B1-X35S is a SINE with insulator activity. (A) Wild-type B1-X35S or its mutant forms Xmut35S (AHR site mutated), X35Smut (E-box mutated), and Xmut35Smut (both sites mutated) were transiently transfected and their insulator activity analyzed by EBA. The constructs are illustrated at the top of the figure. Data are presented as fold-enhancer blocking activity normalized to the reference pELuc vector. (B) Occupancy of SLUG and SNAIL on B1-X35S was analyzed by qChIP after transient transfection of either protein. (C) Effects of AHR expression on SLUG and SNAIL recruitment to B1-X35S were determined by qChIP after transient transfection of AHR-specific shRNA or scrambled shRNA as negative control. (D) Simultaneous presence of AHR and SLUG on B1-X35S was analyzed by sequential qChIP. A first ChIP used anti-SLUG antibody and the immunoprecipitated DNA was re-ChIPed with anti-AHR antibody. Re-ChIP for GAPDH provides a negative control. Data were quantified with respect to input DNA from the first ChIP. Results for Dad1 and Tbc1d1 are shown. (E) The effect of AHR, SLUG, and SNAIL expression on the insulator activity of wild-type B1-X35S was analyzed by EBA and quantified as above. (F) The experiment in E was done using the double-mutant B1-Xmut35Smut. Human HEK 293 cells were used in A, E, and F, while mouse Hepa-1 cells were used in B, C, and D. (B,C) The average data from the five B1-X35S containing genes Dad1, Lpp, Cabin1, Tbc1d1, and Rtl1. Individual results for each gene and experimental condition (B,C) are detailed in Supplemental Table 1. Data are shown as mean ±SD. (*) P = 0.033 and (**) P = 0.009 with respect to B1-X35S.
