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Cryo-EM structures of the human INO80 chromatin-remodeling complex.

ABSTRACT: Access to chromatin for processes such as transcription and DNA repair requires the sliding of nucleosomes along DNA. This process is aided by chromatin-remodeling complexes, such as the multisubunit INO80 chromatin-remodeling complex. Here we present cryo-EM structures of the active core complex of human INO80 at 9.6?Å, with portions at 4.1-Å resolution, and reconstructions of combinations of subunits. Together, these structures reveal the architecture of the INO80 complex, including Ino80 and actin-related proteins, which is assembled around a single RUVBL1 (Tip49a) and RUVBL2 (Tip49b) AAA+ heterohexamer. An unusual spoked-wheel structural domain of the Ino80 subunit is engulfed by this heterohexamer; both, in combination, form the core of the complex. We also identify a cleft in RUVBL1 and RUVBL2, which forms a major interaction site for partner proteins and probably communicates these interactions to its nucleotide-binding sites.

SUBMITTER: Aramayo RJ

PROVIDER: S-EPMC5777635 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Cryo-EM structures of the human INO80 chromatin-remodeling complex.

Aramayo Ricardo J RJ Willhoft Oliver O Ayala Rafael R Bythell-Douglas Rohan R Wigley Dale B DB Zhang Xiaodong X

Nature structural & molecular biology 20171204 1

Access to chromatin for processes such as transcription and DNA repair requires the sliding of nucleosomes along DNA. This process is aided by chromatin-remodeling complexes, such as the multisubunit INO80 chromatin-remodeling complex. Here we present cryo-EM structures of the active core complex of human INO80 at 9.6 Å, with portions at 4.1-Å resolution, and reconstructions of combinations of subunits. Together, these structures reveal the architecture of the INO80 complex, including Ino80 and ...[more]

PMID: 29323271

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Synergy and antagonism in regulation of recombinant human INO80 chromatin remodeling complex.

Project description:We have purified a minimal core human Ino80 complex from recombinant protein expressed in insect cells. The complex comprises one subunit each of an N-terminally truncated Ino80, actin, Arp4, Arp5, Arp8, Ies2 and Ies6, together with a single heterohexamer of the Tip49a and Tip49b proteins. This core complex has nucleosome sliding activity that is similar to that of endogenous human and yeast Ino80 complexes and is also inhibited by inositol hexaphosphate (IP6). We show that IP6 is a non-competitive inhibitor that acts by blocking the stimulatory effect of nucleosomes on the ATPase activity. The IP6 binding site is located within the C-terminal region of the Ino80 subunit. We have also prepared complexes lacking combinations of Ies2 and Arp5/Ies6 subunits that reveal regulation imposed by each of them individually and synergistically that couples ATP hydrolysis to nucleosome sliding. This coupling between Ies2 and Arp5/Ies6 can be overcome in a bypass mutation of the Arp5 subunit that is active in the absence of Ies2. These studies reveal several underlying mechanisms for regulation of ATPase activity involving a complex interplay between these protein subunits and IP6 that in turn controls nucleosome sliding.

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Multiple modes of regulation of the human Ino80 SNF2 ATPase by subunits of the INO80 chromatin-remodeling complex.

Project description:SNF2 family ATPases are ATP-dependent motors that often function in multisubunit complexes to regulate chromatin structure. Although the central role of SNF2 ATPases in chromatin biology is well established, mechanisms by which their catalytic activities are regulated by additional subunits of chromatin-remodeling complexes are less well understood. Here we present evidence that the human Inositol auxotrophy 80 (Ino80) SNF2 ATPase is subject to regulation at multiple levels in the INO80 chromatin-remodeling complex. The zinc finger histidine triad domain-containing protein Ies2 (Ino Eighty Subunit 2) functions as a potent activator of the intrinsic catalytic activity of the Ino80 ATPase, whereas the YL-1 family Ies6 (Ino Eighty Subunit 6) and actin-related Arp5 proteins function together to promote binding of the Ino80 ATPase to nucleosomes. These findings support the idea that both substrate recognition and the intrinsic catalytic activities of SNF2 ATPases have evolved as important sites for their regulation.

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Feedback Modulation between Human INO80 Chromatin Remodeling Complex and miR-372 in HCT116 Cells.

Project description:Human INO80 chromatin remodeling complex (INO80 complex) as a transcription cofactor is widely involved in gene transcription regulation and is frequently highly expressed in tumor cells. However, few reports exist on the mutual regulatory mechanism between INO80 complex and non-coding microRNAs. Herein, we showed evidence that the INO80 complex transcriptionally controls microRNA-372 (miR-372) expression through RNA-Seq analysis and a series of biological experiments. Knocking down multiple subunits in the INO80 complex, including the INO80 catalytic subunit, YY1, Ies2, and Arp8, can significantly increase the expression level of miR-372. Interestingly, mimicking miR-372 expression in HCT116 cells, in turn, post-transcriptionally suppressed INO80 and Arp8 expression at both mRNA and protein levels, indicating the existence of a mutual regulatory mechanism between the INO80 complex and miR-372. The target relationship between miR-372 and INO80 complex was verified using luciferase assays in HCT116 colon cancer cells. As expected, miR-372 mimics significantly suppressed the luciferase activity of pMIR-luc/INO80 and pMIR-luc/Arp8 3'-UTR in cells. In contrast, the miR-372 target sites in the 3'-UTRs linked to the luciferase reporter were mutagenized, and both mutant sites lost their response to miR-372. Furthermore, the mutual modulation between the INO80 complex and miR-372 was involved in cell proliferation and the p53/p21 signaling pathway, suggesting the synergistic anti-tumor role of the INO80 complex and miR372. Our results will provide a solid theoretical basis for exploring miR-372 as a biological marker of tumorigenesis.

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Cryo-EM Structures of Human Drosha and DGCR8 in Complex with Primary MicroRNA.

Project description:Metazoan microRNAs require specific maturation steps initiated by Microprocessor, comprising Drosha and DGCR8. Lack of structural information for the assembled complex has hindered an understanding of how Microprocessor recognizes primary microRNA transcripts (pri-miRNAs). Here we present a cryoelectron microscopy structure of human Microprocessor with a pri-miRNA docked in the active site, poised for cleavage. The basal junction is recognized by a four-way intramolecular junction in Drosha, triggered by the Belt and Wedge regions that clamp over the ssRNA. The belt is important for efficiency and accuracy of pri-miRNA processing. Two dsRBDs form a molecular ruler to measure the stem length between the two dsRNA-ssRNA junctions. The specific organization of the dsRBDs near the apical junction is independent of Drosha core domains, as observed in a second structure in the partially docked state. Collectively, we derive a molecular model to explain how Microprocessor recognizes a pri-miRNA and accurately identifies the cleavage site.

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The INO80 ATP-dependent chromatin remodeling complex is a nucleosome spacing factor.

Project description:The mobilization of nucleosomes by the ATP-dependent remodeler INO80 is quite different from another remodeler (SWI/SNF) that is also involved in gene activation. Unlike that recently shown for SWI/SNF, INO80 is unable to disassemble nucleosomes when remodeling short nucleosomal arrays. Instead, INO80 more closely resembles, although with notable exceptions, the nucleosome spacing activity of ISW2 and ISW1a, which are generally involved in transcription repression. INO80 required a minimum of 33 to 43 bp of extranucleosomal DNA for mobilizing nucleosomes, with 70 bp being optimal. INO80 prefers to move mononucleosomes to the center of DNA, like ISW2 and ISW1a, but does so with higher precision. Unlike ISW2/1a, INO80 does not require the H4 tail for nucleosome mobilization; instead, the H2A histone tail negatively regulates nucleosome movement by INO80. INO80 moved arrays of two or three nucleosomes with 50 or 79 bp of linker DNA closer together, with a final length of ?30 bp of linker DNA or a repeat length of ?177 bp. A minimum length of >30 bp of linker DNA was required for nucleosome movement and spacing by INO80 in arrays.

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The mammalian INO80 chromatin remodeling complex is required for replication stress recovery.

Project description:A number of studies have implicated the yeast INO80 chromatin remodeling complex in DNA replication, but the function of the human INO80 complex during S phase remains poorly understood. Here, we have systematically investigated the involvement of the catalytic subunit of the human INO80 complex during unchallenged replication and under replication stress by following the effects of its depletion on cell survival, S-phase checkpoint activation, the fate of individual replication forks, and the consequences of fork collapse. We report that INO80 was specifically needed for efficient replication elongation, while it was not required for initiation of replication. In the absence of the Ino80 protein, cells became hypersensitive to hydroxyurea and displayed hyperactive ATR-Chk1 signaling. Using bulk and fiber labeling of DNA, we found that cells deficient for Ino80 and Arp8 had impaired replication restart after treatment with replication inhibitors and accumulated double-strand breaks as evidenced by the formation of ?-H2AX and Rad51 foci. These data indicate that under conditions of replication stress mammalian INO80 protects stalled forks from collapsing and allows their subsequent restart.

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human Ino80 chromatin remodeling complex downstream gene

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2015-09-26 | GSE68655 | GEO

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Cryo-EM structures of human RNA polymerase I.

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