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Antisense promoter mapping. Inhibitory methods of transcriptional analysis
1Department of Cell Biology, Vanderbilt University, Nashville, Tennessee 37232.
Abstract:
We have employed antisense methods to study the transcriptional functions of c-fos protein (Fos). Clones expressing inducible anti-fos RNA have been employed to inhibit c-fos expression, resulting in activation of c-fos transcription by inhibiting its normal repressor function. The sites of negative regulation by Fos have been mapped using this antisense mapping method which demonstrates that the serum response element represents the major site of repression by endogenous c-fos protein. A similar strategy (antisense cloning) has been employed to clone four target genes that are Fos dependent. These cDNAs encode mRNAs that are rapidly induced by serum (although this induction is blocked by cycloheximide) but are blocked by induction of anti-fos RNA. These inhibitory methods of studying transcription factor function are extremely useful for transcription factors (like Fos) that require cooperation with other factors to modulate gene transcription.
Insights
Antisense methods reveal that c-fos protein (Fos) represses transcription at the serum response element. This approach also identified four Fos-dependent genes, advancing our understanding of transcription factor regulation.
Area of Science:
- Molecular Biology
- Gene Regulation
- Transcription Factors
Background:
- The c-fos protein (Fos) is a transcription factor involved in cellular responses to stimuli.
- Understanding Fos's transcriptional functions and regulatory mechanisms is crucial for deciphering gene expression control.
Purpose of the Study:
- To investigate the transcriptional functions of the c-fos protein (Fos) using antisense methodologies.
- To identify target genes regulated by Fos and map its sites of negative regulation.
- To demonstrate the utility of antisense approaches for studying transcription factor functions.
Main Methods:
- Employing antisense RNA to inhibit c-fos expression and study its repressor function.
- Mapping sites of negative regulation by Fos using antisense mapping.
- Utilizing antisense cloning to identify Fos-dependent genes.
Main Results:
- Inhibition of c-fos expression using antisense RNA led to the activation of c-fos transcription, indicating Fos's repressor role.
- The serum response element was identified as the primary site of repression by endogenous c-fos protein.
- Four novel Fos-dependent genes were cloned, encoding mRNAs rapidly induced by serum but blocked by anti-fos RNA.
Conclusions:
- Antisense methods are effective for studying transcription factors like Fos that require co-factors.
- Fos protein plays a significant role in repressing transcription, particularly at the serum response element.
- The identification of Fos-dependent genes provides new insights into serum-induced gene expression pathways.