Fig. 1

CTCF and BORIS co-binding to intronic regions of GAL3ST1 and FER genes associated with cancer-testis-specific transcription. a,b Above: Schematic representation of the gene structure of GAL3ST1 (a) and FER (b). Arrows denote somatic (black) and testis-specific (red) TSSs. Below: In the upper part, ChIP-seq peaks illustrate CTCF (red) and BORIS (blue) co-binding in BORIS-positive (BORIS +) K562 cells across GAL3ST1 and FER (exon 7-10). The co-binding coincides with the enrichment of active histones/marks H3K4me3 (purple), H2A.Z (magenta), and RNAPII (brown). RNA-seq peaks (indigo) and CAGE-seq peaks (pink) highlight alternative transcription in K562 cells. In the lower part, CTCF binding alone in BORIS-negative (BORIS −) NHEK cells does not activate testis-specific promoters. CAGE-seq for human testes (pink) is shown between the two panels, with red boxes highlighting testis-specific TSSs. c–e In the upper part, Western blots from whole cell lysates show BORIS protein detection in c K562 wild-type (WT1-total culture, WT2 and WT3 – single-cell wild-type clones transfected with control RNA) versus BORIS knockdown (kd) K562 single-cell clones (#3,4,7), obtained by zinc finger nuclease (ZFN) treatment. d HEK293T and e MDA-MB-435 cells transfected with empty (EV) or BORIS-expressing vector. Tubulin is used as a loading control. Numbers (1,2,3) indicate different single-cell clones. The middle part displays RT-qPCR results indicating the relative expression of GAL3ST1 and FERT in K562, HEK293T, and MDA-MB-435 cell lines. Statistical analysis was performed using two-tailed Student’s t test (*, p < 0.0005). Error bars indicate mean ± SD (n = 3), ns – non-significant. In the bottom part, ChIP-seq peaks show CTCF and BORIS occupancy in K562 (clone#7) BORIS kd cells, HEK293T, and MDA-MB-435 cells. Abbreviations; Ref. TSS (reference transcriptional start site), Alt. TSS (alternative transcriptional start site)