Eviction of CTCF and RAD21 from the −31 element accompanies dissolution of the TAL1 active hub and loss of proximity between the +57 and −31 elements. (A) CTCF occupancy at the +57, +40, and −31 elements 48 and 96 hours after siRNA transfection with GATA1 (KD) or with luciferase (LUC) in K562 cells. (B) RAD21 occupancy at the +57, +40, and −31 elements 48 and 96 hours after siRNA transfection with GATA1 (KD) or with luciferase (LUC) in K562 cells. Gray vertical arrows in panels A and B highlight CTCF and RAD21 occupancy levels at −31. ChIP enrichments (log₂) are shown with SEs. Annotation of test and negative control regions is denoted in black and gray text, respectively. Positive control is a CTCF/RAD21-bound element at the HNF4A locus. The percentage (%) occupancies of each protein in the GATA1 knockdown relative to the level found in the luciferase control are also shown. (C) Bar diagrams of looping interactions, measured as relative ligation frequencies (black bars), between the +57 and −31 elements determined by 3C at 48 and 96 hours after siRNA transfection with GATA1 (KD) or with luciferase (LUC) in K562 cells. Locations of 3C “bait” regions are denoted by vertical gray arrows. Loss of interactions due to GATA1 knockdown at 96 hours is shown by the dotted gray lines with arrowheads (and marked with an “X”). P values are indicated for relative ligation frequencies which are significantly reduced between the +57 and −31 elements in KD conditions when compared with the corresponding LUC controls. (D) Schematic model of looping interactions between CTCF/RAD21-bound elements at the TAL1 locus, and during GATA1 siRNA knockdown, in K562 cells. Loss of GATA1 is accompanied by (1) loss of CTCF and RAD21 from the −31 element and (2) loss of looping interactions between +57 and −31 (96 hours GATA1 knockdown) as the TAL1 erythroid hub is disassembled. *P < .05; **P < .01; ***P < .001.