The retinoblastoma tumor suppressor protein (RB) is an important regulator of the cell cycle and development. Significantly, RB is inactivated in a majority of human cancers. Thus, elucidating the function of RB will give us a better understanding of how it prevents cancer. Many decades of research have yielded a detailed understanding of the role of RB in cell proliferation through transcriptional repression of target genes. However, the precise mechanisms of its action in many cellular pathways are poorly understood, including the control of DNA replication and post-transcriptional control of gene expression. Drosophila melanogaster presents a simplified genetic system to study cancer genes. Several published observations have suggested a role for RB in regulating DNA replication. Interestingly, other data indicate that RB associates with RNA processing factors. I have characterized novel protein-protein interactions with the Drosophila retinoblastoma tumor suppressor homologue Rbf, with an emphasis on its poorly characterized N-terminal domain. I describe the interaction of Rbf with the origin recognition complex, indicating a unique connection to DNA replication control. I also show that Rbf interacts with the RNA binding protein Squid, and review the literature that suggests potential role of RB/E2F in the control of RNA processing. The ability to control RNA processing may be an additional, unappreciated mode of gene regulation by RB. A focused study of the uncharacterized amino-terminal domain of Rbf has revealed new details about the retinoblastoma tumor suppressor in cell regulation, including DNA replication and RNA processing.

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第24326号 / エネ博第454号 / 新制||エネ||85(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 片平 正人, 教授 森井 孝, 教授 杤尾 豪人 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

Chez les animaux, la conformation unique du noyau du spermatozoïde dont la chromatine est organisée avec des protéines chromosomiques spécifiques telles que les protamines le rend totalement inactif. Le remodelage de la chromatine paternelle à la fécondation par des activités d'origine maternelle sont donc des processus essentiels à la formation d'un embryon diploïde, dont les mécanismes restent très mal connus. Lors de ma thèse j'ai essayé de mieux comprendre ces processus par l'étude, chez la drosophile, d'un mutant létal embryonnaire à effet maternel : maternal haploid (mh). Ce mutant affecte l'incorporation des chromosomes paternels à la première division zygotique menant à la formation d'embryons haploïdes gynogénétiques. L'identification du gène de mh comme CG9203 m'ont permis de caractériser sa fonction. Dans les œufs mh, les chromosomes paternels se condensent anormalement et ne parviennent pas à se diviser correctement lors de la première mitose de l'embryon. Récemment, des études sur son orthologue humain, appelé Spartan/DVC1, ont montré qu'il était impliqué dans la synthèse translésionnelle (TLS), un mécanisme de tolérance aux dommages d'ADN. J'ai pu démontrer que dans les cellules somatiques, la fonction de Spartan dans le TLS est conservée chez la drosophile. Cependant, la fonction maternelle de MH ne relève pas du TLS canonique, mais permet de maintenir l'intégrité de l'ADN paternel avant la réplication. Ensemble, mes travaux soulignent la singularité du pronoyau mâle et la complexité que présente le maintien de son intégrité à la fécondation / In animals, sexual reproduction requires the union between two distinct parental gametes: the spermatozoon and the oocyte. The unique nuclear conformation of the sperm, in which the chromatin is organized with sperm-specific chromosomal protein like protamines, abolishes its activity. The paternal chromatin remodeling and the maintenance of its integrity at fertilization by maternal activities are therefore essential processes for zygote formation. However, although their mechanisms are crucial, they remain poorly understood. During my thesis, I tried to better understand the processes involved during de novo paternal chromatin assembly in Drosophila through the study of a maternal embryonic lethal mutation: maternal haploid (mh). The mutant affects the incorporation of paternal chromosomes during the first zygotic division, leading to the development of gynogenetic haploid embryos. The identification of the mh gene as CG9203, and the generation of the null allele mh2 allowed me to characterize its function. In eggs led by mh mutant females, paternal chromosomes abnormally condense and fail to divide leading to the formation of chromatin bridges at the first embryonic division. Recently, its human ortholog Spartan/DVC1, has been described to be involved in translesion synthesis (TLS), a DNA damage tolerance pathway that ensures replication fork progression. Combining genetic and cytological approaches, I demonstrated that the Spartan function in TLS is conserved in Drosophila. However, I discovered that the critical function of MH during the first embryonic division, was not consistent with a canonical TLS. Alternatively, it is specifically required to maintain paternal integrity and to allow its proper replication at the first cycle. The mh phenotype characterization, led me to compare it with others phenotypes induced by the knock-down of replication factors and to study parental chromosome condensation in the zygote. Surprisingly, one of the proteins allowing the establishment of the pre-replication complex is dispensable for the proper paternal chromosome segregation contrarily to the maternal counterpart. Altogether, these works highlight the difference that exists between the two parental pronuclei and the complexity of maintaining their integrity at fertilization

<p>The initiation of two major processes in the eukaryotic cell, gene transcription and DNA replication, is regulated largely through interactions between proteins or protein complexes and DNA. Although a lot is known about the interacting proteins and their role in regulating transcription and replication, the specific DNA binding motifs of many regulatory proteins and complexes are still to be determined. For this purpose, many computational tools for DNA motif discovery have been developed in the last two decades. These tools employ a variety of strategies, from exhaustive search to sampling techniques, with the hope of finding over-represented motifs in sets of co-regulated or co-bound sequences. Despite the variety of computational tools aimed at solving the problem of motif discovery, their ability to correctly detect known DNA motifs is still limited. The motifs are usually short and many times degenerate, which makes them difficult to distinguish from genomic background. We believe the most efficient strategy for improving the performance of motif discovery is not to use increasingly complex computational and statistical methods and models, but to incorporate more of the biology into the computational techniques, in a principled manner. To this end, we propose a novel motif discovery algorithm: PRIORITY. Based on a general Gibbs sampling framework, PRIORITY has a major advantage over other motif discovery tools: it can incorporate different types of biological information (e.g., nucleosome positioning information) to guide the search for DNA binding sites toward regions where these sites are more likely to occur (e.g., nucleosome-free regions). </p><p>We use transcription factor (TF) binding data from yeast chromatin immunoprecipitation (ChIP-chip) experiments to test the performance of our motif discovery algorithm when incorporating three types of biological information: information about nucleosome positioning, information about DNA double-helical stability, and evolutionary conservation information. In each case, incorporating additional biological information has proven very useful in increasing the accuracy of motif finding, with the number of correctly identified motifs increasing with up to 52%. PRIORITY is not restricted to TF binding data. In this work, we also analyze origin recognition complex (ORC) binding data and show that PRIORITY can utilize DNA structural information to predict the binding specificity of the yeast ORC. </p><p>Despite the improvement obtained using additional biological information, the success of motif discovery algorithms in identifying known motifs is still limited, especially when applied to sequences bound in vivo (such as those of ChIP-chip) because the observed protein-DNA interactions are not necessarily direct. Some TFs associate with DNA only indirectly via protein partners, while others exhibit both direct and indirect binding. We propose a novel method to distinguish between direct and indirect TF-DNA interactions, integrating in vivo TF binding data, in vivo nucleosome occupancy data, and in vitro motifs from protein binding microarrays. When applied to yeast ChIP-chip data, our method reveals that only 48% of the ChIP-chip data sets can be readily explained by direct binding of the profiled TF, while 16% can be explained by indirect DNA binding. In the remaining 36%, we found that none of the motifs used in our analysis was able to explain the ChIP-chip data, either because the data was too noisy or because the set of motifs was incomplete. As more in vitro motifs become available, our method can be used to build a complete catalog of direct and indirect TF-DNA interactions.</p> / Dissertation