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. 2019 Apr 24;47(11):5603–5616. doi: 10.1093/nar/gkz275

Figure 5.

Figure 5.

Ectopic H3K27me3 accumulation at highly expressed genes in drh-3(ne4253). (A) Open chromatin is associated with H3K36me3 histone modification (pink), which opposes silencing histone mark H3K27me3 (blue). Highly expressed genes (among them the CSR-1 targets ncl-1, lin-13, snu-23) are enriched in H3K36me3 and depleted of H3K27me3. H3K27me3 is abundant at low expressed/silent regions. Highly expressed genes are in red, low expressed genes (expression quantile 1 and 2, see Supplementary Figure S4A) are in blue. The peaks were generated with MACS2 peak calling algorithm (Galaxy version 2.0.10.2) (50). (B) ChIP-seq normalized coverage for H3K36me3 (pink) and H3K27me3 (blue) demonstrating a decrease in H3K36me3 in drh-3(ne4253) compared to wild type and an increase in H3K27me3 modification at the highly expressed CSR-1 target gene bet-2. H3K9me3 levels do not change at the presented locus (brown). The directionality of transcription is marked with a blue arrow. (C) Global increase in H3K27me3 and reduction in H3K36me3 at highly expressed CSR-1 target genes in drh-3(ne4253), but not in csr-1(WM193) (scatterplots obtained by differential peak analysis), is concordant with the degree of nascent transcript reduction (bottom box plots, GRO-seq reads from (38)) in the mutants compared to wild type. For histone modification analysis, normalization of both replicas was performed as described in Materials and Methods. Only the subclass of highly active genes (CSR-1 targets) is represented. The colors reflect differential enrichment (red) and depletion (blue) of a histone mark observed in both biological replicates. The GRO-seq data (38) were normalized as described in Materials and Methods, and log2 transformed RPKM was plotted. P-values for GRO-seq data: drh3(ne4253) versus wt, P-value < 2.2 × 10−16; csr-1(WM193) versus wt, P-value = 1.092 × 10−11 (Wilcoxon test). (D) A heatmap demonstrating an increase in the heterochromatic modification H3K27me3 and a decrease in the activating modification H3K36me3 at actively transcribed gene coding regions in drh-3(ne4253) compared to wild type. Each row corresponds to a CSR-1 target gene body between transcription start site (TSS) and transcription termination site (TTS) ±100 bp. Gene length was approximated to be 1000 bp for representation purposes. The mean value from two biological replicates was plotted. (E) A heatmap demonstrating an absence of uniform changes in random sets of genes in drh-3(ne4253) compared to wild type. Each row corresponds to gene coding regions between transcription start site (TSS) and transcription termination site (TTS) ± 100 bp. Gene length was approximated to be 1000 bp for representation purposes. The mean value from two biological replicates is shown. A list of the random set of genes is presented in Supplementary Table S3.