Spreading of H3K4me3 to gene bodies leads to defects in gene expression during ES cell differentiation. Scatter plot of gene expression measured by RPKM for shLuc and shKdm5b ES cells differentiated in the absence of LIF for (A) 48 h (day 2) and (B) 72 h (day 3). Log2-adjusted differentially expressed genes (>1.5-fold; FDR <0.001; RPKM >1) are shown in black. (C) Empirical cumulative distribution for the spreading index of H3K4me3 (top panel), H3K4me2 (middle panel), and H3K4me1 (bottom panel) across all genes for shKdm5b knockdown cells (red line) and shLuc control cells (black line) following differentiation for three days after LIF withdrawal. Y-axis shows the percentage of genes that exhibit a spreading index 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). (D) Spreading of H3K4me into gene body regions is associated with defects in gene expression during differentiation of KDM5B-depleted ES cells. Genes were ordered on the x-axis from left to right based on the fold change in their SI (shKdm5b/shLuc) from low to high. A sliding window of 1,000 genes was used to calculate the percentage of differentially expressed (DE) genes (y-axis) in KDM5B-depleted cells. The analyses were performed independently using SIs calculated from H3K4me3 (top panel), H3K4me2 (middle panel), and H3K4me1 (bottom panel) marks following two and three days of ES cell differentiation after LIF withdrawal.