Fig. 4
BORIS binding epigenetically reprograms transcriptionally inert CTCF binding sites into active promoters. a Genome browser view of ChIP-seq data and RNA-seq data for NIH3T3 + EV versus NIH3T3 + BORIS (clone#2, soft-agar derived) cells. CTCF (red) and BORIS (blue) co-occupancy in NIH3T3 + BORIS cells leads to the activation of Oct4 (Pou5f1) gene expression from an alternative intronic promoter (highlighted by red arrow and open box) in all three BORIS-expressing clones (RNA-seq data), compared to no expression in NIH3T3 + EV cells. b Upper panel shows the exon/intron structure of Pou5f1. Black and red arrows indicate reference and alternative promoters of Pou5f1, respectively. Black and white boxes represent coding exons specific to isoforms 4A and 4B, respectively; grey boxes represent exons shared by both isoforms. Lower panel compares the relative expression of Pou5f1 isoforms in three BORIS-expressing soft-agar-derived NIH3T3 clones to NIH3T3 + EV cells, revealing upregulation of only isoform 4B upon BORIS expression. c,d Genome browser view showing that CTCF and BORIS co-binding in NIH3T3 + BORIS (clone#2) cells is associated with the enrichment of active histones/marks (H2A.Z, H3K4me3, H3K27ac), RNAPII, CAGE-seq reads and the activation of alternative transcription (RNA-seq) from Slc6a19 (c) and Hck (d) genes. The alternative promoters are silent under CTCF-only occupancy in NIH3T3 + EV cells. e–g Left panel shows scatter plots of normalized read counts (log10) for H2A.Z e, H3K4me3 f, and CAGE g occupancy at BORIS-bound sites in NIH3T3 + BORIS (clone#2) cells compared to the same genomic sites in NIH3T3 + EV cells. BORIS sites with gain or loss of active histone marks or CAGEs are highlighted by red or green colors, respectively. The right panel displays a heatmap of H2A.Z e, H3K4me3 f, and CAGE g occupancy at BORIS-bound sites that gained active marks in NIH3T3 + BORIS (clone#2) cells compared to NIH3T3 + EV cells. Red arrows connect the left and right panels, indicating the number of gained active histone marks at BORIS-bound sites. h,i Heatmap representation of CTCF (red), BORIS (blue), RNAPII (pink), H2A.Z (yellow), H3K4me3 (purple), and H3K27ac (green) occupancy in NIH3T3 + EV cells (h) compared to NIH3T3 + BORIS (clone#2) cells (i) at the 5871 CTCF/BORIS binding sites, which gained occupancy of at least one active mark of transcription