibility. Together,</p><p>these indicate that species-specific regulatory elements contribute to transcriptional and</p><p>phenotypic differences among primate species.</p><p>Another fundamental function of regulatory elements is to define different cell-types in</p><p>multicellular organisms. Regulatory elements recruit transcription factors that modulate</p><p>gene expression distinctly across cell-types. In a study of 112 human cell-types, I classified</p><p>regulatory elements into clusters based on regulatory signal tissue specificity. I then used</p><p>these to uncover distinct associations between regulatory elements and promoters, CpG-</p><p>islands, conserved elements, and transcription factor motif enrichment. Motif analysis</p><p>identified known and novel transcription factor binding motifs in cell-type-specific and</p><p>ubiquitous regulatory elements. I also developed a classifier that accurately predicts cell-</p><p>type lineage based on only 43 regulatory elements and evaluated the tissue of origin for</p><p>cancer cell-types. By correlating regulatory signal and gene expression, I predicted target</p><p>genes for more than 500k regulatory elements. Finally, I introduced a web resource to</p><p>enable researchers to explore these regulatory patterns and better understand how expression</p><p>is modulated within and across human cell-types.</p><p>Regulation of gene expression is fundamental to life. This dissertation uses identified</p><p>regulatory DNA to better understand regulatory systems. In the context of either evolution-</p><p>ary or developmental biology, understanding how differences in regulatory DNA contribute</p><p>to phenotype will be central to completely understanding human biology.</p> / Dissertation

The mitochondrial control region (MCR) has played an important role as a population genetic marker in many taxa but sequencing of complete eukaryotic genomes has revealed that nuclear integrations of mitochondrial DNA (numts) are abundant and widespread across many taxa. If left undetected, numts can inflate mitochondrial diversity and mislead interpretation of phylogenetic relationships. Comparative analyses of complete genomes in humans, orangutans and chimpanzees, and preliminary studies in gorillas have revealed high numt prevalence in great apes, but rigorous comparative analyses across taxa have been lacking. The present study aimed to systematically compare the evolutionary dynamics of MCR numts in great apes. Firstly, an inventory numts derived from the region containing the MCR subdomains was carried out by genomic BLAST searches. Secondly, presence/absence of each candidate numt was determined in great ape taxa to estimate numt insertion rate. Thirdly, alternative mechanisms of numt insertion, either through direct mitochondrial integration or post-insertional duplications, were also assessed. Fourthly, the effect of nuclear and mitochondrial environment on patterns of nucleotide composition and substitution was assessed through sequence comparisons of nuclear and mitochondrial paralogous sequences. Finally, numts in the gorilla genome were identified through two experimental methods and their use as polymorphic genetic markers was then evaluated in a sample of captive gorillas from U.S. zoos. A deficit of MCR numts covering two particular mitochondrial subdomains was detected in all three apes examined, and is largely attributed to rapid loss of mitochondrial and nuclear sequence identity in the mitochondrial genome. Insertion rates have varied during the great ape evolution and exhibit substantial differences even between related taxa. The most likely mechanism of numt insertion is direct mitochondrial integration through Non-Homologous-End-Joining Repair. Transition/transversion ratios differed significantly between both mitochondrial and nuclear sequences and between numts from coding and non-coding mitochondrial regions. A previously documented upward bias in the GC content of the primate mitochondrial genome was confirmed and the extent of this bias relative to the corresponding numt sequences increased with numt age. Five gorilla-specific numts were isolated, including three exhibiting insertional polymorphisms that will be used in future population genetic studies in free-range gorilla.

abstract: Fundamental hypotheses about the life history, complex cognition and social dynamics of humans are rooted in feeding ecology - particularly in the experiences of young animals as they grow. However, the few existing primate developmental data are limited to only a handful of species of monkeys and apes. Without comparative data from more basal primates, such as lemurs, we are limited in the scope of our understanding of how feeding has shaped the evolution of these extraordinary aspects of primate biology. I present a developmental view of feeding ecology in the ring-tailed lemur (Lemur catta) using a mixed longitudinal sample (infant through adult) collected at the Beza Mahafaly Special Reserve in southwestern Madagascar from May 2009 to March 2010. I document the development of feeding, including weaning, the transition to solid food, and how foods are included in infant diets. Early in juvenility ring-tailed lemurs efficiently process most foods, but that hard ripe fruits and insects require more time to master. Infants and juveniles do not use many of the social learning behaviors that are common in monkeys and apes, and instead likely rely both on their own trial and error and simple local enhancement to learn appropriate foods. Juvenile ring-tailed lemurs are competent and efficient foragers, and that mitigating ecological risks may not best predict the lemur juvenile period, and that increases in social complexity and brain size may be at the root of primate juvenility. Finally, from juvenility through adulthood, females have more diverse diets than males. The early emergence of sex differences in dietary diversity in juvenility that are maintained throughout adulthood indicate that, in addition to reproductive costs incurred by females, niche partitioning is an important aspect of sex differential feeding ecology, and that ontogenetic studies of feeding are particularly valuable to understanding how selection shapes adult, species-typical diets. Overall, lemur juvenility is a time to play, build social relationships, learn about food, and where the kernels of sex-typical feeding develop. This study of the ontogeny of feeding ecology contributes an important phylogenetic perspective on the relationship between juvenility and the emergent foraging behaviors of developing animals / Dissertation/Thesis / Ph.D. Anthropology 2012

Duplicação genica é uma das principais forças levando a evolução dos genomas eucarioto. O impacto de duplicações gênicas/genômicas vem sendo investigado a muito tempo em humanos e outros primatas. Um segundo mecanismo de duplicação gênica, a retrotransposição baseada em RNA maduros, vem sendo menos estudada devido ao seu potencial menor de gerar cópias funcionais. No entanto, recentemente, publicações descreveram retrocópias funcionais em humanos, roedores e mosca de fruta. Nesta tese, para investigar sobre retrocópias causando variabilidade genética no genoma de primatas, nós desenvolvemos a implementamos os métodos para detectar estas inserções. Utilizando nove genomas e transcriptomas publicamente disponíveis (sete primatas e dois roedores) nós confirmamos um número similar, porém, com origem independente, de retrocópias em primatas e roedores. Nós também encontramos um enriquecimento de retrocópias no genoma de Platyrrhini, possivelmente explicado pela expansão de L1PA7 e L1P3 nestes genomas. Posteriormente, nós analisamos a ortologia de retrocópias no genoma de primatas e encontramos 127 eventos específicos à linhagem humana. Nós também exploramos dados do projeto 1000 Genomes para detectar retrocópias polimórficas (retroCNVs germinativos) e encontramos 17 eventos, presentes no genoma referência humano, mas ausentes em mais de um indivíduo. Similarmente, nós investigamos novas retroduplicações de mRNAs no genoma humano, detectando 21 eventos ausentes do genoma referência. Finalmente, investigamos a existência de retroCNVs somáticos e descrevemos sete possíveis retrocópias somáticas. Apesar de sua possível insignificância, nós encontramos que algumas retrocópias compartilhadas entre todos os primatas, espécie específicas, e polimórficas podem ser expressas per se ou como transcritos quiméricos com genes hospedeiros. Sobretudo, nós encontramos que retrocópias são um fator importante da variabilidade genética inter-espécie, intra-espécie e intra-indivíduo e podem estar influenciando a evolução de mamíferos ao criar reservatórios de duplicações potencialmente funcionais. / Gene duplication is a major driving force of evolution in eukaryotic genome. The impact of gene/genomic duplication has long been investigated in human and other primates. A second mechanism of gene duplication, retrotransposition, which is based on mature RNA, has been traditionally less studied due to their lower potential to generate functional copies. Recently, however, publications described functional retrocopies in humans, murines and drosophila. Here, to gain insights of the genetic variability arising from retrocopies on primate genomes, we developed and implemented the methods to detect these insertions. Using nine publicly available reference genomes and transcriptomes (seven primates and two rodents) we described a similar number independently arisen retrocopies in primates and rodents. We also found an enrichment of retrocopies in Platyrhinni genomes, putatively explained by the expansion of L1PA7 and L1P3 in these genomes. Next, we evaluated the orthology of retrocopies in primate genomes and found 127 events specific to human lineage. We also explored 1000 Genomes Project data to detect polymorphic events (germinative retroCNVs) on human populations and found 17 events, present on the reference genome, absent in more than one individual. Conversely, we also investigated new insertions of mRNA retroduplications in the human genome, detecting 21 events absent to the human reference genome. Finally, we evaluated the existence of somatic retroCNVs and described seven putative somatic retrocopies. Despite their putative insignificance, we found that some of these shared, specie-specific and polymorphic events may be expressed per se and as chimeric transcripts within host genes. Taken together, we found that retrocopies are a great factor of genetic variation interspecie, intraspecie e intraindividual and may be affecting mammal evolution by creating reservoirs of potentially functional duplications

Duplicação genica é uma das principais forças levando a evolução dos genomas eucarioto. O impacto de duplicações gênicas/genômicas vem sendo investigado a muito tempo em humanos e outros primatas. Um segundo mecanismo de duplicação gênica, a retrotransposição baseada em RNA maduros, vem sendo menos estudada devido ao seu potencial menor de gerar cópias funcionais. No entanto, recentemente, publicações descreveram retrocópias funcionais em humanos, roedores e mosca de fruta. Nesta tese, para investigar sobre retrocópias causando variabilidade genética no genoma de primatas, nós desenvolvemos a implementamos os métodos para detectar estas inserções. Utilizando nove genomas e transcriptomas publicamente disponíveis (sete primatas e dois roedores) nós confirmamos um número similar, porém, com origem independente, de retrocópias em primatas e roedores. Nós também encontramos um enriquecimento de retrocópias no genoma de Platyrrhini, possivelmente explicado pela expansão de L1PA7 e L1P3 nestes genomas. Posteriormente, nós analisamos a ortologia de retrocópias no genoma de primatas e encontramos 127 eventos específicos à linhagem humana. Nós também exploramos dados do projeto 1000 Genomes para detectar retrocópias polimórficas (retroCNVs germinativos) e encontramos 17 eventos, presentes no genoma referência humano, mas ausentes em mais de um indivíduo. Similarmente, nós investigamos novas retroduplicações de mRNAs no genoma humano, detectando 21 eventos ausentes do genoma referência. Finalmente, investigamos a existência de retroCNVs somáticos e descrevemos sete possíveis retrocópias somáticas. Apesar de sua possível insignificância, nós encontramos que algumas retrocópias compartilhadas entre todos os primatas, espécie específicas, e polimórficas podem ser expressas per se ou como transcritos quiméricos com genes hospedeiros. Sobretudo, nós encontramos que retrocópias são um fator importante da variabilidade genética inter-espécie, intra-espécie e intra-indivíduo e podem estar influenciando a evolução de mamíferos ao criar reservatórios de duplicações potencialmente funcionais. / Gene duplication is a major driving force of evolution in eukaryotic genome. The impact of gene/genomic duplication has long been investigated in human and other primates. A second mechanism of gene duplication, retrotransposition, which is based on mature RNA, has been traditionally less studied due to their lower potential to generate functional copies. Recently, however, publications described functional retrocopies in humans, murines and drosophila. Here, to gain insights of the genetic variability arising from retrocopies on primate genomes, we developed and implemented the methods to detect these insertions. Using nine publicly available reference genomes and transcriptomes (seven primates and two rodents) we described a similar number independently arisen retrocopies in primates and rodents. We also found an enrichment of retrocopies in Platyrhinni genomes, putatively explained by the expansion of L1PA7 and L1P3 in these genomes. Next, we evaluated the orthology of retrocopies in primate genomes and found 127 events specific to human lineage. We also explored 1000 Genomes Project data to detect polymorphic events (germinative retroCNVs) on human populations and found 17 events, present on the reference genome, absent in more than one individual. Conversely, we also investigated new insertions of mRNA retroduplications in the human genome, detecting 21 events absent to the human reference genome. Finally, we evaluated the existence of somatic retroCNVs and described seven putative somatic retrocopies. Despite their putative insignificance, we found that some of these shared, specie-specific and polymorphic events may be expressed per se and as chimeric transcripts within host genes. Taken together, we found that retrocopies are a great factor of genetic variation interspecie, intraspecie e intraindividual and may be affecting mammal evolution by creating reservoirs of potentially functional duplications

Analysis of rhesus monkey alphoid DNA suggests that it arose by tandem duplication of an ancestral monomer unit followed by independent variation within two adjacent monomers (one becoming more divergent than the other) before their amplification as a dimer unit to produce tandem arrays. The rhesus monkey alphoid DNA is a tandemly repeated, 343-bp dimer; the consensus dimer is over 98% homologous to the alphoid dimers reported for baboon and bonnet monkey, 81% homologous to the African green monkey alpha monomer, and less than 70% homologous to the more divergent human alphoid DNAs. The consensus dimer consists of two wings (I and II, 172 and 171 bp, respectively) that are only 70% homologous to each other, but share seven regions of exact homology. These same regions are highly conserved among the consensus sequences of the other cercopithecid alphoid DNAs. The three alpha-protein binding sites reported for African green monkey alpha DNA by F. Strauss and A. Varshavsky (Cell 37: 889-901, 1984) occur in wings I and II, but with one site altered in wing I. Two cloned dimer segments are 98% homologous to the consensus, each containing 8 single-base-pair differences within the 343-bp segment. Surprisingly, 37% of these differences occur in regions that are evolutionarily conserved in the alphoid consensus sequences, including the alpha-protein binding sites. Sequence variation in this highly repetitive DNA family may produce unique nucleosomal architectures for different members of an alphoid array. These unique architectures may modulate the evolution of these repetitive DNAs and may produce unique centromeric characteristics in primate chromosomes.

The chimpanzee is humankind’s closest living relative and the two species diverged ~6 million years ago. Comparative studies of the human and chimpanzee genomes and transcriptomes are of great interest to understand the molecular mechanisms of speciation and the development of species-specific traits. The aim of this thesis is to characterize differences between the two species with regard to their genome sequences and the resulting transcript profiles. The first two papers focus on indel divergence and in particular, indels causing premature termination codons (PTCs) in 8% of the chimpanzee genes. The density of PTC genes is correlated with both the distance to the telomere and the indel divergence. Many PTC genes have several associated transcripts and since not all are affected by the PTC we propose that PTCs may affect the pattern of expressed isoforms. In the third paper, we investigate the transcriptome divergence in cerebellum, heart and liver, using high-density exon arrays. The results show that gene expression differs more between tissues than between species. Approximately 15% of the genes are differentially expressed between species, and half of the genes show different splicing patterns. We identify 28 cassette exons which are only included in one of the species, often in a tissue-specific manner. In the fourth paper, we use massive parallel sequencing to study the chimpanzee transcriptome in frontal cortex and liver. We estimate gene expression and search for novel transcribed regions (TRs). The majority of TRs are located close to genes and possibly extend the annotations. A subset of TRs are not found in the human genome. The brain transcriptome differs substantially from that of the liver and we identify a subset of genes enriched with TRs in frontal cortex. In conclusion, this thesis provides evidence of extensive genomic and transcriptomic variability between human and chimpanzee. The findings provide a basis for further studies of the underlying differences affecting phenotypic divergence between human and chimpanzee.

La sensibilité des primates aux pathogènes et aux maladies inflammatoires chroniques varie considérablement. Par exemple, les singes (tels que les humains et les chimpanzés) sont très sensibles à de très petites doses de lipopolysaccharide (LPS), une molécule mimétique d'agent pathogène, qui cause de graves lésions tissulaires en raison de l'immunopathologie tandis que les singes africains et asiatiques clades soeurs AAM (tels que les macaques et les babouins) sont beaucoup plus tolérants à des doses beaucoup plus élevées de LPS. Cet écart entre l'homme et les autres primates est connu pour être, au moins partiellement, dû à la différence interspécifique de la réponse immunitaire. Dans cette thèse, j'ai effectué une analyse comparative de la réponse immunitaire à travers différentes lignées de primates pour obtenir des informations supplémentaires sur l'évolution de la réponse immunitaire. J'ai trouvé que les singes provoquent une réponse immunitaire beaucoup plus forte aux stimulants (bactériens ou viraux) par rapport aux AMM. Une telle réponse plus élevée s'est également avérée corrélée avec la phylogénie du primate, la plus élevée chez le primate supérieur (humain) et la plus faible chez le primate basal (lémurien). Une réponse aussi élevée peut être bénéfique pour la médiation d'une destruction efficace des agents pathogènes, mais elle est probablement accompagnée de lésions tissulaires plus élevées, ce qui pourrait expliquer pourquoi les humains sont plus sensibles aux maladies immunopathologiques telles que la septicémie. J'ai également caractérisé le paysage réglementaire de la réponse immunitaire chez ces primates. J'ai trouvé que l'activité des éléments régulateurs était significativement différente entre les différentes espèces de primates après une stimulation immunitaire mettant en évidence le rôle de l'épigénétique dans la conduite du changement de la réponse immunitaire chez les primates. De plus, j'ai trouvé une signature d'évolution adaptative sur les régions actives associées aux gènes qui ont la réponse la plus élevée chez l'homme par rapport aux AMM révélant le rôle de la sélection naturelle sur le façonnement de la réponse immunitaire chez les primates. / Primates vary remarkably in their disease susceptibility to pathogens and chronic inflammatory diseases. For instance, apes (such as humans and chimps) are highly sensitive to very small doses of lipopolysaccharide (LPS), a pathogen mimicry molecule, that causes severe tissue damage due to immunopathology while sister clade African and Asian monkeys AAMs (such as macaque and baboon) are far more tolerant to much higher doses of LPS. This discrepancy between humans and other primates is known to be, at least partially, due to interspecies differences of the immune response. In this dissertation, I performed comparative analyses of immune responses across different primate lineages to gain further insights on the evolution of immune response. I found that apes elicit a much stronger immune response to stimulants (bacterial or viral) relative to AMMs. Such a higher response was also found to be correlated with the primate phylogeny, highest in the higher primate (human) and lowest in the basal primate (lemur). Moreover, this high response may be beneficial in mediating effective pathogen killing but it is likely accompanied by higher tissue damage, which might explain why humans are more susceptible to immunopathological diseases such as sepsis. I also characterized the regulatory landscape of immune response across these primates. I found the regulatory elements activity to be significantly different between different primate species after immune stimulation highlighting the role of epigenetics in driving the immune response change across primates. In addition, I found a signature of adaptive evolution on active regions associated with genes that have the highest response in humans versus AMMs revealing the role of natural selection in shaping the immune response in primates.

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