Spreading of H3K4me3 to gene bodies leads to defects in gene expression in ES cells. (A) Schematic representation describing the calculation used to determine the SI of genes marked by H3K4 methylation. The promoter bin is defined as a 1 kb, 1.5 kb, or 3 kb window around the TSS of genes marked by H3K4me3, H3K4me2, and H3K4me1, respectively, while the transcribed region (gene body) is defined as the region extending to the TES. The SI is calculated from the ratio of the density of H3K4 methylation in the gene body bin to the density of H3K4 methylation in the promoter bin. (B) Empirical cumulative distribution for the SI of H3K4me3 (top panel), H3K4me2 (middle panel), and H3K4me1 (bottom panel) across all genes for shLuc (black) and shKdm5b (red) ES cells. Y-axis shows the percentage of genes that exhibit a SI less than the value specified by the x-axis. A line shifted to the right means a systematic increase in the spreading index. P-value for all <1E-5 (Kolmogorov-Smirnov test). Note the increased SI for genes marked by H3K4me3, H3K4me2, and H3K4me1 in shKdm5b ES cells. (C) Scatter plot of gene expression measured by RPKM (reads per kilobases of exon model per million reads). Log2-adjusted differentially expressed (DE) genes (>1.5-fold; false discovery rate <0.001; RPKM >11) are shown in black. (D) Spreading of H3K4 methylation into gene bodies is associated with defects in gene expression in Kdm5b knockdown ES cells. Genes are ordered on the x-axis from left to right based on the fold change in their SI (shKdm5b/shLuc; ES cells) from low to high. A sliding window of 1,000 genes was used to calculate the percentage of DE genes (y-axis). The analysis was performed independently using SIs calculated for H3K4me3 (red), H3K4me2 (green), and H3K4me1 (blue) marks.