Distal enhancer elements regulate the tissue-specific expression of their target genes via the establishment of physical interactions with the gene promoter. In mice, a cluster of five enhancers, jointly classified as a super-enhancer, specifically upregulate α-globin gene expression during erythroid differentiation. Aside from the Nprl3 gene, whose promoter is located inside this enhancer region, expression-levels of other genes within a short distance (&lt,50kb) of the enhancer region are not affected by the activation of the enhancer in erythroid cells, despite being located within the same sub-TAD in erythroid cells. The CCCTC-binding factor (CTCF) is implicated in the organisation of chromosome topology through the formation of interactions between its binding sites in an orientation-dependent manner. In this thesis, I demonstrate that CTCF functions in vivo as a boundary to maintain α-globin enhancer-promoter specificity in erythroid cells. The study of the local chromatin architecture by next-generation Capture-C reveals that α-globin enhancer and promoter interactions are constrained to a compartment of roughly 70kb. The unidirectional interaction profiles of the α-globin enhancers are delimited by the interactions between two genomic domains flanking the α-globin cluster. Further investigation shows that each of these domains contains several CTCF binding sites orientated in tandem, such that CTCF binding orientation between domains is convergent. Although CTCF binding across the α-globin locus is identical between mouse embryonic stem (ES) cells and erythroid cells, interaction between these domains occurs only in erythroid cells suggesting it is dependent on the formation of tissue-specific α-globin enhancer-promoter interactions. By generating a series of mouse models, deleting CTCF binding sites at the α-globin enhancers singly and in combination, I show that the deletion of two CTCF binding sites directly flanking the enhancer cluster results in a shift in interactions between flanking domains, away from the enhancer region. This leads to an expansion of enhancer interactions to include two genes directly upstream of the α-globin enhancers: Rhbdf1 and Mpg. Despite the Rhbdf1 gene being subject to polycomb group protein-mediated gene repression in erythroid cells, ablation of CTCF binding results in increased interactions between both the Rhbdf1 and Mpg gene promoters and the α-globin enhancers and concurrent strong transcriptional upregulation of both genes. The Rhbdf1 gene promoter acquires the active histone mark H3K4me3, but doesn't lose Polycomb Repressive Complex 2 (PRC2) mark H3K27me3 or binding of its catalytic component Ezh2. Despite the presence of this repressive mark, robust levels of Rhbdf1 expression are detected at levels higher than those in ES cells where this gene is actively expressed under the influence of its own enhancer. I conclude that regulation of the direction of enhancer interactions by CTCF is required for the promoter specificity of enhancers and the maintenance of transcriptional states of nearby genes.

La cohésion des chromatides sœurs est assurée par un complexe protéique en forme d’anneau assurant leur capture topologique. Ce complexe est constitué par des protéines conservées de la levure à l’Homme regroupées sous le terme « cohésine » : Smc1, Smc3 et la phosphoprotéine Scc1 fermant l’anneau (respectivement Psm1, Psm3 et Rad21 chez Schizosaccharomyces pombe). Les protéines régulatrices Rad61-Wapl, Pds5 et Scc3 (Wpl1,Pds5 et Psc3 respectivement chez S. pombe) interagissent avec l’anneau via Scc1. Il a été proposé que la capture de l’ADN par les cohésines nécessite l’ouverture transitoire de l’interface Smc1/Smc3. La réaction de dissociation fait quant à elle intervenir le sous-complexe Wapl/Pds5/Scc3 entraînant vraisemblablement l’ouverture de l’interface Scc1/Smc3. Le mécanisme par lequel la cohésion est créée et celui par lequel Wapl promeut la dissociation des cohésines des chromosomes, sont encore inconnus. Parmi les mutants de cohésion chez Saccharomyces cerevisiae, la mutation thermosensible eco1-1 affecte le gène ECO1 codant une acétyl-transférase, essentielle à la viabilité cellulaire, conservée de la levure à l’Homme (Eco1 « Establishment of Cohesion » chez S. cerevisiae, Eso1 chez S.pombe, ESCO1-2 chez l’Homme) et ayant Smc3 pour substrat. Il a été montré que l’acétyl-transférase s’oppose à l’action de dissociation de Wapl. C’est un crible génétique réalisé par plusieurs équipes, visant à trouver des mutants suppresseurs d’eco1-1, qui a permis d’identifier les gènes codant les protéines Wapl, Pds5, Scc3 et Smc3 comme composants du mécanisme d’ouverture de l’anneau de cohésine. Un crible similaire a été réalisé chez S.pombe dans notre laboratoire, dans le but de trouver des suppresseurs de la mutation thermosensible eso1-H17. Ce crible a identifié les gènes orthologues à ceux trouvés chez la levure : wpl1, pds5, psc3 et psm3 mais aussi le gène codant la sous-unité catalytique du complexe sérine/thréonine phosphatase de type IV (PP4), noté pp4c. Nous avons alors mis en œuvre des expériences pour caractériser PP4c ainsi que sa sous-unité régulatrice Psy2 qui s’est révélée être également impliquée dans la cohésion des chromatides soeurs. Nous avons également identifié la protéine Rad21 comme substrat du complexe PP4, puis identifié les phosphosites potentiellement cibles de PP4, pour ensuite cribler et analyser des phosphomutants de Rad21 récapitulant l’effet suppresseur de la délétion de PP4. / Sister-chromatid cohesion is ensured by a ring shape protein complex which is in charge of their topological embrace. This complex consists of proteins which are conserved from yeast to human and grouped under the term “cohesin”: Smc1, Smc3 and the phosphoprotein Scc1 which closes the ring (respectively Psm1, Psm3 and Rad21 in Schizosaccharomyces pombe). The regulatory proteins Rad61-Wapl, Pds5 and Scc3 (Wpl1,Pds5 and Psc3 respectively in S. pombe) interact with the ring via Scc1. It has been suggested that DNA capture by the cohesin complex involves the transient opening of the Smc1/Smc3 interface. The dissociation reaction involves the sub-complex Wapl/Pds5/Scc3 which likely causes the opening of the Scc1/Smc3 interface. The mechanisms by which cohesion is created and by which Wapl promotes the cohesin dissociation from chromosomes are still unknown. Among the cohesion mutants in Saccharomyces cerevisiae the thermosensitive eco1-1 mutation affects the ECO1 gene encoding an acetyl-transferase essential for cell viability and conserved from yeast to human (Eco1 « Establishment of Cohesion » in S.cerevisiae, Eso1 in S. pombe and ESCO1-2 in human) and whose substrate is Smc3. It has been shown that the acetyl-transferase counteracts the dissociation action of Wapl. A genetic screen carried out by several teams in order to find suppressors of the eco1-1 mutation has led to the identification of the genes encoding the Wapl, Pds5, Scc3 and Smc3 proteins as components of the opening mechanism of the cohesin ring. A similar screen was carried out in S. pombe in our lab to find suppressors of the thermosensitive mutation eso1-H17. This screen identified the orthologous genes to those found in the budding yeast: wpl1,pds5, psc3 and psm3 and also the gene encoding the catalytic subunit of the type 4 serine/threonine phosphatase complex (PP4) named pp4c. We have therefore carried out experiments to characterize PP4c and its regulatory subunit Psy2 which has also been found to be involved in sister-chromatid cohesion. We have likewise identified the Rad21 subunit as a PP4 substrate and identified phosphosites as potential targets of PP4. We have then screened and analyzed Rad21 phosphomutants which were able to mimic the suppressor effect of the deletion of pp4c.

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.

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Following the discovery of the one-dimensional sequence of human DNA, much focus has been directed on microscopy and molecular techniques to learn about the spatial organization of chromatin in a 3D cell. The development of these powerful tools has enabled high-resolution, genome-wide analysis of chromosome structure under many different conditions. In this thesis, I focus on how the organization of interphase chromatin is established and maintained following mitosis. Mitotic chromosomes are folded into helical loop arrays creating short and condensed chromosomes, while interphase chromosomes are decondensed and folded into a number of structures at different length scales ranging from loops between CTCF sites, enhancers and promoters to topologically associating domains (TADs), and larger compartments. While the chromatin organization at these two very different states is well defined, the transition from a mitotic to interphase chromatin state is not well understood. The aim of this thesis is to determine how interphase chromatin is organized following mitotic chromosome decondensation and to interrogate factors potentially responsible for driving the transition. First, I determine the temporal order with which CTCF-loops, TADs, and compartments reform as cells exit mitosis, revealing a unique structure at the anaphase-telophase transition never observed before. Second, I test the role of transcription in reformation of 3D chromosome structure and show that active transcription is not required for the formation of most interphase chromatin features; instead, I propose that transcription relies on the proper formation of these structures. Finally, I show that RNA in the interphase nucleus can be degraded with only slight consequences on the overall chromatin organization, suggesting that once interphase chromatin structures are achieved, the structures are stable and RNA is only required to reduce the mixing of active and inactive compartments. Together, these studies further our understanding of how interphase structures form, how these structures relate to functional activities of the interphase cell, and the stability of chromatin structures over time.