Function and Regulation of the Y-Linked Axonemal Dynein Genes During Drosophila Spermatogenesis

The germline is considered to be immortal, meaning an organism’s germ cells have the potential to give rise to all subsequent generations. With so much at stake, the germline goes to great lengths to protect itself while also maintaining reproductive potential, resulting in fascinating and innovative biology. This thesis focuses on two aspects of germ cell development in Drosophila males that appear disadvantageous yet are prevalent across drosophilids. The first is the expression of the Y chromosome gigantic genes – these genes are essential for fertility yet they are riddled with megabases of repetitive DNA. The second is the assembly of the long cilia found within the sperm’s tail – Drosophila have some of the longest sperm in the animal kingdom, yet little is known about how these long cilia are assembled. This thesis will describe the innovations that have allowed germ cells to overcome these challenges and will go on to discuss how these burdens may benefit the fly. In Drosophila, the Y chromosome is largely heterochromatic, encoding only a handful of genes, which are essential for male fertility. Intriguingly, some of these genes are amongst the largest genes identified to date, spanning several megabases. For example, the gene kl-3, which encodes an axonemal dynein motor protein required for sperm motility, spans 4.3Mb with only 14kb of coding sequence. The introns of these genes contain megabases of simple satellite DNA repeats (e.g. (AATAT)n) that comprise over 99% of the locus. Although this “intron gigantism” has been observed in several genes across species, including the mammalian Dystrophin gene, its regulation and functional relevance remains elusive. The transcription/processing of such gigantic genes/RNA transcripts poses a significant challenge. I identified that the Y-linked gigantic genes require a unique gene expression program in order to overcome these challenges. By monitoring Y-linked gene expression over developmental time, I found that transcription of these loci takes 80-90 hours. I further identified two RNA-binding proteins that specifically bind to Y-linked gene transcripts. Loss of either RNA binding protein resulted in sterility due to the loss of Y-linked gene products. I found that this unique gene expression program functions on two fronts: it increases the ability of RNA polymerase to transcribe the repetitive introns, and it aids in processing the large transcripts. I speculate that this program may be utilized to modulate gene expression patterns during development. During Drosophila spermatogenesis, germ cells undergo drastic morphological changes to yield a 1.9mm sperm. The cilia found within the sperm tail are cytoplasmic cilia – a specialized type of cilia where the axoneme (the microtubule structural component) resides within the cytoplasm instead of within a specialized ciliary compartment. Cytoplasmic cilia likely allow for efficient assembly of longer cilia, however, the mechanism for their assembly remains unknown. I found that mRNAs encoding four axonemal dynein heavy chain genes (three of which are Y-linked gigantic genes) colocalize in a novel ribonucleoprotein (RNP) granule, which localizes near the site of axoneme assembly during sperm elongation. Precise localization of this RNP granule mediates incorporation of the axonemal dynein motor proteins into the axoneme. This work is the first to uncover how cytoplasmic cilia are efficiently assembled to allow for the production of 1.9mm sperm, and highlights that there are other cilia assembly mechanisms besides the ancient and conserved mechanism by which traditional cilia assemble.PHDCellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163043/1/jaclynmf_1.pd

La familia génica FTF determina el patrón de colonización y la virulencia en Fusarium oxysporum

[ES] La familia génica FTF (Fusarium Transcription Factor) comprende un gen de copia única, FTF2, que está presente en todos los ascomicetos filamentosos analizados, y varias copias de FTF1, que es exclusivo de Fusarium oxysporum. Un sistema de silenciamiento de genes mediado por ARN ha sido desarrollado para atenuar específicamente los ARNm producidos por todos los genes FTF, probándose en dos formae speciales: F. oxysporum f. sp. phaseoli (cuyo hospedador es la judía) y F. oxysporum f. sp. lycopersici (cuyo hospedador es el tomate). La disminución de la expresión de FTF da lugar a una marcada reducción en la virulencia, una expresión reducida de varios genes SIX (Secreted In Xylem), la familia de efectores mejor estudiada en F. oxysporum, y menores niveles de ARNm de SGE1 (SIX Gene Expression 1), el presunto regulador de la expresión de algunos genes SIX. Por otra parte, los mutantes silenciados muestran un patrón de colonización de la planta huésped similar al mostrado por las estirpes carentes de copias de FTF1 (estirpes poco virulentas), caracterizado por una colonización de los haces xilemáticos lenta y de baja eficiencia, y una fuerte y temprana inducción de la resistencia sistémica adquirida (SAR) en el huésped

Exploration génétique et moléculaire de défauts post-méiotiques sévères de la spermatogenèse entrainant une infertilité masculine

Infertility is currently considered by the World Health Organization (WHO) as a major health concern affecting more than 50 million couples worldwide. In western countries, the majority of infertile couples seek assisted reproductive technologies (ART) to achieve a pregnancy. Despite the success of these techniques, almost half of these couples fail to obtain a child. Part of these failures are explained by the alteration of gametogenesis. In humans, spermatogenesis involves hundreds of genes specifically expressed in the testis. The abundance of these genes suggests that spermatogenic defects are associated with a strong genetic component. Recently, technical advances have led to the identification of numerous causative genes, but the vast majority of male infertility cases remain idiopathic. The aim of the present thesis is to identify new genetic causes responsible for male infertility and to elucidate the physiopathological mechanisms associated with these anomalies.During my thesis, I participated with the team GETI (genetics, epigenetics and therapies of infertility) in the genetic exploration of two phenotypes of male infertility related to post-meiotic defects of spermatogenesis: a rare form of non-obstructive azoospermia and the phenotype of multiple morphological abnormalities of the sperm flagella (MMAF). I have also played a key role in creation and analysis of transgenic mice to better characterize the pathogeny of the identified genetic causes in Human.Genetic analyses performed on two infertile brothers born form consanguineous parents and presenting an-idiopathic non-obstructive azoospermia associated with a post-meiotic arrest of spermatogenesis allowed us to identify a homozygous variant in the SPINK2 gene that encodes a serine-protease inhibitor. Phenotypic analysis of Spink2-/- adult male mice showed that they are infertile and perfectly mimic the sperm and testicular phenotypes observed in our patients. We showed that Spink2 protein is expressed from the round spermatid stage and localized in the acrosome, a lysosomal-like vesicle rich in proteases that play a key role during fertilization. When Spink2 is absent, the deregulated proteolytic activity of the targeted proteases such as acrosin leads to the fragmentation of the Golgi apparatus and arrest of spermiogenesis at the round spermatid stage. We also showed that sperm from heterozygous human and mice present a high level of morphological abnormalities and a decrease of progressive motility leading to a variable subfertility. These results showed for the first time that oligo-teratozoospermia and azoospermia could present a pathological continuum due to the same pathogeny.We also performed exome sequencing in a cohort of 78 non related MMAF subjects and identified in 49 cases deleterious bi-allelic mutations in a total of 11 candidate genes including DNAH1, CFAP43, CFAP44, WDR66 and FSIP2 giving a genetic diagnosis yield of 63%. These results confirm the genetic heterogeneity of MMAF and the efficiency of high throughput sequencing in genetic exploration of this phenotype. We also demonstrated the pathogenic implication of certain candidate genes (n=4) using knock-out mice created by the new technology of genome editing, CRISPR/Cas9.Overall, this work demonstrates the interest and effectiveness of combining exome sequencing and CRISPR/Cas9 system to study spermatogenesis disorders and male infertility.L’infertilité est considérée actuellement par l’organisation mondiale de la santé (OMS) comme une préoccupation majeure de santé affectant plus de 50 millions de couples dans le monde. Dans les pays occidentaux, la majorité des couples infertiles ont recours aux techniques d’assistance médicale à la procréation (AMP) pour obtenir une grossesse. Malgré le succès de ces techniques, près de la moitié des couples qui ont recours à l’AMP sortent du parcours de soin sans enfant. Une partie de ces échecs est expliquée par l’altération de la gamétogenèse. Chez l’homme, la spermatogenèse fait interagir des centaines de gènes spécifiquement exprimés dans le testicule. L’abondance de ces gènes suggère que les troubles de la spermatogenèse présentent une forte composante génétique. Récemment, les avancées techniques ont favorisé l’identification de gènes responsables de ces anomalies mais la grande majorité des cas d’infertilité masculine reste classée comme idiopathique. L’objectif de la thèse est d’identifier de nouvelles causes génétiques responsables d’infertilité masculine et d’élucider les mécanismes physiopathologiques associés à ces anomalies.Au cours de ma thèse j’ai participé avec l’équipe GETI (génétique, épigénétique et thérapies de l’infertilité) à l’exploration génétique et moléculaire de deux phénotypes distincts d’anomalies spermatiques liés à des défauts post-méiotiques de la spermatogenèse : une forme rare d’azoospermie non obstructive (ANO) et le phénotype d’anomalies morphologiques multiples du flagelle spermatique (AMMF). Enfin j’ai joué un rôle important dans la création et l’analyse de modèles murins pour caractériser la pathogénie de ces anomalies.L’analyse génétique de deux frères infertiles nés de parents consanguins et présentant une ANO idiopathique associée à un arrêt post-méiotique de la spermatogenèse nous a permis d’identifier un variant homozygote délétère dans le gène SPINK2 qui code pour un inhibiteur de sérine-protéases. L’étude des souris KO pour ce gène nous a permis d’observer que les souris mâles adultes sont infertiles et miment parfaitement les phénotypes spermatique et testiculaire observés chez nos patients. Nous avons montré que la protéine codée par ce gène est exprimée dans l’acrosome à partir du stade de spermatide ronde. En l’absence de Spink2, l’activité protéolytique non-neutralisée des protéases cibles qui transitent par le Golgi cause sa fragmentation et bloque la spermiogénèse au stade de spermatide ronde. Nous avons également pu observer que les spermatozoïdes provenant de patients et de souris hétérozygotes présentent un taux élevé d’anomalies morphologiques et une baisse de la mobilité progressive conduisant à une hypofertilité à expressivité variable. Ces résultats montrent pour la première fois que l’oligo-tératozoospermie et l’azoospermie peuvent constituer un continuum pathologique dû à une même pathogénie.Nous avons également réalisé le séquençage exomique complet d’une cohorte de 78 individus AMMF non apparentés et avons identifié chez 49 sujets des mutations bi-alléliques délétères dans 11 gènes candidats dont DNAH1, CFAP43, CFAP44, WDR66 et FSIP2, soit un rendement diagnostique de 63%. Ces résultats confirment l’hétérogénéité génétique du phénotype MMAF et l’efficacité diagnostique du séquençage haut débit dans son exploration. Nous avons également validé l’implication de certains gènes candidats (n=4) dans ce phénotype chez le modèle murin knock-out créé par la nouvelle technologie d’édition du génome, CRISPR/Cas9.Dans son ensemble, ce travail montre l’intérêt et l’efficacité de la combinaison du séquençage exomique et de la technique de CRISPR/Cas9 pour étudier les troubles de la spermatogenèse et l’infertilité masculine

Defining the functions and mechanisms of mRNA targeting to the mitotic apparatus

La localisation des ARNm dans différents compartiments subcellulaires est conservée dans un large éventail d'espèces et de divers types cellulaires. Le trafic est médié par l'interaction entre les protéines de liaison à l'ARN (RBP) et l'ARNm. Les RBP reconnaissent les éléments cis-régulateurs de l'ARNm, également appelés éléments de localisation. Ceux-ci sont définis par leur séquence et/ou leurs caractéristiques structurelles résidant dans la molécule d'ARNm. La localisation des ARNm est essentielle pour la résolution subcellulaire et temporelle. De plus, les ARNm se sont avérés enrichis dans de nombreux compartiments cellulaires, notamment les mitochondries, l'appareil mitotique, et le réticulum endoplasmique. En outre, des études ont démontré que les RBP et les ARNm sont associés aux structures de l'appareil mitotique. Cependant, le rôle que joue la localisation de l'ARNm au cours de la mitose reste largement inexploré. Ma thèse de doctorat vise à comprendre comment le trafic d'ARNm est impliqué lors de la mitose. La première partie de cette thèse porte sur l'interaction post-transcriptionnelle qui se produit entre les deux ARNm, cen et ik2. Les gènes qui se chevauchent sont une caractéristique frappante de la plupart des génomes. En fait, il a été constaté que le chevauchement des séquences génomiques module différents aspects de la régulation des gènes tels que l'empreinte génomique, la transcription, l'édition et la traduction de l'ARN. Cependant, la mesure dans laquelle cette organisation influence les événements réglementaires opérant au niveau post-transcriptionnel reste incertaine. En étudiant les gènes cen et ik2 de Drosophila melanogaster, qui sont transcrits de manière convergente avec des régions 3' non traduites qui se chevauchent, nous avons constaté que la liaison physique de ces gènes est un déterminant clé dans la co-localisation de leurs ARNm aux centrosomes cytoplasmiques. Le ciblage du transcrit ik2 dépend de la présence et de l'association physique avec l'ARNm de cen, qui est le principal moteur de la co-localisation centrosomale. En interrogeant les ensembles de données de séquençage de fractionnement, nous constatons que les ARNm codés par des gènes qui se chevauchent en 3' sont plus souvent co-localisés par rapport aux paires de transcrits aléatoires. Ce travail suggère que les interactions post-transcriptionnelles des ARNm avec des séquences complémentaires peuvent dicter leur destin de localisation dans le cytoplasme. La deuxième partie de cette thèse consiste à étudier le rôle que jouent les RBP au cours de la mitose. Auparavant, les RBP se sont avérés être associés au fuseau et aux centrosomes. Cependant, leur rôle fonctionnel au niveau de ces structures reste à étudier. Grâce à un criblage par imagerie avec plus de 300 anticorps, nous avons identifié 30 RBP localisés dans les structures mitotiques des cellules HeLa. Ensuite, pour évaluer les rôles fonctionnels de ces RBP, nous avons utilisé l'interférence ARN (ARNi) pour évaluer si la fidélité du cycle cellulaire était compromise dans les cellules HeLa et les embryons de Drosophila melanogaster. Fait intéressant, nous avons identifié plusieurs candidats RBP pour lesquels le knockdown perturbe la mitose et la localisation de l'ARNm dans les cellules HeLa. De plus, la perte des orthologues a entraîné des défauts de développement chez l'embryon de mouche. Grâce à ce travail, nous avons démontré que les RBP sont impliquées pour assurer une mitose sans erreur. En résumé, les travaux que j'ai menés mettent en lumière l'implication de la régulation post-transcriptionnelle au cours de la mitose. En définissant les fonctions et le mécanisme de localisation des ARNm en mitose, ce travail permettra de définir de nouvelles voies moléculaires impliquées dans la régulation de la mitose. Puisque la division cellulaire non contrôlée peut mener à des maladies tel le cancer, étudier le contrôle du cycle cellulaire sous cet angle « centré sur l'ARN » peut aider à développer de nouvelles approches thérapeutiques pour trouver des solutions aux problèmes de santé.The localization of mRNAs to different subcellular compartments is conserved in a wide range of species and diverse cell types. Trafficking is mediated by the interaction between RNA binding proteins (RBPs) and mRNA. RBPs recognize mRNA cis regulatory motifs, otherwise known as localization elements. These are defined by their sequence and/or structural features residing within the mRNA molecule. Localization of mRNAs is essential for subcellular and temporal resolution. Furthermore, mRNAs have been found to be enriched in many cellular compartments including the mitochondria, mitotic apparatus, and endoplasmic reticulum. Moreover, studies have demonstrated that RBPs and mRNAs are associated with mitotic apparatus structures. However, the role that mRNA localization plays during mitosis remains largely unexplored. My PhD thesis aims to understand how the trafficking of mRNAs is implicated during mitosis. The first part of this thesis encompasses the post-transcriptional interaction that occurs between the two mRNAs, cen and ik2. Overlapping genes are a striking feature of most genomes. In fact, genomic sequence overlap has been found to modulate different aspects of gene regulation such as genomic imprinting, transcription, RNA editing and translation. However, the extent to which this organization influences regulatory events operating at the post-transcriptional level remains unclear. By studying the cen and ik2 genes of Drosophila melanogaster, which are convergently transcribed with overlapping 3’untranslated regions, we found that the physical linkage of these genes is a key determinant in co-localizing their mRNAs to cytoplasmic centrosomes. Targeting of the ik2 transcript is dependent on the presence and physical association with cen mRNA, which serves as the main driver of centrosomal colocalization. By interrogating global fractionation-sequencing datasets, we find that mRNAs encoded by 3’overlapping genes are more often co-localized as compared to random transcript pairs. This work suggests that post-transcriptional interactions of mRNAs with complementary sequences can dictate their localization fate in the cytoplasm. The second part of this thesis involves investigating the role that RBPs play during mitosis. Previously, RBPs have been found to be associated with the spindle and centrosomes. However, their functional role at these structures was yet to be investigated. Through an imaging screen with >300 antibodies, we identified 30 RBPs localized to mitotic structures in HeLa cells. Then, to assess the functional roles of these RBPs, we used RNA interference (RNAi) to assess whether cell cycle fidelity was compromised in HeLa cells and Drosophila melanogaster embryos. Interestingly, we identified several RBP candidates for which the knockdown disrupted mitosis and mRNA localization in HeLa cells. Furthermore, loss of the orthologs led to developmental defects in the fly embryo. Through this work, we demonstrated that RBPs are involved in ensuring an error-free mitosis. In summary, the work that I have conducted sheds light on the involvement of post-transcriptional regulation during mitosis. By defining the functions and mechanism of mRNA localization in mitosis, this work will help define new molecular pathways involved in mitosis regulation. As uncontrolled cell division can lead to diseases such as cancer, studying cell cycle control from this ‘RNA-centric’ angle may help to develop new therapeutic approaches to find solutions to health problems

The function of the WHIRLY1 protein in photosynthetic light acclimation of barley

WHIRLY1 is a chloroplast-nucleus located DNA-binding protein. Its association to nucleoids makes this protein an ideal candidate to be involved in responses to environmental changes, which are signalled to the nucleus by retrograde signalling. The latter consequently leads to orchestrating gene expression in the plastid and nucleus in response to environmental changes. In this study, light acclimation was compared between transgenic barley plants with an RNAi-mediated knockdown of HvWHIRLY1 (W1) and wild-type plants (WT). Whereas WT plants showed the typical light acclimation responses, e.g. higher photosynthetic capacity and thicker leaves, W1 did not respond to high irradiance at any developmental stage. The results revealed a systemic role of WHIRLY1 in light acclimation by coordinating responses at both levels of photosynthesis and leaf morphology. Intriguingly, W1 plants did not show symptoms of photoinhibition after chloroplast development. Therefore, their survival under light energy exceeding their photosynthetic capacity with compromised HL acclimation must have been owed to different photoprotective strategies. Among many possible mechanisms, this study revealed some. An extremely large xanthophyll cycle pool, mostly present as zeaxanthin, was in accordance with enhanced NPQ which compensated their low photosynthetic efficiency. Beyond its role in NPQ, enhanced zeaxanthin content together with a high lutein content was assumed to function as an antioxidant. Besides, the dihydroxyflavone lutonarin was highly induced in W1 plants. Due to its antioxidant properties, lutonarin was proposed to be involved in the photoprotection of W1 plants under HL conditions in addition to the carotenoids. Taken together, our results suggest that WHIRLY1 might be a scaffold protein whose ‘optimal abundance’ rather than ‘high or low abundance’ is required for the light acclimation of barley plants

Chromatin and Epigenetics

Genomics has gathered broad public attention since Lamarck put forward his top-down hypothesis of 'motivated change' in 1809 in his famous book "Philosophie Zoologique" and even more so since Darwin published his famous bottom-up theory of natural selection in "The Origin of Species" in 1859. The public awareness culminated in the much anticipated race to decipher the sequence of the human genome in 2002. Over all those years, it has become apparent that genomic DNA is compacted into chromatin with a dedicated 3D higher-order organization and dynamics, and that on each structural level epigenetic modifications exist. The book "Chromatin and Epigenetics" addresses current issues in the fields of epigenetics and chromatin ranging from more theoretical overviews in the first four chapters to much more detailed methodologies and insights into diagnostics and treatments in the following chapters. The chapters illustrate in their depth and breadth that genetic information is stored on all structural and dynamical levels within the nucleus with corresponding modifications of functional relevance. Thus, only an integrative systems approach allows to understand, treat, and manipulate the holistic interplay of genotype and phenotype creating functional genomes. The book chapters therefore contribute to this general perspective, not only opening opportunities for a true universal view on genetic information but also being key for a general understanding of genomes, their function, as well as life and evolution in general

Current Frontiers and Perspectives in Cell Biology

A numerous internationally renowned authors in the pages of this book present the views of the fields of cell biology and their own research results or review of current knowledge. Chapters are divided into five sections that are dedicated to cell structures and functions, genetic material, regulatory mechanisms, cellular biomedicine and new methods in cell biology. Multidisciplinary and often quite versatile approach by many authors have imposed restrictions of this classification, so it is certain that many chapters could belong to the other sections of this book. The current frontiers, on the manner in which they described in the book, can be a good inspiration to many readers for further improving, and perspectives which are highlighted can be seen in many areas of fundamental biology, biomedicine, biotechnology and other applications of knowledge of cell biology. The book will be very useful for beginners to gain insight into new area, as well as experts to find new facts and expanding horizons