Il y a presque 20 ans, la mise en évidence de mutations bi-alléliques inactivatrices du gène SMARCB1 dans les tumeurs rhabdoïdes établissait la première démonstration d’altérations du complexe SWI/SNF de remodelage de la chromatine en oncologie. Depuis, l’avènement des techniques d’analyse moléculaire à haut débit appliquées à la cancérologie a permis de montrer que des altérations dans d’autres gènes du complexe SWI/SNF était présentes dans un très grand nombre de cancers. A travers la présentation de plusieurs types de tumeurs SWI/SNF déficientes et de nos modèles d’étude des tumeurs rhabdoïdes, nous montrons que la perte de SMARCB1 est associée à une augmentation de la biosynthèse de la sérine et des voies métaboliques en aval importantes pour l’oncogenèse. Ces résultats pourraient aboutir à une option thérapeutique pour les tumeurs rhabdoïdes voire, plus généralement, pour d’autres modèles de tumeurs SWI/SNF-déficientes. Enfin, la mise en perspective de ces changements métaboliques avec les altérations épigénétiques observées dans les tumeurs SWI/SNF déficientes pourrait se révéler pertinente pour continuer d’approfondir nos connaissances sur ces tumeurs. / Nearly 20 years ago, the demonstration of truncated bi-allelic mutations in the SMARCB1 gene in rhabdoid tumors established the first demonstration of alterations in the SWI/SNF chromatin remodeling complex in oncology. Since then, the advent of high-throughput molecular analysis techniques applied to oncology has shown that alterations in other genes of the SWI/SNF complex are present in a wide variety of cancers. Through the presentation of several types of SWI/SNF deficient tumors and our models of rhabdoid tumors, we show that the loss of SMARCB1 is associated with an increase of the serine biosynthesis pathway and the downstream metabolic pathways important for oncogenesis.These results could lead to a therapeutic option for rhabdoid tumors or, more generally, for other models of SWI/SNF-deficient tumors. Finally, the prospect of these metabolic changes with the epigenetic alterations observed in SWI / SNF deficient tumors may be relevant to continue to deepen our knowledge of these tumors.

Chromatin remodelers are ATP-dependent multisubunit assemblies that regulate transcription and other processes by altering DNA-histone contacts. The mechanism of action is based on the transduction of energy released by ATP hydrolysis to translocation on DNA and ultimately the movement of histones in cis or trans. Though the critical ATP burning and translocation activities are fulfilled by a conserved ATPase domain in the catalytic subunit, there are accessory domains and subunits that are speculated to regulate these activities. Important questions in the field center around the identification of these domains and subunits, whether they affect complex formation, substrate affinity or a critical step in remodeling. If they do affect remodeling, what is the structural basis of the regulatory activity. In this study, these questions have been addressed using the prototype remodeler SWI/SNF from budding yeast. ySWI/SNF is a 12 subunit complex that includes the catalytic subunit Swi2/Snf2. It affects 6% of the yeast genome being primarily involved in gene activation. We employed a systematic protein or domain deletion strategy and characterized the mutant complexes in vitro and in vivo. A key finding was that SWI/SNF is organized in distinct structural modules and that the Snf2 module regulates most of its activities. Snf2 is a central subunit in this module and the function of conserved regions within Snf2 were studied. The N terminus preceding the HSA and ATPase domain has three major roles - complex assembly, recruitment and regulation of catalytic activity. A novel SnAC domain located C terminal to ATPase domain was identified to play critical role in coupling ATP hydrolysis to nucleosome movement by acting as a histone anchor. Finally the tandem AT-hooks between SnAC and bromodomain serve as DNA binding domains but also affect ATPase activity and nucleosome mobilization independent of its binding activity. Taken together, this study provides a comprehensive overview of the function of regulatory domains in SWI/SNF.

Thesis (Ph.D.)--Indiana University, 2009. / Title from screen (viewed on December 1, 2009). Department of Cellular and Integrative Physiology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): B. Paul Herring, Anthony B. Firulli, Frederick M. Pavalko, Simon J. Rhodes. Includes vitae. Includes bibliographical references (leaves 138-149).

No description available.

In higher eukaryotes, DNA is packaged with histones and other proteins into chromatin. While this is important in the control of unwanted gene expression, chromatin also serves as a barrier to many vital functions in the cell. Therefore, cells have evolved many different types of chromatin remodeling enzymes to contend with this inhibitory structure and enable gene expression and other functions. The Saccharomyces cerevisiae PHO5 gene is triggered in response to phosphate starvation. In this study, I evaluate the chromatin remodeling requirements of this gene with respect to the multisubunit complexes SWI/SNF and SAGA. I show, for the first time, physical recruitment of SWI/SNF to the PHO5 promoter. I also demonstrate the role of promoter occupancy in influencing requirements for chromatin remodeling enzymes. Further, I describe various interactions between these two complexes at the PHO5 promoter. This study presents evidence for the first instance of excess recruitment of an ATP-dependent remodeler potentially compensating for the lack of a histone acetyltransferase.

No description available.

AN ABSTRACT OF THE DISSERTATION OF SOLOMON G. HAILU, for the Doctor of Philosophy degree in Molecular Biology, Microbiology and Biochemistry, presented on August 22, 2017, at Southern Illinois University, School of Medicine. TITLE: DISTINCT GENOME WIDE FUNCTIONS OF CHROMATIN REMODELERS IN NUCLEOSOME ORGANIZATION AND TRANSCRIPTION REGULATION MAJOR PROFESSOR: Dr. Blaine Bartholomew Chromatin remodelers are conserved from yeast to humans and are the gatekeepers of chromatin. They regulate transcription by occluding or exposing DNA regulatory elements globally. They are crucial for DNA processes such as DNA replication, repair and recombination. In addition, they are critical in developmental processes and differentiation. Chromatin remodelers are categorized into several families based on their conserved ATPase domain, an essential component required for their DNA translocation ability. In this study, we investigated the role yeast ISWI and SWI/SNF family of chromatin remodelers play on nucleosome rearrangement and transcription regulation by targeted mutagenesis of domains in accessory subunits and at the C-terminus of the catalytic subunit. All members of the ISWI family (ISW1a, ISW1b, ISW2) share a conserved C-terminal HAND, SANT and SLIDE domains, which are important for sensing linker DNA. We find an auto-regulation of ISWI complexes by the SLIDE domain, independent of the histone H4 Nterminal tail. Our protein-protein chemical crosslinking and mass spectrometry (CX-MS) analysis indicate that the SLIDE domain regulates the ATPase core through N terminal domains of the accessory subunit Itc1. Moreover, we show that the accessory subunits of ISWI modulate the ATPase activity and specificity of ISWI complexes. The DNA sensing ability of the SLIDE domain is required for the in vivo nucleosome spacing and transcription regulation by ISWI. We find that while ISW2 primarily regulates transcription at the 5’ end of genes, ISW1a is important in transcription elongation by rearranging nucleosomes starting at the +2 nucleosome and through the rest of the body of genes towards the 3’ end. ISW1b on the other hand rearrange nucleosomes in the gene body to facilitate suppression of cryptic transcription. For the first time, we show the potential division of labor between ISW1a and ISW1b during transcription elongation. On the other hand, SWI/SNF chromatin remodelers are essential epigenetic factors that are frequently mutated in cancer and neurological disorders. They harbor a C-terminal SnAC and AT hook domains that positively regulate their DNA dependent ATPase activity and nucleosome mobilizing capabilities. By deleting the AT hook motifs, we have identified the role of SWI/SNF in organizing the -1 and +1 nucleosomes at transcription start sites flanking the nucleosome free region (NFR). Our RNA-seq analysis shows SWI/SNF positively regulates the bi-directional transcription of non-coding RNA (ncRNA) which are activated when the AT hook motifs are deleted. Moreover, AT hooks regulate such activities of SWI/SNF through direct protein-protein interactions with the ATPase core as evidenced by our chemical crosslinking and mass spectrometry (CX-MS) analysis.

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.

Coordination of gene expression within the cell requires the integrated actions of various multi-protein, gene regulatory complexes. The Mediator and BRG1 Associate Factor (BAF) complexes are large, dynamic regulatory cofactors (COF) that are made up of multiple different submodules, and play key roles in regulating gene expression. Gene-specific regulation requires that transcription factors (TFs) recruit these COF complexes to gene promoters. How separate subdomains in each complex interact with distinct sets of TFs in each cell remains an important question. In this study, to address this question, we sought to apply the nuclear extract protein-binding microarray (nextPBM) technology being developed in our lab to study interactions between TFs and subunits of the Mediator and BAF complexes. To facilitate this, we cloned, expressed and purified subdomains of proteins from the Mediator and BAF complexes. We then used the nextPBM technology to study the interactions of our subdomains with TFs in human macrophages. We identified several new interactions with TFs, and demonstrate the utility of this approach to student TF-COF interaction.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21645号 / 医博第4451号 / 新制||医||1034(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 羽賀 博典, 教授 小西 靖彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM