Browsing by Author "Wang, Jinhua"

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    Alpha-defensin gene single nucleotide polymorphism shapes the gut microbiota
    (2019-04-24) Rashidi, Armin; Herman, Adam; Wang, Jinhua; Blazar, Bruce; Weisdorf, Daniel; aherman@umn.edu; Herman, Adam; Division of Hematology, Oncology, and Transplantation - Department of Medicine
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    Structure and mechanism of chromatin remodeler SWI/SNF
    (2019-12) Wang, Jinhua
    SWI/SNF complex, the ATP-dependent chromatin remodeler, alters the interactions between DNA and histones using the energy of ATP hydrolysis, modifying gene expression by controlling the accessibility of DNA to transcription factors. The gene-specific transcription is regulated through functional interactions within different subunits of SWI/SNF and associated co-factors. Genes encoding subunits of the SWI/SNF are mutated in more than 20% cancers, which has attracted a great deal of interest. Despite the huge amount of genetic, biochemical and structural biology data published, the mechanism by which SWI/SNF complex binds and remodels nucleosomes remains to be fully elucidated. High-resolution structure determination will certainly be invaluable for understanding the process in mechanistic detail. Given the dimensions, the complexity and the flexibility of the complex, single-particle cryogenic electron microscopy (cryo-EM) would be the optimal method of choice. However, it is difficult to purify and solve the structure of the whole multi-subunit macromolecular SWI/SNF complex. The highly conserved subunits, BRG1, BAF155, and BAF47 form a stable complex that retains nearly full remodeling activity and therefore have been regarded as the core components of SWI/SNF complex. Here we expressed the core sub-complex composed of BRG1, BAF155 and BAF47, with baculovirus expression system in Sf9 insect cells, and purified the complex with size exclusion chromatography. The result of negative staining electron microscopy showed that the protein sample of core SWI/SNF complex formed homogenous single particles after stabilization by Gradient Fixation (GraFix) method. We reconstituted nucleosome by wrapping DNA around recombinant histone octamer and achieved nucleosome structure by cryo-EM. The core SWI/SNF complex had the capability to bind nucleosome assessed by electrophoretic mobility shift assay (EMSA). In future direction, the purified recombinant core SWI/SNF complex will be used for binding to reconstituted nucleosome and applied to cryo-EM for structure determination. Our work will illustrate the structure of chromatin remodeler engaging with its substrate, shedding light on the chromatin remodeling mechanism of SWI/SNF complex.