CTCF occupancy and chromatin loop formation over 2 Mb of chromosome 11. (A) CTCF localization on chr11:4,000,000–6,000,000 in K562 and 293T cells derived from ChIP-chip experiments. CTCF peaks were predicted using the ACME algorithm optimized to the training set (C1–C31) of sites validated by ChIP. RefSeq genes are depicted at the bottom in red (globin genes), blue (odorant receptor genes), or gray (other genes). (B) Long-range interactions among CTCF/cohesin sites vary between cell types. (Upper) 3C was carried out with K562 cell chromatin. Specific interactions between and among CTCF sites (black circles; gray circles represent CTCF sites not analyzed by 3C) are depicted by blue lines with the height of the curve corresponding to the cross-linking frequency. (Lower) Interactions among CTCF sites in 293T cells are depicted by red curves.Cross-linking is plotted relative to the signal for two fragments in the α-tubulin gene. Note the log scale of the y axis. Error bars represent SD.

Reduction of CTCF results in loss of long-range interactions between and among its binding sites. (A) Western blot indicating reduction of CTCF protein by shRNA without alteration of Rad21 or SMC3 levels. (B) K562 cells were transduced with control or CTCF shRNA. ChIP was performed with an antibody to CTCF. Error bars represent SEM. (C) 3C was carried out using chromatin from K562 cells transduced with control or CTCF shRNA. Interactions between and among CTCF sites are indicated by blue curves, and reduction of these interactions after knock-down of CTCF is indicated by gray curves. Error bars represent SD. **P < 0.01, *P < 0.05.

Reduction of cohesin components diminishes long-range interactions among CTCF sites. (A) Western blots showing reduction of Rad21 by shRNA and lack of effect of this reduction on CTCF protein. (B) K562 cells were transduced with control or Rad21 shRNA. ChIP was performed with an antibody to Rad21. Error bars represent SEM. (C) ChIP was carried out as described in B with antibodies to CTCF. (D) 3C was carried out using chromatin from K562 cells transduced with control or Rad21 shRNA. Interactions between and among CTCF sites are indicated by blue curves, and reduction of interactions after knock-down of Rad21 is indicated by gray curves. Error bars represent SEM. **P < 0.01, *P < 0.05.

Effect of CTCF shRNA on K562 cells. (A) RT–PCR analysis of globin gene transcription in K562 cells after transduction with control or CTCF shRNA. Results of three RNA preparations are shown ±SEM. (B) ChIP performed on chromatin from K562 cells after treatment with control or CTCF shRNA using an antibody against RNA pol II. Error bars represent SEM. Amplicons include ERVin and ERVdn, within and downstream (unique amplicon) of ERV9-LTR; HS5 and 3′HS1, globin locus-flanking CTCF sites; HS1–HS4, locus control region DNase I hypersensitive sites; globin gene promoters and exons as indicated; and OR51V1, odorant receptor gene in the globin locus 3′ flank. (C) ChIP was performed using K562 cell chromatin as described in B with antibodies to H3K4me2, H3K9me2, CTCF, and SMC1. H3 methylation is plotted with the highest value for each modification set to 1 in the line graphs. CTCF or cohesin ChIP enrichment is represented by superimposed bars. Diagram at the top: orange lines, globin genes; gray box, LCR; blue lines, odorant receptor genes; gray lines, other silent genes; green lines, ERV9-LTR. Note the depiction of chromosome 11 in the 5′ to 3′ direction, which is reversed compared to the Genome browser view in Figs. 1–3. Panel C is drawn to scale but the numbers below the graph are arbitrary. (D) ChIP was performed as described in B with antibodies to H3K4me2. (E) ChIP was performed as described in B using an antibody to H3K9me2.