Fig. 2: Fully recombinant genome-wide reconstitution of nucleosome positioning by INO80.

a Overview of genome-wide in vitro chromatin reconstitution system. b Heat maps of MNase-seq data for SGD chromatin assembled with embryonic or recombinant histones from the indicated species (H.s. Homo sapiens, S.c. Saccharomyces cerevisiae) and remodeled with endogenous (GSM1855399)²⁹ or recombinant S. cerevisiae INO80 complex as indicated. Heat maps are aligned at in vivo +1 nucleosome positions and sorted by NDR length. Single replicates were plotted, see Supplementary Fig. 1c and Supplementary Data 1 for all replicates. c Left panel: schematic of INO80 complex submodule and subunit organization (middle) with zoom into Nhp10 (top) or Arp8 module (bottom) showing three mutant versions each. Right panel: composite model of INO80 based on high-resolution cryoEM structure of ctINO80 (Chaetomium thermophilum INO80) core in complex with a mononucleosome⁴⁴ and X-ray structure of Arp8 module⁴⁷ modeled on 70 bp linker DNA. The AAA⁺ ATPase hetero-hexamer Rvb1/2 (omitted for clarity and indicated by a dotted line in the left representation) acts as a stator for the Ino80 ATPase motor and the nucleosome gripping subunit Arp5. The direction of entry DNA translocation is indicated. d Heat maps of MNase-seq data of individual replicates for SGD chromatin incubated with the indicated recombinant WT (WT) or mutant INO80 complexes (as in c, left, and also with combinations of HMGII and HQ1 or HQ2 mutations) from S. cerevisiae or C. thermophilum (ctINO80^∆N). e Composite plots of MNase-seq data of individual replicates for SGD chromatin incubated with the indicated recombinant WT (WT) or mutant INO80 complexes as in d. Each color represents an independent replicate (n = 3 for SGD (“none”), WT, HQ1, HQ2, ctINO80^∆N; n = 2 for HQ1/2, HMGII-HQ1, HMGII-HQ2, Ino80^∆N). Composite plots of replicates from d are shown as purple traces. f Distributions of distances between +1 nucleosome positions determined by paired-end sequencing after reconstitution by the indicated combinations of INO80 complexes and histones at the indicated histone-to-DNA mass ratio relative to in vivo +1 nucleosome positions. Dots mark the medians, vertical lines the interquartile distances. Alternating white and gray vertical zones group replicates of the indicated remodeler/histone combinations. g Density distributions of MNase-seq reads relative to in vivo +1 nucleosome positions of biological replicates with INO80 WT (yellow and orange area), HQ1 (pink areas), and HMGII-HQ1 (purple areas) mutant complexes as in f.