Loss of BGLT3 affects RNA Pol II recruitment to γ-globin genes. (A) 3C was carried out using CRISPRi K562 cells targeted with (dCAS9/gRNA) or without (dCAS9) a specific gRNA with anchor fragment BGLT3. (B) 3C was carried out as in panel A except using K562 cells treated with a BGLT3 specific ASO or a control version (ctrol). Primer sequences are 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 both (supplemental Figure 4). (C) RNA-ChIP was carried out using K562 cells and antibodies to CBP, MED12, or RAD21. MALAT1, highly expressed in erythroid cells, and eRNA LINC00853, lowly expressed in K562 cells, served as positive controls for MED12 binding.^18,21,55  (D) ChIP-qPCR was carried out for K562 cells with primers across BGLT3 using antibodies to MED12. Locations of BGLT3 primers shown in Figure 1G; primer sequences are listed in supplemental Table 1; antibodies are listed in supplemental Table 2. (E) Model of the BGLT3-containing locus interaction (solid lines) with the γ-globin genes and with the LCR before or after deletion of BGLT3 sequences. Dotted lines represent lost interactions; red triangle represents deletion of BGLT3; wavy orange lines, γ-globin transcripts; wavy purple lines, BGLT3 transcripts.