Le complexe de remodelage de la chromatine NuRD, composé des sous-unités catalytiques CHD3 et CHD4, est un régulateur épigénétique de l’expression génique. Nos résultats montrent que NuRD s’associe avec les facteurs de transcription essentiels du mélanome que sont MITF et SOX10. Cependant, malgré une association physique et une co-localisation génomique, CHD4/NuRD ne semble pas agir comme un cofacteur important pour MITF ou SOX10. Néanmoins, la répression de CHD4 conduit à un ralentissement de la prolifération et déréprime l’expression des enzymes PADI1 et PADI3 dans les cellules de mélanome ainsi que dans de nombreux types de cellules cancéreuses. Ainsi, l’induction de ces enzymes, responsables de la conversion des arginines en citrullines, entraîne la citrullination spécifique de PKM2, une enzyme glycolytique essentielle, diminuant ainsi sa sensibilité aux inhibiteurs allostériques, et donc altérant l’équilibre physiologique entre activateurs et inhibiteurs de l’enzyme. L’ensemble de ce travail de thèse a permis de mettre en évidence une nouvelle voie reliant, d’une part la régulation épigénétique de l’expression de PADI1 et PADI3 par CHD4/NuRD ainsi que la reprogrammation de la régulation allostérique de PKM2 via la citrullination d’arginines, au flux glycolytique et au contrôle de la prolifération des cellules cancéreuses d’autre part. / The Nucleosome Remodelling and Deacetylation (NuRD) complex is an epigenetic regulator of gene expression that includes two mutually exclusive ATPase subunits CHD3 and CHD4. Our results show that NuRD associates with essential melanoma cell transcription factors namely MITF and SOX10. However, despite their physical association and genomic co-localization, CHD4-NuRD does not appear to act as a cofactor for MITF or SOX10 regulated gene expression. Nevertheless, CHD4 silencing leads to a slow growth phenotype and de-represses the expression of PADI1 (Protein Arginine DeIminase 1) and PADI3, two enzymes involved in converting arginines to citrullines in melanoma and multiple types of cancer cells. Increased expression of PADI1 and PADI3 enhances citrullination of arginines within the key glycolytic regulatory enzyme PKM2 then promoting excessive glycolysis, lowering ATP levels and slowing down proliferation. PKM2 citrullination lowers its sensitivity to allosteric inhibitors thus shifting equilibrium towards allosteric activators thereby bypassing the normal physiological regulation of glycolysis. Overall, our results lead to describe a novel pathway linking, epigenetic regulation of PADI1 and PADI3 expression by CHD4/NuRD and reprogramming of PKM2 allosteric regulation through arginines citrullination, to glycolytic flux and cancer cell proliferation.

Defective chromatin remodeling proteins are associated with both germline and acquired human disease. PHF6 is encoded by an X-linked gene that is predominantly expressed in the brain and thymus. Structurally, PHF6 contains nuclear and nucleolar localization sequences as well as two ZaP domains, which bind dsDNA. Germline mutations in PHF6 are the cause of BFLS, an XLID, while somatic PHF6 mutations have been identified in T-ALL, AML, and CML. Indeed, screening of a pediatric cohort of nine T-ALL patients revealed a novel H329Q mutation. In a further clinical analysis, T-ALL onset occurred in a 9-year old male BFLS patient with an R342X mutation, suggesting that BFLS might be a cancer predisposition syndrome. To better understand its protein function, recombinant PHF6 was co-immunoprecipitated for a mass spectrometry based proteomic screen. Notably, PHF6 co-purified with multiple constituents of the NuRD complex, an important transcriptional regulator during embryogenesis and lineage commitment with particularly well characterized responsibilities during lymphogenesis. PHF6-NuRD localization was restricted to the nucleoplasm, however PHF6 also co-purified with several ribosomal and splicing proteins. When examined further, PHF6 was found to be recruited to the nucleolus by an RNA-mediated interaction and co-localized within the subnucleolar FC and DFC compartments. ChIP-qPCR revealed that PHF6 binds to transcribed regions of rDNA, resulting in the repression of rRNA. These data thus present a model of PHF6 acting as a tumour suppressor by mediating both nucleoplasmic and nucleolar transcriptional events.

Purpose: Determination of genotypic/phenotypic features of GATAD2B-associated neurodevelopmental disorder(GAND). Methods: Fifty GAND subjects were evaluated to determine consistentgenotypic/phenotypic features. Immunoprecipitation assays utilizing in vitrotranscription–translation products were used to evaluate GATAD2B missensevariants’ ability to interact with binding partners within the nucleosomeremodeling and deacetylase (NuRD) complex. Results: Subjects had clinical findings that included macrocephaly,hypotonia, intellectual disability, neonatal feeding issues, polyhydramnios,apraxia of speech, epilepsy, and bicuspid aortic valves. Forty-one novelGATAD2B variants were identified withmultiple variant types (nonsense, truncating frameshift, splice-site variants,deletions, and missense). Seven subjects were identified with missense variantsthat localized within two conserved region domains (CR1 or CR2) of the GATAD2Bprotein. Immunoprecipitation assays revealed several of these missense variantsdisrupted GATAD2B interactions with its NuRD complex binding partners. Conclusions: A consistent GAND phenotype was caused by a range of geneticvariants in GATAD2B that includeloss-of-function and missense subtypes. Missense variants were present inconserved region domains that disrupted assembly of NuRD complex proteins.GAND’s clinical phenotype had substantial clinical overlap with other disordersassociated with the NuRD complex that involve CHD3 and CHD4, with clinicalfeatures of hypotonia, intellectual disability, cardiac defects, childhoodapraxia of speech, and macrocephaly.

La régulation de la transcription est un processus dynamique faisant intervenir le recrutement de complexes protéiques impliqués dans le remodelage de la chromatine. Parmi eux, mon travail s’est focalisé sur le complexe NuRD (Nucleosome Remodeling and histone Deacetylation) et sa sous-unité de liaison à l’ADN CpG MBD3. Pour cela une approche de biologie structurale intégrative combinant la préparation biochimique, la caractérisation biophysique et l’étude structurale par cryo-EM et cristallographie aux rayons-X a été mise en place. Les caractérisations biophysiques de MBD3 ont permis de mettre en évidence son interaction avec un ADN non-modifié CpG et des cristaux diffractant jusqu’à 3.9 Å ont été obtenu. De plus la région désordonnée en aval du domaine de liaison a été identifiée et son impact dans la formation de complexe caractérisé. Des cristaux pour les différentes constructions en complexe avec l’ADN ont été obtenus et sont actuellement optimisés. Enfin l’optimisation de la purification et la préparation du complexe, ont permis la visualisation du complexe NuRD et mettent en avant pour la première fois une organisation en domaines du complexe. / Transcription regulation of chromatin is a very dynamic process regulated through the recruitment of chromatin-remodeling complexes. My work focuses on NuRD for Nucleosome remodeling and histones deacetylation complex a 1 MDa multi-subunit protein complex and its subunit MBD3 a CpG-binding protein and more precisely on an integrated biology approach of this molecular assembly and its interaction with DNA. It combines biochemical preparation, biophysical characterization, single particle cryo-eletron microscopy and x-ray crystallography. Biophysical analysis show that MBD domain of MBD3 interacts with unmodified CpG DNA, a crystal diffracting up to 3.9 Å were obtained. Moreover a C-terminal intrinsically disordered region of MBD3 were identified and despite is inherent disorder seems to increase the binding affinity of MBD3 for DNA. Crystals were obtained for both constructs in complex with DNA and are currently optimized.Cryo-EM study of NuRD complex allows us to develop and optimized purification and grids preparation for the visualization of the complex. The present results reveal a domain organization of the complex never identify before.

Une approche de biologie structurale intégrative a été mise à profit pour l'étude de l’organisation structurale du complexe NuRD. Mon travail s'est focalisé essentiellement sur trois sous-unités du complexe: MBD3, RbAp46 et RbAp48. J'ai mis en place les protocoles de production et de purification de ces différentes sous-unités, et les ai caractérisé biophysiquement par diverses méthodes. Nous avons ensuite entrepris des études de liaisons sur des nucléosomes reconstitués au laboratoire. Pour MBD3, l'optimisation du complexe nous a permis d'obtenir des cristaux diffractant jusqu'à 7 A de résolution. Parallèlement, une reconstruction 3D préliminaire à partir de données de cryo-microscopie électronique a pu être obtenue à 25A de résolution. Pour RbAp46/48, nous avons pu montrer que ces protéines formaient un complexe stable avec le nucléosome, pavant la voie pour leur future étude structurale par cryo-microscopie électronique ou cristallographie aux rayons-X. / An integrative structural biology approach has been used to study the structural organization of the NuRD complex.My work focused especially on three subunits of this complex: MBD3, RbAp46 and RbAp48. I set up the preparation of the individual subunits and characterized them by various biophysical methods. We next carried out binding assays with homemade human nucleosomes. For MBD3, optimization of the complex led to crystals diffracting up to 7 Å. In parallel, a preliminary 3-D reconstruction at 25 Å resolution has been solved in cryo-EM. For RbAp46/48, crystal we were able to show that these proteins form stable complexes with the nucleosome, paving the way for future structural analysis by cryo-EM or X-ray crystallography.

Chromodomain Helicase DNA-Binding Protein 4 (CHD4) is an ATPase that alters the phasing of nucleosomes on DNA and has recently been implicated in DNA double stranded break (DSB) repair. Here, we show that depletion of CHD4 in Acute Myeloid Leukemia (AML) blasts induces a global relaxation of chromatin that renders cells more susceptible to DSB formation, while concurrently impeding their repair. Furthermore, CHD4 depletion renders AML blasts more sensitive both in vitro and in vivo to genotoxic agents used in clinical therapy: daunorubicin (DNR) and cytarabine (ara-C). Sensitization to DNR and ara-C is mediated in part by activation of the ATM pathway, which is preliminarily activated by a Tip60-dependent mechanism in response to chromatin relaxation and further activated by genotoxic-agent induced DSBs. This sensitization preferentially affects AML cells, as CHD4 depletion in normal CD34+ hematopoetic progenitors does not increase their susceptibility to DNR or ara-C. Unexpectedly, we found that CHD4 is necessary for maintaining the tumor formatting behavior of AML cells, as CHD4 depletion severely restricted the ability of AML cells to form xenografts in mice and colonies in soft agar. Taken together, these results provide evidence for CHD4 as a novel therapeutic target whose inhibition has the potential to enhance the effectiveness of genotoxic agents used in AML therapy.

How tumor cells migrate and metastasize from primary sites requires four major steps: invasion, intravasation, extravasation and proliferation from micrometastases to malignant tumor. The initiation of tumor cell invasion requires Epithelial-Mesenchymal Transition (EMT), by which tumor cells lose cell-cell interactions and gain the ability of migration. The gene expression profile during the EMT process has been extensively investigated to study the initiation of EMT. In our studies, we indicated that tyrosine phosphorylation of human p68 RNA helicase positively associated with the malignant status of tumor tissue or cells. Studying of this relationship revealed that p68 RNA helicase played a critical role in EMT progression by repression of E-cadherin as an epithelial marker and upregulation of Vimentin as a mesenchymal marker. Insight into the mechanism of how p68 RNA helicase represses E-cadherin expression indicated that p68 RNA helicase initiated EMT by transcriptional upregulation of Snail. Human p68 RNA helicase has been documented as an RNA-dependent ATPase. The protein is an essential factor in the pre-mRNA splicing procedure. Some examples show that p68 RNA helicase functions as a transcriptional coactivator in ATPase dependent or independent manner. Here we indicated that p68 RNA helicase unwound protein complexes to modulate protein-protein interactions by using protein-dependent ATPase activity. The phosphorylated p68 RNA helicase displaced HDAC1 from the chromatin remodeling MBD3:Mi2/NuRD complex at the Snail promoter. Thus, our data demonstrated an example of protein-dependent ATPase which modulates protein-protein interactions within the chromatin remodeling machine.

No description available.

Central to the control of virtually all cellular activity is the regulation of gene expression. In eukaryotes, this regulation is greatly influenced by chromatin structure, which is itself regulated by numerous chromatin-remodeling complexes. These are typically large protein complexes with interchangeable subunits that allow for highly specialized functions in different cell types. Moreover, additional specificity can be gained through complexes formed from different subunit isoforms. Histone modifications also regulate chromatin by recruiting remodeling complexes to particular genomic regions. In this thesis we characterize MBD3C, an isoform of the Nucleosome Remodeling and Deacetylase (NuRD) complex subunit MBD3. MBD3 is essential for pluripotency and development, but MBD3C appears to be expressed only in embryonic stem cells (ESCs), and whether it forms a distinct NuRD complex, how its expression is regulated, and its precise function(s) remain unknown. We show that MBD3C forms a complete NuRD complex that functions redundantly with the other MBD3 isoforms in ESC gene regulation. Furthermore, MBD3C binds the SET/MLL complex subunit WDR5 through a conserved motif within its unique N-terminal region, and this interaction is necessary for the regulation of >2,000 ESC genes. Together, these findings indicate that ESCs can utilize isoforms of the same protein to achieve similar functions through diverse mechanisms. The second part of this thesis focuses on the role of the histone modification H3.3K56ac in pluripotency and differentiation. Although H3K56ac is well-studied in yeast, in mammalian cells it is far less abundant and its functions are largely unknown. Our data indicate that the H3.3K56R mutant is largely normal for ESC maintenance and loss of pluripotency markers during differentiation, but H3.3K56ac is necessary for proper lineage commitment. Ongoing studies will characterize the H3.3K56Q phospho-mimetic mutant during differentiation, and examine H3.3K56ac function at lineage-specific genes.

An understanding of the human fetal to adult hemoglobin switch offers the potential to ameliorate β-type globin gene disorders such as sickle cell anemia and β-thalassemia through activation of the fetal γ-globin gene. Chromatin modifying complexes, including MBD2-NuRD and GATA-1/FOG-1/NuRD play a role in γ-globin gene silencing, and Mi2β (CHD4) is a critical component of NuRD complexes. In the studies presented in Chapter 2, we observed that the absence of MBD2 in a sickle cell mouse model leads to a decrease in the number of sickled cells observed in the peripheral blood, and significantly increases survival in these mice. Although further studies will be necessary to fully understand the effect of MBD2 knockout in sickle cell disease mice, absence of MBD2 appears to partially ameliorate the sickle cell anemia phenotype in vivo. In the studies presented in Chapter 3, we observed that knockdown of Mi2β relieves γ-globin gene silencing in β-YAC transgenic murine CID hematopoietic cells and in CD34+ progenitor derived human primary adult erythroid cells. We show that independent of MBD2-NuRD and GATA-1/FOG-1/NuRD, Mi2β binds directly to and positively regulates both the KLF1 and BCL11A genes, which encode transcription factors critical for γ-globin gene silencing during β-type globin gene switching. Remarkably, less than 50% knockdown of Mi2β is sufficient to significantly induce γ-globin gene expression without disrupting erythroid differentiation of primary human CD34+ progenitors. These results indicate that Mi2β is a potential target for therapeutic induction of fetal hemoglobin.