Chromatin remodelers utilize the energy derived from ATP hydrolysis to mobilize nucleosomes. ISWI remodelers mobilize and evenly space nucleosomes to regulate gene expression. ISW2, an ISWI remodeler in yeast, has been shown to reposition nucleosome near promoter regions and represses both mRNA and antisense non coding RNA transcription. ISW2 is composed of four subunits and the catalytic Isw2 subunit consists of several conserved domains. The highly conserved ATPase domain is present at the N-terminus whereas the conserved HAND, SANT and SLIDE domain are towards the carboxyl terminal end of Isw2. Nucleosome mobilization by ISW2 requires both extranucleosomal DNA and the N-terminal tail of histone H4. DNA crosslinking and peptide mapping revealed that the ATPase domain contacts nucleosome two helical turns away (SHL2) from dyad to a site close to the H4 tail, whereas the HAND, SANT and SLIDE domain contact a 30bp stretch of DNA comprising the edge of nucleosome and ~20bp of extranucleosomal DNA. The ATPase domain and the C-terminal domains were investigated for their role in regulation of ISW2 activity both in-vitro and in-vivo. It appears that there are distinct modes of ISW2 regulation by these domains. Mutation of a patch of five acidic amino acids on the region of ATPase domain that contact SHL2 was found to be crucial for both ISW2 remodeling and nucleosome stimulated ATPase activity. Acidic patch mutant ISW2 was unable to mobilize nucleosome or hydrolyze ATP in absence of H4 tail. This indicates that the region of ATPase domain contacting nucleosome at SHL2 and H4 tail act in two separate and independent pathways to regulate ISW2 remodeling. Both HAND and SLIDE domain were shown to crosslink entry/exit site and linker DNA respectively. The roles of C-terminal domains were investigated either by deletion of the individual domain or mutation of conserved basic residues on the surface of these domains that are suspected to interact extranucleosomal with DNA. Deletion of HAND domain had minimal effect on in vitro ISW2 activity, however whole genome transcription analysis revealed one key role of this domain in ISW2 regulation. In absence of HAND domain, ISW2 had minimal role on repression of genes that were RPD3 (co-factor) dependent, however significantly derepressed genes that were RPD3 independent. At these loci, nucleosome positions were altered and ISW2 recruitment was reduced in absence of a functional HAND domain. Thus the HAND domain regulates recruitment and remodeling of ISW2 at those genes where ISW2 acts independent of other cofactors. The SANT domain, C-terminal to HAND domain, appears to control the "step size" of nucleosome remodeling and was found to be required for processive nucleosome remodeling by ISW2. Both H4 tail and SANT domain appear to control two distinct stages of ISW2 remodeling. A long alpha helical spacer separates SANT domain from SLIDE domain. SLIDE domain was found to be the protein-protein interaction domain that interacts with accessory Itc1 subunit to maintain ISW2 complex integrity. The two ways by which SLIDE domain regulate ISW2 is by binding or recruitment of ISW2 to promoter regions and additionally by binding independent regulation of both ATPase and remodeling activity. The remodeling mechanism of ISW2 was further compared with another ISWI type remodeler in yeast, Isw1a; using time resolved nucleosome remodeling combined with high resolution site specific histone DNA crosslinking at six different nucleosomal positions to track the movement of the nucleosomes. Nucleosome remodeled by the same remodeler showed discontinuous nucleosome movement between two tracking points indicating formation of small "bulges". One key difference in remodeling mechanism was that although both ISW2 and Isw1a moved nucleosomes towards longer linker DNA, only Isw1a remodeled nucleosomes "backtracked" ~11bp during remodeling. Backtracking of remodeling was prominently observed at nucleosomal regions in close proximity to translocase binding sites suggesting the potentially different mechanisms shared by similar remodeling complexes.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24791号 / 医博第4983号 / 新制||医||1066(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 小林 恭, 教授 小濱 和貴 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Les sarcomes épithélioides sont caractérisés dans 85% des cas par une perte d'expression nucléaire de la protéine SMARCB1, codée par un gène suppresseur de tumeurs situés en 22q11 impliqué dans la génèse des tumeurs rhabdoides malignes. L'exploration par BAC-FISH (Bacterial Artificial Chromosome- Fluorescence In Situ Hybridization) d'une série de 40 sarcomes épithélioides a permis d'établir que cette perte d'expression était secondaire dans 85% des cas à des délétions homozygotes et a mis en évidence le premier cas de sarcome épithélioide associé à une délétion germinale de SMARCB1, altération jusqu'alors uniquement identifiée dans les tumeurs rhabdoides malignes. Nous avons par la suite testé le gène suppresseur de tumeurs SMARCA4 comme gène candidat impliqué dans les sarcomes épithélioides SMARCB1-conservés à partir d'une série rétrospective de 16 cas. SMARCA4 code la sous-unité ATPase du complexe BAF dont SMARCB1 représente une sous unité. Ce screening initial a permis d'identifier 6 cas de sarcomes SMARCA4-inactivés dont la localisation était exclusivement thoracique et dont les caractéristiques clinique et anatomopathologique stéréotypées ont permis le recrutement prospectif et rétrospectif de nouveaux cas. L'étude par RNA-sequencing d'une fraction de notre cohorte (n=13/19) a confirmé leur homogénéité transcriptomique et souligné leur parenté avec les tumeurs rhabdoides SMARCB1 et SMARCA4 déficientes. L'absence de mutation germinale fréquente (n=1/11) a fait proposer le terme de sarcome thoracique SMARCA4-déficient (SMARCA4-DTS) en proscrivant l'utilisation du qualificatif « rhabdoide ». La parenté transcriptomique de ces tumeurs laisse entrevoir des vulnérabilités thérapeutiques communes qui restent à identifier / Epithelioid sarcomas (ES) display loss of SMARCB1 nuclear expression in 85% of cases. SMARCB1 is encoded by a tumor suppressor gene located in 22q11 which was first linked to cancer in malignant rhabdoid tumors. While investigating a series of 40 epithelioid sarcomas with BAC-FISH (Bacterial Artificial Chromosome-Fluorescence In Situ Hybridization), we demonstrated that SMARCB1 loss in ES occurred through genomic deletions in 85% of cases. We were also able to highlight the first case of ES associated with a heterozygous SMARCB1 deletion in the germ line, which feature was previously thought to be restricted to malignant rhadboid tumors (MRT). We subsequently investigated a series of 16 SMARCB1-retained ES to identify its underlying culprit gene with a focus on the candidate tumor suppressor gene SMARCA4. SMARCA4 encodes one of the ATPase subunit of BAF complexes. Interestingly, SMARCB1 is also a core submit of these complexes which regulate chromatin remodeling. We were able to identify a set of 6 cases displaying SMARCA4 inactivation with this discovery cohort. The review of medical records highlighted these cases had similar presentation : all tumors presented with large compressive and aggressive mediastinopulmonary masses. We further recruited 13 cases based on these characteristics including 5 prospective cases. The characterization of their transcriptomes by RNA-sequencing (n=13/19) confirmed their remarkable homogeneity, all our samples clustering together with MRT. However our variant diverge from malignant rhabdoid tumors as it lacks SMARCA4 alteration in the germline (n=0/11) and displays complex polyploidy genetic profiles. We therefore called this new tumor variant “SMARCA4-deficient thoracic sarcoma” (SMARCA4-DTS). The transcriptomic vicinity of SMARCA4-DTS and MRT let foresee they share common therapeutic vulnerabilities

Durant l’hématopoïèse, les cellules souches hématopoïétiques peuvent soit s’autorenouveler soit se différencier dans les divers types de cellules matures constituant le système hématopoïétique. Un des modèles prédominants sur le développement du système hématopoïétique postule une différenciation par étape des différentes lignées le constituant. Ce modèle débute avec les cellules souches hématopoïétiques qui donnent naissance à des précurseurs multipotents qui peuvent à leur tour se différencier en précurseurs dédiées à la lignée lymphoïde ou myéloïde. Bien que la dernière décennie ait apporté de nombreuses connaissances sur les principales signalétiques transcriptionnelles impliquées dans le développement hématopoïétique, le détail des mécanismes moléculaires en jeu expliquant comment les cellules souches hématopoïétiques sont initialement amorcées puis complètement engagées vers une voie de différenciation reste toujours à élucider. Le travail de notre laboratoire indique que l’assemblage combinatoire du complexe de remodelage de la chromatine SWI/SNF est un élément clé parmi les mécanismes épigénétiques qui gouvernent le destin cellulaire et notamment la famille de sous-unités Smarcd qui comporte 3 membre alternatifs Smarcd1/2/3. Des analyses du transcriptome par séquençage haut débit ont montré que l’expression de la sous-unité Smarcd1 du complexe est élevée dans le compartiment des cellules souches, les précurseurs multipotents et les progénitures lymphoïdes tandis que la sous-unité Smarcd2 est principalement exprimée dans les précurseurs myéloïdes. En utilisant des modèles de délétion conditionnelle dans des modèles murins, nous avons démontré que Smarcd1 et Smarcd2 jouent des rôles critiques et lignés spécifiques durant l’hématopoïèse. Dans un premier temps, nous avons pu montrer que Smarcd1 collabore avec le facteur de transcription de la famille bHLH E2A pour spécifier le destin lymphoïde des précurseurs multipotents et qu’elle est donc absolument essentielle pour la lymphopoïèse. Notre travail sur les mécanismes moléculaires en jeu a pu montrer que Smarcd1 interagit directement avec E2A et est nécessaire pour l’accessibilité de la chromatine sur un ensemble de régions enrichies avec les modifications d’histones H3K27ac/H3K3me1 qui marquent des régions activatrices (« enhancer ») impliquées dans l’activation d’une signature lymphoïde dans les précurseurs multipotents. Le blocage de l’interaction entre Smarcd1 et E2A inhibe l’amorce de cette signature lymphoïde et bloque l’émergence de précurseurs destinés à la voie lymphocytaire. Concernant la fonction de Smarcd2, nous avons pu montrer que cette sous-unité est absolument nécessaire pour la granulopoïèse. Les souris ayant subi une délétion génétique de Smarcd2 deviennent très rapidement neutropéniques. Ce phénotype découle d'un blocage au stade de différenciation myélocyte/métamyélocyte, tandis que les autres lignées hématopoïétiques restent non affectées par la délétion. Nous avons pu identifier le facteur de transcription C/ebpƐ comme un partenaire essentiel de Smarcd2 qui interagit avec le complexe SWI/SNF sur les promoteurs de gènes de granules secondaires afin d’en activer la transcription. Les analyses du transcriptome que nous avons effectué lorsque l’interaction de Smarcd2 et C/ebpƐ est interrompue dans des précurseurs de granulocytes ont montré une diminution de l’expression des gènes de granules secondaires liée à une maturation incomplète des granulocytes menant au développement d’un syndrome de myélodysplasie au court du temps. / During hematopoiesis, hematopoietic stem cells (HSCs) either selfrenew or differentiate into all mature blood cell types through successive rounds of binary cell fate decisions. The prevailing model of hematopoiesis predicts a step-by-step model of lineage differentiation in which HSCs first give rise to multipotent progenitors that subsequently differentiate into myeloid and lymphoid restricted progenitors. Although key transcriptional pathways controlling hematopoietic development are beginning to be deciphered, detailed molecular mechanisms explaining how HSCs and progenitors are initially primed and then commit to the different hematopoietic cell lineages are lacking. Work from our laboratory indicates that combinatorial assembly of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex is a key epigenetic mechanism that governs cell fate decisions. Transcriptomics analyses revealed that expression of the Smarcd1 subunit is enriched in hematopoietic stem/progenitors and early lymphoid cells, while Smarcd2 is mainly expressed in myeloid progenitors. Using conditional knock-out mouse models, we demonstrated that Smarcd1 and Smarcd2 subunits perform critical and lineage-specific roles during hematopoiesis. First, we found that Smarcd1 collaborates with the bHLH transcription factor E2A to specify lymphoid cell fate during hematopoiesis and, therefore, is absolutely required for lymphopoiesis. Mechanistically, we showed that Smarcd1 physically interacts with E2A and is required for chromatin accessibility of a set of H3K27ac/H3K4me1-enriched enhancers that coordinate activation of the early lymphoid signature in hematopoietic stem cells. Impairing the interaction between Smarcd1 and E2A inhibits lymphoid lineage determination and the emergence of lymphoid-primed multipotent progenitors. Conversely, we showed that Smarcd2 is absolutely required for granulopoiesis. Smarcd2-deficient mice quickly become neutropenic due to a XIII block at the myelocyte/metamyelocyte stage of granulocyte maturation while other lineages remain unaffected. We discovered that Smarcd2 interacts with the transcription factor C/ebpε to recruit the mSWI/SNF complex on the promoter of secondary granule genes, thus inducing their transcriptional activation. As shown by transcriptomic analysis, impairing this interaction results in decreased expression of secondary granule genes, improper granulopoietic maturation, and development of a myelodysplastic-like syndrome over time. Altogether, this work identifies the Smarcd1 and Smarcd2 subunits of SWI/SNF complexes as master chromatin remodelers allowing the recruitment of lineage-specific transcription factors at key regulatory loci controlling lymphoid lineage priming and granulocyte development, respectively. More globally, these studies highlight that combinatorial assembly of alternative subunits of mSWI/SNF complexes is a key epigenetic mechanism controlling cell fate decisions during hematopoiesis.

<p>The production of developmentally competent in vitro derived embryos is necessary to decreasing both economic and emotional losses. Epigenetic abnormalities/insults have been shown to occur at a higher incidence in in vitro embryos. An increased prevalence of epigenetic derived disorders such as Parkinson’s disease, Prader-Willi syndrome, and α-thalassemia as well as elevated preimplantation embryo arrest and reduced developmental rates are theorized to be caused by errors in the mediation of chromatin remodeling. Chromatin remodeling refers to the restructuring of packaged DNA so that transcription factors are either given more or less access to specific sequences. This can be done by covalent modification through histone methylation, acetylation, and phosphorylation as well as noncovalent modifications which employ ATP dependent chromatin remodeling complexes. The purpose of this thesis was to characterize two structurally integral core subunits, BAF155 and BAF170, of the SWI/SNF chromatin remodeling complex in porcine oocytes and preimplantation embryos. </p> <p>The first study concentrated on the transcript abundance of BAF155 and BAF170 in porcine oocytes and embryos. First, BAF155 and BAF170 transcript sequences were identified in porcine muscle and heart tissues. Those sequences were used to create quantitative polymerase chain reaction (qPCR) primers. mRNA from pools of GV oocytes (100-800) was converted to cDNA for transcript abundance measurements. However, transcript abundance remained too low for either BAF155 or BAF170 to be accurately quantified. </p> <p>The second study focused on developmental competency of embryos post interfering RNA (RNAi) knockdown of BAF155, BAF170, or both BAF155/BAF170 combined. After 7 days of culture, an analysis of variance (ANOVA) was performed to determine differences in mean nuclei numbers and morphological blastocyst percentages across the three groups. No significant difference was seen between means of treatment groups vs. both control groups. Significant differences were seen between siRNA and Non-Injected groups as well as Non-Injected and Scramble RNA groups. However this indicates that loss of BAF155, BAF170, or a combination of the two transcripts is not the driving force of the significant differences, rather the microinjection itself caused the differences.</p> <p>The third study examined the process by which BAF155 and BAF170 proteins are imported from the cytoplasm into the nucleus. It was hypothesized that karyopherin α 7 (KPNA7), a nuclear importer known to be prevalent in the porcine oocyte and early embryo, is the main importer of both subunits. A dominant-negative KPNA7 construct missing the importin beta binding (IBB) domain was microinjected into parthenogenetically activated embryos to outcompete competent wild-type KPNA7. No change in protein localization was seen at the 4-cell stage of development (48 hours post-injection) for either BAF155 or BAF170. To reinforce these results, an RNAi targeting KPNA7 was also microinjected into parthenogenetically activated embryos. Again, no change was shown in protein localization at the 4-cell stage (48 hours post-injection), indicating that KPNA7 was not the main nuclear importer of either BAF155 or BAF170.</p> <p>Further study is necessary to determine transcript abundance and the mechanism of nuclear import of both BAF155 and BAF170.</p><div><br></div>

Dans les cellules eucaryotes l'ADN nucléaire est organisé sous la forme de chromatine, dont l'unité de répétition est le nucleosome. En règle générale, la chromatine est considérée comme répressive pour les processus nécessitant un accès à l'ADN tels que la transcription, la réplication ou la réparation. Le nucléosome représente une forte barrière pour des protéines nécessitant l'accès à l'ADN. Pour surmonter cette barrière, la cellule a développé des méthodes variées, dont la plus importante semble être le remodelage des nucléosomes dépendant de l'ATP. Une propriété commune à tous ces facteurs de remodelage est leur capacité de repositionner les nucléosomes le long de l'ADN.<br /><br />Dans ce travail, nous avons étudié le mécanisme de déplacement des nucléosomes par RSC et SWI/SNF, deux facteurs de remodelage de levure bien caractérisés. Nous avons combiné des approches basées sur la visualisation à haute résolution, notamment la microscopie à force atomique (AFM) et la cryo-microscopie électronique, avec des approches nouvelles à pointe de la biochimie et de la biologie moléculaire. <br /><br />Nous avons montré que la mobilisation des nucléosomes par RSC ou SWI/SNF implique des espèces réactionnelles intermédiaires métastables dont l'existence et la structure étaient jusqu'alors inconnues. Ces particules nucléosomales, que nous avons nommé ‘remosomes', possèdent certaines propriétés structurales distinctes des nucléosomes canoniques. En particulier, les ‘remosomes' contiennent ~180 pb d'ADN associées à l'octamère d'histones au lieu de 147 pb pour les nucléosomes canoniques. En utilisant, l'empreinte à la DNase I nous avons montré que le ‘remosome' représente un ensemble de structures multiples caractérisées par un enroulement fortement perturbé de l'ADN sur l'octamère d'histones. Pour caractériser ces ‘remosomes' avec une grande précision, nous avons mis au point une nouvelle technique « one pot in gel assay » qui consiste à cartographier toutes les 10 pb l'accessibilité d'une enzyme de restriction au ‘remosome' fractionné. L'application de cette technique a révélé que le profil de l'accessibilité du ‘remosome' est très différent de celui du nucléosome. Alors que celui du nucléosome peut être extrapolé par une fonction de type hyperbolique, le profil du ‘remosome' est ajusté par une fonction parabolique. <br /><br />Nous avons voulu répondre à la question du mécanisme de l'inhibition de la mobilisation du nucléosome variant H2A.Bbd par SWI/SNF. En utilisant les techniques décrites plus haut sur des nucléosomes variants ou chimériques (contenant des délétions ou translocations de domaines d'histones) nous avons montré que le domaine d'accrochage (‘docking domain') de l'histone H2A est essentiel pour la mobilisation des nucléosomes. Nous avons aussi montré que l'incapacité du nucléosome à glisser est due à la génération d'états intermédiaires ‘remosomes erronés', distincts de ceux apparaissant dans le cas du nucléosome conventionnel.

abstract: Small Cell Carcinoma of the Ovary Hypercalcemic Type (SCCOHT) is a rare and highly aggressive ovarian cancer that affects children and young women at a mean age of 24 years. Most SCCOHT patients are diagnosed at an advanced stage and do not respond to chemotherapy. As a result, more than 75% of patients succumb to their disease within 1-2 years. To provide insights into the biological, diagnostic, and therapeutic vulnerabilities of this deadly cancer, a comprehensive characterization of 22 SCCOHT cases and 2 SCCOHT cell lines using microarray and next-generation sequencing technologies was performed. Following histological examination, tumor DNA and RNA were extracted and used for array comparative genomic hybridization and gene expression microarray analyses. In agreement with previous reports, SCCOHT presented consistently diploid profiles with few copy number aberrations. Gene expression analysis showed SCCOHT tumors have a unique gene expression profile unlike that of most common epithelial ovarian carcinomas. Dysregulated cell cycle control, DNA repair, DNA damage-response, nucleosome assembly, neurogenesis and nervous system development were all characteristic of SCCOHT tumors. Sequencing of DNA from SCCOHT patients and cell lines revealed germline and somatic inactivating mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 79% (19/24) of SCCOHT patients in addition to SMARCA4 protein loss in 84% (16/19) of SCCOHT tumors, but in only 0.4% (2/485) of other primary ovarian tumors. Ongoing studies are now focusing on identifying treatments for SCCOHT based on therapeutic vulnerabilities conferred by ubiquitous inactivating mutations in SMARCA4 in addition to gene and protein expression data. Our characterization of the molecular landscape of SCCOHT and the breakthrough identification of inactivating SMARCA4 mutations in almost all cases of SCCOHT offers the first significant insight into the molecular pathogenesis of this disease. The loss of SMARCA4 protein is a highly sensitive and specific marker of the disease, highlighting its potential role as a diagnostic marker, and offers the opportunity for genetic testing of family members at risk. Outstanding questions remain about the role of SMARCA4 loss in the biology, histogenesis, diagnosis, and treatment of SCCOHT. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2014