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Specific inhibition of c-fos proto-oncogene expression by triple-helix-forming oligonucleotides
Y Lavrovsky1, V Mastyugin, R A Stoltz
1Rockefeller University, New York, New York 10021, USA.
Abstract:
The promoter region of the c-fos oncogene 5' flanking sequence contains enhancer elements crucial for binding nuclear factors that regulate transcription following cell proliferation and differentiation. Single-stranded deoxyoligonucleotides were chosen for modulation of c-fos protooncogene expression because of their high-affinity binding to specific nucleotide sequences. We designed two oligonucleotides that form a triple-helix complex on the retinoblastoma gene product-responsible element of the c-fos oncogene. Modification of the DNA triplex with dimethyl sulfate and affinity cleaving assays demonstrate that the predicted oligonucleotides form a DNA triplex structure with the c-fos promoter in a sequence-specific manner. Tumorigenic and non-tumorigenic fibroblasts were transiently transfected with fos-CAT plasmid modified with alkylating triplex-forming oligonucleotide reagents. A dramatic depression of CAT activity was found when the cross-linked triple helix complex at the retinoblastoma gene product-related site of the c-fos promoter was used. These experiments suggest that transcription of individual genes can be selectively modulated in cell culture by sequence specific triplex formation in regulatory enhancer sequences.
Insights
Researchers developed DNA triplexes to control the c-fos oncogene. This sequence-specific gene modulation offers a new way to regulate gene expression in cell culture.
Area of Science:
- Molecular Biology
- Gene Regulation
- Oncogene Research
Background:
- The c-fos oncogene promoter contains enhancer elements vital for gene transcription regulation.
- Single-stranded deoxyoligonucleotides offer high-affinity binding for modulating gene expression.
Purpose of the Study:
- To design and validate DNA triplexes for sequence-specific modulation of c-fos protooncogene expression.
- To investigate the potential of triplex formation in regulating gene transcription.
Main Methods:
- Design of two oligonucleotides to form a triple-helix complex on the c-fos promoter.
- Modification of DNA triplexes with dimethyl sulfate and affinity cleaving assays.
- Transient transfection of fibroblasts with fos-CAT plasmid and triplex-forming reagents.
Main Results:
- Demonstrated sequence-specific formation of DNA triplex structures with the c-fos promoter.
- Observed a significant depression of CAT activity when the triple helix complex was formed at a key regulatory site.
- Confirmed selective modulation of gene transcription via triplex formation.
Conclusions:
- Sequence-specific triplex formation can selectively modulate gene transcription in regulatory enhancer sequences.
- This approach offers a novel method for controlling gene expression in cell culture.
- Potential applications in understanding and manipulating oncogene activity.