Figure 3.

Role of the Sp1 binding sites in the recruitment of various pointers to the promoter and on DNA looping. (A) The expression levels of the target/reporter plasmids containing a deletion (×) of the distal (#1) or the proximal (#2) Sp1 site were determined by measuring CAT activities (percent acetylation) after transient transfection (32). For these transfections, 5 μg of the reporter gene and 2 μg of the expression vector for TR were used, and cells were treated with or without T3. The extent (fold, in parenthesis) of silencing with TR and activation with T3/TR is in comparison with the basal level of expression (without TR). The data for CAT activities were obtained from three to six independent transfections. (B) Recruitment of Sp1, TBP, TFIIB, and Cdk7 pointers to the target plasmids [wild type (WT), #1, and #2] were analyzed as described in the legend for Fig. 1B. (C) Analysis of DNA looping in target plasmids #1 and #2. Cleavage in the TRE region was analyzed by performing LM-PCR on the samples from B with primer 2. (D) Summary of pointer recruitment and detectable DNA looping for target plasmids WT, #1, and #2. “US” indicates unstable or transient DNA looping.