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Volume 271, Number 42, Issue of October 18, 1996 pp. 26281-26290
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

Repression of Platelet-derived Growth Factor A-chain Gene Transcription by an Upstream Silencer Element
PARTICIPATION BY SEQUENCE-SPECIFIC SINGLE-STRANDED DNA-BINDING PROTEINS

(Received for publication, May 30, 1996, and in revised form, July 25, 1996)

Bin Liu , Raymond S. Maul and David M. Kaetzel Jr.

From the Department of Pharmacology, University of Kentucky Medical Center, Lexington, Kentucky 40536

Platelet-derived growth factor A-chain is a potent mitogen expressed in a restricted number of normal and transformed cells. Transient transfection and deletion analysis in BSC-1 (African green monkey, renal epithelial) cells revealed that the -1680 to -1374 region of the A-chain gene repressed homologous and heterologous promoter activities by 60-80%. An S1 nuclease-hypersensitive region (5'SHS) was identified within this region (-1418 to -1388) that exhibited transcriptional silencer activity in BSC-1 and a variety of human tumor cell lines (U87, HepG2, and HeLa). Electrophoretic mobility shift assays conducted with 5'SHS oligodeoxynucleotide probes revealed several binding protein complexes that displayed unique preferences for binding to sense, antisense, and double-stranded forms of the element. Southwestern blot analysis revealed that the antisense strand of 5'SHS binds to nuclear proteins of molecular mass 97, 87, 44, and 17 kDa, whereas the double-stranded form of 5'SHS is recognized by a 70-kDa factor. Mutations within 5'SHS element indicated the necessity of a central 5'-GGGGAGGGGG-3' motif for protein binding and silencer function, while nucleotides flanking both sides of the motif were also critical for repression. These results support a model in which silencer function of 5'SHS is mediated by antisense strand binding proteins, possibly by stabilizing single-stranded DNA conformations required for interaction with enhancer sequences in the proximal promoter region of the A-chain gene.


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