<p>Chromatin is a highly dynamic, regulatory component in the process of transcription, repair, recombination and replication. The BRG1 and SNF2H proteins are ATP-dependent chromatin remodeling proteins that modulate chromatin structure to regulate DNA accessibility for DNA-binding proteins involved in these processes. The BRG1 protein is a central ATPase of the SWI/SNF complexes involved in chromatin remodeling associated with regulation of transcription. SWI/SNF complexes are biochemically hetero-geneous but little is known about the unique functional characteristics of the various forms. We have shown that SWI/SNF activity in SW13 cells affects actin filament organization dependent on the RhoA signaling pathway. We have further shown that the biochemical composition of SWI/SNF complexes qualitatively affects the remodeling activity and that the composition of biochemically purified SWI/SNF complexes does not reflect the patterns of chromatin binding of individual subunits. Chromatin binding assays (ChIP) reveal variations among subunits believed to be constitutive, suggesting that the plasticity in SWI/SNF complex composition is greater than suspected. We have also discovered an interaction between BRG1 and the splicing factor Prp8, linking SWI/SNF activity to mRNA processing. We propose a model whereby parts of the biochemical heterogeneity is a result of function and that the local chromatin environment to which the complex is recruited affect SWI/SNF composition.</p><p>We have also isolated the novel B-WICH complex that contains WSTF, SNF2H, the splicing factor SAP155, the RNA helicase II/Guα, the transcription factor Myb-binding protein 1a, the transcription factor/DNA repair protein CSB and the RNA processing factor DEK. The formation of this complex is dependent on active transcription and links chromatin remodeling by SNF2H to RNA processing.</p><p>By linking chromatin remodeling complexes with RNA processing proteins our work has begun to build a bridge between chromatin and RNA, suggesting that factors in chromatin associated assemblies translocate onto the growing nascent RNA.</p>

Chromatin is a highly dynamic, regulatory component in the process of transcription, repair, recombination and replication. The BRG1 and SNF2H proteins are ATP-dependent chromatin remodeling proteins that modulate chromatin structure to regulate DNA accessibility for DNA-binding proteins involved in these processes. The BRG1 protein is a central ATPase of the SWI/SNF complexes involved in chromatin remodeling associated with regulation of transcription. SWI/SNF complexes are biochemically hetero-geneous but little is known about the unique functional characteristics of the various forms. We have shown that SWI/SNF activity in SW13 cells affects actin filament organization dependent on the RhoA signaling pathway. We have further shown that the biochemical composition of SWI/SNF complexes qualitatively affects the remodeling activity and that the composition of biochemically purified SWI/SNF complexes does not reflect the patterns of chromatin binding of individual subunits. Chromatin binding assays (ChIP) reveal variations among subunits believed to be constitutive, suggesting that the plasticity in SWI/SNF complex composition is greater than suspected. We have also discovered an interaction between BRG1 and the splicing factor Prp8, linking SWI/SNF activity to mRNA processing. We propose a model whereby parts of the biochemical heterogeneity is a result of function and that the local chromatin environment to which the complex is recruited affect SWI/SNF composition. We have also isolated the novel B-WICH complex that contains WSTF, SNF2H, the splicing factor SAP155, the RNA helicase II/Guα, the transcription factor Myb-binding protein 1a, the transcription factor/DNA repair protein CSB and the RNA processing factor DEK. The formation of this complex is dependent on active transcription and links chromatin remodeling by SNF2H to RNA processing. By linking chromatin remodeling complexes with RNA processing proteins our work has begun to build a bridge between chromatin and RNA, suggesting that factors in chromatin associated assemblies translocate onto the growing nascent RNA.

The Hedgehog (HH) signalling pathway and its primary transducer, GLI2, regulate cardiomyogenesis in vivo and in differentiating P19 embryonal carcinoma (EC) cells. To further assess the role of HH signalling during mouse embryonic stem (mES) cell differentiation, we studied the effects of GLI2 overexpression during mES cell differentiation. GLI2 overexpression resulted in temporal enhancement of cardiac progenitor genes, Mef2c and Nkx2-5, along with enhancement of Tbx5, Myhc6, and Myhc7 in day 6 differentiating mES cells. Mass spectrometric analysis of proteins that immunoprecipitate with GLI2 determined that GLI2 forms a complex with BRG1 during mES cell differentiation. Furthermore, modulation of HH signalling during P19 EC cell differentiation followed by chromatin immunoprecipitation with an anti-BRG1 antibody determined that HH signalling regulates BRG1 enrichment on Mef2c. Therefore, HH signalling accelerates cardiac progenitor gene expression during mES cell differentiation potentially by recruiting a chromatin remodelling factor to at least one cardiac progenitor gene.

Le récepteur des glucocorticoïdes (GR) est un facteur de transcription, activé par la libération de glucocorticoïdes (GCs) en réponse à un stress. Il est impliqué dans la modulation des comportements influencés par le stress tels que les émotions, les comportements sociaux et l’addiction. L’invalidation du GR dans les neurones dopaminoceptifs (GRD1Cre) entraine une diminution des réponses à la cocaïne. Afin d’identifier les gènes cibles du GR, une analyse comparative du transcriptome de souris contrôles et GRD1Cre, en conditions basales, suite à un traitement à la cocaïne ou aux GCs, a été réalisée. Le GR agit principalement comme activateur de la transcription dans les neurones dopaminoceptifs, quelque soit le traitement. De plus, une corrélation entre les gènes régulés par le GR suite à un traitement aux GCs et ceux régulés suite à un traitement à la cocaïne existe. Le GR contrôle l’expression de ses gènes par des mécanismes variés dont certains impliquent le recrutement du complexe de remodelage de la chromatine SWI/SNF. Ce complexe est composé d’une ATPase catalytique qui peut être soit Brahma (BRM), soit BRG1 (Brahma-Releated Gene 1), deux protéines qui partagent un haut degré d’homologie. Nos résultats montrent que le GR interagit avec l’une et l’autre dans le striatum, structure clé dans l’addiction. En utilisant des souris invalidées pour BRM (BRM-/-) et/ou spécifiquement pour BRG1 (BRM-/-BRG1D1Cre et BRG1D1Cre), un rôle différentiel de BRM et BRG1 a été mis en évidence. Alors que BRM n’est pas nécessaire aux réponses à la cocaïne, BRG1 dans les neurones dopaminoceptifs est essentiel aux réponses à cette drogue. BRG1 est également impliqué dans les réponses locomotrices à la morphine. De manière différente, BRG1 et BRM se compensent dans les comportements émotionnels. / The glucocorticoid receptor (GR) is a transcription factor involved in the stress response, activated by glucocorticoids (GCs). Inactivation of GR in dopaminoceptive neurons (GRD1Cre) induces a decrease of behavioral and cellular responses to cocaine. In order to identify the GR target genes, we performed thorough transcriptome analyses using controls and GRD1Cre mice under basal conditions, after treatment of cocaine or GCs. We highlighted that GR acts predominantly as a transcriptional activator within dopaminoceptive neurons. We then identified a closed link between genes induced in responses to an acute stress and in response to cocaine. GR controls expression of its target genes by numerous mechanisms. We examined the putative contribution of the SWI/SNF chromatin remodeler complexes. This multi-subunit complex contains either BRM (Brahma) or BRG1 (Brahma-related gene 1) as central ATPase subunit. We detected an interaction between GR and BRG1, and between GR and BRM in the striatum. To understand the role of BRM and BRG1 proteins, we developed animals with a constitutive BRM inactivation (BRM-/-) and/or with a specific deletion of BRG1 in dopaminoceptive neurons (BRM-/-BRG1D1Cre, BRG1D1Cre mice). A differential role of theses two proteins has been highlighted. While BRM is not required for behavioral responses to cocaine, BRG1 within dopaminoceptive neurons is essential. BRG1 is also involved in locomotor responses to morphine. Nevertheless, inactivation of either BRM or BRG1 gene in dopaminoceptive neurons did not change anxiety-like behaviors.

CD8+ T cells are essential for host protection against intracellular pathogens and tumors. During antigen-driven responses, CD8+ T cell fate is governed by transcriptional and epigenetic processes that allow naïve CD8+ T cells to develop into a wide range of effector and conventional memory cell subsets. Over the last decades, novel techniques and major efforts led to a better understanding of the origin, nature, and short- and long-term effects of these processes on individual CD8+ T cells. Under certain conditions, naïve CD8+ T cells can acquire memory phenotype and functions in an antigen-independent manner. Although homeostatic cytokines and initial activation pathways that drive the development of these unconventional memory cells had been identified, the ensuing transcriptional profile of these cells and their degree of similarity with conventional memory cells remained ill-defined. The epigenetic events that accompany unconventional memory formation were also not known.Here, we show that innate memory cells, a type of thymic unconventional memory cells, are transcriptionally close to conventional memory cells but only partially epigenetically programmed toward the full memory fate. We also show that the sole overexpression of the transcription factor Eomesodermin (EOMES), a master regulator of effector and conventional memory cells, is able to drive many of the phenotypical, functional, transcriptional, and epigenetic features of innate memory cells, and to induce the recruitment of BRG1, a member of chromatin remodeling complexes, to innate memory gene regulatory regions. We further show that the in vivo interleukine-4-dependent development of innate memory cells is largely dependent on BRG1. We bring to light that, in innate memory cells, EOMES is recruited in many instances to genomic regions previously bound by the transcription factor RUNX3. Overall, we provide insights into the mechanisms that allow memory cell formation and T cell receptor stimulation to be uncoupled. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

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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

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

SMARCA4 är en gen som kodar för ett protein vid namn Brahma-related gene 1 (BRG1). BRG1 utgör en väsentlig katalytisk subenhet av SWI/SNF-komplexet, vilket är ett proteinkomplex utvecklat för kromatinremodellering. Att modulera kromatinstrukturen bidrar till att viktiga funktioner, såsom transkription och celldifferentiering, kan utföras. En mutation i SMARCA4-genen kan ge upphov till en lungtumör med benämningen Thoracic SMARCA4-deficient undifferentiated tumor (SMARCA4-UT), vilken kännetecknas av en förlust av uttrycket BRG1 i de maligna cellerna. Då tumören kan variera i morfologiskt utseende och i många fall påminna om andra tumörtyper, kan tillämpning av immunhistokemiska metoder vara till stor hjälp vid den medicinska diagnosticeringen. Syftet med denna studie var att vidareutveckla ett befintligt protokoll för att uppnå en optimal immunhistokemisk infärgning av SMARCA4 (BRG1), för att diagnostiskt kunna differentiera mellan olika lungtumörer. För detta ändamål testades flera olika infärgningsprotokoll och kontrollvävnader, samt två antikroppar av olika kloner från företagen Abcam och Santa Cruz. Infärgning med Abcam-antikroppen (klon EPNCIR111A) uppvisade intensiva och specifika resultat, medan Santa Cruz-antikroppen (klon G-7) gav upphov till negativa infärgningar, trots flera försök till optimering. I samråd med en patolog fastställdes det infärgningsprotokoll som gav bäst resultat, vilket inkluderade en förbehandling bestående av buffert CC1 (64 min, 95° C), antikroppsinkubation med Abcam-antikroppen (32 min, 36° C, spädning 1:100), detektion med ultraView Universal DAB Detection Kit, samt kontrastfärgning med hematoxylin (8 min) och blåning (4 min). Denna immunfärgning kommer att införas som ny metod och användas inom den kliniska verksamheten. Däremot kommer metoden att fortsätta prövas under en tid framöver, för att helt säkerställa dess pålitlighet och kunna valideras inför framtida bruk.

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