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Okadaic acid induces transcription of junB through a CCAAT box and NF-Y
J S Finch1, S F Rosenberger, J D Martinez
1Arizona Cancer Center, University of Arizona, 1515 N. Campbell Avenue, Tucson, AZ 85724-5024, USA.
Abstract:
The shellfish toxin, okadaic acid (OA), is a potent tumor promoter that induces expression of the proto-oncogene junB in mouse keratinocyte 308 cells. Here we show, through deletion analysis of the junB promoter, that sequences near the TATA box conferred transcriptional induction by OA. Transient transfections of luciferase constructs bearing the junB promoter with single mutations in various cis elements demonstrated that a promoter containing a mutated CCAAT box could not be induced by OA. When this CCAAT box was inserted into a heterologous promoter construct, OA induction was dependent on an intact CCAAT box. Flanking cis elements located near the CCAAT box, although not required for OA inducibility, did play a role in the basal level of transcription. NF-Y was shown by EMSA to bind to the CCAAT box. OA induction from the junB CCAAT box was blocked by dominant negative NF-YA as well as the CCAAT box-dependent anticancer drug, ET-473. Expression of a lexA/NF-YA chimeric protein demonstrated that OA induction was dependent on the binding of NF-Y family members. These studies demonstrate that OA can mediate transcriptional activation of junB through the classical CCAAT box and that transcription factor NF-Y plays a functional role in the induction.
Insights
Okadaic acid, a shellfish toxin, promotes tumors by increasing junB gene expression. This induction relies on the CCAAT box and NF-Y transcription factor binding, crucial for tumor promoter activity.
Area of Science:
- Molecular Biology
- Cancer Research
- Toxicology
Background:
- Okadaic acid (OA) is a known tumor promoter.
- Proto-oncogene junB expression is induced by OA in mouse keratinocytes.
- The regulatory mechanisms of junB induction by OA are not fully understood.
Purpose of the Study:
- To elucidate the role of specific promoter elements in OA-induced junB transcription.
- To identify the transcription factors involved in OA-mediated junB induction.
- To investigate the functional significance of the CCAAT box and NF-Y in this process.
Main Methods:
- Deletion analysis of the junB promoter.
- Transient transfection assays using luciferase reporter constructs.
- Site-directed mutagenesis of cis-acting elements.
- Electrophoretic mobility shift assays (EMSA) to assess transcription factor binding.
- Use of dominant-negative NF-YA and a chimeric protein to study NF-Y function.
Main Results:
- Transcriptional induction by OA was conferred by sequences near the TATA box.
- A mutation in the CCAAT box abolished OA induction of junB.
- OA induction of a heterologous promoter was dependent on the inserted CCAAT box.
- NF-Y was identified as the transcription factor binding to the CCAAT box.
- OA induction was blocked by dominant-negative NF-YA and the drug ET-473, confirming NF-Y's role.
Conclusions:
- Okadaic acid mediates transcriptional activation of the junB gene through the CCAAT box.
- Transcription factor NF-Y plays a critical functional role in OA-induced junB expression.
- These findings highlight a molecular mechanism by which the tumor promoter OA acts.