FIG. 6.

Analysis of enhancer-blocking and silencing activity of the 200-bp CTCF-binding fragment by transient transfection of linearized (A) or circular (B) plasmids into HD3 cells. Constructs are shown on the left and CAT activity is shown on the right. The chicken adult β-globin promoter (gray boxes) and a minimal β^A/ɛ enhancer (black boxes) were used to control expression of the reporter CAT gene (construct 1). As a negative control, the same construct with a deleted β^A/ɛ enhancer (construct 2) was used. The 200-bp DNA fragment (white boxes) was cloned in the SalI or HindIII sites of pAcatE in different orientations (indicated by arrows showing the direction of α-globin gene transcription). The filled circle inside the 200-bp fragment indicates the approximate position of the CTCF-binding site. CAT activity was normalized considering the efficiency of transfection. Error bars show the standard error of the mean (SEM) from three independent experiments.