Composite CTCF Binding Sites Figure 4. F1F2 and ESRRa composite elements mediate hormone sensitive enhancer blocking on episomes. (A) Schematic representation of the insulator-enhancer-reporter arrangement of the episomal vectors. The composite insulator sequence (CTCF/TR) is usually integrated as a single insert, or as 4x or 5x multimers as indicated for the constructs used. Cells transfected with the indicated episomes were incubated for 48 h in the absence or presence of T3. (B) Efficient enhancer blocking depends on the presence of the enhancer. The repressive effects cannot be attributed to direct promoter repression. Transfection was in N2ab cells. (C) TSA and 3-ABA were added 8 h or 28 h after transfection, respectively. Transfection was in N2ab cells. (D–F) HepG2 cells were additionally cotransfected with 0.2 mg TRb expression vector. (F) Comparison of the enhancer-less constructs with the enhancer constructs demonstrate that the enhancer blocking activity is dependent on the enhancer. doi:10.1371/journal.pone.0010119.g004 PLoS ONE | www.plosone.org 6 April 2010 | Volume 5 | Issue 4 | e10119