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. 2018 Nov 23;7:e41497. doi: 10.7554/eLife.41497

Figure 2. ASH1 and SET-2 specificities segregate the genome into compartments of poorly and robustly transcribed genes.

(A) Representative IGV tracks of H3K36me2 ChIP-seq in WT, Δset-2, and ash1(Y888F) backgrounds. Gene location, DNA methylation (to highlight constitutive heterochromatin), and ‘input’ tracks are included for reference. All of linkage group (LG) III is shown. (B) H3K36me2 profiles as determined by ChIP-seq in set-2 and ash1(Y888F) backgrounds. Metaplots divide the H3K36me2 profile across gene quartiles determined by WT expression (i.e., ‘1 st’=genes in the lowest 25% of WT expression). Heatmaps were independently sorted by signal intensity in descending order. (C) IGV tracks of H3K36me2 and H3K36me3 ChIP-seq in WT, Δset-2, and ash1(Y888F) backgrounds. Gene location, WT RNA abundance, DNA methylation, and input tracks are included for reference. Representative SET-2-rich and ASH1-rich regions are presented in the left and right panels, respectively.

Figure 2.

Figure 2—figure supplement 1. (A) Representative IGV tracks of H3K36me3 ChIP-seq in WT, ash1(Y888F), and Δset-2 backgrounds are shown for LGIII.

Figure 2—figure supplement 1.

Gene location and DNA methylation are included for reference. All of LGIII is shown. (B) Average H3K36me2 signal was determined for each gene in the Δset-2 background and the distribution of signal intensity is represented in a histogram (red). Average expression level (RPKM) in WT is overlayed as a Whisker plot (black) for each quintile of H3K36me2 intensity. Pseudo-reads of 1 were assigned to genes with expression values <1. Whisker plot shows median, 25th, 75th, and 99th percentiles, and outliers. (C) Average genic H3K36me2 levels (reads/bp) were normalized to ‘background’ as defined by average H3K36me2 level across each centromere. Log2 values are plotted in the ash1(Y888F) background (X-axis) and Δset-2 background (Y-axis). Genes with positive X- or Y-axis values were defined as SET-2-marked or ASH1-marked, respectively. (D) H3K36me3 ChIP results are shown for wt, Δset-2, and Δset-2; ash1(Y888F) strains (each performed in triplicate) at three genomic regions with distinct H3K36me profiles. 8:G3 is a constitutive heterochromatin region that lacks H3K36me and is used to assess ‘background’ in the ChIP. hH4 is an actively expressed gene that is marked by SET-2-catalyzed H3K36me but not ASH1-catalyzed H3K36me. NCU07152 in a silent ‘uncharacterized gene’ that is densely marked by ASH1-catalyzed H3K36me and H3K27me2/3.