Identification of a cell type-specific enhancer in the distal 5'-region of the platelet-derived growth factor A-chain gene

Abstract

Transient transfection analysis of DNA subfragments from the distal 5'- flanking region of the human platelet-derived growth factor A-chain gene (- 18.3 to -1.8 kilobase pairs (kb)) revealed enhancer and silencer elements that contribute significantly to transcriptional regulation. Two adjacent regions (-8.2 to -7.5 kb and -7.5 to -7.0 kb) enhanced transcription of both A-chain and heterologous thymidine kinase promoters, whereas repression was observed in two other nearby regions (-9.9 to -8.2 kb and -7.0 to -5.9 kb). The -7.5 to -7.0-kb fragment, or J, was the strongest enhancer, and its activity was localized to a 66-base pair element (A-chain cell type-specific enhancer (ACE 66)). ACE66 activity was highly cell type-specific, with greatest activity seen in choriocarcinoma cell lines (4-10-fold enhancement). Progressive 5'- and 3'-deletions of the ACE66 revealed distribution of activity across the element, with nucleotides 1-33 being critical for function. Electrophoretic mobility shift assays revealed cell type-specific patterns of high affinity protein binding to the element. Ethylation interference footprinting of JEG-3 extract localized guanine contacts on nucleotides 1-18 of both strands of the ACE element, whereas more extensive contacts were made with the phosphate backbone (nucleotides 1-32). The ACE66 element is a potent transcriptional regulator in placental cells and represents a valuable model of long distance regulation in a growth factor gene.