BGLT3 deletion affects interaction of γ-globin genes with BGLT3 but not with the LCR. (A) 3C was carried out for ΔBGLT3 K562 cells and control cells with no gRNA (ctrol). The interaction frequency of each fragment with the LCR anchor fragment (yellow bar) is plotted in the middle of the fragment (dotted vertical lines). Blue diamonds, EcoR1 sites. (B) 3C was carried out as in panel A except the anchor fragment was BGLT3. Primer sequences listed in supplemental Table 1. Note that the Gg primer does not distinguish between ^Gγ and ^Aγ, and the values presented represent the average normalized signals from both³¹  (supplemental Figure 4). (C) Control and ∆BGLT3 K562 cells were subjected to ChiP using antibodies against total RNA Pol II (N20) and primers amplifying regions as indicated below the graph. (D) ChIP was carried out as in panel A with antibodies to TBP. (E) ChIP was carried out as in panel A with antibodies to H3K4me3. The results were normalized to the ChIP signal for total histone H3. Error bars indicate standard deviation; n = 3 biological replicates. **P < .01 by Student t test. Primer sequences are listed in supplemental Table 1; antibodies are listed in supplemental Table 2.