<p>Male mammals must simultaneously produce prodigious numbers of sperm and maintain an adequate reserve of stem cells to ensure continuous production of gametes throughout life. Failures in the mechanisms responsible for balancing germ cell differentiation and spermatogonial stem cell (SSC) self-renewal can result in infertility. We discovered a novel requirement for Androgen Receptor Trapped clone-27 (ART-27) in spermatogenesis by developing the first knockout mouse model for this gene. Constitutive deletion of ART-27 is embryonic lethal between e5.5 and 7.5 due to defects in extra-embryonic tissues. Conditional knockout in the male germline results in a rapid decline in pre-meiotic germ cell number that starts around day 6-7 post-partum, eventually leading to a Sertoli cell-only phenotype that does not recover in the adult. Gene expression analysis revealed that ART-27 deletion downregulates the transcription of genes governing SSC self-renewal, differentiation, and meiosis. These data are consistent with spermatogenic arrest before meiotic entry and the total lack of germ cells after day 23. Sertoli cell-specific knockout of ART-27 also results in germ cell loss, and we hypothesize this is due to disruption of androgen receptor signaling. Our study has revealed the first in vivo function for ART-27 in the mammalian germline as a regulator of distinct transcriptional programs in SSCs and differentiating spermatogonia.

<p> Sperm mobility is a major determinant of male fertility in chicken. In spite of low heritability of reproductive traits, sperm mobility has high heritability index which suggests presence of quantitative trait loci (QTLs) governing the trait. Our research focused on three objectives: i) to identify the QTLs affecting low mobility phenotype in chicken, ii) to understand the impact of Sertoli-cells and germ cells interactions in influencing the mobility phenotype and iii) to identify the genes and gene networks differentially expressed in male and female PGCs. To detect the QTLs, genome wide association studies (GWAS) was conducted which revealed the presence of multiple minor alleles influencing the trait and indicated the role of epistasis. The second section of research involved isolation, culture and transfer of primordial germ cells (PGCs) to create high line germ line chimera chicken carrying low line PGCs. We established the culture of chicken PGCs isolated from the embryonic blood in a feeder free culture conditions but could not detect the presence of low line genotype in the semen of transgenic males. Our final study involved RNA-sequencing (RNAseq) of male and female PGCs to identify differentially expressed genes from their transcriptomes. We identified five candidate genes: 3-hydroxy-3-methylglutaryl CoA reductase (HMGCA), germ cell-less (GCL), SWIM (zinc finger SWIM domain containing transcription factor), SLC1A1 (solute carrier family 1 member 1), UBE2R2L (ubiquitin conjugating enzyme) and validated their expression level in male and female PGCs by RT-qPCR. GCL was exclusively expressed in males while SLC1A1 &amp; UBE2R2L were expressed only in female cPGCs. This present study provides novel gender specific germ cell markers in the broiler chicken. These results will help in elucidating the genetic programming of gender specific germ line development in broilers.</p><p>

Genome-wide binding and expression studies in Drosophila melanogaster have revealed widespread roles for Dichaete and SoxNeuro, two group B Sox proteins, during fly development. Although they have distinct target genes, these two transcription factors bind in very similar patterns across the genome and can partially compensate for each other's loss, both phenotypically and at the level of DNA binding. However, the inherent noise in genome-wide binding studies as well as the high affinity of transcription factors for DNA and the potential for non-specific binding makes it difficult to identify true functional binding events. Additionally, externalfactors such as chromatin accessibility are known to play a role in determining binding patterns in Drosophila. A comparative approach to transcription factor binding facilitates the use of evolutionary conservation to identify functional features of binding patterns. In order to discover highly conserved features of group B Sox binding, I performed DamID-seq for SoxNeuro and Dichaete in four species of Drosophila, D. melanogaster, D. simulans, D. yakuba and D. pseudoobscura. I also performed FAIRE-seq in D. pseudoobscura embryos to compare the chromatin accessibility landscape between two fly species and to examine the relationship between open chromatin and group B Sox binding. I found that, although the sequences, expression patterns and overall transcriptional regulatory targets of Dichaete and SoxNeuro are highly conserved across the drosophilids, both binding site turnover and rates of quantitative binding divergence between species increase with phylogenetic distance. Elevated rates of binding conservation can be found at bound genomic intervals overlapping functional sites, including known enhancers, direct targets of Dichaete and SoxNeuro, and core binding intervals identified in previous genome-wide studies. Sox motifs identified in intervals that show binding conservation are also more highly conserved than those in intervals that are only bound in one species. Notably, regions that are bound in common by SoxNeuro and Dichaete are more likely to be conserved between species than those bound by one protein alone. However, by examining binding intervals that are uniquely bound by one protein and conserved, I was able to identify distinctive features of the targets of each transcription factor that point to unique aspects of their functions. My comparative analysis of group B Sox binding suggests that sites that are commonly bound by Dichaete and SoxNeuro, primarily at targets in the developing nervous system, are highlyconstrained by natural selection. Uniquely bound targets have different tissue expression profiles, leading me to propose a model whereby the unique functions of Dichaete and SoxNeuro may arise from a combination of differences in their own expression patterns and the broader nuclear environment, including tissue-specific cofactors and patterns of accessible chromatin. These results shed light on the evolutionary forces that have maintained conservation of the complex functional relationships between group B Sox proteins from insects to mammals.

La régulation de l’expression génique chez les mammifères dépend de l’organisation tridimensionnelle des chromosomes, en particulier à l’échelle des communications entre les séquences régulatrices et leurs promoteurs cibles. Ainsi, les chromosomes sont organisés en une nouvelle architecture consistant en domaines d’interactions topologiques (TADs, acronyme anglais). Mon projet de thèse avait pour but de caractériser les mécanismes moléculaires impliqués dans cette architecture et leurs importances au cours du développement embryonnaire, pour un locus bien particulier, le Xic (acronyme anglais pour X-inactivation centre). Le Xic contient les éléments régulateurs nécessaires pour initier l’inactivation du chromosome X (ICX), un phénomène épigénétique spécifique du développement des mammifères femelles, rendant l’un des deux chromosomes X inactif du point de vue transcriptionnelle. L’ICX permet d’égaliser l’expression des gènes liés au X entre les sexes chez les mammifères. Le Xic est organisé au moins en deux TADs mais une partie du locus reste encore non identifiée. Je présente ici une analyse fonctionnelle approfondie des différents éléments régulateurs au sein du Xic, comprenant des enhancers, des gènes d’ARNs non codants et des éléments structurels. Après avoir créé une série d’allèles mutés chez la souris et les cellules souches embryonnaires murines, j’ai caractérisé l’impact de ces réarrangements génomiques sur le paysage structurel et transcriptionnel du Xic. J’ai identifié des nouveaux acteurs dans la régulation de ce locus, en particulier des séquences régulatrices conservées chez les mammifères placentaires et des éléments structurels importants pour la formation d’une frontière entre les deux TADs du Xic, importante pour leur séparation et régulation. Je décris aussi la découverte de communication entre ces TADs, ce qui constitue un mécanisme inédit de régulation génique pendant le développement. Ce travail contribue à un nouveau niveau de compréhension des lois qui régissent l’organisation des TADs dans le contexte de la régulation génique chez les mammifères. / Mammalian gene regulatory landscapes rely on the folding of chromosomes in the recently discovered topologically associating domains (TADs), which ensure appropriate communication between cis-regulatory elements and their target promoters. The aim of my PhD project was to characterise the molecular mechanisms that govern this novel architecture and its functional importance in the context of a critical and developmentally regulated locus, the X-inactivation centre (Xic). The Xic contains the necessary elements to trigger X-chromosome inactivation, an epigenetic phenomenon that occurs during the development of female mammals to transcriptionally silence one of the X-chromosomes and equalise X-linked gene expression between sexes. The Xic is partitioned into at least two TADs, but its full extent is unknown. Here, I present a comprehensive functional analysis of different cis-regulatory elements within the Xic, including enhancer-like regions, long noncoding RNA loci and structural elements. Upon generating a series of mutant alleles in mice and murine embryonic stem cells, I characterised the impact of these genomic rearrangements in the structural and transcriptional landscape of the Xic and identified novel players in the regulation of this locus, including cis-acting elements conserved across placental mammals and structural elements critical for the insulation between the Xic TADs. I also found evidence for communication across TADs at this locus, which provides new insights into how regulatory landscapes can work during development. This study also extends our understanding of the rules governing the organisation of TADs and their chromatin loops in the context of mammalian gene regulation. / Nos mamíferos, a regulação da expressão genética depende da organização tridimensional dos cromosomas, em particular ao nível da comunicação regulatória entre promotores e enhancers. A esta escala, descobriu-se recentemente que os cromossomas estão organizados em domínios de interações topológicas (conhecidos como TADs, no acrónimo inglês) que se pensa providenciarem uma base estrutural para as paisagens de regulação transcricional dos genes. O meu projecto de tese teve como objectivo caracterizar os mecanismos moleculares responsáveis por esta arquitectura e a sua importância funcional no contexto de um locus crítico para o desenvolvimento embrionário, o centro de inactivação do cromossoma X (Xic, acrónimo inglês). O Xic contém os elementos genéticos necessários e suficientes para iniciar a inactivação do cromossoma X, um fenómeno epigenético que ocorre durante o desenvolvimento das fêmeas de mamíferos para silenciar um dos cromosomas X e igualar a expressão dos genes do X entre indivíduos XX e XY. O Xic está organizado em pelo menos dois TADs, mas o seu intervalo genético completo permanece desconhecido. Apresento nesta tese uma análise funcional e detalhada de diferentes sequências reguladoras presentes no Xic, incluindo regiões do tipo enhancer, genes de ARNs não codificantes e elementos estruturais. Após a criação de diversos alelos mutantes (deleções, inserções, inversões) em ratinho e em células estaminais embrionárias, através das recentes técnicas de engenharia genética, TALENs e CRISPR/Cas9, caracterizei o impacto destes rearranjos genéticos na paisagem topológica e transcricional do Xic, o que permitiu a identificação de novos actores moleculares na regulação deste locus. Em particular, descobrimos sequências de regulação transcricional altamente conservadas em mamíferos placentários e elementos estruturais importantes para a formação da fronteira entre os dois TADs do Xic. Descrevo também evidência de que há comunicação entre os dois TADs neste locus, o que compromete os modelos actuais do modus operandis dos TADs, e por isso contribui para um novo nível de compreensão dos mecanismos que regulam a expressão genética durante o desenvolvimento.