Figure 6: RepID is present in a complex between LCR and Rep-P in early replicating HBB loci.

(a) Schematic illustration of the beta-globin locus and the outline of ChIP-3C procedure. Cells were lysed and digested with HindIII. Crosslinked chromatin fragments were immunoprecipitated with anti-RepID antibody and ligated. Crosslinks were reversed and DNA was isolated and amplified by PCR. The primers and TaqMan probe used for the ChIP-3C analysis are listed in the ‘Methods’ section. The red arrows indicate the location of AG and HS2 on the beta-globin locus, which are more than 50 kb away from each other. (b) ChIP-3C analysis of long-distance RepID-associated chromatin interactions at the HBB locus in U2OS WT and RepID knockout cells. The 3C primers correspond to sequences near the downstream sticky ends of the 3C fragments. Primer/probe combinations were designated p6 to p13, and their locations are indicated as half arrows. The x axis shows the positions of the restriction fragments on the genomic scale (vertical bars). The diagram represents the extent of PCR amplification of each primer/probe pair. Values represent the average of triplicate samples (bars represent s.e.’s). Data were obtained after subtraction of no ligation controls and were normalized to the AG site loading control. Primer efficiencies were normalized using a single bacterial artificial chromosome (BAC) clone covering the genome segment under study.