The INO80 Complex Requires the Arp5-Ies6 Subcomplex for Chromatin Remodeling and Metabolic Regulation

Abstract

ATP-dependent chromatin remodeling complexes are essential for transcription regulation, and yet it is unclear how these multisubunit complexes coordinate their activities to facilitate diverse transcriptional responses. In this study, we found that the conserved Arp5 and Ies6 subunits of the Saccharomyces cerevisiae INO80 chromatin-remodeler form an abundant and distinct subcomplex in vivo and stimulate INO80-mediated activity in vitro. Moreover, our genomic studies reveal that the relative occupancy of Arp5-Ies6 correlates with nucleosome positioning at transcriptional start sites and expression levels of >1,000 INO80-regulated genes. Notably, these genes are significantly enriched in energy metabolism pathways. Specifically, arp5Δ, ies6Δ, and ino80Δ mutants demonstrate decreased expression of genes involved in glycolysis and increased expression of genes in the oxidative phosphorylation pathway. Deregulation of these metabolic pathways results in constitutively elevated mitochondrial potential and oxygen consumption. Our results illustrate the dynamic nature of the INO80 complex assembly and demonstrate for the first time that a chromatin remodeler regulates glycolytic and respiratory capacity, thereby maintaining metabolic stability.

FIG 1

Arp5 and Ies6 form an independent subcomplex. (A to C) Silver-stained 6% (top) and 15% (bottom) SDS-PAGE gels of FLAG-purified proteins. Subunits are designated on the right of each gel. M, molecular mass markers. (A to C) Ino80-FLAG, Arp5-FLAG, and Ies6-FLAG purified from the wild type (WT) and the indicated deletion strains and mock purification from cell lysate lacking FLAG-tagged protein. (D) Anti-Flag Western blot of Ies6-FLAG in WT and the indicated deletion strain lysates. An antihexokinase Western blot was used as a loading control. (E) Ten to 25% sucrose gradient sedimentation of complexes purified from Ino80-FLAG, Arp8-FLAG, Arp5-FLAG, and Ies6-FLAG. (F) Fifteen to 20% sucrose gradient sedimentation of complexes purified from Ies6-FLAG-expressing cells. Samples were electrophoresed on 6% and 15% SDS-PAGE gels to resolve Arp5 and Ies6, respectively. The relative protein amount was quantified using Oriole fluorescent protein stain.

FIG 2

Arp5-Ies6 function together to maintain cellular fitness. (A) Serial dilution fitness assays of wild-type (WT) and ino80Δ, arp5Δ, ies6Δ, and arp8Δ mutant strains grown at 30 or 37°C or on medium lacking inositol. (B) Serial dilutions of WT, arp5Δ, ies6Δ, and arp5Δ ies6Δ strains. Tetrad segregation of arp5Δ and ies6Δ heterozygous diploids, indicating an epistatic genetic interaction between ARP5 and IES6, as cells with either single deletion or the double deletion exhibit similar growth. (C) Fitness assays of WT and indicated deletion strains show nonepistatic genetic interactions between ARP8 and ARP5 or IES6, as the growth defect is additive of single mutants.

FIG 3

Arp5-Ies6 subcomplex associates with chromatin in an INO80-dependent manner and modulates nucleosome sliding. (A) The illustration at the top shows the experimental design for in vivo chromatin fractionation with micrococcal nuclease (MNase). Below is the chromatin fractionation of wild-type (WT) and ino80Δ cells. Arp5 was detected by anti-Arp5 antibody. Hexokinase and histone H3 are indicative of cytoplasmic and chromatin fractions, respectively. MNase treatment solubilizes H3 chromatin fraction. (B) In vivo chromatin fractionation of WT and indicated deletion strains expressing Ino80-FLAG, Arp5-FLAG, or Ies6-FLAG. Arp5 and Ies6 are predominantly present in the chromatin fraction in WT cells and the soluble fraction in ino80Δ cells. Ies6-FLAG protein is undetectable in arp5Δ strains, as shown in Fig. 1D. A strain with both Ies6-FLAG and arp8Δ is nonviable. (C) Native PAGE gels of DNA (left panel) and nucleosome containing 60 bp extranucleosomal DNA (Nuc, right panel). A 2 nM concentration of DNA or nucleosome was incubated with the indicated molar equivalents (Mol Eq) of INO80 complex or Arp5-Ies6 subcomplex. (D) The illustration at the top depicts the experimental setup, wherein INO80 complex deficient of the Arp5-Ies6 subcomplex (INO80^d) is preincubated with end-positioned (N1) nucleosomal substrate. Upon addition of the Arp5-Ies6 subcomplex and ATP, INO80^d activity is stimulated, and the nucleosome is moved to the center position (N2).

FIG 4

Arp5 occupancy at promoters influences the transcription of >1,000 yeast genes. (A) Distribution of significant gene expression changes determined by RNA-seq, shown on a log₂ scale, in the indicated deletion strains relative to the wild type (WT). (B) PCA, log-transformed gene expression values from ino80Δ, arp5Δ, ies6Δ, and arp8Δ samples were projected onto primary (PC1) and secondary (PC2) principal components. Vectors represent projections of the indicated sample onto principal components, whereas black dots are log-transformed expression values of individual genes transformed into principal components. Note the close proximity of arp5Δ, ies6Δ, and ino80Δ vectors. (C) Micrococcal nuclease-ChIP (MNase-ChIP) maps of nucleosomes, Arp5, and Ino80 on 1,029 Arp5 and Ino80-regulated genes aligned by their TSSs. Genes are ordered by the total Arp5 occupancy at the +1 nucleosome (+1 Nuc) and are illustrated by the vertical red gradient strip. The NFR and promoter proximal nucleosomes are illustrated. (D) RNA-seq gene expression profiles in WT, ino80Δ, and arp5Δ strains are ordered by wild-type Arp5 occupancy (horizontal red gradient strip) and Ino80 occupancy (horizontal blue gradient strip) at the +1 Nuc. Note, in the ino80Δ and arp5Δ plots, that the gene expression in mutant cells is plotted according to Arp5 or Ino80 occupancy in wild-type cells. The fragments per kilobase of transcript per million (FPKM) mapped reads are shown. The cross-validated (CV) spline function (solid, black line) and 95% confidence bands for the spline fit (dashed, gray lines) are shown. Genes within the interquartile range of Arp5 occupancy (dotted, black lines) have relatively higher expression levels. The red box outline highlights the asymmetric trend line in wild-type (WT) expression that is not observed in deletion strains.

FIG 5

Arp5 abundance at +1 nucleosomes regulates promoter architecture. (A) As in Fig. 4D, genes are ordered by Arp5 occupancy at +1 nucleosomes, as indicated by the horizontal red gradient strip. Nucleosome fuzziness, which is the base pair standard deviation from the mean position (mean bp deviation) in wild-type (WT), ino80Δ, and arp5Δ strains are shown. The cross-validated spline function (solid, black line) and 95% confidence bands for the spline fit (dashed, gray lines) are shown. Horizontal dotted lines indicate the interquartile range. The red box outline highlights the asymmetric trend line in WT nucleosome fuzziness that is not observed in deletion strains. (B) NFR length profiles of Arp5-regulated genes are shown as in panel A. (C) Distribution of +1 nucleosome fuzziness values of all Arp5-regulated genes in WT, arp5Δ, and ino80Δ cells.

FIG 6

Arp5-Ies6-dependent genes are enriched in metabolic pathways. A TreeMap of enriched gene ontology (GO) categories (P < 10⁻³) grouped by biological process for significantly differentially expressed upregulated (A) or downregulated (B) genes in arp5Δ mutants (according to the statistical analysis described in Materials and Methods) is shown. GO terms are hierarchically clustered into REVIGO-determined parent categories indicated by color key below TreeMap. Ambiguous parents categories include “single organism cellular process,” “single organism metabolism,” and “biosynthesis,” which were manually curated to indicate the largest constituent group. The size of the box is scaled to represent the number of genes in the indicated category. *, “de novo” IMP biosynthetic process.

FIG 7

Arp5-Ies6 regulates INO80-mediated glycolytic function and respiration capacity. (A) Genes with ≥1.5-fold change (FC) in arp5Δ, ies6Δ, and ino80Δ mutants compared to the wild type are shown in glycolysis, citric acid cycle, ethanol fermentation (dashed lines), and the electron transport chain. Gene expression in each mutant is color coded as indicated. (B) Mitochondrial potential, in the wild type (WT) and the indicated mutant cells grown in glucose or ethanol-containing media, measured as TMRE fluorescence that accumulates in active mitochondria. Values are given relative to the WT signal within each growth condition. (C) Oxygen consumption (pmol/min/million cells) in wild-type (WT) and the indicated mutant cells grown in glucose or ethanol-containing media, measured by using an XF96 extracellular flux analyzer (Seahorse Biosciences). (D) Doubling time in the mid-log-phase growth of WT and indicated mutant cells in glucose-, ethanol-, and 2-DG-containing media. Fitness is given as relative to the WT growth in glucose-containing media.