FIG. 1.

Glutamine-rich activation domains are dependent on a distal enhancer in human cells. (A) HeLa cells were cotransfected with expression plasmids where the DBD of Gal4 (amino acids 1 to 93) was fused to glutamine-rich activation domains of Sp1, Oct1, or Oct2 (Transactivators), along with a reporter plasmid under the control of two proximal Gal4 binding sites, with or without a distal SV40 enhancer (Reporters). As an internal control, a reference plasmid expressing the 5′-truncated form of the β-globin gene was cotransfected. (B) Total RNA was harvested 2 days after transfection of HeLa cells, and S1 nuclease analysis was performed. Lanes 7 to 12, due to the stronger reporter signals, were exposed for a shorter time than lanes 1 to 6. Quantification of the reporter signal (Rep.) relative to the corresponding reference (Ref.) was carried out by using a PhosphorImager. Without a distal enhancer, the glutamine-rich activation domains stimulate reporter gene expression only slightly above the background level (lanes 3 to 6 versus lanes 1 and 2). In combination with the remote SV40 enhancer, the glutamine-rich activation domains synergistically activated gene expression (lanes 9 to 12) when compared to the influence of the SV40 enhancer alone (lanes 7 and 8).