Genetic interactions that underlie developmental processes such as cell differentiation and pattern formation are complex and difficult to elucidate. Neural Crest (NC) cells and their derivatives offer an optimal system in which to probe for these complex interactions as they acquire different cell fates and constitute a variety of structures. The transcription factors Sox10 and Pax3 as well as the transmembrane receptor Endothelin receptor b (Ednrb) are temporally and spatially co-expressed early in NC cells and mutations in these genes lead to similar hypopigmentation phenotypes due to a reduced number of NC-derived melanocyte precursors, the melanoblasts. The goal of this study was to establish whether Sox10 and Ednrb or Pax3 and Ednrb interact to promote normal murine melanocyte development. Crosses of Sox10 or Pax3 with Ednrb heterozygous mutants showed that the double heterozygous hypopigmentation phenotype was significantly more pronounced than phenotypes of single heterozygotes, implying that a synergistic interaction exists between Sox10 and Ednrb and Pax3 and Ednrb. This interaction was further explored by the attempt to rescue the Sox10 and Pax3 hypopigmentation phenotypes by the transgenic addition of Ednrb to melanoblasts. Pigmentation was completely restored in the Sox10 and partially restored in the Pax3 mutant mice. The comparison of the number of melanoblasts in transgenic and non-transgenic Sox10 mutant embryos showed that the transgenic rescue occurred as early as E11.5, a critical time for melanoblast population expansion. Cell survival assays indicated that the rescue was not due to an effect of the transgene on melanoblast survival. A novel phenotype arose when studying the interaction between Ednrb and Pax3. Newborns appeared normal but by 3.5 weeks of age, the affected pups were smaller than normal littermates and developed a dome-shaped head; some also developed thoracic kyphosis. Affected pups were dead by 4 weeks of age: 80% were Pax3Sp/+ and 75% were female. When compared to normal littermates, affected mice had brains with enlarged 4th ventricles and more glia while skeletal staining showed kyphosis, wider rib cages and pelvic differences. An epistatic interaction resulting from the mixing of genetic backgrounds that is exacerbated in the presence of Pax3 heterozygosity is suspected.

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Genetics is among the most rewarding fields of biology for the theoretically inclined, offering both room and need for modeling approaches in the light of an abundance of experimental data of different kinds. Many aspects of the field are today understood in terms of physical and chemical models, joined by information theoretical descriptions. This thesis discusses different mechanisms and phenomena related to genetics, employing tools from statistical physics along with experimental biomolecular methods. Five articles support this work. Two articles deal with interactions between proteins and DNA. The first one reports on the properties of non-specific binding of transcription factors proteins in the yeast Saccharomyces cerevisiae, due to an effective background free energy which describes the affinity of a single protein for random locations on DNA. We argue that a background pool of non-specific binding sites is filled up before specific binding sites can be occupied with high probability, thus presenting a natural filter for genetic responses to spurious transcription factor productions. The second article describes an algorithm for the inference of transcription factor binding sites for proteins using a realistic physical model. The functionality of the method is verified on a set of known binding sequences for Escherichia coli transcription factors. The third article describes a possible genetic feedback mechanism between human cells and the ubiquitous Epstein-Barr virus (EBV). 40 binding regions for the major EBV transcription factor EBNA1 are identified in human DNA. Several of these are located nearby genes of particular relevance in the context of EBV infection and the most interesting ones are discussed. The fourth article describes results obtained from a positional autocorrelation analysis of the human genome, a simple technique to visualize and classify sequence repeats, constituting large parts of eukaryotic genomes. Applying this analysis to genome sequences in which previously known repeats have been removed gives rise to signals corroborating the existence of yet unclassified repeats of surprisingly long periods. The fifth article combines computational predictions with a novel molecular biological method based on the rapid amplification of cDNA ends (RACE), coined 5’tagRACE. The first search for non-coding RNAs encoded in the genome of the opportunistic bacterium Enterococcus faecalis is performed here. Applying 5’tagRACE allows us to discover and map 29 novel ncRNAs, 10 putative novelm RNAs and 16 antisense transcriptional organizations. Further studies, which are not included as articles, on the monitoring of secondary structure formation of nucleic acids during thermal renaturation and the inference of genetic couplings of various kinds from massive gene expression data and computational predictions, are outlined in the central chapters. / QC 20110316

Background: Multiple lines of evidence implicate the insulin-like growth factor(IGF) group of proteins in human type 2 diabetes. The actions of IGF-I and IGF-IIare modulated through their interaction with IGF binding proteins. A holisticapproach to study the IGF system is preferable to analyses of individual proteininteractions as the inter-relationships between these proteins are complex. Inparticular, the associations of IGF-II and its associated binding proteins withcardiovascular risk have been inadequately studied. This study aimed to study indetail the genotype and phenotype interactions of the IGF system with longitudinalcardiovascular risk factor trends and phenotypic outcomes in type 2 diabetes.Methods: 1000 subjects of predominantly Caucasian origin from the SalfordDiabetes Cohort were studied. Measurements of IGF proteins (IGF-I, IGF-II,IGFBP-1, IGFBP-2 and IGFBP-3) were performed in 554 of these patients. 991Caucasian subjects were successfully genotyped for 76 single nucleotidepolymorphisms (SNPs) related to ten genes in the IGF system. In this project weanalysed associations of the studied SNPs with the measured IGF proteins as well aslongitudinal risk factor trends. In addition, the baseline concentrations of themeasured proteins were studied for associations with cardiovascular risk factortrends and vascular outcomes.Results: This project demonstrates for the first time that high serum IGF-IIconcentration at baseline predicts longitudinal increases in high-density lipoproteincholesterol. High baseline IGF-II was also observed to predict longitudinal weightloss. High baseline concentration of IGFBP-2 (which has a preferential associationof IGF-II over IGF-I) was associated with a number of favourable longitudinalcardiovascular risk trends like increased HDL cholesterol and decreased diastolicblood pressure. However high IGFBP-2 was also associated with deterioration inrenal function and increased all-cause and cardiovascular mortality. The IGF2 geneand the genes encoding IGFBP-2 and IGFBP-5 (proteins with IGF-II bindingaffinity) were also associated with longitudinal trends in renal function, bloodpressure and cholesterol concentration.Discussion: This study is the most detailed exploration to date of the genotype andphenotype interactions of the IGF system in a Caucasian population with type 2diabetes. Results from this study strongly hint that changes in IGF-II bioavailabilitymay influence inter-individual variations in cardiovascular risk. The precisebiological role of IGF-II merits clarification in future expression studies in renal,adipose and vascular tissues. Replication of significant results in an independentdiabetes cohort and measurement of other IGF binding proteins will be performed inthe next stage of this study.

Paralogs are present during ribosome biogenesis as well as in mature ribosomes in form of ribosomal proteins, and are commonly believed to play redundant functions within the cell. Two previously identified paralogs are the protein pair Ssf1 and Ssf2 (94% homologous). Ssf2 is believed to replace Ssf1 in case of its absence from cells, and depletion of both proteins leads to severely impaired cell growth. Results reveal that, under normal conditions, the Ssf paralogs associate with similar sets of proteins but with varying stabilities. Moreover, disruption of their pre-rRNP particles using high stringency buffers revealed that at least three proteins, possibly Dbp9, Drs1 and Nog1, are strongly associated with each Ssf protein under these conditions, and most likely represent a distinct subcomplex. In this study, depletion phenotypes obtained upon altering Nop7, Ssf1 and/or Ssf2 protein levels revealed that the Ssf paralogs cannot fully compensate for the depletion of one another because they are both, independently, required along parallel pathways that are dependent on the levels of availability of specific ribosome biogenesis proteins. Finally, this work provides evidence that, in yeast, Nop7 is genetically linked with both Ssf proteins. / Les paralogues sont présents lors de la biogenèse des ribosomes ainsi que dans les ribosomes matures sous forme de protéines ribosomiques, et sont généralement censées jouer des fonctions redondantes dans la cellule. Deux paralogues précédemment identifiées sont la paire de protéines Ssf1 et Ssf2 (94 % d'homologie). Ssf2 remplacerait Ssf1 en cas d’absence du dernier dans la cellule, et l’absence des deux protéines diminue la croissance cellulaire. Nos résultats révèlent que, dans des conditions normales, les paralogues Ssf s’associent à des ensembles de protéines similaires, mais avec différentes stabilités. De plus, la perturbation de leurs particules pré-rRNP à l’aide de tampons de haute stringence a révélé qu'au moins trois protéines, probablement Dbp9, Drs1 et Nog1, sont fortement associées à chaque protéine Ssf dans ces conditions, et très probablement représentent des sous-complexes distincts. Dans cette étude, les phénotypes cellulaires observés lors de la déplétion des protéines Nop7, Ssf1 et/ou Ssf2 ont révélé que les paralogues Ssf ne peuvent pas compenser entièrement pour la diminution de l'autre, car ils sont, indépendamment l’un de l’autre, nécessaires le long de voies de biogénèse ribosomale parallèles qui dépendent des niveaux de protéines impliqués dans la biogénèse des ribosomes disponibles. Enfin, ce travail fournit des preuves que, dans la levure, Nop7 est génétiquement lié aux deux protéines Ssf.

Oxidative stress represents a complex and intensely studied phenomenon tightly linked to a range of human diseases, and to aging in many organisms. A plethora of key cellular regulators, including the Notch signaling pathway, have been recently described to respond to the cellular redox status. We have characterized the role of CSL (CBF1/Su(H)/LAG-1) proteins, the effectors of Notch signaling pathway in metazoa, in oxidative stress response in fission yeast. Schizosaccharomyces pombe contains two CSL paralogs, Cbf11 and Cbf12, that have antagonistic functions in the regulation of cell cycle and cellular adhesion. Both proteins are able to bind the canonical CSL motif and activate transcription and, thus, function as genuine CSL transcription factors. We have determined that the strain lacking cbf11 is resistant to hydrogen peroxide but not to menadione, a source of superoxide anion radical. Using double knock-outs to assess genetic interactions we have revealed that the resistance of cbf11 knock-out is dependent on the antioxidants catalase and sulfiredoxin. Genes encoding these antioxidants are under transcriptional control of the Sty1 MAP kinase pathway and the Pap1 transcription factor which are also required for the resistance of Δcbf11 cells. Cbf12 is believed to play only a minor role in...

Les facteurs de transcription Pitx ont été impliqués dans la croissance et la détermination de l’identité des membres postérieurs. D’abord, l’inactivation de Pitx1 chez la souris résulte en la transformation partielle des membres postérieurs en membres antérieurs. Ensuite, la double mutation de Pitx1 et de Pitx2 a montré l’activité redondante de ces facteurs pour la croissance des membres postérieurs. Ainsi, les souris mutantes Pitx1-/-;Pitx2néo/néo montrent une perte des éléments squelettiques proximaux et antérieurs. Des travaux récents ont impliqué les gènes de la famille des Iroquois dans le développement des membres. Tout particulièrement, les souris Irx3-/-;Irx5-/- montrent la perte des éléments squelettiques proximaux et antérieurs, exclusivement au niveau des membres postérieurs. Cette phénocopie entre les souris mutantes pour Pitx1/2 et Irx3/5 nous a amenés à poser trois hypothèses : (1) les Pitx sont responsables de l’expression de Irx dans les bourgeons postérieurs ; (2) à l’inverse, les Irx dirigent l’expression des Pitx ; (3) les Pitx et les Irx participent ensemble au programme génétique de croissance des bourgeons postérieurs. Nous avons pu conclure que les Pitx et les Irx font partie de cascades de régulation indépendantes l’une de l’autre et qu’ils sont capables d’interaction transcriptionnelle autant sur un promoteur générique que sur des régions conservées du locus de Tbx4. Enfin, autant l’inactivation Pitx que celle des Irx mène à un retard d’expression de Pax9 exclusivement dans les bourgeons postérieurs. Ainsi, les Pitx et les Irx semblent agir sur des programmes génétiques parallèles impliqués dans la croissance et le patterning des membres postérieurs. / The Pitx transcription factors have been implicated in growth and specification of hindlimb identity. First, Pitx1 gene inactivation in mice results in partial transformation of hindlimbs into forelimbs. Further, Pitx1 and Pitx2 have redundant activity for hindlimb growth as demonstrated in double mutant mice for Pitx1 and Pitx2. Indeed, Pitx1-/-;Pitx2neo/neo mice show loss of proximal (femur) and anterior (ilium, tibia and first digit) skeletal structures. A collaborator, Dr C. C. Hui of Toronto University, has demonstrated the importance of the Iroquois (Irx) family transcription factors in hindlimb development. Irx3-/-;Irx5-/- mice show loss of proximal and anterior skeletal structures, exclusively in hindlimbs, even if Irx are expressed in the two limb pairs. This phenocopy between Pitx and Irx mutants led us to investigate the interaction between these transcription factors with three hypotheses : (1) Pitx control Irx expression in hindlimb buds ; (2) the Irx direct Pitx gene expression in hindlimb bud ; (3) Pitx and Irx participate together in a growth regulation cascade in hindlimb buds. Our analyses led us to conclude that Pitx and Irx are in independant genetic cascade in hindlimb buds. However, we observed that Pitx and Irx transcription factors are capable of transcriptionnal interactions assessed using either a generic promoter or conserved regions of the Tbx4 locus. Finally, inactivation of either Pitx or Irx led to a delay of Pax9 expression exclusively in hindlimb buds. In sum, Pitx and Irx gene appear to act on parallel genetic programs involved in control of hindlimbs growth and patterning.

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