Bi-directional Activity of the RARa Promoter is Inhibited by SV40-associated GC Repeats

Abstract

Retinoic acid receptors (RARs), are nuclear transcription factors that interact with other nuclear hormone receptors. Based on published data, the RARa promoter was PCR-amplified, ligated into the pGL3 basic luciferase reporter vector and transfected into Jurkat cells. Results showed that the RARa promoter was active in both the sense (RP) and the antisense (AsRP) orientations. Also, the promoter was inhibited in both orientations when ligated into the pGL3 enhancer vector. The mechanism of inhibition was further investigated. The enhancer portion of the pGL3 enhancer vector contains two different components of the SV40 virus DNA. One is the SV40 enhancer, which is composed of two 72 bp repeats (En). The other component is three 21 bp repeats that are elements of the SV40 early promoter (GC3). Each 21 bp repeat contains two binding sites for the transcription factor Sp1. Removal of the 21 bp repeats in the pGL3 enhancer resulted in enhancement of the AsRP activity, demonstrating that the repeats have a role in inhibition of the promoter. Bi-directional activity of the RARa promoter was also observed in MCF7, HepG2, RD131, CEM-SS and HeLa cell lines. However, the enhancement and inhibition caused by En and EnGC3 in Jurkat cells were found to be cell-type specific. The inhibition by the 21 bp repeats was further investigated by changing the arrangement of 21 bp repeats. Removing the first 21 bp repeat resulted in a 7-fold increase in promoter activity. Separation of the remaining two 21 bp repeat units by 6 bp resulted in inhibition of the promoter. A point mutation in this inhibitory region, causing disruption in one of the Sp1 binding sites, resulted in a 20-fold enhancement of promoter activity. These results indicate that the enhancement and inhibition of the RARa promoter is affected by the arrangement of Sp1 binding sites in the enhancer region. It is proposed that the mechanism of enhancement is due to physical association of Sp1 protein bound to the sites in the promoter and to sites in the enhancer, allowing interaction between the enhancer and the promoter. This association can stabilize the transcription apparatus. It is thought that the inhibition observed is due to changes in the spatial conformation of the Sp1 proteins bound to the GC units. These changes could alter the distance between the Sp1 domains that associate together such that association of the enhancer-bound Sp1 protein cannot occur with the Sp1 protein bound to the promoter region of the vector.