The Saccharomyces cerevisiae iron-responsive transcription factor, Aft1p, has a well established role in regulating iron homeostasis through the transcriptional induction of iron-regulon genes. However, recent studies have implicated Aft1p in other cellular processes independent of iron-regulation such as chromosome stability. In addition, chromosome spreads and two-hybrid data suggest that Aft1p interacts with and co-localizes with kinetochore proteins, however the cellular implications of this have not been established. Here, we demonstrate that Aft1p associates with the kinetochore complex through Iml3p. Furthermore, we show that Aft1p, like Iml3p, is required for the increased association of cohesin with the pericentromere and that aft1Δ cells display sister chromatid cohesion defects in both mitosis and meiosis. Our work defines a new role for Aft1p in the sister chromatid cohesion pathway.

Le complexe de la cohésine est requis pour de nombreuses transactions chromosomiques, la cohésion des chromatides soeurs, la réparation des dommages à l'ADN, la régulation de la transcription et le contrôle de l'architecture de la chromatine en 3D. La manière dont la cohésine engage la chromatine est restée une question majeure. Les sous-unités de base de cohesin, Smc1, Smc3, Scc1 assemblent un complexe en forme d’anneau via la connexion des domaines SMC «charnières» hétérodimères fournis par Smc1 et Smc3, et par la liaison des domaines SMC ATPase par Scc1. D'autres facteurs jouent un rôle dans différents aspects de la fonction de la cohésine, tels que Scc3, qui favorise l'association de la cohésine à l'ADN, les complexes de chargement et de déchargement, Scc2-Scc4 et Pds5-Wapl, respectivement responsables du chargement de la cohésine et de sa dissociation de la chromatine. . Au cours de la phase S, une acétyltransférase appelée Eco1 acétyle le domaine ATPase de Smc3 et déclenche l’établissement de la cohésion. Pour augmenter davantage la cohésion, un facteur métazoaire supplémentaire, la sororine forme un complexe avec Pds5 pour empêcher la liaison de Wapl. Pendant la métaphase, la cohésine centromérique est protégée par shugoshin-PP2A. Chez les métazoaires, la cohésine est libérée des chromosomes en deux étapes. La première nécessite la phosphorylation de la cohésine et permet à Wapl de se lier à nouveau à Pds5 afin d'assurer la médiation de la libération de cohésine indépendante du clivage à partir des bras des chromosomes. La seconde se produit lors de la réalisation de l'assemblage de la broche et nécessite l'activation d'une protéase appelée séparase, ce qui entraîne le clivage Scc1, libérant ainsi des chromatides soeurs à séparer dans des cellules filles. Au-delà de la cohésion, il devient également évident que la cohésine joue des rôles plus divers en interagissant avec une multitude d'autres facteurs, notamment la CTCF, une protéine connue comme un isolant, dont il a été rapporté qu'elle collabore à la détermination du génome 3D. structure.Pour comprendre comment la cohesine engage l'ADN, j'ai étudié les propriétés de liaison à l'ADN de sous-complexes précédemment identifiés. En déterminant une structure cristalline de la levure Scc3 liée à un fragment de la sous-unité Scc1 kleisin et de l'ADN, j'ai pu démontrer que Scc3 et Scc1 forment un module d'interaction composite de l'ADN. Le sous-complexe Scc3-Scc1 engage un ADN double brin à travers une surface conservée, chargée positivement. Nous démontrons que ce domaine est requis pour la liaison à l'ADN par Scc3-Scc1 in vitro, ainsi que pour l'enrichissement de la cohésine sur des chromosomes et pour la viabilité cellulaire. Ces résultats suggèrent que l'interface de liaison à l'ADN Scc3-Scc1 joue un rôle central dans le recrutement des complexes de la cohésine sur les chromosomes et donc que cette dernière exécute fidèlement ses fonctions lors de la division cellulaire.Pour étudier les bases moléculaires de la collaboration fonctionnelle signalée entre la cohésine et le CTCF dans la définition de la structure chromosomique 3D, j'ai identifié et déterminé la structure d'un complexe ternaire composé de SA2 humain (un orthologue de Scc3), de Scc1 et de CTCF. La structure révélait un motif de liaison SA2-Scc1 très répandu qui était présent non seulement dans le CTCF, mais aussi dans d’autres facteurs connexes fonctionellement, tels que shugoshin et Wapl. Les tests de compétition déroulants ont indiqué que la liaison de ces facteurs à SA2-Scc1 était mutuellement exclusive, ce qui suggère fortement qu'ils interagissent avec la cohésine via des mécanismes similaires. Pour démontrer ce principe, j'ai pu déterminer une structure de shugoshin en complexe avec SA2-Scc1, ce qui a confirmé que tant le shugoshin que le CTCF se lient à la même surface conservée sur la cohésine. / The cohesin complex is required for numerous chromosomal transactions including sister chromatid cohesion, DNA damage repair, transcriptional regulation and control of 3D chromatin architecture. How cohesin engages chromatin has remained a major question. The basic subunits of cohesin, Smc1, Smc3, Scc1 assemble a ring-shaped complex via connection of the heterodimeric SMC ‘hinge’ domains contributed by of Smc1 and Smc3, and through linkage of the SMC ATPase domains by Scc1. Additional accessory factors play important roles in different aspects of cohesin function, such as Scc3, which promotes the association of cohesin with DNA, the loading and unloading complexes, Scc2-Scc4 and Pds5-Wapl respectively, responsible for cohesin loading and its disassociation from chromatin. During S phase, an acetyltransferase called Eco1 acetylates the ATPase domain of Smc3 and triggers the stabilization, or establishment, of cohesion. To further augment cohesion, an additional metazoan factor, sororin forms a complex with Pds5 to prevent Wapl binding. During metaphase, centromeric cohesin is protected by the shugoshin-PP2A complex. In metazoans, cohesin is released from chromosomes in two major steps. The first requires cohesin phosphorylation and allows Wapl to bind Pds5 again to mediate cleavage-independent release of cohesin from chromosome arms. The second transpires upon fulfilment of spindle assembly and requires activation of a protease called separase, resulting in Scc1 cleavage, thus releasing sister chromatids to be segregated into daughter cells. Beyond cohesion, it is also becoming apparent that cohesin plays more diverse roles by interacting with a plethora of other factors, most notably CTCF, a zinc finger protein that is known as an insulator, which has been reported to collaborate with cohesin in determining 3D genome structure.To understand how cohesin engages DNA, I investigated the DNA binding properties of previously identified globular sub-complexes. By determining a crystal structure of the budding yeast Scc3 bound to a fragment of the Scc1 kleisin subunit and DNA, I could demonstrate that Scc3 and Scc1 form a composite DNA interaction module. The Scc3-Scc1 subcomplex engages double-stranded DNA through a conserved, positively charged surface. We demonstrate that this conserved domain is required for DNA binding by Scc3-Scc1 in vitro, as well as for the enrichment of cohesin on chromosomes and for cell viability. These findings suggest that the Scc3-Scc1 DNA-binding interface plays a central role in the recruitment of cohesin complexes to chromosomes and therefore for cohesin to faithfully execute its functions during cell division.To investigate the molecular basis of the reported functional collaboration between cohesin and CTCF in defining 3D chromosome structure, I identified and determined the structure of a ternary complex composed of human SA2 (an orthologue of Scc3), Scc1 and CTCF. The structure revealed a wide-spread SA2-Scc1 binding motif which was found to be present not only in CTCF, but also other functionally related factors, including shugoshin and Wapl. Competition pulldown assays indicated that binding of these factors to SA2-Scc1 was mutually exclusive, which strongly suggested that they interact with cohesin via similar mechanisms. To demonstrate this principle, I was able to determine a structure of shugoshin in complex with SA2-Scc1, which confirmed that both shugoshin and CTCF bind the same conserved surface on cohesin.

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The cohesin complex is responsible for the fidelity of chromosomal segregation during mitosis. It consists of four core subunits namely Rad21/Mcd1/Sccl, Smc1, Smc3 and one of the yeast Scc3 orthologs SA1 or SA2. Sister chromatid cohesion is formed by the cohesin complex during DNA replication and maintained until the onset of anaphase. Among the many proposed models of how cohesin holds sister chromatids together, the 'core' cohesin subunits Smc1, Smc3 and Rad21/Mcd1/Scc1 are almost universally displayed as forming a contiguous ring. However, other than its supportive role in the cohesin ring, little is known about the fourth core protein SA1/SA2 - despite its physical association to the cohesin ring. To gain deeper insight into how physically and physiologically SA2 interacts with the cohesin complex, we performed structural characterization of SA2 and Rad21 and mapped the interaction region of the two proteins in vitro and ex vivo . We found SA2 interacts with Rad21 at multiple domains while Rad21 only interacts with SA2 through a 10 amino acid α-helical motif from 383-392aa. Deletion of these 10 amino acids or mutation of three conserved amino acids (L385, F389, and T390) in this α-helical motif prevents Rad21 from physically interacting with SA2/SA1 and causes premature sister chromatid separation in mitotic cells that often leads to aneuploidy. Our studies provide a model for how SA2 structurally strengthens the cohesin ring through its interaction with Rad21. Results from our structural characterization of these two proteins also provided directions for further investigation of the structural basis of protein-protein interaction in the cohesin complex.

The CCCTC-binding factor (CTCF) is a versatile, multifunctional zinc-finger protein involved in a broad spectrum of cellular functions. In mammalian cells, CTCF functions together with the Cohesin complex, an essential regulator of sister chromatid cohesion. Together, CTCF and Cohesin have been shown to regulate gene expression at a genome-wide level in mammalian cells. In the yeast Saccharomyces pombe, Cohesin has been implicated in transcription termination of convergently transcribed genes, in a cell cycle dependent manner. The aim of this thesis was to investigate the possibility of direct transcriptional involvement of CTCF and Cohesin in human cells. The first model system applied for this experimental purpose was the β-globin gene with introduced canonical CTCF-binding sites replacing the endogenous Co- Transcriptional Cleavage (CoTC) element downstream of β-globin. The results obtained indicate that recruitment of CTCF to the β-globin 3` flanking region does not prevent read-through transcription. However, CTCF-binding does mediate RNA Polymerase II (Pol II) pausing at the site of recruited CTCF. This results in more efficient pre-mRNA 3` end processing and therefore rescues β-globin mRNA to wild type levels. Cohesin was not detected at the introduced CTCF-binding sites. These results are a contribution to our understanding of the spatio-temporal requirements for cotranscriptional events like 3` end pre-mRNA processing and Pol II kinetics. The second part of my thesis presents an investigation on the involvement of CTCF and Cohesin in lipopolysaccharide (LPS)-induced Tumor Necrosis Factor α (TNFα) gene expression regulation in human monocytes and differentiated M1- and M2-type macrophages. These studies provide first evidence of Cohesin recruitment to the TNFα gene body and its regulatory NFκB-binding sites. Differences in the recruitment profiles obtained indicate potential regulatory differences of TNFα among the three cell types. Preliminary data provide an insight into the effects on TNFα mRNA levels upon down-regulation of Cohesin subunits.

Polyadenylation signals in yeast are not very well defined and are believed to be largely degenerate. Here, we present a computational and experimental genome-wide analysis of polyadenylation signals in Schizosaccharomyces pombe (S. pombe), identifying the canonical AATAAA motif as the most frequent and functional signal. RNA-Seq data from cells grown under various physiological conditions were used to map 3’UTRs, which classify as commonly heterogenic. We have shown that many genes have alternative 3’UTRs. Our results are summarised and can be accessed in a user-friendly online database Pomb(A). It has been shown that convergent genes require trans elements, like Cohesin, for efficient transcription termination. We demonstrate that convergent genes lacking Cohesin are generally associated with longer overlapping transcripts. Furthermore, we analysed ChIP-chip data of Rad21 and Mis4 as well as other Cohesin and loading complex subunits and show that regions of Rad21/Mis4 co-localisation are generally associated with highly transcribed genes. They are also cohesive, while sites with Rad21 only are less cohesive. Rad21/Mis4 co-localisation sites are in close proximity to annotated origins of replication, suggesting that cohesive sites may facilitate replication. microRNAs (miRNAs) are well studies in higher eukaryotes and participate on post-transcriptional gene silencing by degrading target mRNA or blocking translation. It is believed that miRNAs do not exist in yeast. We reanalyzed miRNA presence in yeast using recently available small RNA data sets. Potential miRNA genes and targets in S. pombe were computationally predicted based on the described alternative 3’UTR data and further experimentally tested. Dicer is an enzyme, which recognizes long dsRNA substrates and cleaves them into siRNA e↵ector molecules, essential for gene silencing. Dicer has been thought to be a purely cytoplasmic protein. However, we employed ChIP-Seq and dsRNA RNA-Seq data to show that Dicer localises in the nucleus of mammalian cells and associates with the chromatin on numerous loci. Furthermore, we present evidence that Dicer processes long dsRNA into siRNA in the nucleus and the lack of Dicer causes the accumulation of long dsRNA. This consequently induces the interferon response pathway, which ultimately leads to apoptosis and cell death.

Lignocellulosic biomass is an abundant renewable resource targeted for biofuel production. Cellulose and hemicellulose from biomass both contain fermentable sugars and other moieties that can be converted to biofuels or other commodity chemicals. Enzymatic hydrolysis of these biopolymers is a critical step in the liberation of sugars for fermentation into desired products. In nature, anaerobic microbes produce protein nanostructures called cellulosomes that efficiently degrade cellulose substrates by combining multiple enzyme activities onto a scaffolding protein. However, current enzyme cocktails used in industry contain secretomes of aerobic microbes and are not efficient enough to be highly economical. Furthermore, most bio-processes focus on cellulose, rendering hemicellulose under-utilized. The three main objectives of this dissertation are to 1) develop multi-functional, self-assembling protein nanostructures for hemicellulose degradation using the architecture provided by cellulosomes, 2) understand the self-assembly mechanism at conditions for consolidated bioprocessing applications, and 3) compare the effectiveness of structured to non-structured hemicellulases in the hydrolysis of biomass. Xylan is a major type of hemicellulose in biomass feedstocks targeted for biofuel production. Six different xylanosomes were designed for hydrolysis of xylan within multiple biomass substrates using the cohesin-dockerin domain systems from Clostridium thermocellum, Clostridium cellulovorans, and Clostridium cellulolyticum. Each two-unit structure contained a xylanase for internal cleavage of the xylan backbone and one side-chain acting enzyme, either a ferulic acid esterase or bi-functional arabinofuranosidase/xylosidase. Expansion to three-unit xylanosomes included a family 10 or 11 xylanase, a bi-functional arabinofuranosidase/xylosidase, and bi-functional ferulic acid esterase/acetylxylan esterase. These multi-functional biocatalysts were used to degrade hemicellulose-rich wheat arabinoxylan and cellulose-containing destarched corn bran. Synergistic release of soluble sugars and ferulic acid was observed with select xylanosomes and in some cases required addition of an endoglucanase and cellobiohydrolase for enhanced hydrolysis. Furthermore, a putative ferulic acid esterase gene from the soil bacterium Cellvibrio japonicus was characterized and its role in xylan hydrolysis investigated. Information for the development of stable and functional cellulosome-like biocatalysts in metabolically-engineered microbes was collected using surface plasmon resonance. The protein-protein interaction of cohesin and dockerin domains for xylanosome self-assembly was examined at various temperatures and in the presence of ethanol to mimic different hydrolysis and fermentation processes and found to retain high affinities at the selected conditions. Moreover, the high-affinity interaction of cohesin and dockerin domains in the presence of non-specific proteins eliminated the need for protein purification for xylanosome construction. In addition to development of the first cellulosome-like biocatalysts targeted for hemicellulose degradation, this dissertation provides insight on possible improvements for the enzymatic hydrolysis of biomass, as well as the applicability of xylanosomes in consolidated bioprocessing.

Lors des réponses immunitaires, les lymphocytes B diversifient leur répertoire par l’hypermutation somatique (HMS) et la commutation isotypique (CI). Ces deux mécanismes sont dépendant de l’activité de « activation-induced cytidine deaminase » (AID), une enzyme qui déamine les cytosines de l’ADN en uraciles générant des mésappariements qui sont processés différemment dans le cas de l’HMS et de la CI. Au cours de la CI, le locus de la chaîne lourde des immunoglobulines subit un changement de conformation qui rapproche les promoteurs, les enhancers et les régions de switch afin de permettre la recombinaison des régions de switch. Cependant, les mécanismes moléculaires sous-jacents n’ont pas encore été identifié. Dans le but de comprendre les mécanismes de régulation d’AID, nous avons réalisé un criblage protéomique et identifié CTCF ainsi que les complexes médiateur et cohésine qui constituent des facteurs préalablement impliqués dans les interactions longues distances. Au cours de ce travail de thèse, nous avons montré que le complexe médiateur est requis pour la transcription de la région de switch acceptrice, pour l’interaction de cette dernière avec l’enhancer Eµ et pour le recrutement d’AID au locus des IgH. D’un autre côté, nous avons montré que le complexe cohésine est impliqué dans la réparation des cassures induites par AID et qu’il pourrait être impliqué dans la recombinaison des régions de switch. / During immune responses, B cells diversify their repertoire through somatic hypermutation (SHM) and class switch recombination (CSR). Both of these mechanisms are dependent on the activity of activation-induced cytidine deaminase (AID), an enzyme that deaminates cytosines into uracils generating mismatches that are differentially processed to result in SHM and CSR. During CSR, the Ig heavy chain (IgH) locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers and switch regions are brought into close proximity. Nevertheless, little is known about the underlying mechanisms. To gain insight into the molecular mechanism responsible for AID regulation during CSR, we performed a proteomic screen for AID partners and identified CTCF, cohesin and mediator complexes, which are factors previously implicated in long-range interactions. We showed that during CSR, the mediator complex is required for acceptor switch region transcription, long-range interaction between the enhancer and the acceptor switch region and AID recruitment to the IgH locus whereas the cohesin complex is required for proper AID-induced breaks repair and might favor switch regions synapsis.

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