Figure 6. CTCF-containing loops in the IgH locus.

(A) A schematic of the IgH locus as described in Figure 2A. J558/3609, S107 and 7183 refer to V_H gene families. Triangles show positions of oppositely-oriented primers labeled V_H3 and V_H10 used in ChIP-loop and 4C assays. The nearest V_H gene segment to these primers is indicated.

(B) ChIP-loop 4C assays were performed using D345 pro-B cells. Cross-linked chromatin was immunoprecipitated with anti-CTCF antibodies, followed by digestion of the associated chromatin with Nla III. After re-ligation the DNA was amplified with V_H3 or V_H10 primers. Sequences amplified within V_H3 primers (red trace) or V_H10 primers (blue trace) were hybridized to Affymetrix chromosome 12 tiling arrays and quantitated as described in Experimental Procedures. Asterisk indicates position of anchor primers; labeled arrows indicate positions of sequences identified in the assay. Data shown is representative of two independent experiments with each anchor primer.

(C) Conventional 4C assay using Nla III restriction enzyme was carried out using V_H3 (red trace) or V_H10 (blue trace) anchor primers. Asterisks mark the position of anchor primers; arrows indicate interacting regions shared between ChIP-loop and 4C arrays.

(D) CTCF binding to sites identified by anti-CTCF ChIP-loop assays. Cross-linked chromatin from D345 cells was immunoprecipitated with anti-CTCF or anti-Rad21 antibodies. Co-precipitated genomic DNA was amplified with primers close to regions identified in ChIP-loop and 4C assays. V_H1, 2, 3, V_H3-1 to V_H3-5 lie in the cluster of interacting sequences identified with V_H3 primers. V_H10-1 to V_H10-4 correspond to interacting sequences identified with V_H10 primers. V_H3 and V_H10 amplicons are close to the corresponding anchor primers used in 4C assays. CTCF binding to the 3′ DNase I hypersensitive site (3′HS1) in the β globin locus was used as the positive control. CTCF binding within the IgH locus is Eμ-independent (Figure S5A). Error bars represent the standard deviation between three independent experiments.

(E) Top line shows the location of five short probes used in 3D FISH (see also Figure S5B). Three color 3D-FISH was carried out using bone marrow pro-B cells with wild type and P⁻E⁻ IgH alleles on a RAG2-deficient background. Probes were labeled as follows: a and c, DFL-red, V3-green; b and d, V10-3-red, V10-green. Distances between probes were determined as described in Figure 4C and shown in Table S1. Right panels show quantitation of FISH data as described in Figure 4C, obtained from the analyses of 60-90 nuclei from 2 independent cell preparations. 3C analysis of V3-DFL loops in WT and Eμ⁻ pro-B cells is shown in Figure S5C, D.