Over the past five years the role of epigenetic modifiers in brain development has become increasingly evident. In this regard, Snf2l, a homolog of the chromatin remodeling protein ISWI, was shown to have enriched expression in the brain and be important for neuronal differentiation. Mice lacking functional Snf2l have hypercellularity of the cerebral cortex due to increased cell cycle re-entry. In this thesis I demonstrate the effects of Snf2l-ablation on cortical progenitor cells including increased proliferation and cell cycle deregulation, the consequence of which is a delay in neuronal migration and altered numbers of mature cortical neurons. This phenotype arises from increased expression of Foxg1, a winged-helix repressor expressed in the forebrain and anterior optic vesicle. Moreover, genetically reducing its overexpression rescues the Snf2l-ablated phenotype. Snf2l is bound directly to a promoter region of Foxg1 suggesting that it acts as a repressive regulator in vivo and is an important factor in forebrain differentiation.

The packaging of genomic DNA into nucleosomes creates a barrier to transcription which can be relieved through ATP-dependent chromatin remodeling via complexes such as the switch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complex. The SWI/SNF complex remodels chromatin via conformational or positional changes of nucleosomes, thereby altering the access of transcriptional machinery to target genes. The SWI/SNF complex does not possess intrinsic DNA binding ability, and therefore its recruitment to target loci requires interaction with DNA-associated transcription factors. The Cdx family of homeodomain transcription factors (Cdx1, Cdx2 and Cdx4) are essential for a number of developmental programs in the mouse. Cdx1 and Cdx2 also regulate intestinal homeostasis throughout life. Although a number of Cdx target genes have been identified, the basis by which Cdx members impact their transcription is poorly understood. We have found that Cdx members interact with the SWI/SNF complex and make direct contact with Brg1, a catalytic member of SWI/SNF. Both Cdx2 and Brg1 co-occupy a number of Cdx target genes, and both factors are necessary for transcriptional regulation of such targets. Finally, Cdx2 and Brg1 occupancy occurs coincident with chromatin remodeling at certain of these loci. Taken together, our findings suggest that Cdx transcription factors regulate target gene expression, in part, through recruitment of Brg1-associated SWI/SNF chromatin remodeling activity.

Chromatin remodeling enzymes are instrumental for neural development as evidenced by their identification as disease genes underlying human disorders characterized by intellectual-disability. In this regard, the murine Snf2h and Snf2l genes show differential expression patterns during embryonic development, with a unique pattern in the brain where Snf2h is predominant in neural progenitors, while Snf2l expression peaks at the onset of differentiation. These observations led me to investigate the role of Snf2h and Snf2l in brain development by using conditionally targeted Snf2h and Snf2l mice. I selectively ablated Snf2h expression in cortical progenitors, cerebellar progenitors, or postmitotic Purkinje neurons of the cerebellum, while Snf2l was deleted in the germline. I found that Snf2h plays diverse roles in neural progenitor expansion and postmitotic gene expression control, while Snf2l is involved in the precise timing of neural differentiation onset. Gene expression studies revealed that Snf2h and Snf2l co-modulate the FoxG1 and En1 transcription factors during cortical and cerebellar neurogenesis, respectively, to precisely control the transition from a progenitor to a differentiated neuron. Moreover, Snf2h is essential for the postmitotic neural activation of the clustered protocadherin genes, and does so by functionally interacting with the matrix-attachment region protein Satb2. My neurobehavioral studies also provided insight into how Snf2h loss in cerebellar progenitors results in cerebellar ataxia, while Snf2h loss in cortical progenitors, or in postmitotic Purkinje neurons of the cerebellum, resulted in learning and memory deficits, and hyperactive-like behavior. Molecularly, Snf2h plays an important role in linker histone H1e dynamics and higher order chromatin packaging, as evidenced by loss of chromatin ultrastructure upon Snf2h deletion in progenitor and postmitotic neurons. I further demonstrated that Snf2h loss in a neuronal cell culture model results in reduced H1e deposition, and that overexpression of human SNF2H or SNF2L upon Snf2h knockdown rescues this biochemical dysfunction. My experiments suggest that Snf2h and Snf2l are regulatory nucleosome remodeling engines that co-modulate the gene expression programs necessary for proper brain development, maturation and function.

Eukaryotes organize their genomes by wrapping DNA around positively charged proteins called histones to form a structure known as chromatin. This structure is ideal for keeping the genome safe from damage, but also becomes an obstacle for the transcriptional machinery to access information stored in the DNA. To facilitate a balance between storage and accessibility, eukaryotes utilize a family of enzymes known as ATP-dependent chromatin remodelers to directly manipulate chromatin structure. The diverse activities of these chromatin remodeling enzymes range from simply sliding nucleosomes to reveal transcription start sites, to editing the composition of a nucleosome by exchanging canonical histones for histone variants. Chromatin remodeling enzymes recognize features of the nucleosome that activate their ATPase domains and enable proper remodeling function. One nuclear epitope that has been extensively studied is the nucleosomal acidic patch. This negatively charged region on the face of the nucleosome has been shown to be essential for remodeling enzymes like Chd1, ISWI, and INO80C. The chromatin remodeler SWR1C edits nucleosomes by removing the canonical histone H2A from nucleosomes and exchanges it for the histone variant H2A.Z, but the role of the acidic patch in this process has not been investigated. In this work, I showed that SWR1C has normal binding affinity to acidic patch mutant nucleosomes and retains ATPase stimulation but can no longer exchange dimers on this substrate. This work also identified a novel arginine anchor on the essential SWR1C subunit, Swc5, that binds specifically to the nucleosomal acidic patch. The data in this work suggest a mechanism where SWR1C engages nucleosomes and uses the Swc5 subunit to recognize the nucleosomal acidic patch to couple ATPase activity to histone dimer exchange.

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.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24199号 / 医博第4893号 / 新制||医||1060(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 武藤 学, 教授 斎藤 通紀, 教授 浅野 雅秀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23797号 / 医博第4843号 / 新制||医||1058(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 藤田 恭之, 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

Au sein du noyau, l'ADN est organise en chromatine dont l'unité de base est le nucléosome. La structure de la chromatine est très dynamique, ce qui est nécessaire pour la plupart des opérations qui se produisent dans l'ADN telles que la réplication, la transcription, la réparation et la recombinaison. Le nucléosome est constitué de deux dimères H2A/H2B et deux dimères H3/H4 associés avec 147 paires de bases d'ADN. La protéine Nap1 est un chaperon d'histone H2A/H2B impliquée dans l'assemblage et démontage des nucléosomes. Nap1 protège les interactions non spécifiques entre l'ADN chargé négativement et les dimères H2A/H2B chargés positivement, afin de permettre la formation de la structure ordonnée des nucléosomes. Lors de l'assemblage des nucléosomes, les dimères d'histones H3/H4 sont déposés en premier lieu, suivi par le dépôt de dimères H2A/H2B. Lors du démontage du nucléosome, les dimères H2A/H2B sont retirés avant le retrait des dimères H3/H4. La determination de la structure du complexe Nap1-H2A/H2B pourra permettre une meilleure compréhension du processus d'assemblage du nucléosome. Dans cette étude, nous voulons comprendre comment le chaperon Nap1 cible spécifiquement les dimères d'histones H2A/H2B pour l'assemblage des nucléosomes. Notre objectif est de caractériser la structure et la fonction du complexe de Nap1-H2A/H2B. Ainsi nous nous sommes tout d'abord intéresse à la stoechiometrie de ce complexe. Nous avons trouvé qu'un dimère de Nap1 s'associe à un dimère H2A/H2B (Nap1_2-H2A/H2B). D'autre part, l'analyse par spectrométrie de masse non-dénaturante a montré que ce complexe de base peut s'oligomériser et contenir jusqu'à 6 copies de Nap1_2-H2A/H2B. L'analyse de ce complexe par spectrométrie de masse non-dénaturant a montré que ce complexe peu oligomériser dans un grand complexe contenant jusqu'à 6 copies de Nap1_2-H2A/H2B. Nous avons également obtenu la première structure cristalline à basse résolution de ce complexe. L'analyse du même complexe par microscopie électronique à coloration négative a révélé la présence en solution du même oligomère que dans l'unité asymétrique du cristal, qui contient aussi 6 copies de Nap1_2-H2A/H2B. Ainsi, nous avons pu mettre en évidence de nouvelles interfaces d'interaction entre les différents composants de ce complexe qui nous permettent de mieux comprendre le processus d'assemblage des nucléosomes. Le remodelage de la chromatine permet l'expression des gènes eucaryotes. Ce remodelage nécessite des enzymes telles que des histone acétyltransférases (HAT) et les chaperons d'histones. Les HATs acétylent les chaînes latérales des lysines. Il a été proposé que les HATs et les histones chaperons agissent en synergie pour moduler la structure de la chromatine pendant la transcription. La HAT p300 a été proposé d'interagir avec l'histone chaperon Nap1. Nous avons entrepris de caractériser cette interaction. Malheureusement, nos expériences n'ont pas pu détecter d'interaction directe entre ces protéines. / Assembly of chromatin is an essential process that concerns most DNA transactions in eukaryotic cells. The basic repeating unit of chromatin are nucleosomes, macromolecular complexes that consist of a histone octamer that organizes 147 bp of DNA in two superhelical turns. Although, the structures of nucleosomes are known in detail, their assembly is poorly understood. In vivo, nucleosome assembly is orchestrated by ATP-dependent remodelling enzymes, histone-modifying enzymes and a number of at least partially redundant histone chaperones. Histone chaperons are a structurally diverse class of proteins that direct the productive assembly and disassembly of nucleosomes by facilitating histone deposition and exchange. The currently accepted model is that nucleosome assembly is a sequential process that begins with the interaction of H3/H4 with DNA to form a (H3/H4)2 tetramer-DNA complex. The addition of two H2A/H2B dimers completes a canonical nucleosome. High-resolution structures of histone chaperons in complex with H3/H4 histones have resulted in detailed insights into the process of nucleosome assembly. However, our understanding of the mechanism of nucleosome assembly has been hampered by the as yet limited number of co-crystal structures of histone–chaperone complexes. In particular it remains unclear how histone chaperons mediate H2A/H2B deposition to complete nucleosome assembly. In this work, we have investigated the role of the H2A/H2B chaperon Nap1 (Nucleosome assembly protein 1) in nucleosome assembly. We have determined the crystal structure of the complex between Nap1 and H2A/H2B and analysed the assembly by various biophysical methods. The structure shows that a Nap1 dimer binds to one copy of H2A/H2B (Nap1_2-H2A/H2B). A large ~550 kDa macromolecular assembly containing 6 copies of the Nap12-H2A/H2B complex is seen in the asymmetric crystallographic unit. We confirmed by both non-denaturing mass spectroscopy and negative stain electron microscopy studies that this assembly is the predominant form of the Nap1_2-H2A/H2B complex in solution. We further investigated the potential interplay between p300-mediated histone acetylation and nucleosome assembly. Together, the structure and associated functional analysis provide a detailed mechanism for the Nap1 chaperon activity, its role in H2A/H2B deposition and in nucleosome assembly.

<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>