Identification of a novel CCCTC binding factor (CTCF) binding site near the ribosomal (r)DNA spacer promoter. (A) Outline of mouse rDNA repeat. Transcription (spacer promoter = ncRNA, gene promoter = pre-rRNA, right-pointing arrows), chromatin immunoprecipitation (ChIP) primers (downward-pointing arrows), organization of 47S pre-rRNA (external transcribed spacers (ETS), internal transcribed spacers (ITS), 18S, 5.8S and 28S rRNA, CTCF consensus site (grey box) and enhancer repeats (white boxes)), are indicated. (B) Binding of CTCF, upstream binding factor (UBF) and RNA polymerase I to mouse rDNA was analyzed by ChIP on formaldehyde-fixed mouse embryonic fibroblast (MEF) nuclei. (C) CTCF binding site in mouse rDNA. The R30 sequence is shown, with CTCF consensus site (underlined), highly conserved CTCF site residues (dots), deviation from consensus (asterisk) and CpG dinucleotides (gray). Nucleotides in bold were mutated (R30mut). (D, E) Band-shift analysis. Increasing amounts of HEK293T extracts (non-transfected (n) or transfected with CTCF), were incubated with the indicated probes (a known CTCF binding site (F1, chicken lysozyme, R30 and R30mut). Competitor was added in 300-fold excess. Band shifts were also performed with purified bacterial proteins (glutathione-S-transferase (GST) only (G) and zinc fingers (ZF) of CTCF tagged with GST. (F) CTCF and UBF bound R30 DNA together. HEK293T extracts (increasing amounts indicated, transfected with GFP-CTCF (+) Flag-UBF (1 to 5×) or the C-terminal domain of CTCF tagged with GFP (Ct, see Figure 2C) were incubated with R30. αC = addition of anti-CTCF; pr = addition of anti-UBF and anti-FLAG (preclear). (G) CTCF and UBF bound human rDNA together. HEK293T extracts (increasing amounts indicated, transfected with GFP-CTCF (+) or Flag-UBF (1 to 7×) were incubated with H42.1. Asterisks indicate UBF binding to H42.1. αC = addition of anti-CTCF. (H) UBF weakly bound human rDNA. HEK293T extracts (increasing amounts indicated), transfected with Flag-UBF (1 to 5×) were incubated with H42.1. Asterisks indicate UBF binding to H42.1 Cp = cold probe; UBF = addition of anti-UBF; UBF(p) = addition of anti-UBF and anti-FLAG before incubation with probe (preclear).