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Cornelia de Lange Syndrome (CdLS) is a multisystem genetic disorder caused by mutations in the cohesin complex. It is believed that cohesin is able to regulate gene expression with CTCF by holding chromatin in topological complexes, such as active chromatin hubs, and that CdLS is caused by loss of these complexes causing aberrant gene expression. In order to determine if loss of these complexes in CdLS resulted in a general change in the compaction of chromatin, I undertook a series of analyses of the nucleus in CdLS patient lymphoblastoid cell lines (LCLs), compared to wildtype, and later in RNAi knockdown models of CdLS. By fluorescent in situ hybridisation (FISH) I studied the chromatin compaction of different regions of the genome, and found that in some, but not all, CdLS cell lines, gene-rich regions have less compact chromatin compared to wildtype. RNAi knockdown of two proteins that are mutated in CdLS, NIPBL and SMC1, also resulted in decompaction of regions of the genome, however these were different regions than in the patient LCLs, perhaps due to variation between cell lines. This change was not due to the interaction between cohesin and CTCF, as I found that knockdown of CTCF did not result in changes in chromatin compaction. I have also looked at the published data for gene expression in CdLS, and in mouse and Drosophila models of CdLS, and have found no correlation between the genes misexpressed in CdLS in the three species, nor between three cell lines of the same species. These data suggest that the variation in chromatin compaction observed in CdLS may not be due to an interaction between cohesin and CTCF, and that cohesin can act independently of CTCF to regulate gene expression.

Thesis (Ph. D.)--Miami University, Dept. of Chemistry and Biochemistry, 2005. / Title from second page of PDF document. Document formatted into pages; contains [3], vi, 124 p. : ill. Includes bibliographical references.

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Sister chromatid cohesion is essential for accurate chromosome segregation. Cohesion is generated by cohesin, a conserved multi-subunit protein complex composed of four core subunits: Smc1, Smc3, Scc1, and Scc3. Cohesin holds sister chromatids together in mitotic cells starting from S-phase, when DNA replicates, until their separation at the onset of anaphase where its Scc1 subunit is cleaved. In budding yeast, most Scc1 is destroyed by cleavage at anaphase and is only re-synthesised in late G1, whereupon it associates with the unreplicated chromatin. Although sister chromatid cohesion is known to be mediated by a topological interaction of cohesin complexes around sister DNAs, the nature of cohesin`s interaction with chromatin before DNA replication remains to be elucidated. My project aims to develop a new system in order to find out whether ‘non-cohesive’ cohesin interacts with chromatin topologically. This is important for two main reasons. Firstly, understanding the physical nature of cohesin’s interaction with chromatin before DNA replication is essential for determining how cohesion is established during DNA replication. Another reason is that most cohesin in multicellular organisms is associated with the unreplicated chromatin of post mitotic cells where it regulates transcription. How cohesin mediates gene expression is unknown. Understanding how cohesin binds unreplicated chromatin may therefore bring insights into the mechanisms by which cohesin complex performs its non-canonical functions. In order to address this, we needed a situation where cohesin is already loaded onto chromosomes, but either DNA replication or cohesion establishment is prevented. Therefore, we used a temperature sensitive allele of Eco1 (required for establishment of cohesion). Quantitative measurement of cohesin levels on chromosomes in either wild type allele or temperature sensitive allele of Eco1 showed that the amount of cohesin associated with centromeric and inner pericentromeric regions in both strains are almost indistinguishable from each other throughout the whole cell cycle. Despite normal levels of cohesin, we confirmed by minichromosomal assay that no sister chromatid cohesion is established in the absence of functional Eco1 protein. If “non-cohesive” cohesin interacts with the chromatin in a topological manner when there is no sister chromatid cohesion, then its association with chromatin should be resistant to denaturing conditions in the presence of a modified version of the cohesin complex that can be covalently circularized. To test this prediction, a cross-linkable cohesin molecule was needed, which should be resistant to SDS denaturation and should not have major cohesion defects due to the modifications making it to be cross-linkable. The previously created cross-linkable cohesin molecule had cohesion defects due to the presence of Smc3-Scc1 fusion protein. In addition, this fusion alone could bypass the requirement for Eco1, and therefore we could not test how “non-cohesive” cohesin interacts with chromatin, using this version of cross-linkable cohesin complex. We tried two different methods to conditionally close Smc3/Scc1 interface in a way resistant to protein-denaturants and create a new cross-linkable cohesin complex. In our first attempt, the C-terminus of Smc3 and the N-terminus of Scc1 were fused to FRB and FKBP12 respectively, proteins that can form a complex upon addition of rapamycin. Crystal structure of the ternary complex of FKP12/rapamycin/FRB enabled us to design cysteine pairs for the crosslinking of FRB and FKBP12 only in the presence of rapamycin. A more efficient in vivo crosslinking was achieved between the Smc3 and Scc1 in our second attempt. Amino acids within the coiled coil region of Smc3 were replaced by the unnatural photo-cross-linkable amino acid ρ-benzoyl-phenylalanine that can be induced to form covalent bonds with neighbouring proteins (T.Gligoris, unpublished data). Photo and chemically cross-linkable interfaces of cohesin were then integrated with each other to generate a new version of cross-linkable cohesin molecule.

The three-dimensional organization of the genome is important for various processes such as transcription, replication, and repair. Several studies have shown that the genome is organized into long-range and short-range chromatin loops. Gene loops represent a short-range chromatin loop, synonymous with the juxtaposition of promoter and terminator regions of a gene. In Chapter III, I investigate the mechanism of gene-loop formation in a constitutively expressed gene, mouse serum albumin (Alb). The Alb locus appears to exist in a clover-leaf structure, with the promoter in close physical proximity with an upstream enhancer and downstream genic sequences. Furthermore, Alb forms a promoter-terminator gene loop that is dependent on serine 2 phosphorylation of RNA polymerase C-terminal domain. I also investigate the presence of gene loop conformation at the human Nuclear factor NF-kappa-B (NFκB1) gene. In response to cytokine stimulation, NFκB1 transcription proceeds as a wave, with nascent RNA appearing as RNA polymerase traverses along the gene length. This coincides with formation of transient contacts between NFκB1 promoter and genic regions. The cohesin complex is a key mediator of chromatin loops and sister chromatid cohesion. The association of cohesin with chromatin is dependent on the loading complex, Mis4/Ssl3. In Chapter IV, I provide direct evidence for two functionally different populations of cohesin is Schizosaccharomyces pombe. Cohesin that co-localizes with Mis4 represents "cohesive" cohesin. In contrast, cohesin alone is unable to maintain stable sister chromatid cohesion, therefore, "less-cohesive" cohesin. Cohesive cohesin ensures stable cohesion because it is acetylated by the Eso1 acetyltransferase, which preferentially interacts with Mis4. In contrast, less-cohesive cohesin may function in recombination/repair. In Chapter V, I have identified a novel interplay between cohesin loading and transcription by RNA polymerase II. Inhibition of transcription initiation results in loss of Mis4 and consequently, cohesin binding on chromosomal arms regions. Furthermore, cohesin and Mis4 physically interact with RNA polymerase II. In Chapter VI, I summarize the above findings and propose a model that describes the stepwise loading of cohesin onto chromosomal arms during the fission yeast cell cycle. To conclude, I discuss the importance of understanding cohesin and its functions in health and diseases.

With 50,310 related deaths this year, colorectal cancer (CRC) has emerged as the second largest cause of cancer related deaths among Americans. While 70 million Americans are considered at-risk of developing CRC, it is highly curable if detected early. Cohesin proteins, which hold sister chromatids together during replication, have emerged as a potential biomarker in multiple cancer lines. Because of their probable role in DNA replication, DNA repair, chromatin nano-architecture, and gene expression, this paper assessed whether cohesion proteins could be used as a potential biomarker for colorectal cancer risk stratification. While cohesin protein mutations have been reported in different cancers and involved in chromosomal instability, its role in early cancer formation has yet to be observed. Using immunohistochemical and Quantitative Real Time PCR analysis, this thesis assessed the protein and RNA expression levels of cohesin proteins SA-1, NIPBL, and SMC3 from human biopsies at different stages and locations of colorectal cancer development. The results showed that SA-1, a structural cohesion subunit, was significantly (p<0.01) down regulated in cancerous compared to normal tissue. The SA-1 protein was also down regulated in the involved mucosa adjacent to CRC polyps. The cohesion loading protein, NIPBL, was also significantly (p<0.01) under expressed in cancerous versus normal tissue. The RNA expression analysis of rectal mucosa showed that SMC3 and SA-1 was over expressed two fold in patients harboring hyperplastic and adenomous polyps, giving evidence that cohesin proteins are differentially expressed throughout the field of carcinogenesis. Our results demonstrate for the first time that cohesion dysregulation is an early event in human colorectal cancer development and may serve as an important biomarker of field carcinogenesis.

Le complexe cohésine est un complexe protéique en forme d'anneau composé de quatre sous-unités essentielles très conservées: Smc1, Smc3, Rad21 et Scc3. Par sa capacité à encercler les molécules d’ADN, les cohésines participent à de nombreux processus cellulaires tels que la ségrégation des chromosomes, la signalisation et la réparation des dommages à l’ADN, la régulation de la transcription et l'organisation du génome. Pour assurer ces différentes fonctions biologiques les cohésines doivent être finement régulées à la fois dans le temps et l’espace. Ces régulations reposent en partie sur le contrôle de leur association à la chromatine (capture de l’ADN). Cela nécessite l'action d'un «facteur de chargement » composé de deux protéines conservées et essentielles, Mis4 et Ssl3 chez la levure S. pombe. Comment ce complexe régule la capture de l’ADN par l’anneau de cohésine dans l'espace et le temps demeure à ce jour très mal compris. Afin d’identifier des régulateurs de l’association des cohésines à la chromatine, nous avons réalisé un crible génétique visant à rechercher des suppresseurs de la mutation thermosensible mis4-367. Ce crible a conduit à l’identification de la Cyclin-Dependent Kinase Pef1 qui agit comme un régulateur négatif de la cohésion des chromatides soeurs en contrôlant vraisemblablement négativement l’association des cohésines à la chromatine. De forts arguments expérimentaux indiquent que Pef1 exerce sa fonction en régulant directement la phosphorylation de la sous-unité Rad21 du complexe cohésine. De façon intéressante, via un autre crible génétique, nous avons identifié la phosphatase Pph3/Psy2 qui joue un rôle dans l’établissement de la cohésion des chromatides soeurs en contrôlant la déphosphorylation de Rad21.Ensemble, ces données suggèrent que le contrôle de l’état de phosphorylation de la sous-unité Rad21 du complexe cohésine joue un rôle central dans le processus de cohésion chez la levure S. Pombe. / Cohesin is a highly conserved ring-shaped protein complex made of four essential subunits: Psm1, Psm3, Rad21 and Psc3. By its ability to capture DNA molecules within its ring-like structure, cohesion plays a key role in many cellular processes such as chromosome segregation, DNA damage signalling and repair, transcriptional gene regulation and nuclear organization. To ensure all of its biological functions, cohesin must be tightly regulated in space and time. This regulation relies in part on the control of cohesin binding to chromatin (DNA capture). Cohesin recruitment to chromatin requires the action of a “loading complex” made of two conserved and essential proteins named Mis4 and Ssl3 in the fission yeast. How this complex regulates where and when DNA capture by the cohesin ring must occur remains poorly understood. To identify regulators of cohesin binding to chromatin we have performed a genetic screen for suppressors of the thermosensitive mutation mis4-367. This genetic screen has led to the identification of the cyclin-dependent-kinase Pef1 that acts as a negative regulator of sister chromatids cohesion may be bynegatively controlling cohesin binding to chromatin. Strong experimental evidences indicate that Pef1 exerts its function at least in part by directly phosphorylating the Rad21 subunit of the cohesin complex. Interestingly, a genetic screen made in parallel identified the Pph3/Psy2 phosphatase as implicated in the establishment of sister chromatid cohesion by regulating Rad21 dephosphorylation. Strikingly, the control of Rad21 phosphorylation status appears central to the cohesion process in the fission yeast S. pombe.

When a cell divides into two, it is imperative that each resultant daughter receives a full complement of chromosomes; DNA is ultimately responsible for all cellular processes. Cohesion between sister chromatids from the moment of their generation in S phase is central to ensuring the fidelity of chromosome segregation. Smc1 and Smc3 proteins interact with each other via their hinges and with a bridging kleisin subunit via their heads to form the cohesin ring. It is cohesin, through entrapment of sister chromatid within its ring, that confers sister chromatid cohesion. The process of cohesin’s loading onto DNA is poorly understood. While it is thought to depend on ATP hydrolysis, opening of the ring at one of its three interfaces, and the as yet undefined action of the kollerin complex, comprising Scc2 and Scc4 proteins, the sequence of events as they occur are yet to be defined. A recent screen for suppressors of a thermosensitive scc4 allele in budding yeast revealed a mutation within Smc1’s hinge that could bypass the kollerin subunit. Here, the Smc1 suppressor mutation is investigated. Through targeted mutagenesis, the Smc1D588Y mutant identified in the screen and two additional point mutants, Smc1D588F and Smc1D588W, are herein proven able to bypass Scc4 function completely. Thus we provide the strongest evidence to date to suggest that cohesin’s hinge is a critical factor in its loading. Biochemical evidence shows that isolated Smc1 hinge mutants are defective in their binding to Smc3 hinges. This, together with the genetic link made between the hinge and loading complex, suggests that hinge opening might be a requisite for loading. Through mutagenesis of Scc2 and Scc4 we show that the N-terminus of each protein is responsible for their dimerisation. Furthermore, the N- terminus of Scc2 confers no function other than in its binding to Scc4. Finally, we show that Scc4 is required for the enrichment of both Scc2 and cohesin at centromeres, but not at arm loci. Our results are therefore indicative of there being two different pathways of cohesin loading.