Fig 4.

Stimulation induces enlarging subgene loops to form in 458-kbp ZFPM2 and 312-kbp EXT1. HUVECs were treated with TNF-α for 0 to 85 min, and 3C was performed on templates prepared using HindIII. In some cases, 50 μM DRB was added 25 min before harvesting. Diagrams on the left illustrate regions targeted by 3C primers (green arrow for the reference point; white arrows for the others). (A) Changing contacts detected using 3C-PCR. Details and controls are as for Fig. 3B. Contacts sweep down the gene in time with the traveling wave, and DRB abolishes contacts. Primers b plus g yield no band at 0 min indicative of a whole-gene loop (or at any time, since the pioneering polymerase only reaches the termini well after 85 min). (B) Interaction frequencies determined by 3C-PCR. The positions of standing (green) and traveling (yellow) waves are indicated. Primer b (green) was used in turn with the primers indicated, and the results are given in the same row as the indicated primer. The interaction frequency (arbitrary units [au]] ± the SD; n = 4) is normalized relative to value given by “loading” and “intra-GAPDH” controls. Again, peak interactions (which are DRB sensitive) coincide with the traveling wave, and no whole-gene loop is seen in either gene. *, difference significant (P < 0.01, two-tailed unpaired Student t test) compared to the 0-min sample.