Related Experiment Videos
Repression of platelet-derived growth factor A-chain gene transcription by an upstream silencer element.
1Department of Pharmacology, University of Kentucky Medical Center, Lexington, Kentucky 40536, USA.
Abstract:
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.
Insights
A specific DNA region in the Platelet-derived growth factor A-chain gene acts as a transcriptional silencer. This silencer function is mediated by antisense strand binding proteins interacting with a key G-rich motif.
Area of Science:
- Molecular Biology
- Gene Regulation
Background:
- Platelet-derived growth factor A-chain (PDGF-A) is a mitogen found in normal and cancer cells.
- Understanding PDGF-A gene regulation is crucial for cellular growth and disease research.
Purpose of the Study:
- To identify and characterize regulatory elements within the PDGF-A gene promoter.
- To elucidate the mechanism of transcriptional repression mediated by a specific DNA region.
Main Methods:
- Transient transfection and deletion analysis in BSC-1 cells.
- S1 nuclease hypersensitivity assays.
- Electrophoretic mobility shift assays (EMSA) and Southwestern blot analysis.
Main Results:
- A -1680 to -1374 region of the PDGF-A gene repressed promoter activity by 60-80%.
- A hypersensitive region (5'SHS) within this locus exhibited silencer activity in multiple cell lines.
- The 5'SHS element binds specific nuclear proteins, particularly to its antisense strand and a central G-rich motif, which is critical for repression.
Conclusions:
- The 5'SHS region functions as a potent transcriptional silencer for the PDGF-A gene.
- Antisense strand binding proteins interacting with a specific G-rich motif are key mediators of this silencing.
- This mechanism may involve stabilizing DNA conformations for interaction with enhancer elements.