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. 2010 Nov 15;24(22):2543–2555. doi: 10.1101/gad.1967810

Figure 4.

Figure 4.

Both p68 and SRA are required for CTCF-mediated insulator function. (A) Schematic diagrams of the reporter constructs pIHLIE and pIHLME that were used in enhancer-blocking assays. In pIHLIE, CTCF binds to the H19 ICR and represses luciferase gene expression by blocking enhancer access to the promoter. In pIHLME, CTCF-binding sites have been mutated. (Enh) Enhancer; (H19P) mouse H19 promoter; (Mut) mutated ICR. (B) CTCF or p68 shRNA-depleted HeLa cells were transfected with either pIHLIE or pIHLME and a renilla luciferase control plasmid. Cells were harvested and luciferase activities were measured 48 h after transfection of the luciferase plasmids. Relative luciferase activity was determined for each cell lysate and normalized against control shRNA. (C) HeLa cells were depleted by SRA siRNA and then, 24 h later, transfected with either pIHLIE or pIHLME and a renilla luciferase control plasmid. Cells were harvested and luciferase activities were measured 24 h after transfection of luciferase plasmids. Relative luciferase activity was determined for each cell lysate and normalized against control siRNA. (D) Schematic representation of the pNI control and pNI-5′HS4 core insulator plasmids that were used in insulator assays. The plasmid backbone of pNI or pNI-core is pGEM4Z. pNI-core contains two copies of the chicken β-globin HS4 core insulator fragments, which are located between the mouse HS2 enhancer and Y-neomycin (Neo) reporter gene and which block the contact between the HS2 enhancer and Neo reporter gene. (E) K562 cells were depleted by CTCF or p68 shRNA and transfected with either pNI or pNI-core plasmid together with a GFP plasmid. Cells were harvested and RNAs were isolated for reverse transcription to examine the expression of the Neo reporter gene after 24 h of transfection. The expression of Neo was normalized against the expression of GFP. (F) SRA siRNA-depleted K562 cells were transfected with either pNI or pNI-core. Cells were harvested and RNAs were isolated for reverse transcription to examine the expression of the Neo reporter gene after 24 h of transfection. Relative neomycin expression was determined for each cell lysate and normalized against control shRNA (E) or control siRNA (F) samples. Data were analyzed by Student's t-test. For all panels, bars represent mean ± SD of n = 3. (*) P < 0.01.