Fig. 3

CTCF and BORIS interact at CTCF&BORIS bound regions. a ChIP-seq does not resolve the two closely spaced CTCF binding sites at CTCF&BORIS bound regions: no enrichment of reads between two CTCF binding sites (black dots) in the middle panel compared with the right panel with two CTCF binding regions resolved by ChIP-seq. b Western blot demonstrates co-immunoprecipitation of CTCF and BORIS complexes: K562 nuclear lysates were immunoprecipitated with IgG, anti-CTCF and anti-BORIS antibodies. Precipitated complexes were probed with CTCF antibodies. The lysates were either untreated (−) or treated (+) with ethidium bromide (EtBr) before co-immunoprecipitation. c, d In situ proximity ligation assay (ISPLA) shows the close proximity between CTCF and BORIS proteins in BORIS-positive cells: ovarian cancer cells (OVCAR8) (c) and human testis tissue (d). e A 200-bp ³²P-labeled probe representing human TP53 promoter (2xCTS, the palindromic orientation of CTCF motifs shown by arrows) was incubated with increasing concentrations of the 11 ZF domain of CTCF (11ZFs, left and middle panels) or Pichia recombinant full-length CTCF (FL CTCF, right panel). The 2xCTS was used in EMSA either as wild type with two CTCF binding sites (grey boxes) or as the mutant type with one CTCF mutated site (red cross). The model of 11 ZFs and full-length CTCF occupancies are shown by arrows and red dots (CTCF molecules). f A 200-bp ³²P-labeled probe representing IRF2BP1 3’ untranslated region (UTR) was incubated with nuclear extracts (n.e.) from either K562 or NHDF cells. All lanes contain the indicated nuclear extract, except the first lane for K562, where only free probe is present. Nuclear extracts and probe were also incubated with either control mouse IgG (−), antibodies against CTCF or BORIS. The red and blue arrows point to the super-shifted bands corresponding to CTCF–DNA and BORIS–DNA complexes, respectively. NHDF nuclear extract did not produce a super-shift band with anti-BORIS antibodies. g Gene tracks show that exogenous BORIS expression in MCF7 cells (MCF7 + BORIS) recapitulates endogenous BORIS occupancy in OVCAR8 cells. The name of the molecules against which antibodies were directed and the cell lines used in ChIP-seq are shown in the tracks. h Heatmap of BORIS (blue) occupancy at 50,000 CTCF peaks invariantly mapped in both OVCAR8 and MCF7 + BORIS cells. The tag density was subjected to k-means ranked clustering with two clusters expected. i ISPLA in BORIS-negative MCF7 cells stably transfected with either empty vector (MCF7+EV) or BORIS (MCF7+BORIS). The specific positive ISPLA signal (red) is present only in MCF7 cells transfected with BORIS-expressing vector