Genomes of higher plants comprise a large proportion of repetitive DNAs, where one major class is satellite DNA. Satellite DNA is organized in tandem arrays of basic repeating units, which often occurs in heterochromatin of centromeric/pericentromeric and intercalary as well as subtelomeric regions. Besides these typical satellite repeats, there are also non-typical satellite DNAs, which are organized in short tandem arrays and integrated into a transposable element. The chromosomal localization of non-typical satellites is not in large regions of heterochromatin, but tend to be dispersed along chromosomes. This thesis describes the identification of the major repeat classes including major satellite content in six beet and related species. The focus was on identification and characterization of new satellite families in the beet genomes. In this study, the information regarding repetitive DNA as well as satellite families fraction in six beet and related species was gained based on graph-based clustering of next generation sequenced short sequence reads. The repeat proportion of the six analyzed species ranges from 34.4% in C. quinoa to 65.6% in B. lomatogona, in which the portion of nearly 50% belongs to B. vulgaris, B. nana, P. procumbens, and P. patellaris. Among all classes of repetitive DNAs, LTR retrotransposons are the most abundant repeat type in all analyzed genomes, which is a common feature of higher plant genomes. The other repeat sequences are DNA transposons, rDNA, and satellite DNA with variable portions in different species. A set of satellite families in each species was analyzed in detail and reflects the relationship between six species. The closely related relationship between B. lomatogona and B. nana as well as between P. procumbens and P. patellaris is affirmed by seven and 13 satellite families shared between two species, respectively. Similarly, the closer relationship between B. vulgaris and two species B. lomatogona and B. nana than between B. vulgaris and two species P. procumbens and P. patellaris from the sister-genus Patellifolia is also confirmed. C. quinoa is a distantly related species and this is reflected by vastly different satellite content. Therefore, satellite DNA analysis might be a useful tool to trace species evolution. In the B. lomatogona genome, by the application of RepeatExplorer tool, six novel tandemly repeated DNA sequences were identified and designated BlSat1-BlSat6. The three typical satellite families BlSat1, BlSat5, and BlSat6 are organized in tandem arrays in large heterochromatic blocks. BlSat1 is mainly localized in the pericentric region of the chromosome 3, 5, 6, and 9, while BlSat5 is amplified in the pericentromeric region of the chromosome 3, 5, and 7. BlSat6 is a chromosome-specific satellite and is located in the subtelomeric region on the south arm of the chromosome 8. The other three satellite families BlSat2, BlSat3, and BlSat4 are characterized as non-typical satellite DNA because of their dispersed distribution along chromosomes. BlSat2 and BlSat3 are identified as a tandem repeat domain in Ogre/Tat retrotransposons. The occurrence of one or several short tandem arrays in a transposable element is a common phenomenon in both animals and plants. These short repeats are considered to be continuously evolving and eventually amplifying to new satellite families. Furthermore, the distribution of the six new satellite families in beet and related species was confirmed by comparative PCR, comparative Southern hybridization, and mapping of sequence reads from referent species against each satellite sequence. The BlSat1 and BlSat6 satellite families are specific for the genus Beta, while BlSat5 is only amplified in two sections Corollinae and Nanae of the genus Beta. BlSat4 is an ancient satellite family which exists in all tested species belonging to the genera Beta, Patellifolia, Chenopodium, and Spinacia, whereas BlSat2 and BlSat3 might have evolved before the separation of the genus Beta and Patellifolia but their sequences have been lost or heavily diverged during the species radiation. Comparison of two wild beet genomes P. procumbens and P. patellaris was performed aiming to address the open question whether P. patellaris is auto- or allotetraploid. The high similarity between these two genomes indicates their close relationship. However, the genetic difference between two genomes, in particular the molecular characteristics as well as the chromosomal localization of two satellite families PproSat1 and PpatSat1, might support a hypothesis that P. patellaris is allotetraploid species with a half of its chromosome set derived from P. procumbens. The results obtained in this work might provide comprehensive information of the repetitive classes as well as satellite families in the genomes of beets and related species. The results can be used as the species-specific and chromosome-specific markers in beet genome studies.

Chez les organismes complexes, comme les mammifères, les séquences de régulation génomique, dispersées sur les chromosomes, peuvent interagir à l'intérieur de l'espace nucléaire pour effectuer des actions coordonnées de régulations géniques. La méthylation de l'ADN et les modifications post-traductionnelles des histones, en combinaison avec des séquences de régulation, des facteurs protéiques et des ARNs non codants, conduisent à une organisation supérieure de la chromatine spécifique du type cellulaire. Cependant, l'organisation et la dynamique de la chromatine in vivo à l'échelle supérieure à celle du nucléosome reste encore largement méconnues. L'objectif général des travaux de notre équipe est d'élucider l'organisation de la chromatine à l'échelle supranucléosomale et sa dynamique in vivo, dans différents contextes physiologiques ou pathologiques, afin de comprendre leurs participations au contrôle et à la coordination de l'expression des gènes chez les mammifères. Notre hypothèse de travail est que certains compartiments nucléaires permettent un confinement de contacts chromatiniens spécifiques facilitant les régulations génomiques. L'objectif principal de mon travail de thèse était de développer une nouvelle méthode, simple et directe, permettant de cartographier et d'analyser les régions du génome murin qui sont associées aux compartiments nucléaires importants pour la régulation de l'expression des gènes (lamine nucléaire, les nucléoles, usines à transcription ou corps de Cajal). Le principe de notre méthode repose sur des traitements à haut sel de noyaux cellulaires transcriptionnellement actifs. Des séquençages à haut-débit permettent ensuite d'identifier les régions génomiques retenues dans les complexes nucléaires ainsi rendus d'insolubles. Elle a donc été appelée HRS-Seq : High-salt Recovered Sequences-sequencing (séquençage de séquences récupérées à haut-sel). Mon programme de travail s'est déroulé en 4 étapes distinctes : 1- la mise en œuvre et l'amélioration de la partie expérimentale (test HRS), 2- l'adaptation des techniques de séquençage à haut-débit à notre méthode (collaboration avec L. Journot, H. Parrinello, E. Dubois), 3 – l'application d'une analyse statistiques adéquate afin d'identifier les HRS (collaboration avec C. Reynes et R. Sabatier, statisticiens) et 4- l'analyse bio-informatique de ces régions destinée à les cartographier et à les caractériser (collaboration avec J. Mozziconacci et A. Cournac).Dans un premier temps, nous avons utilisé la méthode HRS-seq sur des noyaux de cellules de foie de souris. L'analyse bioinformatique des HRS nous a permis de réaliser la toute première cartographie de ces régions chez la souris et de découvrir leurs principales caractéristiques. Les régions HRS peuvent être classées en deux catégories distinctes : Les HRS riches en AT sont fortement associées à la lamine nucléaire, tandis que celles riches en GC sont associées aux régions géniques. La présence exceptionnelle, parmi cette dernière catégorie, des gènes codant pour les protéines d'histones, indique que le test HRS permet la rétention des Corps des Loci d'Histones (HLB – Histone Locus Body), un type spécifique de corps de Cajal. De plus, grâce à une analyse croisée avec des données de Hi-C disponibles dans la littérature, nous avons pu montrer que les HRS présentent entre-elles une haute probabilité de contact dans l'espace tridimensionnel du noyau, et qu'elles sont fortement enrichies en certaines séquences répétées (gènes des ARNt). L'ensemble de ces résultats nous permet de valider expérimentalement notre méthode. Dans un second temps, nous avons appliqué cette méthode à 3 autres types cellulaires : des cellules souches embryonnaires, des cellules progénitrices neurales et des neurones (collaboration avec T. Bouschet). Le but de ce travail est de déterminer comment les régions HRS évoluent au cours de la différentiation cellulaire. Les analyses statistiques et bioinformatiques sont en cours. / In complex organisms like mammals, regulatory sequences, dispersed on the chromosomes, can interact together within the nuclear space to tightly coordinate gene expression. DNA methylation and post-translational histone modifications combine with regulatory sequences, proteic factors and non-coding RNA, to provide cell-type specific patterns of higher-order chromatin organization. However, the in vivo organization of the mammalian chromatin beyond the simple nucleosomal array remains largely enigmatic. The general objective of our group is to elucidate the in vivo organization and dynamic of the chromatin at the supranucleosomal scale in diverse physiological and pathological contexts, in order to better understand how they are involved in the maintenance and coordination of gene expression in mammals. Our working hypothesis is that some nuclear compartments are confining specific chromatin contacts in order to facilitate genomic regulations. The principal objective of my thesis was to develop a novel straightforward method to map and to characterize genomic regions that are associated, in the mouse, with nuclear compartments that are important for gene regulation (nuclear lamina, nucleolus, transcription factories, Cajal bodies). The principle of our method is based on high-salt treatments of transcriptionally active cell nuclei. High-throughput sequencings then allow to identify the genomic regions that are retained in the resulting insoluble nuclear complexes. We thus named this method the HRS-seq (High-salt Recovered Sequences-sequencing). My working program was divided into 4 steps: 1- the improvement of the experimental procedure (HRS assay), 2- the adaptation of the NGS techniques to our method (collaboration with L. Journot, H. Parrinello, E. Dubois), 3- the use of an adequate statistical analysis in order to identify the HRS (Collaboration with C. Reynes and R. Sabatier, statisticians), 4- the bioinformatics analysis of these regions in order to map and to characterize them (collaboration with J. Mozziconacci and A. Cournac). We first used the HRS-seq method on mouse liver cells. The bioinformatics analysis allowed us to obtain the first global profiling of HRS in the mouse and to discover their essential characteristics. The HRS can be classified into two categories: the AT-rich HRS are linked to lamina associated domains, while GC-rich HRS are strongly associated to genes. The presence of histone genes amongst this latter category suggests that the Histone Locus Bodies (HLBs), a specific type of Cajal's body, is retained in the HRS assay. Furthermore, thanks to a cross-analysis with Hi-C data available in international databases, we have shown that the HRS display a high contact probability in the tri-dimensional space of the nucleus and that they are highly enriched in some specific repeat sequences (tRNA genes). Globally, these results allow us to validate the experimental approach used in the HRS-seq method. In a second time, we have applied this method to 3 other cell types: mouse embryonic stem cells, neural progenitor cells and neurons (collaboration with T. Bouschet). The aim of this work is to determine how the HRS regions are regulated during cell differentiation. Statistical and bio-informatics analyses are in progress.

A hallmark feature of Dictyostelium development is the establishment and maintenance of precise cell-type proportions. In the case of D. discoideum, roughly 20% of the cells that aggregate form the stalk while the remaining 80% form the spores. In order to identify genes involved in cell-type proportioning Jaiswal et al. (2006) carried out random insertional mutagenesis (REMI) of the D. discoideum genome. This led to the identification of a novel gene, which was named trishanku (triA). A knock-out of triA did not show any defects during growth and early development but multiple defects later during development. To understand the reasons for the multiple developmental defects in the absence of triA, I looked at the genomic organization and the pattern of expression of the triA gene. In silico analysis points to the presence of more than one consensus D. discoideum promoter sequence upstream to exons1 and 2, raising the possibility that the triA gene could code for more than one transcript. Northern blot analysis confirms this prediction and provides evidence for the presence of two transcripts: triA1-2-3 (~ 2.9 kb, containing exons 1+2+3) and triA2-3 (~ 2 kb, containing exons 2+3). Both transcripts have exons 2 and 3 in common. In triA- cells, the REMI cassette is inserted in exon 2, which is common to both transcripts; thus, the absence of triA results in the lack of both. The transcripts are absent in vegetative cells but expressed during development. triA2-3 is expressed earlier, by 3h, while triA1-2-3 is expressed later, by 9h, and both remain till the end of development. triA2-3 and triA1-2-3 are differentially regulated by different aspects of the extracellular environment which include mode of development of cells (solid substratum versus shaken suspension), the presence of a high level of extracellular cAMP and formation of stable cell-cell contacts. The expression of triA2-3 and triA1-2-3 in triA- cells, one at a time under a constitutive promoter (Actin15 promoter), suggests that the two transcripts have both specific as well as overlapping functions in the cell. The triA2-3 transcript can specifically restore spore forming efficiency and stalk thickness, while the triA1-2-3 transcript can rescue the stream break up defect. Both the transcripts can rescue the sub-terminal position of the sorus, spore shape and spore viability. To address the question of stream break-up during mid to late aggregation in triA- cells, I have looked at the cell adhesion profile of triA- cells and compared it with the wild type (Ax2). triA- cells show transient disaggregation in buffer and a 2h delay in agglutination in presence of buffer with 10mM EDTA. This aberrant cell adhesion profile seen in triA- cells is in accordance with the expression pattern of genes encoding known cell adhesion molecules. triA- cells also overproduce an extracellular factor which significantly decreases the aggregate size of both Ax2 and triA-. The nature of the extracellular factor overproduced by in triA- cells is currently unknown, but it is not the same as cell-counting factor which is overproduced by smlA null cells. To look at the mis-expression of cell type-specific genes, I have monitored the movement of prestalk cells into the prespore region and vice versa in both Ax2 and triA- slugs. My studies show that there is extensive movement of prestalk cells into the prespore region and of prespore cells into the prestalk region in triA- slugs, which is absent in Ax2 slugs. Also, cells that move into the ‘wrong’ region show a change their cell fate (transdifferentiate) appropriate to the new location; whether transdifferentiation precedes or succeeds cell movement is not yet clear. Transdifferentiation is observed to a certain extent in Ax2 slugs, but only after prolonged migration; triA- slugs show enhanced transdifferentiation even in the absence of migration. To find out the possible reason(s) for the formation of a sub-terminal spore mass in the absence of triA, I have checked whether the defect lies in the ability of the prespore cells to rise up the stalk or in the ability of the upper cup (cells present above the spore mass contributed by a subset of prestalk cells and anterior like-cells) to pull the spore mass to the top. To see which of the two reasons could be responsible for the formation of a sub-terminal spore mass in triA-, I carried out transplantation experiments where the anterior one-fourth region of an Ax2 or triA- slug is grafted to the posterior four-fifth region of a triA- or Ax2 slug and the morphology of the fruiting body is observed. My studies show that the sub-terminal position of the spore mass in triA- is not due to an inability of the prespore cells to rise to the top but to a defect in the upper cup. The upper cup in triA- remains motile but is unable to remain attached to the prespore mass during culmination. It detaches, rises up the stalk and is present at the tip of the stalk. Mixing a minority of triA- cells (20%) with an excess of Ax2 (80%) results in an upper up formed by Ax2 alone. In this situation, the wild type upper cup is able to lift the triA- prespore mass to the top. Thus, the presence of triA (a prespore-specific gene) is essential for the proper functioning of the upper cup cells (which belong to the prestalk class) in order to enable prespore cells to ascend to the top of the stalk.

Since, its discovery over three decades ago, HIV has wrecked havoc worldwide. According to the UNAIDS report 2011, at present 34 million people is living with HIV and AIDS vaccine with broadly neutralizing activity still remains elusive. The envelope glycoproteins on the virion surface, is the most accessible component to the host immune system and therefore is targeted for vaccine design. However, the virus has employed various strategies to avoid the host immune response. The extremely high rate of mutations, extensive glycosylation of the envelope glycoprotein, conformational flexibility of the envelope, has made all the efforts aimed to design a broadly neutralizing immunogen futile. In Chapter1, we briefly discuss about the structural and genomic organization of the HIV-1 along with various strategies the virus has employed to evade the immune system. We also present the progress and failures encountered in the past three decades, on the way to design protective HIV vaccine and inhibitors. On the host cell surface, HIV-1 glycoprotein gp120 binds to the cell surface receptor CD4 and leads to the fusion of viral and host cellular membranes. CD4 is present on the surface of T-lymphocytes. It consists of a cytoplasmic tail, one transmembrane region, and four extracellular domains, D1−D4. sCD4 has been used as an entry inhibitor against HIV-1. However, this molecule could not neutralize primary isolates of the virus. Previously, from our lab, we had reported the design and characterization of a construct consisting of the first two domains of CD4 (CD4D12), that binds gp120 with similar affinity as soluble 4-domain CD4 (sCD4). However, the first domain alone (CD4D1) was previously shown to be largely unfolded and had 3-fold weaker affinity for gp120 when compared to sCD4 [Sharma, D.; et al. (2005) Biochemistry 44, 16192−16202]. In Chapter 2, we describe the design and characterization of three single-site mutants of CD4D12 (G6A, L51I, and V86L) and one multisite mutant of CD4D1 (G6A/L51I/L5K/F98T). G6A, L51I, and V86L are cavity-filling mutations while L5K and F98T are surface mutations which were introduced to minimize the aggregation of CD4D1 upon removal of the second domain. All the mutations in CD4D12 increased the stability and yield of the protein relative to the wild-type protein. The mutant CD4D1 (CD4D1a) with the 4 mutations was folded and more stable compared to the original CD4D1, but both bound gp120 with comparable affinity. In in vitro neutralization assays, both CD4D1a and G6A-CD4D12 were able to neutralize diverse HIV-1 viruses with similar IC50s as 4-domain CD4. These stabilized derivatives of human CD4 are useful starting points for the design of other more complex viral entry inhibitors. Most HIV-1 broadly neutralizing antibodies are directed against the gp120 subunit of the env surface protein. Native env consists of a trimer of gp120−gp41 heterodimers, and in contrast to monomeric gp120, preferentially binds CD4 binding site (CD4bs)-directed neutralizing antibodies over non-neutralizing ones. One group of cryo-electron tomography studies have suggested that the V1V2 loop regions of gp120 are located close to the trimer interface and the other group claimed that the V1V2 loop region is far from the apex of the trimer. To further investigate the position of the V1V2 region, in the native envelope trimer, in Chapter 3, we describe the design and characterization of cyclically permuted variants of gp120 with and without the h-CMP and SUMO2a trimerization domains inserted into the V1V2 loop. h-CMP-V1cyc is one such variant in which residues 153 and 142 are the N- and C-terminal residues, respectively, of cyclically permuted gp120 and h-CMP is fused to the N-terminus. This molecule forms a trimer under native conditions and binds CD4 and the neutralizing CD4bs antibodies b12 with significantly higher affinity than wild-type gp120. It binds non-neutralizing CD4bs antibody F105 with lower affinity than gp120. A similar derivative, h-CMP-V1cyc1, bound the V1V2 loop-directed broadly neutralizing antibodies PG9 and PG16 with ~15-fold higher affinity than wild-type JRCSF gp120. These cyclic permutants of gp120 are properly folded and are potential immunogens. The data also support env models in which the V1V2 loops are proximal to the trimer interface. HIV-1 envelope (env) protein gp120 has approximately 25 glycosylation sites of which ~4 are located in the inner domain, ~7-8 in the V1/V2 and V3 variable loops and the rest in the outer domain (OD) of gp120. These glycans shield env from recognition by the host immune system and are believed to be indispensable for proper folding of gp120 and viral infectivity. However, there is no detailed study that describes whether a particular potential n-linked glycan is indispensable for folding of gp120.Therefore, in Chapter 4, using rationally designed mutations and yeast surface display (YSD), we show that glycosylation is not essential for the correct in vivo folding of OD alone or OD in the context of core gp120. Following randomization of the remaining four glycosylation sites, we isolated a core gp120 mutant, which contained a single inner domain glycan and retained yeast surface expression and broadly neutralizing antibody (bNAb) binding. Thus demonstrates that most gp120 glycans are dispensable for folding in the absence of gp41. However in the context of gp160, we show that all core gp120 glycans are dispensable for folding, recognition of bNAbs and for viral infectivity. We also show that deglycosylated molecules can serve as a starting point to re-introduce epitopes for specific glycan dependent bNAbs. Several of these constructs will also be useful for epitope mapping and env structural characterization. Glycosylation of env is known to inhibit binding to germline precursors of known bNAbs. Hence the present results inform immunogen design, clarify the role of glycosylation in gp120 folding and illustrate general methodology for design of glycan free, folded protein derivatives. On the virion surface env glycoproteins gp120 and gp41 interact via non-covalent interactions and form trimers of heterodimers. Upon binding cell surface receptor CD4 and co-receptor CCR5/CXCR4, gp120 and gp41 undergo a lot of conformational changes, which ultimately lead to the fusion of viral and cellular membranes by formation of six-helix bundle in gp41. High resolution structural information is available for core gp120 and post-fusion six-helix bundle conformation of gp41. However, the structural information about the native gp120:gp41 interface in the native trimer is lacking. In Chapter 5, we describe the design and characterization of various single chain derivatives of gp120 inner doamin and gp41. Among the designed constructs, gp41-id2b is folded but is a mixture of dimer and monomer under native conditions. To facilitate, trimer formation, two trimerization domains (h-CMP and Foldon) were individually fused to the N-terminus of gp41-id2b to generate h-CMP-gp41-id2b and Foldon-gp41-id2b. Although, these molecules were proteolytically more stable than gp41-id2b, they did not form trimer under native conditions. All the single chain derivatives were designed based on the crystal structure of gp120, which was devoid of C1 and C5 domains (PDBID 1G9M). A new set of constructs to mimic the native gp120:gp41 interface will be designed and characterized based on the recently solved crystal structure of gp120 with the C1 and C5 domains (PDBID 3JWD and 3JWO). Helix-helix interactions are fundamental to many biological signals and systems, found in homo- or hetero-multimerization of signaling molecules as well as in the process of virus entry into the host. In HIV, virus-host membrane fusion during infection is mediated by the formation of six helix bundle (6HB) from homotrimers of gp41, from which a number of synthetic peptides have been derived as antagonists of virus entry. Yeast surface two-hybrid (YS2H) system is a platform, which is designed to detect protein-protein interactions occurring through a secretory pathway. In Chapter 6, we describe the use of aYS2H system, to reconstitute 6HB complex on the yeast surface and delineate the residues influencing homo-oligomeric and hetero-oligomeric coiled-coil interactions. Hence, we present YS2H as a platform for facile characterization of hetero-oligomeric interactions and design of antagonistic peptides for inhibition of HIV and many other enveloped viruses relying on membrane fusion for infection, as well as cellular signaling events triggered by hetero-oligomeric coiled coils. However, using this YS2H platform, the native hetero-oligomeric complex of gp120 and gp41 could not be captured. In Appendix 1, we report cloning, expression and purification of PΔGgp120 and ΔGgp120 from methylotrophic yeast Pichia pastoris. PΔGgp120 was purified as a secreted protein. However, in electrophoretic analyses the molecule ran as a heterogeneous smear. Further optimization of the purification protocol and biophysical characterizations of this molecule will be performed in future. In Appendix 2, gp41 variants were expressed on the yeast cell surface as a C-terminally fused protein and its interaction with externally added gp120 was monitored by FACS. The surface expression of the gp41 constructs was poor and they did not show any interaction with gp120.