Identification of a cell type-specific enhancer in the distal 5'-region of the platelet-derived growth factor A-chain gene.
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.
Maul
R S
RS
Department of Pharmacology, University of Kentucky Medical Center, Lexington, Kentucky 40536, USA.
Zhang
H
H
Reid
J D
JD
Pedigo
N G
NG
Kaetzel
D M
DM
D001483:Base Sequence; D002460:Cell Line; D004247:DNA; D004742:Enhancer Elements, Genetic; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000235:genetics*; D011485:Protein Binding
D009687:Nuclear Proteins; D010982:Platelet-Derived Growth Factor; C075278:platelet-derived growth factor A; D004247:DNA