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

We have prepared recombinant fourteen subunit yeast SWR1 complex from insect cells using a modified MultiBac system. The 1.07 MDa recombinant protein complex has histone-exchange activity. Full exchange activity is realized with a single SWR1 complex bound to a nucleosome. We also prepared mutant complexes that lack a variety of subunits or combinations of subunits and these start to reveal roles for some of these subunits as well as indicating interactions between them in the full complex. Complexes containing a series of N-terminally and C-terminally truncated Swr1 subunits reveal further details about interactions between subunits as well as their binding sites on the Swr1 subunit. Finally, we present electron microscopy studies revealing the dynamic nature of the complex and a 21 Å resolution reconstruction of the intact complex provides details not apparent in previously reported structures, including a large central cavity of sufficient size to accommodate a nucleosome.

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“…We have also seen this for recombinant human Ino80 complex (14). Consequently, this complex was not characterised further.…”

Section: Resultsmentioning

confidence: 56%

Functional characterization and architecture of recombinant yeast SWR1 histone exchange complex

Lin

Chaban

Rees

et al. 2017

Nucleic Acids Research

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“…Crystal and electron microscopy structures of the yeast INO80-C recapitulated its nucleosome-targeting function ( Tosi et al 2013 ; Watanabe et al 2015 ). Additionally, yeast INO80-C subunits have been shown to stimulate or facilitate nucleosome targeting and ATPase activity in vitro ( Jónsson et al 2004 ; Chambers et al 2012 ; Watanabe et al 2015 ; Yao et al 2016 ; Willhoft et al 2016 ). Loss of these proteins confers sensitivity to metabolic stress, polyploidy, and reduces cellular fitness ( Chambers et al 2012 ; Yao et al 2016 ; Gowans et al 2018 ).…”

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confidence: 99%

Identification of Two Distinct Classes of the Human INO80 Complex Genome-Wide

Runge

Raab²,

Magnuson

2018

G3 Genes|Genomes|Genetics

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Chromatin remodeling and histone modifying enzymes play a critical role in shaping the regulatory output of a cell. Although much is known about these classes of proteins, identifying the mechanisms by which they coordinate gene expression programs remains an exciting topic of investigation. One factor that may contribute to the targeting and activity of chromatin regulators is local chromatin landscape. We leveraged genomic approaches and publically-available datasets to characterize the chromatin landscape at targets of the human INO80 chromatin remodeling complex (INO80-C). Our data revealed two classes of INO80-C targets with distinct chromatin signatures. The predominant INO80-C class was enriched for open chromatin, H3K27ac, and representative subunits from each of the three INO80-C modules (RUVBL1, RUVBL2, MCRS1, YY1). We named this class Canonical INO80. Notably, we identified an unexpected class of INO80-C targets that contained only the INO80 ATPase and harbored a repressive chromatin signature characterized by inaccessible chromatin, H3K27me3, and the methyltransferase EZH2. We named this class Non-Canonical INO80 (NC-INO80). Biochemical approaches indicated that INO80-C and the H3K27 acetyltransferase P300 physically interact, suggesting INO80-C and P300 may jointly coordinate chromatin accessibility at Canonical INO80 sites. No interaction was detected between INO80-C and EZH2, indicating INO80-C and EZH2 may engage in a separate form of regulatory crosstalk at NC-INO80 targets. Our data indicate that INO80-C is more compositionally heterogenous at its genomic targets than anticipated. Moreover, our data suggest there is an important link between INO80-C and histone modifying enzymes that may have consequences in developmental and pathological contexts.

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“…The tagged subunit was then cloned with other genes of the hINO80 core complex using a modified MultiBac system (Berger et al, 2004) as described previously (Willhoft et al, 2016). Ino80 subunit DEAQ-box mutant (E563A) was prepared by InFusion-based mutagenesis.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, INO80 is able to centre mono-nucleosomes on short (<250 bp) DNA fragments in vitro (Jin et al, 2005; Udugama et al, 2011; Willhoft et al, 2016) and to space multiple nucleosomes on longer DNA fragments (Udugama et al, 2011). However, the Ino80 subunit lacks AutoN and NegC motifs (Clapier and Cairns, 2012) and is not regulated by binding of H4 tails (Udugama et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Crosstalk within a functional INO80 complex dimer regulates nucleosome sliding

Willhöft

McCormack

Aramayo

et al. 2017

eLife

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Several chromatin remodellers have the ability to space nucleosomes on DNA. For ISWI remodellers, this involves an interplay between H4 histone tails, the AutoN and NegC motifs of the motor domains that together regulate ATPase activity and sense the length of DNA flanking the nucleosome. By contrast, the INO80 complex also spaces nucleosomes but is not regulated by H4 tails and lacks the AutoN and NegC motifs. Instead nucleosome sliding requires cooperativity between two INO80 complexes that monitor DNA length simultaneously on either side of the nucleosome during sliding. The C-terminal domain of the human Ino80 subunit (Ino80CTD) binds cooperatively to DNA and dimerisation of these domains provides crosstalk between complexes. ATPase activity, rather than being regulated, instead gradually becomes uncoupled as nucleosome sliding reaches an end point and this is controlled by the Ino80CTD. A single active ATPase motor within the dimer is sufficient for sliding.DOI: http://dx.doi.org/10.7554/eLife.25782.001

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

Cited by 29 publications

References 36 publications

Functional characterization and architecture of recombinant yeast SWR1 histone exchange complex

Functional characterization and architecture of recombinant yeast SWR1 histone exchange complex

Identification of Two Distinct Classes of the Human INO80 Complex Genome-Wide

Crosstalk within a functional INO80 complex dimer regulates nucleosome sliding

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