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. 2011 Apr 18;108(18):7391–7396. doi: 10.1073/pnas.1018279108

Fig. 4.

Fig. 4.

Chromosome architecture of the human and mouse HOXA locus. Interaction frequencies between the anchor primer (noted by a yellow circle) and other primers in the locus were determined by real-time PCR and normalized to a random ligation library generated from EcoRI restriction digestion fragments from the bacterial artificial clones (BAC) covering the HOXA locus (CTD-3054H22 and CTD-2536K9) of this locus. A shows the interaction frequencies observed with the anchor primer located at CBS5 (blue vertical bar) with the all the other primers in the locus in the top panel. The middle panel shows the interaction frequencies observed with a control anchor primer located 12-Kb downstream from CBS5 with all the other primers in the locus. The bottom panel shows the corresponding H3K27me3 ChIP-seq density across the HOXA locus in IMR90 cells. B is the result of the 3C assay in IMR90 cells infected with either CTCF or RAD21 knockdown (KD) viruses, or control pGIPz virus, tested to determine the interaction between CBS5 and the heterochromatin boundary (p40), which was detected as the strongest specific long-range interaction site in A. C and D show the interaction frequency of CBS5 or control primer (noted by yellow circles) with all the other 3C primers in mES cells and mNPC across the mouse HOXA locus. The interaction frequencies were calculated as in A. The control random ligation library for the mouse HOXA locus was generated using the BAC, RP23-33N14. The H3K27me3 ChIP-Seq densities across the HOXA locus from mES and mNPC are shown in the bottom panel. A strong interaction frequency observed between the close proximal site (within 10-Kb downstream to CBS5) and the anchor site at CBS5 likely results from self-ligation and thus the strong signal may not indicate long-range interaction between the two sites. E–G are the results of ChIP-qPCR at CBS5 performed using CTCF, RAD21, and OCT4 antibodies, respectively, and using mES and mNPC chromatin. H shows the results of ReChIP performed with CTCF and RAD21 antibodies following a primary OCT4 ChIP in ES cells.