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Identification of COUP-TF as a transcriptional repressor of the c-mos proto-oncogene
1Department of Biology, Boston University, Boston, Massachusetts 02215, USA.
Abstract:
The c-mos proto-oncogene is specifically expressed in the male and female germ cells of the mouse and other vertebrates. We previously identified a 15-base pair sequence element (B2) as the binding site of a candidate repressor of c-mos transcription in somatic cells. In the present study, we used the yeast one-hybrid system to isolate HeLa cell cDNAs encoding proteins that specifically bound to the c-mos B2 element. Nucleotide sequencing identified several of the clones isolated in this screen as the orphan nuclear receptors COUP-TFI and COUP-TFII. A COUP-TF-binding site was then identified within the B2 sequence. Complexes formed between purified COUP-TFs and the c-mos B2 probe comigrated in electrophoretic mobility shift assays with those formed using whole nuclear extracts of NIH 3T3 or HeLa cells. Moreover, the complexes formed with NIH 3T3 nuclear extracts and B2 probe were supershifted with antibody against COUP-TF, identifying COUP-TF as the candidate repressor previously detected in these somatic cell extracts. Substitution of a consensus COUP-TF-binding site for the c-mos negative regulatory element suppressed expression from the c-mos promoter in transfected somatic cells, demonstrating the functional activity of COUP-TF as a repressor of c-mos transcription.
Insights
Orphan nuclear receptors COUP-TFI and COUP-TFII bind to the c-mos proto-oncogene B2 element. This binding identifies COUP-TF as a repressor of c-mos transcription in somatic cells.
Area of Science:
- Molecular Biology
- Gene Regulation
- Oncology
Background:
- The c-mos proto-oncogene is crucial for germ cell development.
- A specific DNA sequence (B2 element) in the c-mos gene acts as a binding site for a repressor in somatic cells.
Purpose of the Study:
- To identify proteins that bind to the c-mos B2 element using a yeast one-hybrid system.
- To determine the role of these proteins in regulating c-mos transcription.
Main Methods:
- Yeast one-hybrid screening to isolate binding proteins.
- Nucleotide sequencing to identify isolated cDNAs.
- Electrophoretic mobility shift assays (EMSA) to analyze protein-DNA interactions.
- Supershift assays using antibodies against identified proteins.
- Functional assays involving promoter activity in transfected cells.
Main Results:
- HeLa cell cDNAs encoding orphan nuclear receptors COUP-TFI and COUP-TFII were isolated.
- A functional COUP-TF binding site was identified within the c-mos B2 element.
- COUP-TF was confirmed as the repressor of c-mos transcription in somatic cells via EMSA and supershift assays.
- Replacing the native element with a consensus COUP-TF site suppressed c-mos promoter activity.
Conclusions:
- COUP-TFI and COUP-TFII bind to the c-mos B2 element.
- COUP-TF acts as a functional repressor of c-mos transcription in somatic cells.
- This identifies a key regulatory mechanism for c-mos proto-oncogene expression.