Reorientation of INO80 on hexasomes reveals basis for mechanistic versatility

Abstract

Unlike other chromatin remodelers, INO80 preferentially mobilizes hexasomes, which can form during transcription. Why INO80 prefers hexasomes over nucleosomes remains unclear. Here, we report structures of Saccharomyces cerevisiae INO80 bound to a hexasome or a nucleosome. INO80 binds the two substrates in substantially different orientations. On a hexasome, INO80 places its ATPase subunit, Ino80, at superhelical location -2 (SHL -2), in contrast to SHL -6 and SHL -7, as previously seen on nucleosomes. Our results suggest that INO80 action on hexasomes resembles action by other remodelers on nucleosomes such that Ino80 is maximally active near SHL -2. The SHL -2 position also plays a critical role for nucleosome remodeling by INO80. Overall, the mechanistic adaptations used by INO80 for preferential hexasome sliding imply that subnucleosomal particles play considerable regulatory roles.

Fig. 1.. Structure of the INO80-hexasome complex reveals large rotation.

(A) Cartoon illustration of a +X Nucleosome and a +X Hexasome. H2A-H2B dimer proximal to the flanking DNA (entry side dimer): cyan; H3-H4: light gray; 601 DNA: dark gray; flanking DNA: orange; additional free (unwrapped) DNA: cyan; super helical locations: yellow dots; DNA from the bottom gyre: dotted line. (B) Two different views of cryo-EM density map of the INO80-hexasome complex (class 3). (C) Atomic model of the INO80-hexasome complex (class 3), viewed in the same orientation as the map is viewed in (B). (D) Cryo-EM density map of Chaetomium thermophilum INO80-nucleosome complex (EMDB: 4277 (14)) displayed with its nucleosome dyad and H3-H4 tetramer aligned with that of the hexasome in the right panel of (B). Note that INO80 on a hexasome rotates ~180° from where it sits on a nucleosome when keeping the nucleosome/hexasome dyad and H3-H4 aligned. (E) Structural comparisons of INO80-nucleosome complex (left), SWR-nucleosome complex (middle) and INO80-hexasome complex (right), with nucleosome/hexasome dyad and H3-H4 aligned.

Fig. 2.. Conformational snapshots of INO80-hexasome complexes.

Comparison of DNA from each INO80-hexasome class (blue) with DNA from an unbound hexasome (PDB: 6ZHY, gray), showing degree of DNA unwrapping (upper row) and binding locations of Ino80^ATPase and Arp5 (bottom row).

Fig. 3.. Inhibition of DNA translocation at specific SHL sites influence nucleosome and hexasome sliding by INO80.

(A) Cartoon illustration of a +80 Nucleosome (left) and a +80 Hexasome (right) with approximate locations of site-specific single base gaps indicated. Colors are the same as in Fig. 1A. (B-C) Example gels and time courses of native gel-based remodeling assays of WT INO80 on +80 nucleosomes with no gap, gap near SHL-2, and gap near SHL-6. (D-E) Example gels and time courses of native gel-based remodeling assays of WT INO80 on +80 hexasomes with no gap, gap near SHL-2, and gap near SHL-6. (F-G) Average observed rate constants of INO80 sliding activity. k_obs (min⁻¹): +80N: 1.551 ± 0.1846; +80N Gap @ SHL-2: 0.005995 ± 0.001054; +80N Gap @ SHL-6: 0.006497 ± 0.0007117; +80H: 1.01 ± 0.1668; +80H Gap @ SHL-2: 0.000379 ± 0.0002849; +80H Gap @ SHL-6: 1.213 ± 0.2209. Data represent the mean ± SEM for three technical replicates performed under single-turnover conditions with saturating enzyme and ATP.

Fig. 4.. The Arp8 module engages different regions of DNA in nucleosomes versus hexasomes.

Overlay of atomic models of the hexasome (class 1 and class 3) and the nucleosome (class 2) with the Arp8 module (PDB: 8A5O), aligned by the H3-H4 tetramer.

Fig. 5.. Model of INO80-induced hexasome and nucleosome sliding.

(A) Hexasome sliding: the Ino80^ATPase samples different positions between SHL-3 and SHL-2 but binds predominantly near SHL-2. The INO80 complex becomes sliding-competent when Ino80^ATPase engages near SHL-2. (B) Nucleosome sliding: INO80 initially binds with Ino80^ATPase at SHL-7 or −6. Upon ATP-hydrolysis, Ino80^ATPase moves toward SHL-2 where INO80 becomes sliding-competent.