(A) Cell fractionation in DU145 cells overexpressing PHF19S followed by Western blot analysis, showing that PHF19L is present on chromatin, and PHF19S in the cytoplasm. Total, total cell extract; Cyt, cytoplasm; Nuc, nucleus; NP, nucleoplasm; Chr, chromatin. (B) Volcano plot of the 1,245 PHF19L significant peaks identified by DiffBind (P value < 0.05 and FDR < 0.2) introducing two biological replicates of each sample (ChIP and Input). The x-axis represents the difference in the number of reads at each peak between ChIPs and the corresponding inputs. The y-axis represents the significance of the peaks (–log P value). (C) Histogram showing the distribution of PHF19L binding sites (ChIP-seq peaks) relative to the nearest TSS. The x-axis represents relative distance (bp) from the peak summit to the TSS, and the y-axis, the number of peaks in each class. (D) Boxplot showing PHF19 ChIP-seq signal intensity at the 1245 binding sites in two independent replicates (R1 and R2) of control (shCTR) and PHF19L knockdown (shPHF19L#4) DU145 cells. PHF19L knockdown cells showed strongly reduced binding, demonstrating the specificity of the antibody. Input signal is shown as negative control. Values associated to the peaks were normalized by the total number of reads of each ChIP-seq experiment. (E) UCSC genome browser screenshots of three PHF19L target genes showing the profiles of the corresponding ChIP-seq experiments in control and PHF19L knockdown (shPHF19L#4) DU145 cells. (F) ChIP-qPCR validation of eight representative PHF19L binding sites and two negative controls (GATA2 and an intergenic region) in control (shCTR) and PHF19L-depleted (shPHF19L#4 and shPHF19L#B) DU145 cells. Data represent the mean ± SD from three biological replicates. (G,H) (Top) TSS (± 5 kb) enrichment plot of the indicated ChIP-seq experiments (Replicate 2) at the 1,010 PHF19L target genes in DU145 cells. Boxplots showing the corresponding distribution of values are shown next to each TSS plot. Enrichment levels are normalized for the total number of reads of each sample. (Bottom) Scatter plots comparing the ChIP-seq enrichment signals (IP/IgG) of PHF19 against EZH2 (G, left panel), SUZ12 (G, right panel), or H3K27me3 (H) in the 1,245 PHF19 peaks for the second set of replicates. CC is the correlation between each pair of variables (P value ≤ 10−16 in all cases). P values were computed using Pearson’s product-moment correlation. (I) Venn diagram showing a significant overlapping between the 1,010 PHF19L target genes and the 4,256 H3K27me3 target genes in DU145 cells (P value ≤ 10−16; Fisher’s exact test).