PML enhances HIRA binding to ISGs following IFN-β stimulation.

Primary fibroblast (IMR-90) cells were stably transduced to express non-targeting control (shCtrl) or PML-targeting (shPML) shRNAs. Cells were either mock treated (no treatment, nt) or stimulated with IFN-β (2000 IU/ml) for 24 h prior to native ChIP-seq using a panel of HIRA mAbs [69, 74]. (A) Western blot analysis of the expression levels of PML in whole cell lysates derived from untreated shCtrl or shPML cells. Actin is shown as a loading control. Molecular mass markers are indicated. (B) Bar chart showing the number of unique sequencing (USeq) peaks identified following HIRA ChIP-seq for each treatment condition (as indicated; S7 Table). (C) Venn diagram showing the intersection in bp for each condition (i, shCtrl (nt); ii, shCtrl + IFN-β; iii, shPML + IFN-β). (D) Representative UCSC plot of the ISG Mx1 showing relative HIRA binding under each condition (as indicated). The Y-axis represents library normalised read count. For the gene track, introns are shown as horizontal lines and exons as vertical boxes. (E) Bar graph showing median gene body HIRA enrichment at 49 core ISGs under each condition (as indicated; S7 Table). 95% confidence interval shown. *** P < 0.001; one-way Anova (tukey), ns; not significant. (F). Composite profile of HIRA enrichment over 49 core ISGs. Shaded grey boxes represent gene body sequences located between transcriptional start and end sites (TSS and TES, respectively). Y-axis indicates mean HIRA enrichment (normalised to input control) per bin width. (G) Bar graph showing the median HIRA enrichment in the gene body of 49 coding non-ISGs equal in size to core ISGs (as in E). 95% confidence interval shown. ns (not significant); one-way Anova (tukey). (H) Composite profile of HIRA enrichment at the same 49 coding non-ISGs as described in G.