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Antisense mapping of the c-fos promoter: role of the serum response element
C Robinson-Benion1, N Kamata, J T Holt
1Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232.
Abstract:
Using an antisense RNA approach to eliminate endogenous expression of the c-fos protein, we have verified by nuclear run-on and transient expression assays that the Fos protein is a negative regulator of its own transcription in vivo. The negative autoregulation of the c-fos promoter by Fos was further confirmed by overexpression of an antisense-resistant c-fos expressing vector. Antisense mapping of the c-fos promoter demonstrated that the serum responsive element (SRE) represents the major site for c-fos suppression only during the first hour, but that additional adjacent DNA sequences are required for suppression at later times. We propose that antisense inhibition of transcriptional repressors provides a useful method for analyzing the significance and mechanism of transcriptional repression in vivo.
Insights
The Fos protein negatively regulates its own transcription. Antisense RNA technology helps study gene regulation and transcriptional repression mechanisms in vivo.
Area of Science:
- Molecular Biology
- Gene Regulation
- Cancer Research
Background:
- The c-fos gene is crucial in cellular responses to stimuli.
- Understanding the regulation of c-fos is key to deciphering cellular signaling pathways.
- Autoregulation mechanisms play a significant role in controlling gene expression.
Purpose of the Study:
- To investigate the role of the Fos protein in regulating its own transcription.
- To identify the specific DNA elements involved in Fos-mediated transcriptional repression.
- To establish antisense RNA as a tool for studying transcriptional repressors in vivo.
Main Methods:
- Utilized antisense RNA to inhibit endogenous Fos protein expression.
- Employed nuclear run-on and transient expression assays for verification.
- Performed antisense mapping of the c-fos promoter, including serum-responsive element (SRE) analysis.
- Confirmed findings using an antisense-resistant c-fos expressing vector.
Main Results:
- Demonstrated that the Fos protein acts as a negative regulator of c-fos transcription in vivo.
- Identified the serum-responsive element (SRE) as a major site for early c-fos suppression.
- Showed that additional DNA sequences are required for later-stage suppression.
- Confirmed negative autoregulation through overexpression of a modified c-fos vector.
Conclusions:
- Fos protein negatively autoregulates its own transcription.
- Antisense inhibition is an effective method for analyzing transcriptional repressors in vivo.
- The c-fos promoter has complex regulatory elements involved in autoregulation.