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. 2014 Aug 7;10(8):e1004507. doi: 10.1371/journal.pgen.1004507

Figure 5. A subset of genes exhibit reduction of H3K4me3 with loss of UTX and JMJD3.

Figure 5

(A) UCSC genome browser view of Crabp2, Pax6, Wnt6, and Ccnd2. Illustrated are Input (black), WT ES H3K27me3 ChIP (dark green), KO ES H3K27me3 ChIP (dark red), WT RA H3K27me3 ChIP (light green), KO RA H3K27me3 ChIP (light red), WT RA H3K4me3 ChIP (blue), KO RA H3K4me3 ChIP (orange), and MACS defined enrichment peaks are illustrated as black bars underneath each track. Regions tested by ChIP-qPCR are noted on the bottom. (B) H3K4me3 ChIP-seq was performed on WT (Utxfl/fl;Jmjd3fl/fl;CreER −TX) or KO (Utxfl/fl;Jmjd3fl/fl;CreER +TX) cells treated with RA. The normalized sequence reads from all promoters (+/−1 KB KB) from RA treated cells were compared by edgeR to identify promoters that exhibit H3K4me3 reductions in KO cells. The log fold change (logFC) is plotted against the average log counts per million reads (Average logCPM). In the plot, 161 KO promoters exhibited H3K27me3 reductions with RA treatment (negative logFC, FDR<0.05, and an identified H3K4me3 MACS peak in WT RA cells). (C) Quantitative RT-PCR of Crabp2, Pax6, Wnt6, and Ccnd2 from Utxfl/fl;Jmjd3fl/fl;CreER ES cells (Diff −) or after 2 days of RA treatment (Diff +) left untreated (−TX, light grey) or pre-treated with tamoxifen (+TX, black). N = 3 samples per treatment. All samples are normalized relative to −TX Differentiation + RA treatment. Significantly reduced expression is demonstrated (p-vale = *0.01, **0.03, ***0.001, ****0.004). (D) Verification of H3K4me3 reductions in KO RA cells by ChIP-qPCR. H3K4me3 ChIP of Utxfl/fl;Jmjd3fl/fl;CreER ES cells (dark green, Diff −) or after 2 days of RA treatment (light green or red, Diff +) left untreated (green) or pre-treated with tamoxifen (red). An IgG control ChIP is illustrated as white bars. Quantitative PCR of a H3K4me3 negative locus (gene desert region, Neg) was utilized for comparison to Npm1 (a positive control), genes exhibiting normal KO gene activation (Hoxb1, Foxa1), and genes with reductions in KO H3K4me3 (Crabp2, Wnt6). Only Crabp2 and Wnt6 demonstrated decreases in KO RA treatment (p-value = *0.01, **0.07). (E) H3K27me3 ChIP of Crabp2 and Wnt6 relative to a negative control (Slc2a8). Both genes demonstrated increases in H3K27me3 in RA KO cells (light red bars, p-value = *0.003, **0.03).