Fig. 4: Eed interacts with Eaf3 and is required for the Ash1-H3K36me2 and Eaf3 occupancy.

a Summary of Eaf3-interactors identified by a mass spectrometry assay in the EAF3-GFP strain. b Eed specifically interacts with CD domain of Eaf3 in yeast cells. Indicated fragments were cloned into pGBKT7 or pGADT7 respectively. AD and BD, plasmid with empty pGADT7 or pGBKT7 used in the experiments. c Co-IP assays were performed with the strains co-expressing EED-GFP and EAF3-Flag. Three independent repeats were performed with similar results. d Relative abundance of H3K36me2 and H3K36me3 in the indicated strains, which were calculated by ImageJ software respectively. Values are means ± SD from five biological replicates. e Metagene plots and heatmaps showing H3K36me2 ChIP-seq signals of the Ash1-H3K36me2-marked regions within 3.0-kb flanking the peak summits in the indicated strains. f Box plots and violin plots showing average transcriptions of the genes with and not Ash1-H3K36me2-marked in the indicated strains. For box-plot, the horizontal lines from top to bottom represent the maximum, first quartile, median, third quartile, and minimum of the total data respectively. g Metagene plots and heatmaps showing H3K36me2 ChIP-seq signals of the Eaf3-occupied regions in the indicated strains. h Box plots and violin plots showing average transcription levels of the genes with and not Eaf3-occupied in the indicated strains. i Relative abundance of Eaf3-GFP in the indicated strains, which calculated by ImageJ software and that of WT was set as 1. Values are means ± SD from five biological replicates. j IGV showing Eaf3-GFP, H3K36me2 and H3K27me3 occupancy in the WT and ∆eaf3 strains. ChIP-qPCR assays were performed to validate the ChIP-seq results. Relative enrichments of Eaf3 (k), H3K36me2 (l), and H3K27me3 (m) are shown. I and II indicated the amplified fragments in the ChIP-qPCR assays. Values are means ± SD from three technical replicates. For d, f, h, i, k–m, statistical significance was assessed by a two-tailed Student’s test (** P < 0.01). For e, g, and j, metaplots, heatmaps, and IGV snapshots are shown with one biological replicate and similar results were gained in another replicate.