Structure of the ISW1a complex bound to the dinucleosome
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE240192 | GEO | 2023/08/09
REPOSITORIES: GEO
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Project description:Nucleosomes, the basic repeating units of chromatin, form evenly spaced arrays inside the cell. Chromatin remodelers alter the positions of nucleosomes, and are vital in regulating chromatin organization and gene expression. Here we report the cryoEM structure of the chromatin remodeler ISW1a complex bound to the dinucleosome. Both subunits of the complex recognize one nucleosome. The motor subunit binds at the canonical position of the nucleosome, with two arginine anchors recognizing the acidic patch. The DNA-binding module interacts with the entry linker DNA at the nucleosome edge, consistent with the protein ruler model in nucleosome spacing. The Ioc3 subunit recognizes the disk face of the adjacent nucleosome, being important for the spacing activity in vitro, and for chromatin organization in vivo. Together, our findings illustrate a new mode of chromatin remodeling in the context of higher-order chromatin
2023-05-15 | GSE203073 | GEO
Project description:Nucleosome arrays begin at nucleosome-free promoter regions (NFRs) and regulate gene expression. Reconstituting such organization throughout a genome with purified proteins is a critical challenge in establishing biochemical mechanisms for chromosome assembly. Here we establish a four-step hierarchical building plan for yeast genomic nucleosome organization using only purified components: genomic DNA, histones, site-specific organizing factors Abf1 and Reb1, and the chromatin remodelers RSC, ISW2, INO80, and ISW1a. First, RSC makes NFRs by translating promoter poly(dA:dT) tracts into directional nucleosome removal. Second, +1 nucleosomes are positioned by INO80 at most genes potentially involving DNA shape, or by ISW2 using gene-specific Abf1 and Reb1. Third, INO80 or ISW2 create arrays with wide spacing. Fourth, ISW1a tightens the spacing and creates properly positioned arrays. We conclude that entire genomes use a simple set of rules and proteins, without transcription, to build a common chromatin architecture. In this study, nucleosomes were assembled using Salt Gradient Dialysis (SGD) on yeast genomic DNA library. Assembled nucleosomes were either left untreated (labelled as "SGD", control), treated with whole cell extract (WCE), mutant extracts (rsc3ts WCE, isw1 isw2 chd1 WCE), purified remodelers; singly or in combinations (RSC, ISW1a, ISW1b, ISW2, INO80, CHD1, SWI/SNF), combinations of mutant extracts and chromatin remodelers or combination of General Regulatory Factors (Abf1, Reb1) and chromatin remodelers. The resulting nucleosome positions were mapped genome-wide using MNase-(anti-H3-ChIP)-Seq.
2016-08-14 | E-GEOD-72106 | biostudies-arrayexpress
Project description:Nuclear proteins bind chromatin to execute and regulate genome-templated processes. While structural and biochemical studies of individual nucleosome interactions have suggested that an acidic patch on the nucleosome disk surface may be a common site for recruitment to chromatin, the pervasiveness of acidic patch binding and whether other nucleosome surface binding hot-spots exist remains unclear. Here, we use nucleosome affinity proteomics with a library of nucleosomes that collectively disrupts all exposed histone surfaces to establish the universal principles of nucleosome binding. We find that the acidic patch and two adjacent surfaces are the primary hot-spots for nucleosome disk binding and are critical for the majority of nucleosome-protein interactions. In contrast, nearly half of the nucleosome disk surface participates only minimally in protein binding. In addition to establishing the fundamental principles of chromatin binding, our screen defines nucleosome surface requirements of nearly 300 nucleosome interacting proteins implicated in diverse nuclear processes including transcription, DNA damage repair, cell cycle regulation, and nuclear architecture. Building from our screen, we demonstrate that the Anaphase-Promoting Complex/Cyclosome directly binds the acidic patch and elucidate a redundant charge-based mechanism of acidic patch binding by nuclear pore protein ELYS. Overall, our interactome screen illuminates a highly competitive nucleosome binding hub for chromatin-targeted activities and curates a list of nucleosome interacting proteins that will enable mechanistic exploration of many unexpected chromatin-templated nuclear processes.
2020-06-24 | PXD018690 | Pride
Project description:Chromatin remodelers are ATP-dependent enzymes that reorganize nucleosomes within all eukaryotic genomes. The Chd1 remodeler specializes in shifting nucleosomes into evenly spaced arrays, a defining characteristic of chromatin in gene bodies that blocks spurious transcription initiation. Linked to some forms of autism and commonly mutated in prostate cancer, Chd1 is essential for maintaining pluripotency in stem cells. Here we report a complex of yeast Chd1 bound to a nucleosome in a nucleotide-free state, determined by cryo-electron microscopy (cryo-EM) to 2.6 Å resolution. The structure shows a bulge of the DNA tracking strand where the ATPase motor engages the nucleosome, consistent with an initial stage in DNA translocation. Unlike other remodeler-nucleosome complexes, nucleosomal DNA compensates for the remodeler-induced bulge with a bulge of the complementary DNA strand one helical turn downstream from the ATPase motor. Unexpectedly, the structure also reveals an N-terminal binding motif, called ChEx, which binds on the exit-side acidic patch of the nucleosome. The ChEx motif can displace a LANA-based peptide from the acidic patch, which suggests a means by which Chd1 remodelers may block competing chromatin remodelers from acting on the opposite side of the nucleosome.
2022-02-23 | PXD025287 | Pride
Project description:ISWI-family chromatin remodelers organize nucleosome arrays, while SWI/SNF-family remodelers (RSC) disorganize and eject nucleosomes, implying an antagonism that is largely unexplored in vivo. Here, we describe two independent genetic screens for rsc suppressors that yielded mutations in the promoter-focused ISW1a complex, or mutations in the ‘basic patch’ of histone H4 (an epitope that regulates ISWI activity), strongly supporting RSC-ISW1a antagonism in vivo. RSC and ISW1a largely co-localize, and genomic nucleosome studies using rsc isw1 mutant combinations revealed opposing functions: promoters classified with a nucleosome-deficient region (NDR) gain nucleosome occupancy in rsc mutants, but this gain is attenuated in rsc isw1 double mutants. Furthermore, promoters lacking NDRs have the highest occupancy of both remodelers, consistent with regulation by nucleosome occupancy, and decreased transcription in rsc mutants. Taken together, we provide the first genetic and genomic evidence for RSC-ISW1a antagonism, and reveal different mechanisms at two different promoter architectures. Genome-wide nucleosome occupancy maps in RSC and rsc null strains were generated by paired-end sequencing of mononucleosomal DNA. Strains carrying the Sth1 degron allele and either pGal-UBR1 (YBC3386) or ubr1 null (YBC3387) represent RSC null and RSC wildtype, respectively.
2015-04-01 | E-GEOD-65593 | biostudies-arrayexpress
Project description:ISWI-family chromatin remodelers organize nucleosome arrays, while SWI/SNF-family remodelers (RSC) disorganize and eject nucleosomes, implying an antagonism that is largely unexplored in vivo. Here, we describe two independent genetic screens for rsc suppressors that yielded mutations in the promoter-focused ISW1a complex, or mutations in the ‘basic patch’ of histone H4 (an epitope that regulates ISWI activity), strongly supporting RSC-ISW1a antagonism in vivo. RSC and ISW1a largely co-localize, and genomic nucleosome studies using rsc isw1 mutant combinations revealed opposing functions: promoters classified with a nucleosome-deficient region (NDR) gain nucleosome occupancy in rsc mutants, but this gain is attenuated in rsc isw1 double mutants. Furthermore, promoters lacking NDRs have the highest occupancy of both remodelers, consistent with regulation by nucleosome occupancy, and decreased transcription in rsc mutants. Taken together, we provide the first genetic and genomic evidence for RSC-ISW1a antagonism, and reveal different mechanisms at two different promoter architectures. Genomic localization of RSC, ISW1a, and SWI/SNF complexes were measured by chromatin immunoprecipitation followed by Illumina paired-end sequencing. Four strains were analyzed, including Rsc8-9xMyc (YBC2882), Sth1-2xFlag (YBC601 p3018), Ioc3-13xMyc (YBC2883), and Snf2-13xMyc (YBC3010). Each sample consists of one chromatin immunoprecipitate and one input chromatin control.
2015-04-01 | E-GEOD-65592 | biostudies-arrayexpress
Project description:Arrays of regularly spaced nucleosomes dominate chromatin and are often phased, i.e., aligned at reference sites like active promoters. How distances between nucleosomes and distances between phasing sites and nucleosomes are determined remained unclear, specifically, the role of ATP dependent chromatin remodelers in it. Here, we used a genome-wide reconstitution system to probe how yeast remodelers generate phased nucleosome arrays. We find that remodelers bear a structural element named the ‘ruler’ that sets nucleosome spacing, in the order Chd1 < ISW1a < ISW2 < INO80. Structure-based mutagenesis confirmed the functional significance of the ruler element in INO80. Differences in the ruler elements of different remodelers explain the observed nucleosome array features. More generally, we propose that remodelers use their rulers to regulate the direction of nucleosome sliding in response to nucleosome density and environment, leading to nucleosome positioning relative to other nucleosomes, DNA bound factors or DNA sequence elements.
2020-02-10 | GSE140614 | GEO
Project description:Chromatin remodelers regulate genes by organizing nucleosomes around promoters, but their individual contributions are obfuscated by the complex in vivo milieu of factor redundancy and indirect effects. Genome-wide reconstitution of promoter nucleosome organization with purified proteins resolves this problem and is therefore a critical goal. Here we reconstitute four stages of nucleosome architecture using purified components: Yeast genomic DNA, histones, sequence-specific Abf1/Reb1, and remodelers RSC, ISW2, INO80, and ISW1a. We identify direct, specific and sufficient contributions that in vivo observations validate. First, RSC clears promoters by translating poly(dA:dT) into directional nucleosome removal. Second, partial redundancy is recapitulated where INO80 alone, or ISW2 at Abf1/Reb1sites, positions +1 nucleosomes. Third, INO80 and ISW2 each align downstream nucleosomal arrays. Fourth, ISW1a tightens the spacing to canonical repeat lengths. Such a minimal set of rules and proteins establishes core mechanisms by which promoter chromatin architecture arises through a blend of redundancy and specialization.
2016-08-14 | GSE72106 | GEO
Project description:ISWI-family chromatin remodelers organize nucleosome arrays, while SWI/SNF-family remodelers (RSC) disorganize and eject nucleosomes, implying an antagonism that is largely unexplored in vivo. Here, we describe two independent genetic screens for rsc suppressors that yielded mutations in the promoter-focused ISW1a complex, or mutations in the ‘basic patch’ of histone H4 (an epitope that regulates ISWI activity), strongly supporting RSC-ISW1a antagonism in vivo. RSC and ISW1a largely co-localize, and genomic nucleosome studies using rsc isw1 mutant combinations revealed opposing functions: promoters classified with a nucleosome-deficient region (NDR) gain nucleosome occupancy in rsc mutants, but this gain is attenuated in rsc isw1 double mutants. Furthermore, promoters lacking NDRs have the highest occupancy of both remodelers, consistent with regulation by nucleosome occupancy, and decreased transcription in rsc mutants. Taken together, we provide the first genetic and genomic evidence for RSC-ISW1a antagonism, and reveal different mechanisms at two different promoter architectures. Genomic localization of RSC and ISW1a complexes were measured by chromatin immunoprecipitation followed by genome-wide microarray hybridization. Two independent biological replicates of two strains, Rsc8-9xMyc (YBC2882) and Ioc3-13xMyc (YBC2883), each grown in two different media, rich glucose media (YPD) or minimal synthetic glucose (SD) media. Each sample consists of one chromatin immunoprecipitate and one input chromatin control. Please note that each sample consists of two replicates (i.e. two raw data files) and the sample data table contains combined quantile normalized data of the replicates.
2015-04-01 | E-GEOD-65589 | biostudies-arrayexpress
Project description:ISWI-family chromatin remodelers organize nucleosome arrays, while SWI/SNF-family remodelers (RSC) disorganize and eject nucleosomes, implying an antagonism that is largely unexplored in vivo. Here, we describe two independent genetic screens for rsc suppressors that yielded mutations in the promoter-focused ISW1a complex, or mutations in the ‘basic patch’ of histone H4 (an epitope that regulates ISWI activity), strongly supporting RSC-ISW1a antagonism in vivo. RSC and ISW1a largely co-localize, and genomic nucleosome studies using rsc isw1 mutant combinations revealed opposing functions: promoters classified with a nucleosome-deficient region (NDR) gain nucleosome occupancy in rsc mutants, but this gain is attenuated in rsc isw1 double mutants. Furthermore, promoters lacking NDRs have the highest occupancy of both remodelers, consistent with regulation by nucleosome occupancy, and decreased transcription in rsc mutants. Taken together, we provide the first genetic and genomic evidence for RSC-ISW1a antagonism, and reveal different mechanisms at two different promoter architectures.
2015-04-01 | GSE65590 | GEO