affects H3K27me3 enrichment of the Xi. (A) Examples of interphase nuclei after H3K27me3 immunostaining (left panel) and graph of percent of nuclei with an H3K27me3 intense staining spot (right panel) in control and Firre knockdown Patski cells. The H3K27me3 spot over the Xi (marked by arrowheads) is no longer visible after knockdown. P value was determined by Chi-square tests. (B) H3K27me3 enrichment is reduced at the 5’ end of X-linked genes but not at autosomal genes after Firre knockdown using siRNAs in Patski cells. Metagene analysis shows average H3K27me3 enrichment 3 kb upstream and downstream of the transcription start site (TSS) for 647 X-linked genes (X) versus 16,141 autosomal genes (A). (C) The distribution of average H3K27me3 enrichment at 1 to 2 kb downstream of the TSS is clearly shifted left to lower values for X-linked genes but not for autosomal genes after Firre knockdown. (D) Scatter plot of H3K27me3 enrichment at 1 to 2 kb downstream of the TSS shows a shift to lower values for most X-linked genes after Firre knockdown (purple) compared to control (red), as indicated by trend lines. Three escape genes, Eif2s3x, Shroom4, and Pbdc1 that show no H3K27me3 enrichment are labeled. Hprt, a gene subject to XCI, shows no H3K27me3 enrichment because it is deleted in Patski cells. (E, F) Same analysis as in (A, B) but for Ctcf knockdown. There is a lesser decrease in H3K27me3 levels on the X than in Firre knockdown.