To investigate the cohesin ATPase module, I sought to generate a series of expression constructs in E.coli. As only Smc1 proved difficult to express, I screened a series of Smc1 heads from different species. I was able to produce the Smc1 head from Chaetomium Thermophilum Smc1 (ctSmc1) and to obtain a crystal structure in complex with a fragment of yeast Scc1 (CScc1) at 2.1 Å resolution. This allowed me to express and co-purify an assembled cohesin head Smc1-Smc3-Scc1 heterotrimer by fusing NScc1 to the C terminal end of ctSmc1 together with the remaining head module components.

To understand how cohesin engages DNA, I determined a crystal structure of the Scc3-Scc1 subcomplex bound to double-stranded DNA, showing that the Scc3-Scc1 subcomplex engages double-stranded DNA through a conserved, positively charged surface. We found that this surface is essential for the enrichment of cohesin complexes on chromosomes.

To investigate the molecular basis for the collaboration between cohesin and CTCF in defining the 3D architecture of the genome, I identified the critical region of CTCF required for binding to the SA2-Scc1 subcomplex. I was able to determine the structure of the ternary complex by x-ray crystallography. The structure revealed the molecular mechanism for CTCF-cohesin interaction. A similar motif is present in Wapl, the cohesin release factor, which binds to the same surface on the SA2-Scc1 complex, as well as in Shugoshin and in a number of established and novel cohesin clients. I was able to determine a crystal structure of the relevant motif of Shugoshin in complex with SA2-Scc1, which confirmed that it binds to the same conserved surface on SA2-Scc1. As binding of each of the factors investigated is mutually exclusive, competition for the conserved surface might be a required aspect of cohesin function in different biological contexts, for example, to counteract Wapl-mediated release. My results thus provide fundamental insights into the molecular mechanism that enables regulation of cohesin function and dynamic chromatin folding. une protéine à doigt de zinc connue comme un isolant, dont il a été rapporté qu'elle collabore à la détermination de la structure tridimensionnelle du génome.

Résumé

The cell cycle

Cell division has been a wide-spread and fundamental study for biologists since the development of the microscope permitted the direct observation of the cell. Genetic material must be faithfully duplicated and divided into daughter cells. From an evolutionary perspective, from bacteria to eukaryotes, this genetic 'pool' expands considerably, ranging from megabase to gigabase, which makes it more and more complicated. Unravelling the mechanisms by which genomes are accurately maintained, duplicated, and readily segregated into daughter cells has eluded researchers for over a century, since Walther Flemming described the congression and longitudinal splitting of chromosomes, or 'mitosis', a Greek word meaning 'thread', during cell division.

In Bacteria, which lack a nucleus, genome replication and segregation are more tightly and temporally coupled, as compared to eukaryotes. The bacterial cell cycle is divided into three periods: B, C and D. B marks the initiation of chromosome replication, which proceeds during C, and terminates with the end of replication and execution of division in period D.

The process of cell division is complex and involves many regulatory steps. Most of these steps are regulated by reversible protein phosphorylation events, which are thought to facilitate crucial checkpoints throughout the cell cycle. In eukaryotes, the cell cycle is mainly divided into two periods: interphase and mitotic phase (Figure 1.1). Interphase consists of G1, G2 and S phases. During G1 phase cells grow and prepare for DNA replication which takes place during S phase. During G2 phase, the cell prepares to divide. During the mitotic (M) phase, several steps to organise and divide the replicated DNAs take place, namely prophase, metaphase and anaphase. There are three main checkpoints that are important either for entering the cell cycle (G1 checkpoint, which makes the key decision on whether the cell should divide), or entry into the proliferative M phase (G2 checkpoint, wherein the nuclear envelop breaks down and individual chromosomes are resolved into distinct, rod-shaped masses). Finally, all chromosomes must be aligned along the metaphase plate, and kinetochore biorientation and attachment to spindle microtubules must be achieved to satisfy the Metaphase checkpoint, which is also known as the spindle assembly checkpoint (SAC)

(Figure 1.1).

Chromosomes are organised by SMC-kleisin enzymes

Chromosomal DNA is typically organised within cellular, or subcellular compartments of dimensions considerably smaller than its linear length [START_REF] Ou | ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells[END_REF]. Chromatin must therefore be organised and stored in a fashion which permits its enclosure within the cell, without restricting its accessibility. Genome maintenance, replication, transcription, and segregation hence present a significant topological problem.

An ancient family of enzymes, SMC (structural maintenance of chromosomes)-kleisins (from the Greek for 'closure'), facilitates chromatin organisation from bacteria to humans. In eukaryotes, three evolutionarily conserved SMC-kleisin complexes have arisen from a single prokaryotic precursor and fulfil multiple distinct roles within the cell cycle. These complexes are called cohesin (Smc1 and Smc3), condensin (Smc2 and Smc4) and Smc5/6. Together they predominantly associate with sister chromatin cohesion, chromosome condensation, and DNA damage repair, respectively [START_REF] Fujioka | Identification of a novel non-structural maintenance of chromosomes (SMC) component of the SMC5-SMC6 complex involved in DNA repair[END_REF][START_REF] Hirano | Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homolog of the Drosophila Barren protein[END_REF][START_REF] Losada | Identification of Xenopus SMC protein complexes required for sister chromatid cohesion[END_REF]. In animal cells, condensin binds to chromosomes at about the same time that most cohesin dissociates: between prophase and prometaphase [START_REF] Losada | Identification of Xenopus SMC protein complexes required for sister chromatid cohesion[END_REF].

A fundamental and conserved property of SMC-kleisin enzymes is their capacity to topologically engage DNA molecules (Cuylen, et al. 2011;[START_REF] Haering | The cohesin ring concatenates sister DNA molecules[END_REF][START_REF] Kanno | The Smc5/6 Complex Is an ATP-Dependent Intermolecular DNA Linker[END_REF][START_REF] Wilhelm | SMC condensin entraps chromosomal DNA by an ATP hydrolysis dependent loading mechanism in Bacillus subtilis[END_REF]. Once engaged, these enzymes are thought to regulate higherorder genome architecture through coordinated cycles of DNA entrapment and release [START_REF] Hassler | Towards a Unified Model of SMC Complex Function[END_REF][START_REF] Hirano | Condensin-Based Chromosome Organization from Bacteria to Vertebrates[END_REF].

As this thesis is primarily concerned with the structural biochemistry of cohesin, I will elaborate predominantly on the function of this complex and the underlying mechanisms, with some reference and comparison to other SMC-kleisin proteins where appropriate.

Due to significant temporal separation between genome duplication and segregation in eukaryotes, the sister chromatids produced during S phase need to be paired from the moment they are generated until their equal division into two daughter cells. The co-entrapment of sister DNA strands by cohesin allows this complex to establish chromosome cohesion, a fundamental prerequisite for the equal distribution of genetic information to daughter cells during eukaryotic cell division [START_REF] Nasmyth | Cohesin: a catenase with separate entry and exit gates?[END_REF].

Concurrent with the cell cycle, cohesin undergoes its own functional cycle. Cohesin is recruited to chromatin during G1 phase, and loaded on to chromatin by a dedicated loading apparatus; cohesion is then established in S phase during the synthesis of sister chromatids. In metazoans, cohesin is released in two steps. Most cohesin along chromosome arms is removed during prophase, allowing the emergence of X-shaped chromosomes. Finally, residual centromeric cohesin, which is necessary for kinetochore biorientation and satisfaction of the SAC, is released at the metaphase-to-anaphase transition by separasemediated proteolysis. The concept of a 'cohesin cycle' is elaborated on further in section 1.4.

Organisation of the core cohesin complex

The core cohesin complex consists of an Smc1-Smc3 heterodimer bound to the kleisin protein Scc1 (sister chromatid cohesion 1) [START_REF] Gligoris | Closing the cohesin ring: structure and function of its Smc3-kleisin interface[END_REF][START_REF] Haering | Molecular architecture of SMC proteins and the yeast cohesin complex[END_REF][START_REF] Haering | Structure and stability of cohesin's Smc1-kleisin interaction[END_REF], which in turn recruits the essential HEAT (Huntingtin elongation factor 3, A subunit of protein phosphatase 2A, TOR) repeat protein Scc3.

Smc1 and Smc3 feature two globular ABC-type ATPase 'head' domains assembled by their N-and C-termini. Each Smc head contains canonical Walker A, Walker B and Signature motifs. Dimerisation of the heads results in the formation of two composite ATPase sites, and is essential for ATP hydrolysis. The remainder of each Smc consists of an extended antiparallel coil-coiled domain of 300-400 amino acids, with a length of approximately 50 nm that intersects the head domains. The central region of these coiled-coils folds into an SMC 'hinge' domain of ~150 amino acids, through which the Smc proteins heterodimerise.

The Scc1 subunit (also called Mcd1, mitotic chromosome determinant 1, or Rad21, radiationsensitivity 21), forms a bridge between the Smc3 and Smc1 ATPase domains with which it interacts through its N-terminal helix-turn-helix and C-terminal winged-helix domains respectively. Thus, the ring is completed, and is able to wrap around sister chromatids. In addition to sealing the complex, Scc1 also provides docking sites for additional regulatory factors which are essential for the proper function and regulation of the complex.

Like other SMC-kleisins, cohesin complexes organise as large individual ring-like assemblies. The ring model was initially developed by visualization of the cohesin holocomplex by electron microscopy and through mapping interactions between its core subunits [START_REF] Anderson | Condensin and cohesin display different arm conformations with characteristic hinge angles[END_REF]Haering, et al. 2002) (Figure 1.4b). Visualisation by EM suggests an annular geometry, while biochemical experiments indicate that a full complement of inter-subunit contacts are essential as the individual disruption of any of the SMC-kleisin interfaces (Figure 1.2a) results in lethality [START_REF] Arumugam | Cohesin's ATPase activity is stimulated by the C-terminal Winged-Helix domain of its kleisin subunit[END_REF][START_REF] Gligoris | Closing the cohesin ring: structure and function of its Smc3-kleisin interface[END_REF][START_REF] Haering | Structure and stability of cohesin's Smc1-kleisin interaction[END_REF][START_REF] White | Genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes[END_REF][START_REF] Xu | Rad21-cohesin haploinsufficiency impedes DNA repair and enhances gastrointestinal radiosensitivity in mice[END_REF]. Hence, the formation of a fully closed cohesin 'ring' is indispensable for its function. Recent evidence strongly supports the hypothesis that individual cohesin complexes are able to encircle individual DNA molecules as well as pairs of sister DNAs by topological embrace [START_REF] Gligoris | Closing the cohesin ring: structure and function of its Smc3-kleisin interface[END_REF][START_REF] Huis In 't Veld | Characterization of a DNA exit gate in the human cohesin ring[END_REF].

Several structures from different species illustrate the molecular determinants of cohesin ring assembly, in which the subunits form a closed circuit connected via the SMC hinge, and bridging of the head domains via the separate termini of Scc1. The structure of the M.musculus hinge domain, consisting of Smc1 and Smc3, is toroidal (Figure 1.2b, Table S1), harbours a positively charged channel between Smc1 and Smc3, and is thought to be required for DNA entry [START_REF] Gruber | Evidence that loading of cohesin onto chromosomes involves opening of its SMC hinge[END_REF][START_REF] Srinivasan | The Cohesin Ring Uses Its Hinge to Organize DNA Using Non-topological as well as Topological Mechanisms[END_REF]. SMC heterodimerization is conferred principally by the hinge domain, but the head domains are able to dimerize transiently via bound nucleotide (as modelled in figure 1.2c). The structure of the Smc3-Scc1 complex shows that the N terminus of Scc1 assembles into two antiparallel helices that pack against the coiled-coild of Smc3 (Figure 1.2c, Table S1 [START_REF] Gligoris | Closing the cohesin ring: structure and function of its Smc3-kleisin interface[END_REF]).

Disconnection of the Smc3-NScc1 interface is thought to release cohesin from chromosomes [START_REF] Chan | Cohesin's DNA exit gate is distinct from its entrance gate and is regulated by acetylation[END_REF][START_REF] Eichinger | Disengaging the Smc3/kleisin interface releases cohesin from Drosophila chromosomes during interphase and mitosis[END_REF]. The structure of the Smc1-Scc1 complex shows that the C-terminal winged helix domain (WHD) of Scc1 binds to the N-lobe of the Smc1

ATPase (Figure 1.2c). The essential HEAT-repeat subunit, Scc3, is recruited through the central region of Scc1.

Since its discovery, Scc3 has been implicated in both cohesin loading and release, and is known as an essential cohesion maintenance factor [START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF][START_REF] Hu | ATP hydrolysis is required for relocating cohesin from sites occupied by its Scc2/4 loading complex[END_REF][START_REF] Murayama | Biochemical reconstitution of topological DNA binding by the cohesin ring[END_REF][START_REF] Roig | Structure and function of cohesin's Scc3/SA regulatory subunit[END_REF][START_REF] Rowland | Building sister chromatid cohesion: smc3 acetylation counteracts an antiestablishment activity[END_REF].

Whilst yeast contains only one Scc3 isoform, there are three Scc3 paralogs in metazoans:

SA1-SA3 (Stromal Antigen) [START_REF] Sumara | Characterization of vertebrate cohesin complexes and their regulation in prophase[END_REF]. Evidence indicates that SA1 and SA2 have different roles in cohesin biology, and SA3 is a meiosis-specific cohesin subunit: SA1

associates with DNA at the telomeres and SA2 mostly at the centromeres. SA1 binds directly to telomeric DNA via its unique AT-hook which is not found in SA2 [START_REF] Bisht | SA1 binds directly to DNA through its unique AT-hook to promote sister chromatid cohesion at telomeres[END_REF]).

SA1-containing cohesin has bee implicated in gene regulation and replication of telomeres [START_REF] Remeseiro | Cohesin-SA1 deficiency drives aneuploidy and tumourigenesis in mice due to impaired replication of telomeres[END_REF]) and its deficiency drives aneuploidy and tumourigenesis in mouse due to its impaired replication of telomeres [START_REF] Remeseiro | Cohesin-SA1 deficiency drives aneuploidy and tumourigenesis in mice due to impaired replication of telomeres[END_REF]). SA2, the dominant form in somatic cells, is mainly responsible for sister chromatid cohesion at the centromere [START_REF] Remeseiro | Cohesin-SA1 deficiency drives aneuploidy and tumourigenesis in mice due to impaired replication of telomeres[END_REF]. Although both SA1 and SA2 are found at CCCTC-binding factor (CTCF) sites, a distinct population of the SA2-cohesin is linked to tissue-specific transcriptional regulation [START_REF] Kojic | Distinct roles of cohesin-SA1 and cohesin-SA2 in 3D chromosome organization[END_REF]. It was also reported that downregulation of these two SA paralogs have different consequences for gene expression and chromosome structure: Downregulation of SA1 mainly impacts topologically associating domain (TAD) boundaries whereas SA2 downregulation mainly impacts cell-type-specific enhancerpromoter contacts [START_REF] Kojic | Distinct roles of cohesin-SA1 and cohesin-SA2 in 3D chromosome organization[END_REF]). SA3 (STAG3) is a germline-cell specific protein and functions during meiosis I [START_REF] Pezzi | STAG3, a novel gene encoding a protein involved in meiotic chromosome pairing and location of STAG3-related genes flanking the Williams-Beuren syndrome deletion[END_REF] and together with meiosis-specific cohesin, it influences meiotic chromosome structure and recombination [START_REF] Adelfalk | Cohesin SMC1beta protects telomeres in meiocytes[END_REF][START_REF] Ding | Meiotic cohesins modulate chromosome compaction during meiotic prophase in fission yeast[END_REF][START_REF] Revenkova | Cohesin SMC1 beta is required for meiotic chromosome dynamics, sister chromatid cohesion and DNA recombination[END_REF]).

Scc3 associates with chromatin during G1/S, and dissociates, with cohesin, in metaphase after being phosphorylated by Plk1. Plk1, a polo-like kinase, has been reported to be required for regulating a variety of mitotic and meiotic events, such as the metaphase/anaphase transition, cytokinesis and the onset of mitosis and spindle formation [START_REF] Golsteyn | Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function[END_REF][START_REF] Goto | Complex formation of Plk1 and INCENP required for metaphase-anaphase transition[END_REF][START_REF] Randall | Polo kinase and cytokinesis initiation in mammalian cells: harnessing the awesome power of chemical genetics[END_REF][START_REF] Van Vugt | Polo-like kinase-1 is required for bipolar spindle formation but is dispensable for anaphase promoting complex/Cdc20 activation and initiation of cytokinesis[END_REF]). Plk1 is thought to phosphorylate cohesin subunit SA1/2 and Scc1 directly during prophase which results in the dissociation of the bulk of cohesin from chromosome arms (Figure 1.5) [START_REF] Hauf | Dissociation of cohesin from chromosome arms and loss of arm cohesion during early mitosis depends on phosphorylation of SA2[END_REF][START_REF] Losada | Identification of Xenopus SMC protein complexes required for sister chromatid cohesion[END_REF][START_REF] Sumara | Characterization of vertebrate cohesin complexes and their regulation in prophase[END_REF][START_REF] Sumara | The dissociation of cohesin from chromosomes in prophase is regulated by Polo-like kinase[END_REF].

The structure of the human SA2-Scc1 complex was determined in 2015, showing that SA2 and Scc1 form a 1:1 heterodimer. Scc1 interacts extensively with a highly conserved surface along the inner hook of SA2 [START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF]. This paper further identified an interaction between SA2-Scc1 and shugoshin1 (Sgo1), a cohesin protector at the centromere (Figure 1.3, Table S1). It was also shown that shugoshin and Wapl, the cohesin release factor, can compete for the same binding interface on SA2-Scc1 [START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF]. It is likely that interaction of Scc3 with cohesin regulators is an integral, albeit not completely understood aspect of Scc3 function [START_REF] Beckouet | Releasing Activity Disengages Cohesin's Smc3/Scc1 Interface in a Process Blocked by Acetylation[END_REF][START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF][START_REF] Roig | Structure and function of cohesin's Scc3/SA regulatory subunit[END_REF].

The binding of Scc3 to cohesin has also been shown to be essential for efficient loading of the complex onto chromatin [START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF][START_REF] Hu | ATP hydrolysis is required for relocating cohesin from sites occupied by its Scc2/4 loading complex[END_REF][START_REF] Murayama | Biochemical reconstitution of topological DNA binding by the cohesin ring[END_REF][START_REF] Orgil | A conserved domain in the scc3 subunit of cohesin mediates the interaction with both mcd1 and the cohesin loader complex[END_REF]. The mechanism by which Scc3 promotes the association of cohesin with chromosomal DNA will be explored extensively in later chapters of this thesis. association [START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF].

Overview of the cohesin cycle

Following the cell cycle, cohesin also has its own cycle. During G1, cohesin is loaded onto chromatin with the help of the Scc2-Scc4 'loading' complex, and is released again by the recruitment of a dissociation (unloading) factor, Wapl, which interacts with Pds5 and Scc3 to open the Smc3-Scc1 interface. Following loading, and during genome replication, an acetyltransferase, Eco1, acetylates the ATPase head domain of Smc3 to trigger the stabilization of cohesion [START_REF] Ben-Shahar | Eco1-dependent cohesin acetylation during establishment of sister chromatid cohesion[END_REF][START_REF] Unal | A molecular determinant for the establishment of sister chromatid cohesion[END_REF]. In vertebrates, acetylated cohesin recruits an additional factor, sororin, which further stabilizes cohesin during the maintenance phase by inhibiting the interaction of Wapl and Pds5 [START_REF] Nishiyama | Sororin mediates sister chromatid cohesion by antagonizing Wapl[END_REF]. In metazoans, cohesin is removed from sister chromatids in two steps. In the first step, at the beginning of prophase, cohesin is phosphorylated by mitotic kinases such as CDK1, Plk and aurora B, which displaces sororin, allowing Wapl to re-bind Pds5 and to release cohesin from chromosome arms [START_REF] Dreier | Regulation of sororin by Cdk1-mediated phosphorylation[END_REF][START_REF] Hauf | Dissociation of cohesin from chromosome arms and loss of arm cohesion during early mitosis depends on phosphorylation of SA2[END_REF]. To permit assembly of the mitotic spindle, residual centromeric cohesion is protected by Sgo1, which counteracts cohesin phosphorylation by recruiting PP2A, and directly inhibits the association of Wapl with Scc3.

Complete dissociation does not occur until all sister kinetochores have been attached to opposite poles of the mitotic spindle [START_REF] Ramos | FGF-1 reverts epithelial-mesenchymal transition induced by TGF-{beta}1 through MAPK/ERK kinase pathway[END_REF], via a second protease-dependent pathway. The attainment of this state satisfies the spindle assembly checkpoint, thus activating separase through APC-mediated degradation of its inhibitor securin [START_REF] Cohen-Fix | Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p[END_REF][START_REF] Yamamoto | Pds1p, an inhibitor of anaphase in budding yeast, plays a critical role in the APC and checkpoint pathway(s)[END_REF]. In this second step, at the metaphase-to-anaphase transition, centromeric cohesin is cleaved by the separase protease at two cleavage sites on Scc1 [START_REF] Uhlmann | Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1[END_REF]) releasing sister chromatids and allowing segregation into daughter cells [START_REF] Nasmyth | Cohesin: its roles and mechanisms[END_REF][START_REF] Peters | The cohesin complex and its roles in chromosome biology[END_REF]) (Figure 1.4, Table 1.1, Figure 1.5).

SMC1 PSM1 SMC1 SMC1 SMC1 SMC3 PSM3 SMC3 SMC3 SMC3 MCD1 (SCC1) RAD21 RAD21 RAD21 RAD21 (SCC1) IRR1 (SCC3) PSC3 SA SA1, SA2 SA1(STAG1),SA2 (STAG2) Loading SCC2 MIS4 NIPBL SCC2 NIPBL SCC4 SSL3 N/C xSCC4 MAU2 (hSCC4) Establishment ECO1 (CTF7) ESO1 San, Deco XECO1, XECO2 EFO1(ESCO1), EFO2 (ESCO2) Maintenance PDS5 PDS5 PDS5 PDS5A, PDS5B PDS5A, PDS5B RAD61 WPL1 WAPL N/C WAPL Dissolution PDS1 CUT2 PIM Securin Securin (PTTG) ESP1 CUT1 Separase (SSE) Separin Separin (ESPL1) CDC5 PLO1 POLO PLX1 Plk1 SGO1 SGO1, SGO2 SGO1(Mei- S332) Shugoshin- like 1 (xSGO1) Shugoshin (hSGOl1)
Table 1.1 Cohesin and its related factors in different species (adapted from [START_REF] Onn | Sister chromatid cohesion: a simple concept with a complex reality[END_REF]))

Cohesin ATPase dimerization

The ATPase domain of cohesin is evolutionarily related to ABC transporters (ATP-binding cassette transporters), which are typically membrane transporters that utilize the energy of adenosine triphosphate (ATP) binding and hydrolysis enable translocation of substrates across membranes. In cohesin, the substrate is chromosomal DNA, suggesting that the ATPase domain of Smc1 and Smc3 provide the motor activity [START_REF] Arumugam | ATP hydrolysis is required for cohesin's association with chromosomes[END_REF][START_REF] Weitzer | A model for ATP hydrolysis-dependent binding of cohesin to DNA[END_REF] for DNA transport as well as loading of DNA into the cohesin ring to enable association with chromosomes [START_REF] Arumugam | ATP hydrolysis is required for cohesin's association with chromosomes[END_REF].

Essential ATP or DNA binding residues are predicted based on the structure of another ABC ATPase called Rad50, which also contains a Walker A-B motif, and shares similar architecture [START_REF] Liu | ATP-dependent DNA binding, unwinding, and resection by the Mre11/Rad50 complex[END_REF]. Further, mutation of amino acid residues in the head domain (SMC1-K39, D1157; SMC3-K38, D1154) abolishes ATP binding while mutation of residues SMC1-E1158 and SMC3-E1155 abolishes ATP hydrolysis and SMC1-S1130 and SMC3-S1128 impair ATP-dependent dimerization [START_REF] Arumugam | ATP hydrolysis is required for cohesin's association with chromosomes[END_REF]. Also, DNA loading is thought to occur through transient opening of the hinge domain as artificial linking of the SMC1 and SMC3 hinge abolishes cohesin loading [START_REF] Gruber | Evidence that loading of cohesin onto chromosomes involves opening of its SMC hinge[END_REF]. The cohesin cycle is dependent on cell cycle-dependent kinases and also the APC/C complex for appropriate temporal regulation. Figure adapted from [START_REF] Haarhuis | Cohesin and its regulation: on the logic of X-shaped chromosomes[END_REF].

The cohesin loading complex

The ability of cohesin to bind chromosomes depends on the Scc2-Scc4 complex, which is the loading factor for cohesin onto DNA [START_REF] Ciosk | Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins[END_REF], and its deletion causes premature sister separation and abnormal chromosome segregation. Scc2-Scc4 forms a separate complex independently of cohesin [START_REF] Kogut | The Scc2/Scc4 cohesin loader determines the distribution of cohesin on budding yeast chromosomes[END_REF]). Scc2-Scc4 associates with cohesin during G1 through Scc1 and becomes dispensable during S and G2 phase when cohesin establishment and maintenance take place respectively. Scc2 contains a hook-shaped C terminal HEAT repeat domain, and a disordered N-terminal domain that snakes through the Scc4 subunit [START_REF] Chao | Structural Studies Reveal the Functional Modularity of the Scc2-Scc4 Cohesin Loader[END_REF][START_REF] Hinshaw | Structural evidence for Scc4-dependent localization of cohesin loading[END_REF][START_REF] Kikuchi | Crystal structure of the cohesin loader Scc2 and insight into cohesinopathy[END_REF]. In budding yeast, cohesin is loaded at particular locations but quickly relocalizes to new locations, and it is supposed that cohesin could slide alone the chromatin fiber to another site after loading [START_REF] Glynn | Genome-wide mapping of the cohesin complex in the yeast Saccharomyces cerevisiae[END_REF][START_REF] Lengronne | Cohesin relocation from sites of chromosomal loading to places of convergent transcription[END_REF][START_REF] Ocampo-Hafalla | Cohesin loading and sliding[END_REF].

Cleavage-independent cohesin unloading

Dynamic cohesin release via Smc3-Scc1 gate opening is generally considered to depend on a 'releasing' subcomplex formed by Pds5 and Wapl [START_REF] Gandhi | Human Wapl is a cohesin-binding protein that promotes sister-chromatid resolution in mitotic prophase[END_REF][START_REF] Kueng | Wapl controls the dynamic association of cohesin with chromatin[END_REF]Murayama and Uhlmann 2015;[START_REF] Ouyang | Structural Basis and IP6 Requirement for Pds5-Dependent Cohesin Dynamics[END_REF][START_REF] Rowland | Building sister chromatid cohesion: smc3 acetylation counteracts an antiestablishment activity[END_REF][START_REF] Sutani | Budding yeast Wpl1(Rad61)-Pds5 complex counteracts sister chromatid cohesionestablishing reaction[END_REF]).

Pds5 (Precocious dissociation of sisters 5, BimD/Spo76), is an evolutionarily conserved from fungi to human [START_REF] Van Heemst | Spo76p is a conserved chromosome morphogenesis protein that links the mitotic and meiotic programs[END_REF], containing a HEAT repeat domain. In vertebrates, deletion of Pds5 results in no discernible defects in chromosome arm cohesion but loosened cohesion at the centromere while overall cohesin levels are not affected. Thus Pds5 might play both positive and negative roles in sister chromatid cohesion [START_REF] Losada | Functional contribution of Pds5 to cohesin-mediated cohesion in human cells and Xenopus egg extracts[END_REF]. Aside from forming the unloading complex with Wapl, Pds5 is also required to establish and maintain cohesion [START_REF] Chan | Pds5 promotes and protects cohesin acetylation[END_REF][START_REF] Hartman | Pds5p is an essential chromosomal protein required for both sister chromatid cohesion and condensation in Saccharomyces cerevisiae[END_REF].

Pds5 forms an elongated HEAT repeat protein that binds to Scc1 via a conserved surface patch and this interface is important for Pds5 recruitment to cohesin and mutations of this binding interface results in loss of sister chromatid cohesion and cell viability (Figure 1.6, Table S1) (Muir, et al. 2016). (purple) interacts with a discrete binding module on Scc1 (green) (PDB: 5FRP), and this interaction is essential to maintain sister chromatid cohesion (Muir, et al. 2016).

As a subunit of the cohesin releasing complex, Wapl (wings apart-like) was originally identified as a gene product that regulates heterochromatin organization in Drosophila [START_REF] Verni | Genetic and molecular analysis of wings apart-like (wapl), a gene controlling heterochromatin organization in Drosophila melanogaster[END_REF]. Its relevance for cohesion was discovered in 2006 [START_REF] Gandhi | Human Wapl is a cohesin-binding protein that promotes sister-chromatid resolution in mitotic prophase[END_REF][START_REF] Kueng | Wapl controls the dynamic association of cohesin with chromatin[END_REF] when it was reported to promote the release of cohesin by direct interaction with Pds5 and Scc3. Therefore the countervailing influences of Wapl-Pds5 and Scc2-Scc4, result in dynamic cohesin association [START_REF] Kueng | Wapl controls the dynamic association of cohesin with chromatin[END_REF]. Further studies suggest that the well-conserved FGF motifs at the N-terminus of Wapl are responsible for the interaction between Pds5 and Scc3 in vertebrates, but not in yeast [START_REF] Shintomi | Releasing cohesin from chromosome arms in early mitosis: opposing actions of Wapl-Pds5 and Sgo1[END_REF].

Wapl proteins from different species have a very divergent N terminal domain with variable lengths, but conserved C terminal domains. The structure of the human Wapl C terminal domain shows an enlongated shape containing HEAT repeats. Mutation of surface amino acids shows that Wapl is essential for cohesin release. It is also been reported that mutation of the FGF motifs of Wapl reduces its binding to Scc3-Scc1. However, truncation of the entire N terminus comprising the FGF motifs (amino acids 1-500) still retained Scc3-Scc1 binding acvitity indicating that aside from the FGFs, there are additional regions that interact with Scc3-Scc1 (Figure 1.7, Table S1) [START_REF] Ouyang | Structure of the human cohesin inhibitor Wapl[END_REF].

SMC3 acetylation (establishment of cohesion)

To establish cohesion, the head domain of Smc3 is acetylated by Eco1 acetyltransferase at two evolutionarily conserved residues, Smc3-K112 and K113 in yeast, K105 and K106 in human, and these modifications arise only during S phase [START_REF] Ben-Shahar | Eco1-dependent cohesin acetylation during establishment of sister chromatid cohesion[END_REF][START_REF] Rowland | Building sister chromatid cohesion: smc3 acetylation counteracts an antiestablishment activity[END_REF][START_REF] Skibbens | Ctf7p is essential for sister chromatid cohesion and links mitotic chromosome structure to the DNA replication machinery[END_REF][START_REF] Toth | Yeast cohesin complex requires a conserved protein, Eco1p(Ctf7), to establish cohesion between sister chromatids during DNA replication[END_REF][START_REF] Unal | A molecular determinant for the establishment of sister chromatid cohesion[END_REF][START_REF] Zhang | Acetylation of Smc3 by Eco1 is required for S phase sister chromatid cohesion in both human and yeast[END_REF]). Eco1, also called Ctf7 (chromosome transmission fidelity) in yeast and ESCO1 and 2 in humans, was first identified for its involvement in cohesion in 1999 [START_REF] Skibbens | Ctf7p is essential for sister chromatid cohesion and links mitotic chromosome structure to the DNA replication machinery[END_REF] and directly interacts with components of the replication machinery [START_REF] Kenna | Mechanical link between cohesion establishment and DNA replication: Ctf7p/Eco1p, a cohesion establishment factor, associates with three different replication factor C complexes[END_REF]. In Eco1 mutants, cohesin still associates with chromosomes but fails to build cohesion, suggesting that Eco1 functions in the establishment of cohesion. Eco1 contains a zinc finger in the N-terminus and an acetyltransferase domain at the C-terminus [START_REF] Toth | Yeast cohesin complex requires a conserved protein, Eco1p(Ctf7), to establish cohesion between sister chromatids during DNA replication[END_REF]. Eco1 not only acetylates Smc3 during S phase, but in vitro evidence shows that Eco1 could also acetylate itself, Scc1 and Pds5 (Heidinger-Pauli, et al. 2010;[START_REF] Ivanov | Eco1 is a novel acetyltransferase that can acetylate proteins involved in cohesion[END_REF][START_REF] Ivanov | The replicative helicase MCM recruits cohesin acetyltransferase ESCO2 to mediate centromeric sister chromatid cohesion[END_REF].

To limit cohesion establishment to S phase, Eco1 is phosphorylated by Cdk1 and thus becomes the substrate of the APC/C complex targeting for it for degradation [START_REF] Lyons | Cdk1-dependent destruction of Eco1 prevents cohesion establishment after S phase[END_REF].

The structure of human Esco1 contains a catalytic acetyltransferase domain, which is shared with other member of the Gcn5-related N-acetyltransferase family (GNAT), and a N terminal zinc finger. An additional 'loop insert' (residues 727-766) is thought to bind Smc3 (Figure 1.8, Table S1).

Cohesin stabilization

To stabilize cohesion, sororin (p35), is recruited via Smc3 acetylation at the DNA replication fork and stays connected throughout S and G2 phase [START_REF] Schmitz | Sororin is required for stable binding of cohesin to chromatin and for sister chromatid cohesion in interphase[END_REF]). To maintain cohesion, sororin forms a stabilizing complex with Pds5 during G2 phase (Figure 1.5) and it is found that sororin shares a FGF-like motif with Wapl [START_REF] Wu | A conserved motif at the C terminus of sororin is required for sister chromatid cohesion[END_REF] and is supposed to be essential for association with Pds5. An additional Tyrosine-Serine-Argnine (YSR) motif found in both Wapl and sororin is also reported to bind to Pds5 at the same conserved site, thus explaining Wapl-sororin antagonism in cohesin regulation [START_REF] Ouyang | Structural Basis and IP6 Requirement for Pds5-Dependent Cohesin Dynamics[END_REF].

Cohesin release at chromosome arms

Upon entry into mitosis, phosphorylation of sororin by Cdk1/CyclinB abolishes its ability to inhibit Wapl so that the Wapl-Pds5 complex can form again, allowing cohesin removal in prophase (Figure 1.5). Phosphorylated sororin then interacts with Plk1 via its Plk1 binding motif. Plk1 is thought to also regulate the dissociation of cohesin by phosphorylating Scc3 [START_REF] Kueng | Wapl controls the dynamic association of cohesin with chromatin[END_REF][START_REF] Zhang | Interaction of Sororin protein with polo-like kinase 1 mediates resolution of chromosomal arm cohesion[END_REF].

Cohesion protection at the centromere

In the Separase cleavage-independent release pathway, the centromeric cohesin is protected by another complex, formed by shugoshin and PP2A [START_REF] Kitajima | Shugoshin collaborates with protein phosphatase 2A to protect cohesin[END_REF][START_REF] Riedel | Protein phosphatase 2A protects centromeric sister chromatid cohesion during meiosis I[END_REF]) (Figure 1.5). Shugoshin, (a Japanese word for 'guardian spirit') is a centromereassociated protein conserved from yeast to humans and acts as a critical regulator of kinetochore assembly and centromeric cohesion [START_REF] Kitajima | The conserved kinetochore protein shugoshin protects centromeric cohesion during meiosis[END_REF][START_REF] Pouwels | Shugoshin 1 plays a central role in kinetochore assembly and is required for kinetochore targeting of Plk1[END_REF][START_REF] Watanabe | Shugoshin protects cohesin complexes at centromeres[END_REF]. Shugoshin is mostly localized at the centromeres by the conserved spindle checkpoint kinase Bub1, and Haspin, which install specific (not shown) that docks onto PP2A complex (multiple green colors) (PDB: 3FGA), and their interaction is essential for both separase cleavage protection and supporting spindle assembly checkpoint [START_REF] Xu | Structure and function of the PP2A-shugoshin interaction[END_REF]).

Aside from this main function of shugoshin protection at the centromere, there is still a small, yet substantial, amount of cohesin bound at chromosome arms, which are protected by Sgo1.

This small fraction of cohesin can only be removed by separase in order to completely remove cohesin [START_REF] Nakajima | The complete removal of cohesin from chromosome arms depends on separase[END_REF]). The chromosome arms might comprise a mixture of cohesin complexes that require different mechanisms for their dissociation and thus coordinating chromosome separation and spindle elongation.

Cohesin cleavage at the centromere

Separase is a caspase-family protease that was initially identified as Esp1 in budding yeast and Cut1 in fission yeast [START_REF] Mcgrew | Requirement for ESP1 in the nuclear division of Saccharomyces cerevisiae[END_REF][START_REF] Uzawa | The fission yeast cut1+ gene regulates spindle pole body duplication and has homology to the budding yeast ESP1 gene[END_REF]). Separase activation requires both its phosphorylation by Cdk1 [START_REF] Stemmann | Dual inhibition of sister chromatid separation at metaphase[END_REF]) and targeted-degradation of securin (Pds1/Cut2). Securin acts as a substrate mimic to inhibit separase activity [START_REF] Boland | Cryo-EM structure of a metazoan separase-securin complex at near-atomic resolution[END_REF][START_REF] Luo | Molecular mechanism for the regulation of yeast separase by securin[END_REF]. Separase associates with securin to stay inactive until the initiation of anaphase, then securin is removed through the ubiquitin protein ligase APC/C, releasing separase to allow cleavage of Scc1. Cohesin cleavage is also further activated by Cdc5, a Plk kinase, which phosphorylates Scc1 and thus enhances the cleavability by separase [START_REF] Alexandru | Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates sister chromatid separation in yeast[END_REF][START_REF] Clyne | Polo-like kinase Cdc5 promotes chiasmata formation and cosegregation of sister centromeres at meiosis I[END_REF][START_REF] Hornig | Preferential cleavage of chromatin-bound cohesin after targeted phosphorylation by Polo-like kinase[END_REF][START_REF] Lee | Role of Polo-like kinase CDC5 in programming meiosis I chromosome segregation[END_REF][START_REF] Lin | Structural basis of cohesin cleavage by separase[END_REF]. The structure of the separase-securin complex from

Saccharomyces cerevisiae shows a tight and extensive interaction, and residues 258-269 of securin occlude the separase active site, thus explaining how securin inhibits separase activity (Figure 1.10, Table S1).

Cohesin beyond cohesion

As discussed, the co-entrapment of sister chromatids by cohesin allows the complex to establish sister chromatid cohesion [START_REF] Nasmyth | Cohesin: a catenase with separate entry and exit gates?[END_REF]. Beyond this activity, cohesin is also thought to regulate higher-order genome architecture through coordinated cycles of intrachromatid DNA loop extrusion [START_REF] Hassler | Towards a Unified Model of SMC Complex Function[END_REF][START_REF] Hirano | Condensin-Based Chromosome Organization from Bacteria to Vertebrates[END_REF].

Several studies have shown that during interphase, cohesin might have additional divergent roles as a regulator of gene expression in different cell types. Cohesin also interacts with additional factors such as CTCF, the most well-known and most intensively studied insulator in vertebrates [START_REF] Bell | The protein CTCF is required for the enhancer blocking activity of vertebrate insulators[END_REF]. CTCF is reported to associate with cohesin through Scc3 [START_REF] Xiao | Specific sites in the C terminus of CTCF interact with the SA2 subunit of the cohesin complex and are required for cohesin-dependent insulation activity[END_REF]. Additional cohesin binding partners include Mediator, a transcriptional coactivator that occupies enhancer and promoter regions at active genes sites and interacts with the cohesin loading factor Scc2 [START_REF] Kagey | Mediator and cohesin connect gene expression and chromatin architecture[END_REF], and PRC1 (polycomb-group repressive complex 1), which is recruited by PRC2 to H3K27 methylated sites for gene silencing and it is proposed to interact with Scc1 [START_REF] Strubbe | Polycomb purification by in vivo biotinylation tagging reveals cohesin and Trithorax group proteins as interaction partners[END_REF]) to downregulate transcription.

CTCF

CTCF (CCCTC-binding factor) is a transcriptional insulator containing a tandem array of 11 zinc fingers and is universally conserved among Bilateria (Figure 1.11). It has sequencespecific DNA binding activity and plays a critical role in organizing genome structure and establishing gene expression patterns. CTCF was originally identified as a negative regulator of Myc expression [START_REF] Klenova | CTCF, a conserved nuclear factor required for optimal transcriptional activity of the chicken c-myc gene, is an 11-Zn-finger protein differentially expressed in multiple forms[END_REF][START_REF] Lobanenkov | A novel sequence-specific DNA binding protein which interacts with three regularly spaced direct repeats of the CCCTC-motif in the 5'-flanking sequence of the chicken c-myc gene[END_REF]) and subsequently shown to function more widely as an insulator protein. CTCF insulation function in general can be divided into two classes: enhancer-blocking insulators which, when placed between enhancers and promoters, prevent distant enhancers from activating a promoter [START_REF] Filippova | CTCF-binding sites flank CTG/CAG repeats and form a methylation-sensitive insulator at the DM1 locus[END_REF][START_REF] Hark | CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus[END_REF][START_REF] Tanimoto | Human beta-globin locus control region HS5 contains CTCF-and developmental stage-dependent enhancer-blocking activity in erythroid cells[END_REF][START_REF] Weth | CTCF function is modulated by neighboring DNA binding factors[END_REF], or barrier insulators which appear to block heterochromatin from spreading [START_REF] Cuddapah | Global analysis of the insulator binding protein CTCF in chromatin barrier regions reveals demarcation of active and repressive domains[END_REF][START_REF] Dowen | Control of cell identity genes occurs in insulated neighborhoods in mammalian chromosomes[END_REF]. to ZF7 bind in the major groove of the 15bp core CTCF sequence motif. CTCF is methylationsensitive at position 2 (close to 3' end, marked with red spot) of the core sequence, but insensitive at position 12 (close to 5' end, marked with red spot). ZF3, but not ZF8, contributes to DNA sequencespecific readout [START_REF] Hashimoto | Structural Basis for the Versatile and Methylation-Dependent Binding of CTCF to DNA[END_REF].

As an insulator, CTCF is involved in a number of different aspects of genome regulation.

One such example is imprinting, in which genes show parent-of-origin-specific monoallelic expression, regulated by an imprinting control element called ICR (imprinting control region) [START_REF] Ripoche | Deletion of the H19 transcription unit reveals the existence of a putative imprinting control element[END_REF]. The maternal chromosome ICR can bind to CTCF, which mediates silencing of the imprinted gene such as the Ifg2 and H19 gene, but in the paternal chromosome, ICR is methylated which in return inhibits CTCF binding. The methylated ICR lacks insulator activity [START_REF] Tremblay | A 5' 2-kilobase-pair region of the imprinted mouse H19 gene exhibits exclusive paternal methylation throughout development[END_REF][START_REF] Xiao | Specific sites in the C terminus of CTCF interact with the SA2 subunit of the cohesin complex and are required for cohesin-dependent insulation activity[END_REF]) (Figure 1.12). Whereas in the paternal chromosome, the ICR is highly methylated ('Me') thus abolishing CTCF binding and insulation function [START_REF] Wallace | We gather together: insulators and genome organization[END_REF].

CTCF is also able to recruit other protein factors. CTCF interacts with CHD8, a chromatin helicase protein, through its zinc finger domains, and this interaction is required for the insulator function [START_REF] Ishihara | CTCF-dependent chromatin insulator is linked to epigenetic remodeling[END_REF][START_REF] Yusufzai | CTCF tethers an insulator to subnuclear sites, suggesting shared insulator mechanisms across species[END_REF]. It was also reported that both Sin3, a transcriptional repressor, and YB-1, a DNA/RNA-binding factor, also bind the zinc finger domain, indicating that the CTCF zinc finger domain is not just a DNA binder, but also promotes protein-protein interactions ( [START_REF] Wallace | We gather together: insulators and genome organization[END_REF].

Recent work identified RNA-binding regions (RBR) at ZF1, ZF10, as well as the C terminus of CTCF, showing that RBR mediates CTCF clustering. Loss of the RBRs appears to disrupt CTCFmediated chromatin loops that are involved in the establishment of cell-type specific topology of chromatin, as well as gene expression profiles [START_REF] Hansen | An RNA-binding region regulates CTCF clustering and chromatin looping[END_REF][START_REF] Saldana-Meyer | RNA interactions with CTCF are essential for its proper function[END_REF].

Cohesin and CTCF

In addition to its crucial role in sister chromatid cohesion, cohesin is also involved in gene regulation [START_REF] Wendt | Cohesin mediates transcriptional insulation by CCCTC-binding factor[END_REF]. It was discovered that most cohesin binding sites in mammalian genomes are located proximal to CTCF motifs [START_REF] Parelho | Cohesins functionally associate with CTCF on mammalian chromosome arms[END_REF][START_REF] Wendt | Cohesin mediates transcriptional insulation by CCCTC-binding factor[END_REF]. CTCF is required for cohesin recruitment to CTCF sites, which in turn is required for formation of higher-order chromatin structure, thus indicating that cohesin has non-canonical functions that go beyond sister chromatid cohesion [START_REF] De Wit | CTCF Binding Polarity Determines Chromatin Looping[END_REF][START_REF] Lengronne | Cohesin relocation from sites of chromosomal loading to places of convergent transcription[END_REF]). Much of our knowledge concerning long-range chromatin interactions comes from chromatin conformation capture (3C) (de Wit and de Laat 2012). 3C is the founding technique of a set of methods used to measure the spatial organization of chromatin, and it quantifies the number of interactions between genomic loci that are close to each other in 3D space but otherwise distant in sequence [START_REF] Hakim | SnapShot: Chromosome confirmation capture[END_REF]. 3C and its derivatives have been applied to characterize many aspects of genome regulation, such as promoterenhancer interactions. The main difference between 3C and its derivatives is their scope, either for quantifying two specific sequence fragments, such as the original 3C method [START_REF] Dekker | Capturing chromosome conformation[END_REF], or to quantify interaction frequencies between all possible pairs of interactions simultaneously, such as Hi-C (high-throughput sequencing) [START_REF] Hakim | SnapShot: Chromosome confirmation capture[END_REF][START_REF] Lieberman-Aiden | Comprehensive mapping of long-range interactions reveals folding principles of the human genome[END_REF]).

The topologically associating domains (TADs), which can be considered primary units of chromosome folding, are defined as spatially coincident regions of chromatin, as detected by 3C-based techniques. 3D genome architecture is then thought to arise as an emergent property of the organisation of many such TADs.

CTCF associates with chromosomes both at arms and centromeres and directly interacts with the Scc3/SA subunit [START_REF] Rubio | CTCF physically links cohesin to chromatin[END_REF]. Most cohesin binding sites in lower organisms are found at regions of convergent transcription, but in many higher organisms possessing CTCF, cohesin-CTCF loops can occur when CTCF binding sites are convergent (Figure 1.13a, b), both in the topological domain borders and the loops within such domains [START_REF] Lengronne | Cohesin relocation from sites of chromosomal loading to places of convergent transcription[END_REF]; Phillips-Cremins and Corces 2013). While genome binding by CTCF is determined by the recognition of specific DNA sequences [START_REF] Hou | Cell type specificity of chromatin organization mediated by CTCF and cohesin[END_REF], the positions of cohesin-CTCF defined loops tend to vary according to cell type. Depletion of CTCF reduces the enrichment of cohesin to specific sites but does not disrupt the presence of cohesin on chromatin [START_REF] Hoque | Cohesin defects lead to premature sister chromatid separation, kinetochore dysfunction, and spindle-assembly checkpoint activation[END_REF][START_REF] Parelho | Cohesins functionally associate with CTCF on mammalian chromosome arms[END_REF].

In terms of evolution, CTCF has only been identified in bilateria but cohesin is conserved from yeast to vertebrates, and is required for genome organisation in Schizosaccharomyces Pombe [START_REF] Mizuguchi | Cohesin-dependent globules and heterochromatin shape 3D genome architecture in S. pombe[END_REF], for example. Cohesin does not exhibit sequence-specific DNA-binding but it does colocalize with CTCF, which presumably confers sequence specificity by proxy. [START_REF] Parelho | Cohesins functionally associate with CTCF on mammalian chromosome arms[END_REF][START_REF] Wendt | Cohesin mediates transcriptional insulation by CCCTC-binding factor[END_REF]). Hence, it is probable that CTCF serves to position cohesin at specific regions within the genome, directing cohesin to modulate higher order chromatin architecture.

Loop extrusion model and different levels of the mammalian genome folding

Currently, the most prevalent model for genome organisation by SMC-kleisins is that these enzymes exploit their ATPase activity to structure chromatin by a process of DNA loop extrusion [START_REF] Ganji | Real-time imaging of DNA loop extrusion by condensin[END_REF]. The loop extrusion model has been further studied in the condensin system demonstrating that this complex is able to enlarge DNA loops in vitro in an asymmetric and processive fashion [START_REF] Ganji | Real-time imaging of DNA loop extrusion by condensin[END_REF]). Notably, this process was found to be dependent on the SMC ATPase and on a DNA anchoring function facilitated by the Ycg1, a paralogue of Scc3, HEAT-repeat subunit of condensin. As these molecular features are well conserved, it is likely that the paralogous cohesin system, and perhaps indeed all SMCkleisins, operate by similar principles.

According to the loop extrusion model, cohesin spontaneously extrudes DNA with energy from ATP binding and hydrolysis by the Smc3 and Smc1 subunits [START_REF] Fudenberg | Formation of Chromosomal Domains by Loop Extrusion[END_REF][START_REF] Sanborn | Chromatin extrusion explains key features of loop and domain formation in wildtype and engineered genomes[END_REF]. Such loop-extruding cohesin complexes accumulate at inward facing CTCF motifs thus forming a loop domain between convergently oriented CTCF motif pairs.

Deletion of cohesin leads to disappearance of TADs [START_REF] Busslinger | Cohesin is positioned in mammalian genomes by transcription, CTCF and Wapl[END_REF][START_REF] Schwarzer | Two independent modes of chromatin organization revealed by cohesin removal[END_REF] but has no apparent effect on CTCF binding to chromatin [START_REF] Rao | Cohesin Loss Eliminates All Loop Domains[END_REF].

Conversely, loss of CTCF causes TADs to disappear but for different reasons: without CTCF, cohesin extrusion continues but is no longer restricted to convergent CTCF binding sites [START_REF] Wutz | Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins[END_REF]. Thus CTCF must function upstream of cohesin to influence its localisation throughout the genome.

During interphase, de-condensed chromatin is organised into nuclear territories with characteristic gene densities and transcriptional activity. Euchromatin tends to cluster towards the center of the nucleus whereas heterochromatin accumulates towards the inner nuclear membrane and is associated with the nuclear lamina via CTCF [START_REF] Cremer | Chromosome territories--a functional nuclear landscape[END_REF][START_REF] Reddy | Transcriptional repression mediated by repositioning of genes to the nuclear lamina[END_REF]. Such segmentation divides chromosomes into two distinct compartments which were first discovered in Hi-C studies: compartment A encompasses transcriptionally active regions, whereas compartment B contains inactive genes [START_REF] Bickmore | Genome architecture: domain organization of interphase chromosomes[END_REF][START_REF] Fortin | Reconstructing A/B compartments as revealed by Hi-C using long-range correlations in epigenetic data[END_REF][START_REF] Lieberman-Aiden | Comprehensive mapping of long-range interactions reveals folding principles of the human genome[END_REF]). These compartments can encompass TADs that are either on the same chromosome (cis) (Figure 1.13) or different chromosomes (trans) [START_REF] Simonis | Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C)[END_REF]. Although both cohesin and CTCF can direct TAD formation, neither cohesin nor CTCF alone affect compartmentalization [START_REF] Seitan | Cohesin-based chromatin interactions enable regulated gene expression within preexisting architectural compartments[END_REF][START_REF] Sofueva | Cohesin-mediated interactions organize chromosomal domain architecture[END_REF][START_REF] Zuin | Cohesin and CTCF differentially affect chromatin architecture and gene expression in human cells[END_REF]. It could only be disrupted by depletion of both CTCF and cohesin, which causes chromatin compaction [START_REF] Haarhuis | Cohesin: building loops, but not compartments[END_REF][START_REF] Tark-Dame | Depletion of the chromatin looping proteins CTCF and cohesin causes chromatin compaction: insight into chromatin folding by polymer modelling[END_REF]. When I began my Phd work, structural information of cohesin was available for the core subunits and some accessory factors. Several models have been proposed for how cohesin rings might hold sister chromatids together, yet the precise topology and mechanism of action remains unclear. Aside from the ring shaped particles that could be observed in EM images, virtually nothing is known about structural arrangement of the holo-cohesin complex or how cohesin coordinates its activity with its accessory factors throughout the cell cycle. Therefore, the aim of my thesis project was to assemble the holo-cohesin complex and to characterize it structurally and biochemically. It is known that cohesin exhibits a weak affinity for single and double stranded DNA yet at the same time does not show any sequence specificity [START_REF] Fernius | Establishment of cohesion at the pericentromere by the Ctf19 kinetochore subcomplex and the replication fork-associated factor, Csm3[END_REF][START_REF] Murayama | Establishment of DNA-DNA Interactions by the Cohesin Ring[END_REF][START_REF] Sakai | Condensin but not cohesin SMC heterodimer induces DNA reannealing through protein-protein assembly[END_REF]. As there is no structural information explaining how cohesin engages DNA or nucleosomes directly, I also set out to investigate how cohesin structurally associates with DNA. (IMAC Sepharose Fast Flow 6 resin (GE Healthcare), For GST-tagged proteins pellets were resuspended in buffer S (same as buffer 1 but without imidazole). For the expression and purification of Pds5fl, media and buffers were supplemented with 20 µM or 5 µM of inositol hexa-kis-phosphate (IP6), respectively [START_REF] Ouyang | Structural Basis and IP6 Requirement for Pds5-Dependent Cohesin Dynamics[END_REF]. CTCF constructs containing Zinc fingers were expressed in media supplemented with 50 µM ZnCl 2 . The purification buffers were supplemented with 5 µM ZnCl 2 .

Selenomethionine-labelled protein expression

Vectors containing Scc3T-Scc1K constructs were transformed into BL21(DE3), cultured in 2 ml LB until OD 600nm = 0.6. Cells were harvested and washed twice with M9 media, then transferred into M9 media supplemented with trace elements and vitamins as preculture (modified from [START_REF] Van Duyne | Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin[END_REF])), and grown overnight at 37 °C. Cells were then transferred into 1L cultures of M9 (supplemented with trace elements and vitamins), and grown at 37 °C until OD 600nm = 0.6, amino acids were added to induce feedback inhibition of methionine synthesis, then IPTG with final concentration of 0.5 mM and 2.5 mM selenomethionine were added for the expression of the seleno-labelled Scc3T-Scc1K.

Protein purification

Cells were lysed using a microfluidiser (Microfluidics) and centrifuged at 15000 rpm for 1h using a JA-20/14 rotor (Beckman). Lysis procedures were identical for all protein purifications.

Purification with His or His-GST tag

The supernatant was loaded on 5 ml Co 2+ -conjugated IMAC beads (GE healthcare) by using a peristaltic pump (GILSON). The column was washed with 10 column volumes of buffer 1 and the protein was eluted with buffer 2 (40 mM TRIS, pH 7.5, 300 mM NaCl, 300 mM imidazole, 0.5 mM TCEP). The His-GST tag was cleaved by addition of His-tagged TEV protease (1:100 w/w) during overnight dialysis against 40 mM TRIS, pH 7.5, 300 mM NaCl, 0.5 mM TCEP at 4°C. For Smc3-NScc1, Smc1-CScc1, and the Smc3-Smc1 hinge, this cleavage step was bypassed. The His-GST tag, protease and uncleaved protein were removed by passing this mixture over Co 2+ IMAC resin, and target proteins recovered from the flowthrough.

Purification with GST tag

Supernatant was loaded on to 5 ml Glutathione Sepharose 4 Fast Flow resin (GE Healthcare), washed with 10 column volumes of buffer S, and protein eluted with GST elution buffer (40 mM HEPES pH 7.5, 0.3 M NaCl, 10 mM -Glutathione and 0.5 mM TCEP). The GST tag was cleaved by His-tagged 3C protease (provided by Protein Expression and Purification Core Facility, EMBL-Heidelberg) during overnight dialysis against 40 mM TRIS, pH 7.5, 300 mM NaCl, 0.5 mM TCEP at 4°C. Protease and uncleaved protein were removed by passing this mixture over Glutathione Sepharose 4 Fast Flow resin.

Ion-exchange purification

The flow-through was concentrated using an Amicon Ultra -15 concentrator (Millipore) and applied onto a MonoQ 5/50 GL column (GE healthcare) in buffer 3 (150 mM NaCl, 40 mM TRIS, pH 7.5, 0.5 mM TCEP). Proteins were eluted using a linear gradient in buffer 4 (1 M NaCl, 40 mM TRIS, pH 7.5, 0.5 mM TCEP). Proteins with a tendency to precipitate (ctSmc1head, all Clink complexes) were treated differently by diluting them in 250 mM NaCl, 40 mM TRIS, pH 7.5, 0.5 mM TCEP prior to loading onto a MonoQ column.

Size exclusion

The final purification step was performed by using a HiLoad 16/60 Superdex 75/200 prepgrade column in buffer 5 (150 mM NaCl, 20 mM TRIS, pH 7.5, 0.5 mM TCEP). Exceptions were ctSmc1hd-CScc1 and all Clink complexes which were eluted with buffer containing 300 mM NaCl, 20 mM TRIS, pH7.5, 0.5 mM TCEP, and human SA2T-Scc1H with buffer containing 300 mM NaCl, 20 mM TRIS, pH7.7, 5 mM TCEP. Purified proteins were concentrated and flash frozen in liquid N2 for storage at -80 °C. The integrity and purity of protein samples were validated by SDS-PAGE.

Crystallisation

All initial crystallisation trials except for Scc3T-Scc1K-DNA complexes were performed by the EMBL Grenoble HTX facility with standard commercial screens. Scc3T-Scc1K-DNA samples were screened using a custom plate setup, optimized for protein-DNA complexes (Table 2.1). Table 2.1 Scc3T-Scc1K crystallisation screen conditions.

2.4.1 Crystals of ctSmc1hd-CScc1 complex were obtained at 4 °C by hanging-drop vapour diffusion, after mixing equal volumes of protein at 15 mg ml -1 and precipitant containing 0.1 M TRIS (pH8.5), 30% PEG300. During harvesting, crystals were transferred into well solution containing 15% Glycerol and flash frozen in liquid nitrogen.

Crystals of the Scc3-Scc1

-DNA complex were obtained by mixing 8 mg ml -1 protein with DNA at 1:1.1 ratio. 1µl of the protein: DNA complex was mixed with 1 µl 10% PEG 8000, 0.1 M Bis-TRIS, pH 6.5 crystallization buffer and equilibrated against the crystallization buffer at 4 °C by hanging-drop vapour diffusion. Initial crystals with a 17 bp DNA substrate were obtained after 5 days. These crystals were used as seeds for crystallization of Scc3-Scc1 bound to a 19 bp DNA fragment using the same crystallization condition.

Crystal seeding

For crystal seeding, 10-20 crystals containing the Scc3-Scc1 complex bound to 17bp DNA were washed and cracked into small pieces within the crystallization buffer in a volume of around 40 µl. Then 0.4 µl of the seeds were mixed with 0.8 µl Scc3-Scc1 with a DNA oligonucleotide of different length and 0.8 µl of crystallization solution followed by equilibration against crystallization buffer at 4 °C. protected by adding 15% glycerol to the crystallization buffer followed by flash freezing in liquid nitrogen.

Structure determination

Structure of the ctSmc1-CScc1 complex

Diffraction data of the ctSmc1-CScc1 crystals were recorded with a Pilatus3 2M detector at the European Synchrotron Radiation facility on beamline ID30A-1/MASSIF-1 [START_REF] Bowler | MASSIF-1: a beamline dedicated to the fully automatic characterization and data collection from crystals of biological macromolecules[END_REF]) using an X-ray wave-length 0.966 Å at 100 K. Location and optimal centering of crystals were determined automatically as described previously [START_REF] Svensson | Fully automatic characterization and data collection from crystals of biological macromolecules[END_REF]. The beam diameter was selected automatically to match the crystal volume of highest homogeneous quality [START_REF] Svensson | Multi-position data collection and dynamic beam sizing: recent improvements to the automatic data-collection algorithms on MASSIF-1[END_REF]. Data were processed with XDS [START_REF] Kabsch | Xds[END_REF] and imported into CCP4 format using AIMLESS [START_REF] Winn | Overview of the CCP4 suite and current developments[END_REF].

Phases were determined using the previously published ySmc1-CScc1 complex [START_REF] Haering | Structure and stability of cohesin's Smc1-kleisin interaction[END_REF]) as a search model for molecular replacement in Phaser [START_REF] Mccoy | Phaser crystallographic software[END_REF]).

Iterative manual model building and refinement in Coot and PHENIX, respectively, were pursued to derive a final model. The final CtSmc1-Scc1 model was refined to 2.09 Å resolution with an R work and an R free of 21% and 24%, respectively (Table 3.1). Analysis of the refined structure by MolProbity showed that there are no residues in disallowed regions of the Ramachandran plot. The MolProbity all atom clash score was 2.5.

Structure of the Scc3-Scc1-DNA complex

Diffraction data for all native and selenomethionine-derivatised (Scc3-Scc1-DNA) crystals were collected at 100 K at an X-ray wavelength of 0.966 Å at beamline ID30A-1/MASSIF-1 [START_REF] Bowler | MASSIF-1: a beamline dedicated to the fully automatic characterization and data collection from crystals of biological macromolecules[END_REF] of the European Synchrotron Radiation Facility, with a Pilatus3 2M detector using automatic protocols for the location and optimal centring of crystals [START_REF] Svensson | Fully automatic characterization and data collection from crystals of biological macromolecules[END_REF]. The beam diameter was selected automatically to match the crystal volume of highest homogeneous quality [START_REF] Svensson | Multi-position data collection and dynamic beam sizing: recent improvements to the automatic data-collection algorithms on MASSIF-1[END_REF]. Access to a fully automatic beamline was important as only a few crystals in a hundred would diffract sufficiently.

Diffraction from the best native crystal was anisotropic, with the best direction extending to a minimal Bragg spacing of ~3.6 Å and only ~5.7 Å in the worst direction. At this resolution we had to confirm the sequence register by crystallisation of Selenomethionine-derivativized protein in order to use the anomalous signal to assign the sequence register. This required further automatic screening on MASSIF-1 to collect the best data possible. In total, I estimate that over 800 crystals were required to produce the final model of the Scc3-Scc1-DNA complex. Data were processed with XDS [START_REF] Kabsch | Xds[END_REF]) and imported into CCP4 format using AIMLESS [START_REF] Winn | Overview of the CCP4 suite and current developments[END_REF]).

Structure of the SA2-Scc1-CTCF complex

Diffraction data for SA2-Scc1-CTCF crystals were collected at 100 K at an X-ray wavelength of 0.966 Å at beamline ID30A-1/MASSIF-1 [START_REF] Bowler | MASSIF-1: a beamline dedicated to the fully automatic characterization and data collection from crystals of biological macromolecules[END_REF] of the European Synchrotron Radiation Facility, with a Pilatus3 2M detector using automatic protocols for the location and optimal centering of crystals [START_REF] Svensson | Fully automatic characterization and data collection from crystals of biological macromolecules[END_REF]. The beam diameter was selected automatically to match the crystal volume of highest homogeneous quality [START_REF] Svensson | Multi-position data collection and dynamic beam sizing: recent improvements to the automatic data-collection algorithms on MASSIF-1[END_REF]. Data were processed with XDS [START_REF] Kabsch | Xds[END_REF]) and imported into CCP4 format using AIMLESS [START_REF] Winn | Overview of the CCP4 suite and current developments[END_REF].

The structure was determined by molecular replacement using Phaser. A final model was produced by iterative rounds of manual model building in Coot and refinement using PHENIX. The CTCF-containing model was refined to 2.6 Å resolution with an R work and an R free of 25% and 27%, respectively (Table 5.1). Analysis by MolProbity showed that there are no residues in disallowed regions of the Ramachandran plot and the all atom clash score was 7.2.

concentration of 1 mM in 20 mM TRIS, pH 7.5, 150 mM NaCl. Successful annealing and purity of the oligonucleotides was confirmed by native PAGE on a 6% gel.

For EMSA experiments, varying concentrations of protein were incubated at the indicated molar ratios with 1 μM of different lengths of DNA oligos or 0.35 µM nucleosome (provided by Amede Larabi) in 150 mM NaCl, 20 mM TRIS, pH 7.5, 0.5 mM TCEP. Samples were incubated on ice for 30 min. Glycerol was added to a final concentration of 5% and the samples were analysed on a pre-run 6% native 1x TRIS-Glycine (25 mM TRIS, 250 mM glycine, pH 8.3, 5% Glycerol) polyacrylamide gel using 1x TRIS-Glycine running buffer.

Gels were stained with SYBR Safe (Thermo Fisher Scientific) to visualize DNA-bound complexes or with Coomassie Blue to stain protein.

Fluorescence Polarization (FP)

32bp 6-FAM labelled DNA was prepared by annealing two complementary DNA strands, essentially as described for the crystallisation of DNA complexes, albeit under low-light conditions (Table 4.1). Fluoresence polarisation assays were conducted in a buffer containing 50 mM TRIS pH 7.5, 150 mM NaCl, 0.1% Tween20 and 0.5 mM TCEP. A series of protein concentrations, ranging from 0.5 to 25 µM, were incubated in the presence of 50 nM DNA for 30 minutes at room temperature in order to attain equilibrium.

Immediately thereafter, fluorescence polarization was recorded using 485 nm and 520 nm excitation and emission filters, respectively (CLARIOstar, BMG Labtech, Germany). The change in fluorescence polarization was then plotted as mean values of three independent replicates and the dissociation constant for each complex determined.

Isothermal Titration Calorimeter (ITC)

ITC was performed using a MicroCal iTC 200 (GE Healthcare) at 25 °C in 20 mM TRIS, pH7.7, 150 mM NaCl, 0.5 mM TCEP (buffer 6). For each titration, 300 l of 50 M SA2-Scc1 was added to the calorimeter cell. The synthetic CTCF and Sgo1 peptides, at a concentration of 500 M, dialysed into buffer 6, were injected as 18 x 2 lsyringe fractions.

Results were analysed and displayed using the Origin software package supplied with the instrument.

In vivo-Yeast methods

All yeast work was performed by the Haering Laboratory at EMBL Heidelberg.

Tetrad analysis

Wild-type or mutant alleles of SCC3 fused to a C-terminal PK 6 epitope tag were integrated into the ura3 locus of a SCC3/scc3::HIS3 heterozygous diploid yeast strain (C1073). Correct integration was confirmed by PCR (Table 4.4). Following sporulation, strains were tetrad dissected and cultured on YPAD media for 3 days at 30 °C before genotyping by replica plating.

Chromatin immunoprecipitation and ChIP-qPCR

Chromatin immunoprecipitation followed by quantitative PCR. ChIP-qPCR was performed from asynchronous yeast cell cultures as described (Cuylen and Haering 2011), except that sonication was performed with a Bioruptor Plus (Diagenode) at 4 °C using 6 cycles of 30 s on, 60 s off at "high" level. Quantitative PCR was performed with primers listed in Table 4.3.

Peptide array

Peptide arrays, with an area of 3cm 2 , were obtained from Rudolf Volkmer (immunologie.charite.de). For each step, 5 ml of the required reagents were used. Arrays were washed with 100% ethanol for 5 minutes on a shaker at 21 °C, followed by 3 washes, for a total of 10 minutes in TBS-T buffer (50 mM TRIS pH 7.5, 150 mM NaCl, 0.05% Tween-20). For the blocking step, arrays were incubated in 1x blocking buffer (Sigma B6429) for 3 h at 21 °C, followed by 3 washes in TBS-T for a total of 10 minutes. SA2-Scc1 and SA2 (F371A)-Scc1H were added to 1x blocking buffer at a final concentration of 1.

Expression of cohesin complex in E.coli

It has been proposed that cohesin contains both a DNA entry and exit gate and that these are located at the SMC hinge and Smc3-NScc1 interfaces, respectively. It has been argued that such physical separation of entry and exit gates allows for independent regulation of sister chromatid entrapment and their subsequent release [START_REF] Chan | Cohesin's DNA exit gate is distinct from its entrance gate and is regulated by acetylation[END_REF][START_REF] Gruber | Evidence that loading of cohesin onto chromosomes involves opening of its SMC hinge[END_REF].

Acetylation of the Smc3 head domain by Eco1 is an essential step to establish cohesion [START_REF] Ben-Shahar | Eco1-dependent cohesin acetylation during establishment of sister chromatid cohesion[END_REF][START_REF] Rowland | Building sister chromatid cohesion: smc3 acetylation counteracts an antiestablishment activity[END_REF][START_REF] Unal | A molecular determinant for the establishment of sister chromatid cohesion[END_REF][START_REF] Zhang | Acetylation of Smc3 by Eco1 is required for S phase sister chromatid cohesion in both human and yeast[END_REF]).

Hence, it is important to understand how cohesin heterodimerizes at its head domains and how the interactions at this interface are fine-tuned by regulatory factors (Eco1, releasing factors Wapl, Pds5) to enable controlled opening and closure of the exit gate. Assembling the Smc1/Smc3 head domain then is a prerequisite for elucidating these questions.

As previously reported, Smc3hd-NScc1, and Smc3hd-Pds5-NScc1 subcomplexes can be produced recombinantly in E.coli [START_REF] Gligoris | Closing the cohesin ring: structure and function of its Smc3-kleisin interface[END_REF]Muir, et al. 2016). We were able to co-express yeast Smc3-Scc1 in E. coli, but not the ySmc1hd-CScc1 complex. We also have previously found that yeast Smc1 cannot be solubly expressed in E.coli (unpublished data).

We therefore decided to perform expression trials of Smc1hd orthologues from different species in order to screen for soluble constructs suitable for structural characterization.

Expression trials of Smc1 head domain of different species in E.coli

First, I tested expression of Smc1 head constructs from three different species: A. gossypii

Structure of the Chaetomium thermophilum Smc1 head in complex with

Saccharomyces cerevisiae Scc1 (ctSmc1hd-CScc1)

The ctSmc1hd-CScc1 complex was set up for crystallization in the presence of ATP-γ-S (1 mM) and Mg 2+ (2 mM). Crystals were obtained using the method described in section 2.4.1.

Initial hits were further refined using manual setup of gradient concentration of the precipitant PEG300 ranging from 20% -35% and TRIS buffer, from pH 8.0 -pH 9.0. We determined the 2.1Å X-ray crystal structure of CtSmc1 bound to yeastCScc1 domain by molecular replacement, using the yeast ortholog [START_REF] Haering | Structure and stability of cohesin's Smc1-kleisin interaction[END_REF]) and found, as expected, high structural conservation (RMSD=0.98 Å 2 ) (Figure 3.2c, Table 3.1). The structure showed more residues of the coiled-coil arm compared to the previously determined ySmc1-Scc1 complex (Figure 3.2c). Interestingly, in contrast to the yeast Smc1-Scc1 structure, we could not find any ATP-γ-S, Mg 2+ bound to the ctSmc1 head domain. Previously, we have attempted to generate engineered head modules for structural studies by fusing the Scc1 C-terminus to Smc3, which would serve as bait to capture Smc1. Ultimately this was unsuccessful (Muir 2016). To obtain a heterotrimeric Smc3-Smc1-Scc1 ATPase

ctSmc1-CScc1 Data collection

Complex assembly trials of Clink159 in complex with Pds5 and Scc3

To incorporate additional cohesin regulators into the complex, I designed a longer NScc1 fusion which extended to the Pds5 binding region (1-159) (Muir, et al. 2016) 4.1).

Crystallization trials of the Clink complex.

Crystallization at both 4°C and 20 °C, crystallisation with different lengths of DNA oligonucleotides, with/without ATP-γ-S, in situ digestion etc was attempted, but no crystals were obtained, except a single non-reproducible crystal which grew following in situ chymotryptic digestion (1:10000 w/w) (Figure 3.3i).

Result_part2 Structural basis for Scc3-dependent cohesin recruitment to chromatin

Pour comprendre comment la cohésine engage l'ADN, j'ai étudié les propriétés de liaison à l'ADN de sous-complexes globulaires précédemment identifiés. En déterminant une structure cristalline de la protéine Scc3 de levure liée à un fragment de la sous-unité Scc1 kleisin et de

Investigating DNA binding by the cohesin complex

To investigate the DNA-binding properties of cohesin, I co-expressed and purified defined globular domains and subcomplexes of the Saccharomyces cerevisiae cohesin ( derivative and refined a model encompassing amino acid residues 134 to 1064 of Scc3 in complex with residues 309 to 400 of Scc1, bound to a 19 bp DNA molecule (Table 4.2).

Scc3T

Table 4.2 Data collection and refinement statistics

As seen in previous structures of Scc3, the protein is hook-shaped in the C-terminal section and contains an N-terminal 'nose' formed by a pair of extended antiparallel α-helices (Figure 4.4a). Similarly to the interaction of human Scc1 with Scc3, the yeast Scc1 in our structure binds along the convex surface of the hook-shaped HEAT-repeat subunit. We detected additional electron density corresponding to dsDNA within the cradle of this hook (Figure 4.5a, b). Whereas the DNA duplexes were aligned to form pseudocontinuous double helices throughout the crystal, the DNA duplex was slightly too short for tight end-to-end stacking (Figure 4.5c). As a result, the DNA density was only partially resolved, apparently due to rotational and translational disorder of the DNA in the binding cavity. To identify amino acid residues potentially involved in DNA binding, we mapped the electrostatic surface potential onto the Scc3-Scc1 structure (Figure 4.4b). This revealed that DNA is nested within an extended cradle spanning the majority of Scc3-Scc1, lined by a set of positively charged residues that directly contact the DNA phosphate backbone. The DNA is aligned almost parallel to the N-terminal 'nose' of Scc3, which interacts through a set of positively charged amino acid residues with the DNA of a neighbouring complex related by crystallographic symmetry (Figure 4.5c). We observed no direct nucleotide base-amino acid interactions, which explains the apparent lack of DNA sequence specificity.

To ascertain the amino-acid register of Scc1, we used an anomalous difference map peak for M373 in the selenomethionine-derivative data ( Mapping of sequence conservation onto the structure revealed that amino acid residues in the DNA binding groove are generally well conserved among yeast Scc3 orthologs (Figure 4.6c).

In particular, amino acid residues that are located proximal to the DNA phosphate backbone showed strong conservation. To further evaluate the contributions made by individual segments of the DNA-binding surface, we subdivided participating residues into a series of three patches, based on their physical proximity to the DNA (Figure 4.8b), and subjected these patches to site-directed mutagenesis.

We measured the DNA equilibrium dissociation constant by fluorescence polarization (FP), using a 32-bp 6-FAM labelled DNA substrate (Table 4.1), which is sufficient to bridge the entire DNA binding surface present in the Scc3-Scc1 crystal structure (Figure 4.8c). Whereas the wild-type Scc3T-Scc1K complex bound this substrate with an equilibrium dissociation constant of 2.2 μM, charge-inversion mutations in patches 1 and 3, located at the crest of and within the Scc3 cradle, had only modest effects on affinity (equilibrium dissociation constants of 9.5 μM and 7.3 μM, respectively). Patch 2 mutants, residing in the Scc3 'nose', exhibited essentially unaltered DNA binding affinity. In contrast, the simultaneous mutation of all three patches (a heptamutant) reduced the binding affinity of the patch 1 or 3 mutants even further (to 29.3 μM, Figure 4.8c). The defect in binding DNA was not due to any impact of the mutations on the structural integrity of Scc3-Scc1, as all mutant complexes eluted indistinguishably from wild-type Scc3-Scc1 during size-exclusion chromatography (Figure 4.6a) and efficiently formed a complex with Scc1 in vitro (

In vivo characterization of the Scc3-Scc1-DNA structure

We then assayed whether DNA binding by Scc3 is important for cohesin function in vivo. We collaborated with Christian H Haering from EMBL Heidelberg. Wild-type or mutant versions of Scc3 were integrated into a diploid yeast strain in which one of the two endogenous SCC3 genes had been deleted (Table 4.4). Using tetrad dissection, these newly generated yeast strains were then tested for their ability to compensate the loss of SCC3 with the integrated mutant or wild type variant ( Targeting of cohesin to the genome is essential for numerous aspects of chromosome biology, including sister chromatid cohesion, DNA damage repair, and transcriptional regulation [START_REF] Uhlmann | SMC complexes: from DNA to chromosomes[END_REF]. In this study, I identified a direct DNA-binding site in the Scc3 subunit of the cohesin complex and determined its interaction with DNA at near-atomic resolution. These findings provide evidence for a site of direct DNA contact in cohesin complexes, which presumably contributes to the initial step of chromosome entrapment and/or DNA translocation (Figure 4.11c).

We were able to determine the DNA-bound structure of Scc3 in complex with a minimal fragment of Scc1 and demonstrated that DNA binding depends on conserved positively charged residues within a composite Scc3-Scc1 interface. We used this structure to derive DNA-binding deficient Scc3 variants, which fail to support cohesin recruitment to chromatin and consequently cell division. In addition to providing a scaffold for the assembly of cohesin regulators [START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF][START_REF] Murayama | Biochemical reconstitution of topological DNA binding by the cohesin ring[END_REF][START_REF] Roig | Structure and function of cohesin's Scc3/SA regulatory subunit[END_REF]) and thereby participating in the maintenance of cohesion, the Scc3-Scc1 subcomplex also plays a key role in DNA substrate recognition, and hence the efficient association of the cohesin holocomplex with chromatin.

The cohesin ring has been proposed to topologically embrace chromosomal DNA, which requires that DNA be loaded into the ring complex during cohesin's catalytic cycle [START_REF] Nasmyth | Cohesin: a catenase with separate entry and exit gates?[END_REF]. As mutations in Scc3 that prevent DNA engagement by the Scc3-Scc1 subcomplex in vitro fail to stably bind to chromatin in vivo, it is likely that direct DNA-cohesin interactions contribute to such DNA capture (Figure 4.11c).

We propose that the Scc3-Scc1 subcomplex provides a dynamic DNA anchoring point that is required for the efficient loading and/or maintenance of cohesin on chromatin (Figure 4.11).

Such a model for Scc3 is supported by previous data, which indicate that Scc3 contributes to cohesin loading [START_REF] Hu | ATP hydrolysis is required for relocating cohesin from sites occupied by its Scc2/4 loading complex[END_REF][START_REF] Murayama | Biochemical reconstitution of topological DNA binding by the cohesin ring[END_REF][START_REF] Orgil | A conserved domain in the scc3 subunit of cohesin mediates the interaction with both mcd1 and the cohesin loader complex[END_REF][START_REF] Roig | Structure and function of cohesin's Scc3/SA regulatory subunit[END_REF]. In agreement with this model, Scc1 deletion mutants that lack the sequence responsible for binding Scc3 fail to load onto yeast chromosomes [START_REF] Hu | ATP hydrolysis is required for relocating cohesin from sites occupied by its Scc2/4 loading complex[END_REF].

Furthermore, Scc3 enhances topological DNA entrapment by cohesin in in vitro loading assays [START_REF] Murayama | Biochemical reconstitution of topological DNA binding by the cohesin ring[END_REF]. Thus, DNA binding by the Scc3-Scc1 subcomplex might be the first step in moving chromosomal DNA into the cohesin ring. The subsequent entrapment reaction is then presumably catalysed by the Scc2-Scc4 complex [START_REF] Murayama | Biochemical reconstitution of topological DNA binding by the cohesin ring[END_REF][START_REF] Stigler | Single-Molecule Imaging Reveals a Collapsed Conformational State for DNA-Bound Cohesin[END_REF]. Surface patch 3 mutations in Scc3, which partially ablate DNA binding by the Scc3-Scc1

subcomplex and reduce cohesin levels on chromatin, do not exhibit any obvious growth defects and are therefore presumably competent to establish sister chromatid cohesion.

Indeed, partial depletion of cohesin does not seem to impact some of its core functions, including sister-chromatid cohesion and chromosome segregation [START_REF] Elbatsh | Cohesin Releases DNA through Asymmetric ATPase-Driven Ring Opening[END_REF][START_REF] Heidinger-Pauli | Systematic reduction of cohesin differentially affects chromosome segregation, condensation, and DNA repair[END_REF]. In contrast, even mutations that only slightly impair DNA binding of the equivalent HEAT-repeat / kleisin module of the condensin complex are sufficient to abolish stable chromatin association and to interfere with cell division [START_REF] Kschonsak | Structural Basis for a Safety-Belt Mechanism That Anchors Condensin to Chromosomes[END_REF]. Such discrepancies might be due to alternate loading and/or maintenance mechanisms of cohesin and condensin. Whilst cohesin is loaded by the Scc2-Scc4 complex, no such independent loading factor has been identified for condensin thus far, which could explain why the latter depends more strongly on the direct DNA binding site formed by its HEAT-repeat and kleisin subunits.

Binding of condensin to DNA is further stabilized by the entrapment of the bound DNA helix within a kleisin peptide loop (Figure 4.11a, b) [START_REF] Kschonsak | Structural Basis for a Safety-Belt Mechanism That Anchors Condensin to Chromosomes[END_REF]. The relevant section of Scc1 that would contribute to such topological DNA entrapment is not present in our structure. As Scc1 is clearly required for DNA binding of the Scc3-Scc1 subcomplex, it is possible that cohesin uses a similar mode of chromatin engagement. These findings thus point towards a conserved molecular mechanism that enables chromatin substrate engagement by condensin and cohesin. This mechanism potentially facilitates topological loading, chromatin looping and tracking along chromatin fibres by these SMC complexes [START_REF] Ganji | Real-time imaging of DNA loop extrusion by condensin[END_REF]. Les résultats d'un "peptide array" contenant un motif de la cohésine impliquent potentiellement le site de liaison SA2-Scc1 plus largement dans la régulation des gènes, la cohésion centromérique, la formation d'éléments latéraux méiotiques, ainsi que la libération de la cohésine.

Result_part 3 Principles of cohesin regulation on chromatin via its SA2-Scc1 subunits

Motivation to work on human SA2

Following the yeast Scc3-Scc1-DNA structure, I wanted to understand how SA2, a human ortholog of yeast Scc3, functions in the regulation of chromosomal cohesin. I therefore coexpressed and purified the SA2-Scc1 complex from E.coli (Figure 5.2). The codon optimized gene corresponding to the SA2 construct (residues 80-1060, named 'SA2T'), originally from Hongtao Yu's lab [START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF], was inserted into the pGEX-6p vector. An Scc1 construct spanning amino acids 281-420 was inserted into the pACYC-Duet vector (Figure 5.2a, c). The two expression plasmids were co-transformed into E.coli and the SA2-Scc1 complex was co-expressed and co-purified via GST affinity purification, anion exchange chromatography and a final size-exclusion chromatography step. The SA2T-Scc1-281 complex showed considerable degradation of Scc1 (Figure 5.2, c). A shorter construct of Scc1 (317-400) which lacked the disordered region of Scc1 as predicted from the published SA2-Scc1 structure (PDB: 4PJU) (Figure 5.2a, Table S1) was designed and co-expressed

Crystallization trials of the SA2-Scc1-CTCF ternary complex

In order to obtain the structure of the SA2-Scc1-CTCF complex, I first tried to co-express SA2T-Scc1-317 with a CTCF fragment spanning 1-267 (CTCF-N) that covers the binding region 227-235 of CTCF (Figure 5.5a). The complex of CTCF-N and SA2T-Scc1-317 dissociated during ion exchange chromatography (Figure 5.5a, left gel). To restore the ternary complex, fractions containing CTCF-N and SA2T-Scc1-317 were mixed again and diluted to lower salt concentration (150 mM NaCl), followed by size exclusion chromatography (Figure 5.5a, right gel). This showed that CTCF and SA2-Scc1 assembled into a ternary complex. As complex assembly was dependent on the ionic strength suggested a possible charge-based interaction between these proteins.

In another attempt to produce the ternary complex, CTCF (CTCF-N2, amino acids 86-267) and SA2T-Scc1-317 were purified separately and then mixed at a 3:1 ratio (Figure 5.5b) prior to size exclusion chromatography. The ternary complex was subjected to crystallization (by the HTX lab EMBL Grenoble), either directly or in the presence of small amounts of trypsin or chymotrypsin for in situ digestion (ratio protease: complex -1 : 5000). However, no crystals were obtained, irrespective of the conditions. Isolated CTCF-N2 digested in situ with trypsin digestion yielded crystals, but they were not investigated further.

SA2-Scc1-CTCF ternary complex is sensitive to DNA binding

In order to investigate if the ternary complex consisting of SA2, Scc1 and CTCF interacts with DNA similar to CTCF alone, SA2T-Scc1H-CTCF-ZF7 was incubated with a 62 bp DNA oligonucleotide containing a consensus CTCF recognition site(labeled in bold) (Table 4.1, Figure 5.5) and the resulting complexes analyzed via EMSA. The CTCF-ZF7 construct covers both the SA2-Scc1 binding region and zinc fingers through to zinc finger 7. The results showed that both CTCF alone as well as the ternary complex are able to interact with DNA (Figure 5.5c). I also observed that CTCF partially dissociated from the ternary complex when bound to its DNA substrate (Figure 5.5c). (5'-ATTG CAGT GCCC ACAG AGGC CAGC AGGG GGCG TAGT GAGG CCTT TTCA AGGA AACG AAAG TG-3') (Table 4.1) was used for an EMSA. A CTCF construct covering both the SA2-Scc1 interaction region and zinc fingers 1-7 (CTCF amino acids 218-460) was able to shift DNA (left). In the presence of SA2T-Scc1H, I observed a supershifted complex presumably corresponding the the SA2T-Scc1H-CTCF-ZF7 complex. Thus, while SA2T-Scc1H does not bind DNA strongly on its own, in contrast to yeast Scc3-Scc1, it does bind CTCF-DNA complexes. (d)

The CTCF peptide is bound to the convex surface of SA2, in close proximity to the Nterminal 'nose' which is formed by a pair of extended antiparallel α9-10 helices ( mapping studies using isolated SA2 are likely to have been misleading [START_REF] Xiao | Specific sites in the C terminus of CTCF interact with the SA2 subunit of the cohesin complex and are required for cohesin-dependent insulation activity[END_REF].

Mutagenesis analysis of the SA2-Scc1-CTCF ternary complex

As expected, mutagenesis of Y226A or F228A in CTCF abolished SA2-Scc1 binding in a GST pulldown assay (Figure 5.7a). Likewise, mutation of critical amino acid residues in SA2, including W334A, F371A and F367A or in Scc1 I337A/L341A, abolished CTCF binding. SA2 contains a 86 amino acid motif, the so-called 'conserved essential surface' (CES) [START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF][START_REF] Roig | Structure and function of cohesin's Scc3/SA regulatory subunit[END_REF], which is conserved from fungi to mammals. For simplicity, henceforth we will refer to the SA2-Scc1 binding pocket as 'CES' collectively.

Correspondingly, mapping sequence conservation onto the structure shows that the CTCF binding site is highly conserved (Figure 5.8) A series of missense mutations are found in SA2, Scc1 and CTCF in various cancer tissues (cancer.sanger.ac.uk; cosmic; [START_REF] Forbes | The Catalogue of Somatic Mutations in Cancer (COSMIC)[END_REF]). Mapping of mutation frequencies onto the structure shows that R370 of SA2, L341 of Scc1 and the key anchoring residue Y226 of CTCF, which are all largely buried upon complex formation, are hotspots in cancer (Figure 5.8c) indicating that this interface is critical for normal cell physiology and that its disruption contributes to pathogenesis. Table 5.1 X-ray data collection and refinement of SA2-Scc1-CTCF and SA2-Scc1-Sgo1 complex.

Bioinformatic prediction of cohesin binding factors

To investigate the prevalence of this linear interaction motif, we compiled an alignment from known cohesin partners (Figure 5.10) and derived a regular expression motif which we used to query the human and budding yeast proteomes [START_REF] Krystkowiak | SLiMSearch: a framework for proteome-wide discovery and annotation of functional modules in intrinsically disordered regions[END_REF]. From the set of nuclear proteins arising from this search, we were able to identify known cohesin regulators as well as several novel potential binding factors. We generated peptide arrays containing these motifs and probed these with either wild-type SA2-Scc1 or a SA2 (F371A)-Scc1 mutant complex as a negative control (Figure 5.13). We observed robust signal for the CTCF peptide spanning 222-231. A CTCF Y226F mutant showed reduced binding indicating that the hydrogen bond between the hydroxyl group of Y226 and D326 of SA2 is required for higher affinity binding. Consistent with our pulldowns, the CTCF Y226A, F228A, and Y226A/F228A mutants all abolished binding to SA2-Scc1. The Wapl FGF motifs showed considerably weaker binding, while we could not detect binding for low binding affinity Our structural and previously published data [START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF] support the notion that a number of cohesin ligands directly interact through a well-defined Y/FxF core motif with a conserved surface on the SA2-Scc1 complex. As the motif is present in a number of different nuclear proteins, binding through SA2-Scc1 is potentially a general mechanism for cohesin recruitment and regulation (Figure 5.14). Furthermore, since Scc3 and its orthologs, i.e. SA1, SA2 and SA3, are very well-conserved but function in different aspects of cohesin biology, e.g. either at chromosome arms or telomeres, or during meiosis, it will be interesting to further investigate how the 'Y/FxF' motif contributes to cohesin function either temporally or spatially through cell cycle.

Cohesin localisation to convergently oriented CTCF binding sites is a central event in TAD

formation. An equilibrium between loop extrusion and release by cohesin at these sites gives rise to dynamic, tunable genome architecture. In this study, we show that CTCF binds cohesin via a short amino acid sequence in its N-terminus. Direct competition for this binding site by the Wapl likely explains how this cohesin release factor restricts TAD formation [START_REF] Busslinger | Cohesin is positioned in mammalian genomes by transcription, CTCF and Wapl[END_REF][START_REF] Haarhuis | Cohesin: building loops, but not compartments[END_REF][START_REF] Wutz | Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins[END_REF]. Weakly binding motifs such as Shugoshin are tuned by addition of complementary negative phosphorylation charges around the motif core (Figure 5.12) [START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF]. Wapl Y/FxF motifs also contain conserved phosphorylation sites C-terminal to the Y/FxF motifs, suggesting that phosphorylation may be an important aspect in the regulation of competitive SA2-Scc1 ligand displacement.

The cohesin binding motif of CTCF is connected by a ~33 amino acid linker to the first of the 11 DNA binding zinc fingers suggesting that the interaction directly enables DNA recruitment of cohesin (Figure 5.9). In this thesis, I have shown that the concave side comprising the 'nose' of SA2 forms a non-specific DNA binding site in the budding yeast ortholog Scc3 [START_REF] Li | Structural basis for Scc3-dependent cohesin recruitment to chromatin[END_REF]. We therefore propose that CTCF interacts with cohesin through the SA2-Scc1 interface to facilitate loop extrusion at CTCF binding sites in the genome. A model for how CTCF and SA2-Scc1 might co-associate on DNA is shown in Figure 5.9. The attachment of cohesin to the N-terminus of CTCF and the capacity of ATPase driven asymmetric DNA loop extrusion by SMC complexes, to 'reel in' DNA from one side [START_REF] Ganji | Real-time imaging of DNA loop extrusion by condensin[END_REF], could explain how TADs are formed and why CTCF sites on the genome are oriented convergently [START_REF] Fudenberg | Formation of Chromosomal Domains by Loop Extrusion[END_REF]. My work hence reveals a mechanism by which CTCF and cohesin interact, and it will be critical to assess what the effect is of disrupting this interaction in the cell. I am grateful to all members of the Panne lab, Kyle Mooo, Plauuu, Amede, Esther, Ziad and Silvia, thanks for all the support and discussion. And as well as Mathilde, Petra, Audrey, Sylvie, thanks for all the kind help.

Scc1-317

Result_part 1

 1 Assembling the cohesin complex in E.Coli ('Clink') ....................................... Résultats_partie 1 ..................................................................................................................... 3.1 Expression of cohesin complex in E.coli ....................................................................... 3.2 Expression trials of Smc1 head domain of different species in E.coli ...........................

Figure 3 . 1

 31 Figure 3.1 Smc1 head boundaries and purification. ......................................................... 3.3 Structure of the Chaetomium thermophilum Smc1 head in complex with Saccharomyces cerevisiae Scc1 (ctSmc1hd-CScc1) ............................................................

Figure 3 . 2

 32 Figure 3.2 Structure of the ctSmc1 head domain in complex with yeast CScc1. ............. Table 3.1 X ray data collection, phasing and refinement statistics .................................. 3.4 ctSmc1/Smc3 head assembly via NScc1 linker -"Clink" ..............................................

Figure 3 . 3

 33 Figure 3.3 Purification and DNA binding of Clink115/120/125. .....................................

Figure 3 . 4

 34 Figure 3.4 Purification and EMSA of Clink159 in complex with either Pds5, Scc3, or both. .................................................................................................................................. 3.6 Crystallization trials of the Clink complex. ...................................................................

Figure 4 .

 4 Figure 4.1 SDS-PAGE analysis of the purified cohesin components and DNA binding analysis by EMSA. ...........................................................................................................

Figure 4 . 2

 42 Figure 4.2 Cohesin subunits that lack DNA binding affinity. ..........................................

Figure 4 .

 4 Figure 4.3 DNA binding by the Scc3-Scc1 subcomplex. ................................................. Table 4.1 DNA oligos used for crystallization, EMSA and Fluorescence Polarization (FP). ..................................................................................................................................

Figure 4 . 4

 44 Figure 4.4 Structure of the Scc3-Scc1 subcomplex bound to DNA. ................................

Figure 4 . 5

 45 Figure 4.5 Electron density for the DNA molecule bound to the Scc3-Scc1 subcomplex. ..........................................................................................................................................

Figure 4 . 6

 46 Figure 4.6 Biochemical analysis of Scc3-Scc1 subcomplexes and Scc3 conservation alignment. .......................................................................................................................

Figure 4 .

 4 Figure 4.7 DNA binding affinity test of both Scc3T-Scc1K wild type and mutants via EMSA. ............................................................................................................................ 4.3 In vivo characterization of the Scc3-Scc1-DNA structure ...........................................

Figure 4 .

 4 Figure 4.8 A conserved DNA binding domain in the Scc3-Scc1 subcomplex is required for cohesin association with chromatin. .........................................................................

Figure 4 . 9

 49 Figure 4.9 In vivo analysis of SCC3 mutants. ................................................................ Table 4.3 ChIP-qPCR primer sequences (5'→3') .......................................................... Table 4.4 Yeast genotypes. .............................................................................................

Figure 4 .

 4 Figure 4.10 Purification of selenomethionine-labeled Scc3T-Scc1K and different Scc3-Scc1 truncations used for crystallization and FP trials. .................................................. 4.4 Screening for possible protein interactions among cohesin subcomplexes .................

Figure 4 .

 4 Figure 4.11 Screening for possible protein interactions among cohesin subcomplexes. ........................................................................................................................................

Figure 4 .

 4 Figure 4.12 A conserved DNA binding interface in cohesin and condensin. ................ Result_part 3 Principles of cohesin regulation on chromatin via its SA2-Scc1 subunits ...... Résultats_partie 3 ................................................................................................................... 5.1 Motivation to work on human SA2 ..............................................................................

Figure 5 . 1

 51 Figure 5.1 Yeast Pds5 and Scc3 do not bind human CTCF ...........................................

Figure 5 . 2

 52 Figure 5.2 SA2-Scc1 constructs, purification and EMSA analysis. ...............................

Figure 5 . 3

 53 Figure 5.3 Biochemical analysis of CTCF binding to SA2-Scc1. .................................. 5.3 Crystallization trials of the SA2-Scc1-CTCF ternary complex ...................................

Figure 5 . 4

 54 Figure 5.4 Purification of SA2-Scc1H mutants, CTCF-N6 mutants and CTCF covering both SA2-Scc1 binding region and zinc finger 1-7 (CTCF-ZF7) .................................. 5.4 SA2-Scc1-CTCF ternary complex is sensitive to DNA binding..................................

Figure 5 .

 5 Figure 5.5 Co-expression and co-purification of the SA2T-Scc1H-CTCF complex and DNA binding assay.........................................................................................................

Figure 5 . 6

 56 Figure 5.6 Structure of hSA2-Scc1 bound to a fragment of CTCF. ...............................

Figure 5 . 7

 57 Figure 5.7 Mutagenesis and in vitro analysis of the SA2-Scc1-CTCF ternary complex. ........................................................................................................................................

Figure 5 . 8

 58 Figure 5.8 Analysis of the SA2-Scc1-CTCF structure. ..................................................

Figure 5 . 9

 59 Figure 5.9 Model for CTCF-mediated cohesin binding and for the generation of TADs by convergently oriented CTCF sites. ............................................................................

Figure 5 .

 5 Figure 5.10 Y/FxF motifs are conserved among CTCF, Wapl, Sororin and Shugoshin. ........................................................................................................................................

Figure 5 .

 5 Figure 5.11 Sgo1 interacts with SA2-Scc1.....................................................................

Figure 5 .

 5 Figure 5.12 Structure of the SA2-Scc1 and Sgo1 ternary complex. .............................. Table 5.1 X-ray data collection and refinement of SA2-Scc1-CTCF and SA2-Scc1-Sgo1 complex. ......................................................................................................................... 5.8 Bioinformatic prediction of cohesin binding factors....................................................

Figure 5 .

 5 Figure 5.13 Novel SA2-Scc1 binding partners screened via peptide array. ...................

Figure 5 .

 5 Figure 5.14 Model of SA2-Scc1 subcomplex bound with different Y/FxF-containing proteins ........................................................................................................................... Discussion_part 3 ................................................................................................................... Discussion_partie 3 ................................................................................................................

Figure

  Figure 3.3 Purification and DNA binding of Clink115/120/125 Figure 3.4 Purification and EMSA of Clink159 in complex with either Pds5, Scc3, or both Figure 4.1 SDS-PAGE analysis of the purified cohesin components and DNA binding analysis by EMSA Figure 4.2 Cohesin subunits that lack DNA binding affinity Figure 4.3 DNA binding by the Scc3-Scc1 subcomplex

Figure 4

 4 Figure 4.4 Structure of the Scc3-Scc1 subcomplex bound to DNA Figure 4.5 Electron density for the DNA molecule bound to the Scc3-Scc1 subcomplex Figure 4.6 Biochemical analysis of Scc3-Scc1 subcomplexes and Scc3

Figure 4 .FigureFigure

 4 Figure 4.10 Purification of selenomethionine-labeled Scc3T-Scc1K and different Scc3-Scc1 truncations used for crystallization and FP trials Figure 4.11 Screening for possible protein interactions among cohesin subcomplexes Figure 4.12 A conserved DNA binding interface in cohesin and condensin Figure 5.1 Yeast Pds5 and Scc3 do not bind human CTCF Figure 5.2 SA2-Scc1 constructs, purification and EMSA analysis Figure 5.3 Biochemical analysis of CTCF binding to SA2-Scc1 Figure 5.4 Purification of SA2-Scc1H mutants, CTCF-N6 mutants and CTCF covering both SA2-Scc1 binding region and zinc finger 1-7 (CTCF-ZF7) Figure 5.5 Co-expression and co-purification of the SA2T-Scc1H-CTCF complex and DNA binding assay Figure 5.6 Structure of hSA2-Scc1 bound to a fragment of CTCF Figure 5.7 Mutagenesis and in vitro analysis of the SA2-Scc1-CTCF ternary complex Figure 5.8 Analysis of the SA2-Scc1-CTCF structure Figure 5.9 Model for CTCF-mediated cohesin binding and for the generation of TADs by convergently oriented CTCF sites Figure 5.10 Y/FxF motifs are conserved among CTCF, Wapl, Sororin and Shugoshin Figure 5.11 Sgo1 interacts with SA2-Scc1 Figure 5.12 Structure of the SA2-Scc1 and Sgo1 ternary complex Table 5.1 X-ray data collection and refinement of SA2-Scc1-CTCF and SA2-Scc1-Sgo1 complex Figure 5.13 Novel SA2-Scc1 binding partners screened via peptide array Figure 5.14 Model of SA2-Scc1 subcomplex bound with different Y/FxF-containing

  notamment la cohésion des chromatides soeurs, la réparation des dommages à l'ADN, la régulation de la transcription et le contrôle de l'architecture tridimensionnelle de la chromatine. Les sous-unités de base de la cohésine, Smc1, Smc3, Scc1 assemblent un complexe en forme d'anneau via la connexion des domaines «hinge» hétérodimériques fournis par Smc1 et Smc3, et par la liaison des domaines SMC ATPase par Scc1. D'autres facteurs accessoires jouent un rôle important dans différents aspects de la fonction de la cohésine, tels que Scc3, 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. Pour établir la cohésion, une acétyltransférase appelée Eco1 acétyle le domaine ATPase de Smc3, qui peut recruter la sororine pour stabiliser la cohésion en faisant concurrence à Wapl et en formant un complexe de stabilisation avec Pds5. Chez les métazoaires, la cohésine est libérée des chromosomes en deux étapes principales. Une étape implique la libération de la cohésine au niveau des bras chromosomiques déclenchée par la phosphorylation de la cohésine et la seconde étape comprend le clivage de la cohésine centromérique par la séparase. Au-delà de la cohésion, la cohésine est également impliquée dans un certain nombre de fonctions biologiques supplémentaires, y compris, en coopérant avec la CTCF, l'organisation de l'architecture tridimensionnelle du génome mammifère.Pour étudier le module ATPase de la cohésine, j'ai généré une série de constructions pour expression dans E. coli. Comme seul Smc1 était difficile à exprimer, j'ai criblé une série de constructions de Smc1 de différentes espèces. J'ai pu produire la tête de Smc1 de Chaetomium Thermophilum (ctSmc1) et obtenir une structure cristallographique en complexe avec le fragment de Scc1 de levure (CScc1) à une résolution de 2,1 Å. Cela m'a permis d'exprimer et de co-purifier un hétérotrimère de la tête de cohésine assemblée Smc1-Smc3-Scc1 en fusionnant NScc1 à l'extrémité C-terminal de CtSmc1 en même temps que les autres composants du module de la tête.Pour comprendre comment la cohésine engage l'ADN, j'ai déterminé une structure cristallographique du sous-complexe Scc3-Scc1 lié à l'ADN double brin, montrant que le sous-complexe Scc3-Scc1 engage l'ADN double brin à travers une surface conservée chargée positivement. Nous avons trouvé que cette surface est essentielle pour l'enrichissement de complexes de la cohésine sur les chromosomes.Pour étudier les bases moléculaires de la collaboration entre la cohésine et la CTCF dans la définition de l'architecture tridimensionnelle du génome, j'ai identifié la région critique du CTCF nécessaire à la liaison au sous-complexe SA2-Scc1. J'ai pu déterminer la structure du complexe ternaire par cristallographie aux rayons X. La structure a révélé le mécanisme moléculaire de l'interaction CTCF-cohésine. De manière frappante, un motif similaire est présent dans Wapl, le facteur de libération de la cohésine, qui se lie à la même surface sur le complexe SA2-Scc1, ainsi que dans Shugoshin et dans un certain nombre de établis et nouveaux clients de la cohésine. J'ai pu déterminer une structure cristallographique du motif pertinent de Shugoshin dans un complexe avec SA2-Scc1, ce qui a confirmé qu'il se lie à la même surface conservée sur SA2-Scc1. Comme la liaison est mutuellement exclusive, la compétition pour la surface conservée pourrait être un aspect requis pour la fonction de la cohésine dans différents contextes biologiques. Mes résultats fournissent des informations fondamentales sur le mécanisme moléculaire qui permet la régulation de la fonction de la cohésine et le repliement dynamique de la chromatine.

Figure 1 . 1

 11 Figure 1.1 Genome cohesion and condensation during the cell cycle. Cohesin is loaded onto

Figure 1 . 2

 12 Figure 1.2 Structure of the cohesin core subunits and domain boundaries. (a) Model of

Figure 1 . 3

 13 Figure 1.3 Structure of the human SA2-Scc1 complex and domain boundaries.

Figure 1 . 4

 14 Figure 1.4 Cartoon illustration of the ring-shape cohesin and its accessory factors. (a)

Figure 1 . 5

 15 Figure 1.5 Cartoon of cohesin functional cycle and regulators. (a) From left to right:

Figure 1 . 6

 16 Figure 1.6 Structure of the yeast Pds5-Scc1 complex and domain boundaries. Pds5

Figure 1 . 7

 17 Figure 1.7 Structure of the human Wapl-C domain and domain boundaries. Wapl

Figure 1 . 8

 18 Figure 1.8 Structure of human Esco1 and domain boundaries. Esco1 acetylates Smc3

Figure 1 . 9

 19 Figure 1.9 Structure of the Sgo1-PP2A complex and domain boundaries. Shugoshin

Figure 1 .

 1 Figure 1.10 Structure of the yeast Separase-Securin complex and domain boundaries.

Figure 1 .

 1 Figure 1.11 Model of CTCF zinc fingers 2-9 in complex with DNA. A tandem array of 11

Figure 1 .

 1 Figure 1.12 Role of CTCF in the regulation of paternal and maternal imprinting. In

Figure 1 .

 1 Figure 1.13 Levels of Genome folding. (a) Elements involved in cohesin-CTCF looping along

L

  'objectif de cette thèse était de tirer des informations structurales et biochimiques sur le complexe de la cohésine. Dans cette section, je décris les matériels et les méthodes relatifs à cet objectif. Pour faciliter l'isolement des protéines et des complexes protéiques produites de manières recombinants, des amorces ont été synthétisées pour amplifier les gènes d'intérêt par PCR, puis les fragments d'ADN digérés par des enzymes de restriction ont été insérés dans des plasmides cibles par ligation enzymatique. Pour obtenir les protéines d'intérêts, l'expression et la purification des protéines, ont été effectuées, les protéines sont ensuite concentrées et congelées dans de l'azote liquide pour le stockage. Pour déterminer la structure des molécules biologiques d'intérêt, des essais de cristallisation des protéines ainsi que plusieurs cycles d'optimisation des différentes conditions et de marquage avec de la sélénométhionine ont été réalisés. Les techniques utilisées pour comprendre les interactions protéine-protéine et protéine-ADN, y compris la co-expression/co-purification de protéines, des expériences de pull-down, des peptides arrays, des expériences EMSA et la polarisation de fluorescence (FP) sont résumées. Afin de comprendre et d'identifier les résidus essentiels pour les interactions protéine-protéine ou ADN-protéine, nous avons effectué de la mutagénèse dirigée. La caractérisation in vivo des mutants déterminés à partir des structures obtenues, notamment les analyses de ChIp-qPCR et de 'tetrad', ont été réalisées en collaboration avec d'autres équipes.

2. 4 . 4

 44 Crystals of the SA2T-Scc1H complex were grown by hanging-drop vapour diffusion at 20 °C by mixing equal volumes (1 ul) of protein at 8 mg ml -1 and crystallization solution (1 µl) containing 0.06 M Morpheus Divalents mix, 0.1 M Morpheus buffer system2 and 48% Morpheus EOD_P8K. Crystals were soaked for 24-48 h with peptides (peptid.de) spanning amino acid residues 220-230 of CTCF (Uniprot ID: Q8NI51, sequence: DVSVYDFEEE) or 331-341 of shugoshin (Uniprot ID: Q5FBB7; SNDAYNFNLEE). The crystals were cryo

  2 μM and incubated with the array overnight at 4 °C under gentle agitation. The membrane was washed 3 times (1x 30s, then 2x 5 minutes) at 21 °C. The anti 6xHistidine antibody was diluted 1:2000 in 1x blocking buffer and incubated with the arrays for 1h at 21 °C. The array was washed 3 times (1x 30s, then 2x 5 minutes) and developed by addition of 3,3′-Diaminobenzidine (Sigma D4293) for 1 minute followed by quenching in deionized H 2 O. To measure non-specific binding of the anti 6x Histidine antibody, all steps were identical except that no SA2-Scc1 protein solution was added to 1x blocking buffer during the overnight incubation step. Arrays were imaged with a BioRad Gel Doc XR+ Documentation system. Afin d'étudier le module ATPase de la cohésine et ses facteurs associés, j'ai généré une série de constructions pour expression dans E. Coli. Comme seul Smc1 s'est avéré difficile à exprimer, j'ai criblé une série de "têtes" de Smc1 d'espèces différentes. J'ai ensuite obtenu la structure cristalline de de la tête de Smc1 (ctSmc1) de Chaetomium Thermophilum en complexe avec le domaine C-terminal de Scc1 (CScc1) de la levure à une résolution de 2,1 Å. Cela m'a permis d'assembler un hétérotrimère de cohésine Smc1-Smc3-Scc1 appelé "Clink" (cohésine "liée") en co-exprimant une fusion de NScc1 au segment C terminal de ctSmc1 avec les composants de module de tête restants (CScc1, le ctSmc1 N-terminal et le complexe NScc1-Smc3) dans E. Coli. En étendant Scc1, j'ai également pu intégrer les facteurs accessoires Pds5 et Scc3.

(

  ag), S. pombe (sp) and C. thermophilum (ct)(Figure 3.1a). As seen on Figure3.1.b, no band for spSmc1 is visible, possibly because it cannot be expressed in E.coli under these conditions. AgSmc1 appears to be degraded, judged by the distribution of bands on the gel.Only the ctSmc1 head (ctSmc1hd) construct remains intact and soluble (Figure3.1b). However, I observed that the ctSmc1hd precipitates during purification in absence of an interaction partner. I therefore increased the salt concentration at the size-exclusion chromatography step from 150 mM to 300 mM and co-expressed it with a C-terminal fragment of budding yeast Scc1 (CScc1) (Figure3.1c).

Figure 3 . 1

 31 Figure 3.1 Smc1 head boundaries and purification. (a) Schematic representation of Smc1 and

Figure 3 . 2

 32 Figure 3.2 Structure of the ctSmc1 head domain in complex with yeast CScc1. (a) Image

Figure 3 . 3

 33 Figure 3.3 Purification and DNA binding of Clink115/120/125. (a) Cartoon model of the

  for coexpression or co-purification with either truncated or full length Pds5 (T5) (Figure 3.4a-d). To include Scc3 in the Clink complex, I made a longer CScc1 covering the Scc3 binding region (309-564). Unfortunately the longer CScc1 was degraded, which did not allow us to co-express Clink159 with Scc3. Clink159 variants with two truncations lacking residues 400-475 and 400-480 on the extended CScc1 were purified (data not shown) and only the one lacking 400-480 (called CScc1Δ or Clink159-Scc3TΔ) showed less or no degradation (Figure 3.4 c, d).

Figure 3 . 4

 34 Figure 3.4 Purification and EMSA of Clink159 in complex with either Pds5, Scc3, or

  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 conservé est requis pour la liaison à l'ADN par Scc3-Scc1 in vitro, ainsi que pour l'enrichissement de la cohésine sur les chromosomes et pour la viabilité cellulaire. Ces résultats suggèrent que l'interface de liaison à l'ADN de Scc3-Scc1 joue un rôle central dans le recrutement de la cohésine sur les chromosomes et donc que cette dernière exécute fidèlement ses fonctions lors de la division cellulaire.

Figure 4

 4 3e). These encompassed Smc3hd-NScc1, ctSmc1hd-CScc1 (Figure4.1a, Figure4.2a), as well as the Scc3-Scc1 subcomplex (Scc3T-Scc1K)(Figure 4.3a,b). In addition, we produced a Smc1-Smc3 hinge heterodimer (Figure4.1b), Pds5 bound to an Scc1 fragment(Muir, et al. 2016) with full-length Pds5 (Pds5-fl) or as a truncated variant (Pds5-T5); as well as full-length Wapl (Wapl-fl) or truncated variant (Wapl-C) (Figure 4.2b, c), and a GST tagged Eco1 full-length (Figure 4.1d).We found that the Smc3hd-NScc1 module and the Smc1-Smc3 hinge heterodimer, consistent with a prior study (Murayama and Uhlmann 2014), bound DNA, as seen by the appearance of slower-migrating species in EMSA assays (Figure 4.1a, b). As expected for non-sequence specific DNA binding factors, longer DNA fragments (>21 bp) bound more efficiently than shorter DNA duplexes (15 bp). Isolated Smc3hd also interacted with DNA but with relatively weak affinity under these conditions (Figure 4.1c). Eco1, containing a C2H2 zinc finger domain, also bound to DNA directly (Figure 4.1d). Conversely, the Pds5 subunit, the ctSmc1hd-CScc1 subcomplex or the Wapl subunit did not interact with DNA in this assay (Figure 4.2). The Scc3T-Scc1K subcomplex showed most robust DNA binding activity (Figure 4.3e). I also observed that DNA binding depended on the presence of Scc1K as in its absence Scc3T showed greatly reduced DNA binding affinity (Figure 4.3e). As cohesin interacts with chromosomal templates I also assessed if the Scc3T-Scc1K subcomplex interacts directly with nucleosomes. I observed robust nucleosome binding indicating that the Scc3T-Scc1K subcomplex not only accommodates regular B-DNA substrates but also apparently the altered DNA geometry of the nucleosome core particle (Figure 4.3f).

Figure 4 .

 4 Figure 4.1 SDS-PAGE analysis of the purified cohesin components and DNA binding

Figure 4 . 2

 42 Figure 4.2 Cohesin subunits that lack DNA binding affinity. (a-c), ctSmc1hd-CScc1, Pds5

Figure 4 .

 4 Figure 4.3 DNA binding by the Scc3-Scc1 subcomplex. (a) Domain structure of Scc3 and

Figure 4 . 4

 44 Figure 4.4 Structure of the Scc3-Scc1 subcomplex bound to DNA. (a) Cartoon

Figure 4 . 5

 45 Figure 4.5 Electron density for the DNA molecule bound to the Scc3-Scc1 subcomplex.

Figure 4 .

 4 5d). The deduced register places Scc1 residue K363 in close proximity to the DNA (Figure 4.4a, Figure 4.5d). To assess if this region of Scc1 contributes to DNA binding, I truncated Scc1 amino acids 355-400, thus deleting the region that was not visible in our structure. This variant failed to co-purify with Scc3 in our expression system indicating that Scc1 (309-355) is essential for Scc3-Scc1 interaction (Figure4.10e). Direct interactions between Scc1 and the DNA phosphate backbone potentially explain why the Scc3T-Scc1K subcomplex has greater DNA binding affinity than does isolated Scc3T (Figure 4.3e).

Figure 4 .

 4 6b) or in vivo (see below). Similar results were observed via EMSA as well (Figure4.7). We conclude that the positively charged Scc3 cradle comprises the major DNA-binding surface of Scc3 and that the positively charged amino acid residues located in patch 1 and 3 constitute a composite DNA binding surface. The distribution of these residues across an extended surface of Scc3 might explain why their significance has thus far eluded cell-biological and genetic characterization.

Figure 4 . 6

 46 Figure 4.6 Biochemical analysis of Scc3-Scc1 subcomplexes and Scc3 conservation

Figure 4 .

 4 Figure 4.7 DNA binding affinity test of both Scc3T-Scc1K wild type and mutants via

Figure 4 .

 4 8e). Whereas all three individual patch mutants could support cell proliferation, cells expressing the heptamutant version as their only source of Scc3 failed to divide, despite expressing the mutant Scc3 protein at wild-type levels (Figure 4.9a). chromosome arm (arm) regions (chromosomes IV, V, and VI as indicated) of untagged (strain C3) or PK 6 -tagged wild-type or mutant versions of Scc3 expressed from an ectopic locus under its endogenous promoter (strains C5013, C5043, C5033). The fractions of immunoprecipitated DNA relative to input DNA are plotted as circles for two biological repeats with two technical repeats each (same colour pairs). Mean values of all four data points are shown as lines. (e) Tetrad analysis of diploid budding yeast strains expressing ectopic wild-type or mutant versions of Scc3 under control of the endogenous promoter in an SCC3/scc3Δ background (strains C5013, C5014, C5015, C5043, C5033). Images were recorded after three days at 30 °C on rich media. Genetic marker analysis identified Scc3(mutant), scc3Δ cells (circles).

Figure 4 . 9

 49 Figure 4.9 In vivo analysis of SCC3 mutants. (a) Protein levels of ectopically expressed wild-

Figure 4 .

 4 Figure 4.10 Purification of selenomethionine-labeled Scc3T-Scc1K and different Scc3-

Figure 4 .

 4 Figure 4.11 Screening for possible protein interactions among cohesin subcomplexes. (a)

Figure 4 .

 4 Figure 4.12 A conserved DNA binding interface in cohesin and condensin. (a) Structure of

  Pour étudier les bases moléculaires de la collaboration fonctionnelle signalée entre la cohésine et le CTCF dans la structure tridimensionnelle du chromosome, 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 la CTCF, mais aussi dans d'autres facteurs connectés, tels que la shugoshin, Wapl, un autre facteur accessoire de la cohésine, soit pour la protection du centromère, soit la libération de la cohésine aux bras du chromosome. Les tests de compétition par "pull-down" 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 la shugoshin et le CTCF se lient à la même surface conservée sur la cohésine.

  with SA2T. This truncated construct showed no degradation(Figure 5.2d). In order to test whether human SA2-Scc1 binds to DNA, EMSA assays were performed with a 32 bp DNA oligonucleotide. However, no DNA binding was detected under equivalent conditions which yield robust DNA binding of yeast Scc3-Scc1 (Figure5.2b).

Figure 5 . 1

 51 Figure 5.1 Yeast Pds5 and Scc3 do not bind human CTCF (a-b), indicating that the yeast

Figure 5 . 4

 54 Figure 5.4 Purification of SA2-Scc1H mutants, CTCF-N6 mutants and CTCF covering

Figure 5 . 5

 55 Figure 5.5 Co-expression and co-purification of the SA2T-Scc1H-CTCF complex and

  Figure 5.6c, d) The CTCF binding surface is predominantly hydrophobic and composed of amino acids contributed by both SA2 and Scc1. The lead 'anchoring' amino acids of CTCF, which bury the largest solvent-accessible surface area upon binding, are Y226 and F228 (Figure 5.6d) F228 inserts into a pocket made up of amino acids from Scc1 (S334, I337, L341) and SA2 (Y297, W334) (Figure 5.6e) The hydroxyl group of Y226 hydrogen bonds with E326 of SA2 in a deep hydrophobic pocket lined by L329, F366 and F367 (Figure 5.6f) E229 and E230 of CTCF, constitute secondary anchoring residues which presumably contribute to binding specificity by forming salt bridges with R298 of SA2 and R338 of Scc1. Because CTCF engages a composite binding surface containing amino acids from Scc1 and SA2, prior

Figure 5 . 6

 56 Figure 5.6 Structure of hSA2-Scc1 bound to a fragment of CTCF. (a-b) ITC progress

Figure 5 . 7 Figure 5 . 8

 5758 Figure 5.7 Mutagenesis and in vitro analysis of the SA2-Scc1-CTCF ternary complex.

Figure 5 . 9

 59 Figure 5.9 Model for CTCF-mediated cohesin binding and for the generation of TADs

Figure 5 .

 5 Figure 5.11 Sgo1 interacts with SA2-Scc1. (a) ITC showing binding isotherms of a truncated

Figure 5 .

 5 Figure 5.12 Structure of the SA2-Scc1 and Sgo1 ternary complex. (a) Model of the SA2-

  ligands such as Sgo1 (Figure 5.13, array position 12). Instead, robust binding was observed for Mcm3, a subunit of the replicative helicase (Figure 5.13, array position 13), SYCP3, a component of the synaptonemal complex (Figure 5.13, array position 17), ZGPAT, a transcriptional repressor (Figure 5.13, array position 23) and CENPU, a component of the inner kinetochore (Figure 5.13, array position 22). Thus, the conserved binding site on the SA2-Scc1 complex potentially effects cohesin recruitment in a number of different genome transactions.

Figure 5 .

 5 Figure 5.13 Novel SA2-Scc1 binding partners screened via peptide array. (a) Peptide array

Figure 5 .

 5 Figure 5.14 Model of SA2-Scc1 subcomplex bound with different Y/FxF-containing

First

  of all, I would like to thank EMBL and my supervisor, Dr. Daniel Panne, for giving me this opportunity to do my Ph.D. here in Grenoble, France. It has been very challenging but I do learn a lot, both on my scientific training and outlook of my life. I would express my appreciation to Dr. Benjamin Rowland, Dr. Camilla Bjorkegren, Dr. Joanna Timmins and Dr. Ramesh Pillai for spending the time to evaluate my thesis. And also my thesis advisory committee (TAC) members, Dr. Stephen Cusack, Dr. Christian Haering, Dr. Carlo Petosa, thanks for all the valuable suggestions and kind help during my Ph.D. As an excellent cohesin team, I would like to express my deepest appreciation to Dr. Kyle Muir who has invested a lot of effort in guiding the cohesin project in achieving the goal, sharing materials and ideas and it turns out extremely useful. We do spend time extensively working together for years. Furthermore, I would also like to acknowledge with much appreciation on the crucial role of all members of the HTX lab for such effective support and MASSIF-1, especially Dr. Matthew Bowler, who helped test almost a thousand of crystals and this yields 5 essential structures in the cohesin field. It would not be possible for us to achieve this in such a short period without their professional and powerful support. I would also deeply thank Dr. Christian Haering and Dr. Benjamin Rowland for the professional collaboration, and also lab members, Jutta Metz from Haering at EMBL Heidelberg, Judith Haarhuis from Rowland lab at the Netherlands Cancer Institute. I would also like to thank people who put a lot of effort in revising my thesis, Dr. Mariya Lukarska, Dr. Kyle Muir and Dr. Paul Sauer for the primary revision, Dr.Ziad Ibrahim, for the French abstracts and thank Dr. Daniel Panne for revising my thesis after.

Table 1

 1

	.2	CTCF interaction partners
	Figure 1.13 Levels of Genome folding

Table 2

 2

	.1	Scc3T-Scc1K crystallisation screen conditions
	Figure 3.1	Smc1 head boundaries and purification
	Figure 3.2	Structure of the ctSmc1 head domain in complex with yeast CScc1

Table 3

 3

	.1	X ray data collection, phasing and refinement statistics

Table 4

 4

	.1	DNA oligos used for crystallization, EMSA and Fluorescence
		Polarization (FP)

Table 4

 4

	.2	Data collection and refinement statistics

Table 4

 4

	.3	ChIP-qPCR primer sequences (5'→3')

Table 4

 4

	.4	Yeast genotypes

Table 1 .2 CTCF interaction partners

 1 Table 1.2).

	Protein name	Protein function	CTCF interaction domain
	DNA-binding proteins and transcription factors
	YB-1	DNA/RNA-binding factor	Zinc fingers
	Kaiso	Zinc-finger transcription factor	C-terminus
	Yy1	Zinc-finger transcription factor	All, but highest affinity for N-
			terminus
	Histones and histone-modifying proteins
	Sin3	Transcriptional repressor, associated with	Zinc fingers
		histone deacetylases
	CHD8	Chromodomain helicase family member	Zinc fingers
	Taf-1/Set	Molecular chaperone, associated with inhibition	N/A
		of histone acetyltransferases
	H2A/H2A.Z	Nucleosome component	N/A

Table 3 .1 X ray data collection, phasing and refinement statistics 3.4 ctSmc1/Smc3 head assembly via NScc1 linker -"Clink"

 3

-Scc1K native Scc3T-Scc1K SeMet Data collection

	Space group	P2 1 2 1 2	P2 1 2 1 2
	Cell dimensions
	a, b, c (Å)	109.9, 115.4, 295.6 109.9, 115.6, 296.2
	Wavelength (Å)	1.282	1.282
	Resolution (Å)	50-3.60	49.9-4.79
	No. reflections	20963	10279
	R merge	5.8 (122.6)*	4.6 (112.3)*
	I / σI	11.9 (1.6)*	10.6 2(.1)
	CC 1/2	0.99 (0.56)	0.99 (0.52)
	Completeness (%)	91.4 (63.5)*	93.6 (71.2)*
	Redundancy	4.4 (6.0)*	1.8 (1.8)
	Refinement
	Resolution (Å)	50-3.60
	R work / R free	0.28 / 0.31
	No. atoms	16465
	Protein	14909
	DNA	1556
	B-factors (mean)
	Protein	254.5
	DNA	266.4
	R.m.s deviations
	Bond lengths (Å)	0.002
	Bond angles ()	0.5
	*Values in parentheses are for highest-resolution shell.

SA2-Scc1-CTCF SA2-Scc1-Sgo1 Data collection

	Space group	P2 1 2 1 2 1	P2 1
	Cell dimensions
	a, b, c (Å)	79.0, 107.2, 176.4	78.8, 181.0, 111.3
	Resolution (Å)	45.8-2.6	47.8-3.2
	No. reflections	46759(4622)	48453(4517)
	R sym or R merge	6.9 (175)*	10.8 (120)*
	I / σI	12.0 (0.8)*	8.1 (0.74)*
	CC 1/2	0.99 (0.33)	0.99 (0.45)
	Completeness (%)	99.6 (99.7)*	97.9 (91.8)*
	Redundancy	4.4 (4.3)*	2.7 (2.7)*
	Refinement
	Resolution (Å)	45.8-2.6	47.8-3.2
	R work / R free	0.24 / 0.28	0.31 / 0.34
	No. atoms	16099	32295
	SA2	14793	29556
	Scc1	1249	2498
	Ligand	157 CTCF	242 Shugoshin
	B-factors (mean; Å 2 )
	SA2	104.4	114.6
	Scc1	87.3	99.6
	Ligand	119.6 CTCF	118.1 Shugoshin
	R.m.s deviations
	Bond lengths (Å)	0.005	0.003
	Bond angles ()	0.97	0.61

*Values in parentheses are for highest-resolution shell.

Acknowledgement

List of Abbreviations

List of Figures / Tables

Abstract

phosphorylation marks on pericentric nucleosomes [START_REF] Kerrebrock | Mei-S332, a Drosophila protein required for sister-chromatid cohesion, can localize to meiotic centromere regions[END_REF][START_REF] Kitajima | Human Bub1 defines the persistent cohesion site along the mitotic chromosome by affecting Shugoshin localization[END_REF][START_REF] Marston | A genome-wide screen identifies genes required for centromeric cohesion[END_REF][START_REF] Tang | Human Bub1 protects centromeric sister-chromatid cohesion through Shugoshin during mitosis[END_REF][START_REF] Yamagishi | Two histone marks establish the inner centromere and chromosome bi-orientation[END_REF] (Figure 1.5). Shugoshin contains two highly-conserved domains, an N-terminal coiled coil and a C-terminal basic region. Shugoshin forms homo-oligomers through the coiled coil domain and recruits PP2A, which in turn dephosphorylates Scc3 at the centromere and prevents Plk1-dependent removal of shugoshin [START_REF] Clarke | POLO kinase regulates the Drosophila centromere cohesion protein MEI-S332[END_REF][START_REF] Tang | PP2A is required for centromeric localization of Sgo1 and proper chromosome segregation[END_REF]. Phosphorylation of the human shugoshin (Sgo1) at residue T346 also allows to Sgo1directly compete with Wapl to protect Scc3 [START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF][START_REF] Liu | Phosphorylation-enabled binding of SGO1-PP2A to cohesin protects sororin and centromeric cohesion during mitosis[END_REF]).

The PP2A holo-enzyme contains a catalytic C, a scaffold A subunit, which binds to C directly, and a more varied B subunit. The strucutre of the Sgo1-PP2A complex reveals that the coiled coil domain of Sgo1 binds to C and B subunits of PP2A, and it turns out to be essential for centromere cohesion protection (Figure 1.9, Table S1).

B: inactive genes tend to be located toward the periphery of the nucleus associated with the nuclear lamina) [START_REF] Merkenschlager | CTCF and Cohesin in Genome Folding and Transcriptional Gene Regulation[END_REF].

Recent studies have provided greater mechanistic detail, suggesting that cohesin is required for the formation of TADs and loops but restricts compartmentalization. Wapl and Pds5 antagonize these functions: Wapl and Pds5 detemine the size and the boundary of the DNA loops, and also facililate the proper orientation of CTCF sites. The loading complex Scc2-Scc4 in turn promotes the extension of the loops which makes loop formation dynamic but more elaborate (Haarhuis, et al. 2017;[START_REF] Wutz | Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins[END_REF].

Scientific aims

Materials and Methods

Construct Cloning

Most yeast genes were amplified from yeast Genomic DNA (Millipore) by PCR, unless specified otherwise. Codon optimised (for E.coli) genes comprising A. gossypii (ag), S. pombe (sp), and C. thermophilum (ct) Smc1head, and the ySmc3 head were produced by gene synthesis (Thermofisher), Pds5 and Wapl were cloned and expressed as described previously (Muir, et al. 2016), human SA2 inserted into pGEX-6p vector was provided by Hongtao Yu (UT Southwestern, Dallas) and is codon optimized for expression in E.coli. Full length human CTCF was ordered from Addgene, and different constructs amplified by PCR.

The gene encoding human Shugoshin was synthesised and codon optimised for expression in E.coli (Thermofisher). Vectors containing cDNAs encoding human Scc1 and Wapl were a gift from Jan-Michael Peters (IMP, Vienna); constructs arising were subcloned into pACYC and pGEX-6p respectively.

For a list of all vectors, tags, linkers used and construct boundaries or mutants generated, please see Table S2. Site-directed mutagenesis, where required, was performed with the QuikChange® Lightning kit (Agilent Technologies).

Protein expression

Protein expression

All proteins were expressed in E.coli BL21(DE3). Expression was induced by auto-induction [START_REF] Studier | Protein production by auto-induction in high density shaking cultures[END_REF]. Cells were grown at 37°C until OD 600nm = 0.6 and then shifted to 18°C for 16 hours. Cells were harvested in a JLA-8.1 (Beckman) centrifuge at 4000xg and washed once with ice-cold PBS buffer. For protein containing His tags pellets were resuspended in buffer 1 (40 mM TRIS, pH 7.5, 500 mM NaCl, 20 mM imidazole, 0.5 mM TCEP) containing one tablet of complete mini EDTA-free protease inhibitors (Roche) and loaded on Co 2+ beads head module, I fused Saccharomyces Cerevisiae (hereafter 'yeast') NScc1 to the C-lobe of the ctSmc1hd (as described below) and co-expressed this construct in E.coli with the remaining head module constituents: the yeast Smc3 ATPase, the N-lobe ctSmc1 and its binding partner, yeast CScc1 (Figure 3.3a). Based on this idea, I generated three different constructs, which contained different lengths of the NScc1 linker: Scc1 (1-115), (1-120), and (1-125) followed by the prescission cleavage sites (SPGLEVLFQGPRG) (Figure 3.3a, b), Co-purification of the Smc3-ctSmc1-Scc1 head module, termed c-link ('linked' cohesin via a His-tag on the ctSmc1 C-terminus,), showed that a direct physical interaction of NScc1 with the coiled coil of Smc3 is maintained (Figure 3.3c-e, Table S2). Clink complexes containing longer NScc1 (Scc1 1-125) tended to have higher yield in protein expression when compared to Clink115 and Clink120 (Figure 3.3c-e) and higher DNA binding affinity as shown by Electrophoretic mobility shift assay (EMSA), (Figure 3.3f-h).

Crystal structure of the Scc3-Scc1 in complex with a 19 bp DNA

To determine the molecular basis of how cohesin subunits interact with DNA, I screened all cohesin subcomplexes that interacted with DNA using commercial crystallisation screening conditions (EMBL Grenoble HTX facility), i.e. SMC3hd-NScc1, Smc3/Smc3 hinge, Scc3T-Scc1K in complex with different lengths of DNA oligonucleotides as indicated in table S2.

None of the complexes crystallized. I then tried conditions which are typically suitable for crystallization of DNA complexes, based on the collation of conditions reported on the RSCB-PDB (Materials and methods, Table 2.1). I obtained crystals of the Scc3T-Scc1K in complex with a 17 bp DNA substrate. Despite testing hundreds of these crystals for diffraction at the automated Massif beamline (ESRF) the resolution was found to be severely limited. To improve the resolution, I tested different Scc1 truncations, containing Scc3T with either shorter (residues 309-388), or longer Scc1 (residues 273-400, 240-400 or 170-400). I also tested shorter DNA oligonucleotides ranging from 12 to 16 bp (data not shown). While I obtained crystals (data not shown), they did not show improved diffraction. I then used the crystals containing the 17 bp DNA as seeds and rescreened the crystallisation conditions but with different lengths DNA oligonucleotides. I was thus able to reproducibly obtain crystals of Scc3T-Scc1K in complex with a 19 bp DNA fragment.

The optimized crystals diffracted anisotropically to a minimum Bragg spacing of 3.9 Å in the best, and ~5.7 Å in the worst direction (Table 4.2). We determined the structure by molecular replacement, using the structures of a Scc3 ortholog from Zygosaccharomyces rouxii and a C-terminal fragment of Saccharomyces cerevisiae Scc3 as search models [START_REF] Roig | Structure and function of cohesin's Scc3/SA regulatory subunit[END_REF].

The resulting electron-density map provided a continuous trace of the polypeptide main chain, but with a limited level of detail owing to the anisotropy of the data. Despite these drawbacks, we successfully traced the amino-acid register using a selenomethionine

Mapping SA2-Scc1 binding regions on CTCF

SA2 has been reported to interact with CTCF [START_REF] Xiao | Specific sites in the C terminus of CTCF interact with the SA2 subunit of the cohesin complex and are required for cohesin-dependent insulation activity[END_REF], a zinc finger protein with strong sequence-specific DNA binding affinity [START_REF] Hashimoto | Structural Basis for the Versatile and Methylation-Dependent Binding of CTCF to DNA[END_REF]. As demonstrated in figure 5.2b, SA2-Scc1 does not interact directly with DNA. One possibility therefore is that human SA2-Scc1 associates with DNA through accessory factors such as CTCF (Figure 5.

Analysis of additional cohesin ligands containing Y/FxF motifs

Previous data indicate that the SA2-Scc1 complex interacts with multiple cohesin regulators [START_REF] Beckouet | Releasing Activity Disengages Cohesin's Smc3/Scc1 Interface in a Process Blocked by Acetylation[END_REF][START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF][START_REF] Roig | Structure and function of cohesin's Scc3/SA regulatory subunit[END_REF]. The opposing functions of two such factors, Wapl, the general cohesin inhibitor, and Shugoshin (Sgo1) is crucial for the protection of centromeric cohesion during mitosis [START_REF] Gandhi | Human Wapl is a cohesin-binding protein that promotes sister-chromatid resolution in mitotic prophase[END_REF][START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF][START_REF] Kueng | Wapl controls the dynamic association of cohesin with chromatin[END_REF][START_REF] Liu | Phosphorylation-enabled binding of SGO1-PP2A to cohesin protects sororin and centromeric cohesion during mitosis[END_REF]. This antagonism potentially arises as a result of direct competition for binding to SA2-Scc1 [START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF]. As mutants previously reported to interfere with both Sgo1 and Wapl binding cluster in the CTCF binding site on SA2-Scc1, we investigated whether these proteins are recruited to the CES by a comparable mechanism. In Sgo1, the reported CES-binding domain (amino acids 313-353) contains a conserved Y/FxF motif which strongly resembles that of the CTCF peptide [START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF]. Vertebrate Wapl also contains several FGF motifs in its N-terminal region which are potentially involved in cohesin regulation [START_REF] Ouyang | Structural Basis and IP6 Requirement for Pds5-Dependent Cohesin Dynamics[END_REF][START_REF] Shintomi | Releasing cohesin from chromosome arms in early mitosis: opposing actions of Wapl-Pds5 and Sgo1[END_REF]. A minimal fragment of Wapl capable of competing with Sgo1 for access to the CES (amino acids 410-590) contains two such FGF repeats [START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF]. To determine whether Wapl and Sgo1 are able to compete against CTCF for access to the SA2-Scc1, we performed GST-pulldown competition assays. As predicted, a GST-Wapl fusion protein spanning amino acids 1-600 bound to SA2-Scc1 and excluded CTCF from SA2-Scc1(Figure 5.5d).

Similarly, titration of a Sgo1 phospho-T364 peptide, previously reported to preclude Wapl binding [START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF], also displaced CTCF from SA2-Scc1 (Figure 5.7b, lane 17&18).

To further understand this interaction, I synthesized a phosphorylated Sgo1 peptide (pSgo1, Wapl [START_REF] Haarhuis | Cohesin: building loops, but not compartments[END_REF][START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF], and opens up the possibility that this may be a general phenomenon underpinning cohesin regulation and genome recruitment.