CCCTC binding factor (CTCF) binding to ribosomal (r)DNA is methylation-sensitive. (A) Influence of methylation on the binding of CTCF to rDNA. Band-shift assays using human H37.9 and H42.1 rDNA probes, either completely methylated with SssI methyltransferase (methylated probe) or non-methylated (control probe) on HEK293T extracts, transfected or not with CTCF. Competition was assessed by adding increasing amounts of non-labeled probe. In some cases, extracts were incubated with the indicated antibodies. (B) CTCF prefers non-methylated rDNA in vivo. Chromatin from K562 cells was immunoprecipitated with anti-CTCF (CTCF ChIP). Purified DNA was left uncut (mock digestion), or digested with Hpa II or Msp I. Quantitative PCR was then performed with H42.1 primers, both on non-precipitated K562 DNA (input) and on DNA enriched for CTCF binding sites (CTCF ChIP). Note the high content of Hpa II-resistant H42.1 rDNA in K562 cells (input), which represents methylated rDNA. In the CTCF-enriched sample, the Hpa II-resistant rDNA was not present, suggesting that CTCF does not bind well to methylated rDNA. (C) ChIP analysis on (left panel) undifferentiated and (right panel) differentiated 3T3L1 cells. Nuclei were fixed with 1% formaldehyde, and protein-DNA complexes were immunoprecipitated with antibodies against the indicated proteins (the large subunit of RNA polymerase I (RPA194), CTCF, upstream binding factor (UBF) and acetylated histone H4). The position of the primer sets (upward arrows, see also ChiP2 in Figure 6A), spacer promoter (right-pointing arrow with ncRNA (part A), enhancer repeats (white rectangles) and gene promoter (right-pointing arrow with pre-rRNA)) is indicated on the rDNA. The horizontal axis of the panels is co-aligned with the rDNA underneath and shows distance in base pairs.