Nickel insertion into the [NiFe]-hydrogenase requires the accessory protein HypB, which is a GTPase. The GTPase domain of Escherichia coli (E. coli) HypB undergoes dimerization in the presence of GTP. To determine the role of HypB dimerization in hydrogenase biosynthesis, a double mutation L242A/L246A was introduced into full-length E. coli HypB, and the protein was expressed and characterized both in vitro and in vivo. Gel filtration experiments demonstrated that L242A/L246A HypB was monomeric as expected. The inability of L242A/L246A HypB to dimerize does not abolish its GTPase activity and the monomeric L242A/L246A HypB has a similar Ni(II)-binding behavior as that of wild type HypB. Upon the expression of L242A/L246A HypB in vivo the hydrogenase activity is approximately half of the activity of the wild-type control. These experimental results suggest that dimerization of HypB does have a, but not critical, role in hydrogenase biosynthesis.

Nickel insertion into the [NiFe]-hydrogenase requires the accessory protein HypB, which is a GTPase. The GTPase domain of Escherichia coli (E. coli) HypB undergoes dimerization in the presence of GTP. To determine the role of HypB dimerization in hydrogenase biosynthesis, a double mutation L242A/L246A was introduced into full-length E. coli HypB, and the protein was expressed and characterized both in vitro and in vivo. Gel filtration experiments demonstrated that L242A/L246A HypB was monomeric as expected. The inability of L242A/L246A HypB to dimerize does not abolish its GTPase activity and the monomeric L242A/L246A HypB has a similar Ni(II)-binding behavior as that of wild type HypB. Upon the expression of L242A/L246A HypB in vivo the hydrogenase activity is approximately half of the activity of the wild-type control. These experimental results suggest that dimerization of HypB does have a, but not critical, role in hydrogenase biosynthesis.

This study aimed to identify the genetic basis of the Aspergillus nidulans hypB5 mutant phenotype. A. nidulans is a filamentous fungus that is widely used as a cell biological and molecular genetic model system. Its hyphae grow by localized polar secretion, producing tubular cells. A. nidulans hypercellular strains define five unlinked genes, hypA1-hypE2, which cause hyphal morphogenesis defects at 42°C. hypA is orthologous to Saccharomyces TRS120, which mediates Golgi transit and is widely conserved. The hypB5 restrictive phenotype resembles hypA1: wide hyphae, short basal cells and small nuclei. Like hypA1, shifting hypB5 mutants from 28°C to 42°C causes cessation of tip growth but isotropic expansion of basal cells. A hypA1, hypB5 double mutant was impaired for growth at 28°C, suggesting these genes have related roles, but neither was epistatic at 37°C so they function in different pathways. The A. nidulans pRG3-AMA1 genomic library was used to clone hypB5 complementing DNA by phenotype rescue, and subcloned to a 5 kb KpnI fragment, pYY2. pYY2 was disrupted and sequenced by Tn1000 insertional mutagenesis. The pYY2 sequence is 4975 bp and encodes a putative Sec7 domain which has 81% identity to the Saccharomyces SEC7 domain. The Sec7 domain is highly conserved from yeasts to mammals. Saccharomyces SEC7 encodes a guanine nucleotide exchange factor involved in COPI vesicle formation and Golgi biogenesis. Insertions in the pYY2 non-Sec7 domain coding region complemented hypB5 efficiently, whereas those in the Sec7 domain did not, indicating that the Sec7 domain is sufficient for function. A point mutation was found in the hypB5 strain Sec7 domain, which could explain temperature sensitivity. However, the pYY2 sequence is found on chromosome I whereas hypB maps to chromosome VII. Although the origin and functional role of the point mutation in the hypB5 strain Sec7 protein remains unresolved, it appears that pYY2 contains an extragenic suppressor. Thus hypB likely encodes an element in the COPI vesicle assembly pathway.

This study aimed to identify the genetic basis of the Aspergillus nidulans hypB5 mutant phenotype. A. nidulans is a filamentous fungus that is widely used as a cell biological and molecular genetic model system. Its hyphae grow by localized polar secretion, producing tubular cells. A. nidulans hypercellular strains define five unlinked genes, hypA1-hypE2, which cause hyphal morphogenesis defects at 42°C. hypA is orthologous to Saccharomyces TRS120, which mediates Golgi transit and is widely conserved. The hypB5 restrictive phenotype resembles hypA1: wide hyphae, short basal cells and small nuclei. Like hypA1, shifting hypB5 mutants from 28°C to 42°C causes cessation of tip growth but isotropic expansion of basal cells. A hypA1, hypB5 double mutant was impaired for growth at 28°C, suggesting these genes have related roles, but neither was epistatic at 37°C so they function in different pathways. The A. nidulans pRG3-AMA1 genomic library was used to clone hypB5 complementing DNA by phenotype rescue, and subcloned to a 5 kb KpnI fragment, pYY2. pYY2 was disrupted and sequenced by Tn1000 insertional mutagenesis. The pYY2 sequence is 4975 bp and encodes a putative Sec7 domain which has 81% identity to the Saccharomyces SEC7 domain. The Sec7 domain is highly conserved from yeasts to mammals. Saccharomyces SEC7 encodes a guanine nucleotide exchange factor involved in COPI vesicle formation and Golgi biogenesis. Insertions in the pYY2 non-Sec7 domain coding region complemented hypB5 efficiently, whereas those in the Sec7 domain did not, indicating that the Sec7 domain is sufficient for function. A point mutation was found in the hypB5 strain Sec7 domain, which could explain temperature sensitivity. However, the pYY2 sequence is found on chromosome I whereas hypB maps to chromosome VII. Although the origin and functional role of the point mutation in the hypB5 strain Sec7 protein remains unresolved, it appears that pYY2 contains an extragenic suppressor. Thus hypB likely encodes an element in the COPI vesicle assembly pathway.

La thèse décrit, dans le premier chapitre, les effets opposés sur la régulation de l'expression du gène du facteur tissulaire (TF) par la voie PI3K/Akt et la voie Erk1/2 in vitro. TF est une molécule clé pour initier la coagulation du sang. Son rôle est maintenant connu au développement embryonnaire, le maintien de l'intégrité vasculaire et la réparation tissulaire. De fait que divers cancers expriment des niveaux aberrantes du TF qui sont corrélés avec le pronostic, TF pourrait réellement favorise la croissance tumorale, l'angiogenèse et la métastase. En utilisant une lignée cellulaire épithéliale de cancer du sein MDA-MB-231, nous avons quantifié l'expression du gène du TF par le test luminescent, qPCR, western blot, et d'activité TF associée aux cellules in vitro. Nous avons constaté que 1) PI3K/Akt est la principale voie qui active l'expression des gènes TF. 2) L'activité Erk1 / 2 inhibe l'expression du gène TF; 3) le blocage de la voie Erk1/2par PD98059 induit une expression aberrante du gène du TF via sur-activation du récepteur du EGF; 4) cette sur-expression du TF peut être neutralisé par le blocage de l'EGFR et de la voie PI3K/Akt; 5) cette sur-expression induite par l'inhibition de Erk1/2est une caractéristique commune pour les lignées de cellules épithéliales cancéreuses testés, comme SKOV-3-3 et OVCAR ; 6) la forme soluble du TF suite à l'épissage alternatif représente une faible proportion de l'ARNm de TF totale et 7) Le niveau d’expression duTF des cellules MDA-MB-231 est corrélée à une activité procoagulante cellulaire et à l'invasivité des cellules in vitro. Cette étude a révélé une boucle de régulation négative de l’Erk1/2 vis-à-vis du l’activité du EGFR, ce qui suggère un effet indésirable des agents thérapeutiques ciblant l’Erk dans la clinique.La thèse décrit, dans le deuxième chapitre, les éléments de preuve de la fonction angiogénique de Hypb, une H3K36 méthyltransférase avec Hypb-/ - knockout souris.Ces souris ont montré une létalité embryonnaire à E10.5-E11.5 et de graves anomalies vasculaires dans le sac vitellin d'embryons et le placenta. Les expériences avec des cellules endothéliales HMEC-1 in vitro utilisant l’anti-Hypb siARN ont démontré défaut de migration et d’invasion cellulaire. En outre, les cellules traitées ont perdu la capacité de former des vaisseaux. Ces données sont bien cohérente avec l'analyse histologique de embryons Hypb-/- de souris dont le réseau complexe de ramification vaisseaux embryonnaires et la circulation sanguine étaient absentes. L'analyse génétique sur sac vitellin avec microarray ont suggéré une association entre le défaut de l'angiogenèse dans Hypb-/ - souris et la déréglementation de la sécrétion des protéines Angptl3 etCyr61, qui pourraient se lier à avß3. Il a également souligné le rôle de l'angiogénine,Angptl3 et Gja4 dans ce défaut, parce que lien de ces gènes à l'angiogenèse étaient démontrés. Mécanismes plausibles sont explorés. La régulation épigénétique de l'angiogenèse est une question importante parce qu'elle contrôle la régulation spatiale et temporelle de l'expression de milliers de gènes. Notre étude suggère clairement un rôle clé de Hypb et H3K36 méthylation et Hypb mécanismes liés aux processus de vascularisation chez les mammifères (vasculogenèse et l'angiogenèse). L'angiogenèse est importante pour la recherche fondamentale et application médicale dans les maladies cardio-vasculaires et de la thérapie anti-cancereuse. Nous espérons que de nouvelles stratégies thérapeutiques ciblant les voies épigénétiques permettra d'offrir de nouvelles voies thérapeutiques. / The thesis described, in the first chapter, an opposite regulatory effects of PI3K/Akt pathway and Erk1/2 pathway on tissue factor(TF) gene expression in vitro. TF is a keymolecule required to initiate blood coagulation, and is now accepted to be essential forembryo development, maintenance of vascular integrity and tissue repair. Since a variety of cancers show aberrant prognostics-correlated high levels of TF expression, it is believed that TF promotes tumor growth, angiogenesis and metastasis. Using an epithelial breast cancer cell line MDA-MB-231, we quantified TF gene expression by luminescent test, qPCR, western blot, and cell-associated TF activity in vitro. We found that 1) PI3K/Akt is the major pathway that activates TF gene expression. 2) Erk1/2activity inhibits TF gene expression; 3) blocking Erk1/2 by PD98059 aberrant lyupregulates TF gene expression via enhancing EGFR activity; 4) this enhanced TF expression can be neutralized by blocking EGFR and PI3K/Akt pathway activation; 5) TF upregulation induced by Erk inhibition is a common feature in the tested epithelial cancer cell lines SKOV-3 and OVCAR-3; 6) Soluble form of TF due to alternative splicing represents a small proportion of total TF mRNA and 7) The level of TF gene expression in MDA-MB-231 cells is correlated to cell procoagulant activity and cell invasiveness in vitro. This study revealed a negative feedback loop of Erk-mediated EGFR inhibitions,suggesting an undesirable effect of the agents targeting Erk in clinic.The thesis described, in the second chapter, the evidence of angiogenic function of Hypb,a H3K36 methyltransferase with Hypb-/- knockout mice. These mice demonstrated embryonic lethality at E10.5-E11.5 and severe vascular defects in the Hypb−/− embryo,yolk sac, and placenta. The experiments with endothelial cells HMEC-1 in vitro using anti-Hypb siRNA demonstrated defective cell migration and invasion. Furthermore, the treated cells lost the capacity of vessel formation. These data were well coherent with histological analysis of Hypb-/- mice embryos that lacked the intricate network of branching embryonic vessels and showed disrupted blood flow. The genetic microarray analysis on yolk sac suggested an association between the defect of angiogenesis inHypb-/- mice and deregulated Angptl3 and Cyr61 protein release, since both of which could bind to αvβ3. It also suggested the roles of angiogenin, Angptl3 and Gja4 in this defect because these angiogenesis-related genes were downregulated. Plausible mechanisms are discussed. The epigenetic regulation of angiogenesis is an important issue because it controls the spatial and temporal regulation of expression of thousands of genes. Our study clearly suggests a key role of Hypb and H3K36 methylation and Hypb-related mechanisms in the processes of mammalian vascularization (vasculogenesis and angiogenesis). Angiogenesis is important for both basic researchand medical application in cardiovascular diseases and cancer therapeutics. We believe that novel therapeutic strategies targeting epigenetic pathways will achieve real benefit in medical practices.