A PROMOTER-LINKED COUPLING REGION REQUIRED FOR STIMULATION OF ALPHA-FETOPROTEIN TRANSCRIPTION BY DISTANT ENHANCERS

We previously demonstrated that the rat alpha-fetoprotein (AFP) gene h as three upstream enhancers that stimulate the AFP promoter additively in HepG2 cells (1). In this paper, deletion analysis demonstrated tha t a promoter-linked segment from - 178 to - 155 was required for full activity when the enhancers were distant from the promoter, even at le ss than their normal genomic distances, but dispensable when the enhan cers were moved close to the promoter. This 'promoter-coupling element ' appears to interact simultaneously with all three enhancers. Deletio n analysis also localized a transcription stimulatory and a negative r egion in the promoter. Though these latter regions controlled the stre ngth of the isolated promoter, they did not affect 'coupling' to the d istant enhancers, and transcription stimulation by these distal promot er elements was small compared to the distant enhancers. Overall, the distant enhancers, acting through the promoter-coupling element, accou nted for 70% of the activity of the transfected AFP gene. Footprint an alysis with HepG2 nuclear extracts demonstrated protein binding at two sites near the promoter-coupling element. The data indicate a positiv e transcription control mechanism by which distant enhancers stimulate the AFP promoter through a specific promoter-linked element.