Figure 3. EEN is a subunit of the INO80 chromatin remodeling complex.

(A) Graphical representation of H2A.Z and EEN occupancy at the EIN2 gene determined with ChIP-seq. Sequencing reads of two merged H2A.Z ChIP-seq’s (Ler (gray), ein6-1 een-1 (blue)) and one EEN ChIP-seq (Col-0 35S:EEN-FLAG (red)) using 3-day-old etiolated seedlings were used. The occupancy is displayed as log₂ fold change and was calculated as the ratio between the respective ChIP and the respective IgG control in 100 bp bins from 2.4 kb upstream to 7.7 kb downstream of the transcriptional start site (TSS) of EIN2. Occupancies that were lower in the ChIP sample compared to the IgG sample were set to zero. The region with significant EEN enrichment determined with SICER is indicated as well. (B) FLAG-pull-down assays using FLAG-tagged EEN and Halo-tagged ARP5 that were in vitro translated in rabbit reticulocyte extract. Anti-FLAG and anti-Halo antibodies were used to confirm EEN/ARP5 expression, EEN immunoprecipitation and EEN-ARP5 interaction. (C) Heatmap illustrates the log₂ fold change in mRNA expression of INO80-dependent genes in untreated 3-day-old etiolated ino80-8, een-2 and arp5-1 seedlings relative to their expression in Col-0 seedlings. INO80-dependent genes were selected as genes that show a significant differential expression in ino80-8 mutant seedlings compared to Col-0 seedlings (787 genes up, 1079 genes down). (D) Genome browser screenshot shows the occupancy of H3K27me3 (right) and H2A.Z (left) at the EIN2 gene in untreated 3-day-old etiolated Col-0, ref6-1, een-2, ref6-1 een-2, ino80-1, ref6-1 ino80-1, arp5-1 and ref6-1 arp5-1 seedlings. Genome-wide occupancy of H3K27me3 and H2A.Z was determined by ChIP-seq. To ensure an accurate comparison of individual chromatin features between genotypes, the tracks were normalized to the respective sequencing depth. Normalization was separately done for each chromatin feature.

Figure 3—figure supplement 1. Proteomic and genetic validation of EEN as a subunit of the Arabidopsis INO80 chromatin remodeling complex.

(A) Histochemical localization of GUS expression driven by a 2 kb EEN promoter fragment in 7-day-old Ler EEN:GUS seedlings. (B) GFP fluorescence visualizes subcellular EEN localization in roots of 10-day-old Col-0 35S:GFP-EEN seedlings using confocal laser scanning microscopy. 20 µm scale bar is shown. (C) Genome browser screenshot visualizes the in vivo binding of EEN to the gene body of EIN2, At4g11280 and At4g01250. H2A.Z occupancy in Col-0 and ref6-1 een-2 seedlings at these loci is indicated as well. (D) Mass spectrometry analysis of immuno-purified FLAG-tagged EEN from Col-0 35S:EEN flower tissue revealed ARP5 as an interactor. Results from two independent IPs are shown. Flower tissue of non-transgenic Col-0 was used as a control. Both IPs also detected the helicase RVB2 which is part of the yeast INO80 complex as an interaction partner of EEN. (E), (F) Gene models of EEN and INO80 indicate the localization of the T-DNA insertion in een-2 (E), ino80-1 (F) and ino80-8 (F) mutants. (G), (H) Expression of EEN (G) and INO80 (H) in untreated 3-day-old etiolated Col-0, een-2 and ino80-8 seedlings using RNA-seq is shown. Expression is measured in TPM (Transcripts Per Kilobase Million) and results from two biological replicates are shown (mean ±S.D.). (I) (J) Quantification of H3K27me3 levels (I) and H2A.Z levels (J) in the 5’UTR intron, gene body and 3’UTR of the EIN2 gene in untreated 3-day-old etiolated Col-0, ref6-1, een-2, ref6-1 een-2, ino80-1, ref6-1 ino80-1, arp5-1 and ref6-1 arp5-1 seedlings. Occupancy of H3K27me3 and H2A.Z was determined by ChIP-seq. The H3K27me3 and H2A.Z occupancy in the three regions was calculated as the ratio between the respective ChIP-seq sample and Col-0 IgG control sample. (K) Triple response phenotype of 3-day-old etiolated Col-0, ref6-1, een-2, ref6-1een-2, ino80-1, ref6-1 ino80-1, arp5-1, ref6-1 arp5-1, ein2-5, pie1-1 and ref6-1 een-2 pie1-1 seedlings. Seedlings were grown on control LS media or on LS media supplemented with 10 µM ACC.