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Altered DNA/protein complexes specific for the beta-interferon regulatory region observed in murine embryonal
1Department of Microbiology, Immunology, and Molecular Genetics, Albany Medical College, New York 12208.
Abstract:
Murine embryonal carcinoma (EC) F9 cells do not produce interferon (IFN) at the protein or RNA level in response to inducing agents, while retinoic acid differentiated F9 cells do produce IFN. A probe was constructed spanning positions -104 to -39 of the human beta-IFN upstream regulatory region to examine this developmental control at the level of a transcriptional regulatory mechanism. Gel mobility shift analyses were used to examine this molecular mechanism to determine whether the differential expression of positive or negative trans-acting factors may act to control beta-IFN expression in undifferentiated EC cells. These analyses showed that while nuclear extracts from poly-I,C induced L929 cells, in the IFN producing cell line, showed two shifted bands, nuclear extracts from both induced and uninduced F9 cells showed only one shifted band using the -104/-39 probe. While this single shifted band co-migrated with the faster migrating species of L929 cell extracts, competition analysis revealed differences between the two complexes. An oligonucleotide representing the positive regulatory domain PRDII competed efficiently for the probe when induced F9 cell extracts were examined, but failed to compete when induced L929 cell extracts were examined. In contrast, an oligonucleotide representing the positive regulatory domain PRDI competed very well when induced L929 cell extracts were examined but had only a minimal effect when induced F9 cell extracts were examined. These data suggest the involvement of developmentally regulated transcriptional factor(s) which have yet to be characterized.
Insights
Undifferentiated F9 cells lack interferon (IFN) production, but retinoic acid treatment enables it. This study investigates the transcriptional regulation of this developmental control, identifying distinct DNA-binding factors in F9 cells compared to IFN-producing cells.
Area of Science:
- Molecular Biology
- Developmental Biology
- Immunology
Background:
- Murine embryonal carcinoma (EC) F9 cells exhibit a unique lack of interferon (IFN) production, even when stimulated.
- Retinoic acid differentiation induces IFN production in F9 cells, suggesting a developmental regulation of this process.
- Understanding the transcriptional mechanisms controlling IFN expression is crucial for developmental biology and immunology.
Purpose of the Study:
- To investigate the transcriptional regulatory mechanisms underlying the differential expression of beta-interferon (IFN) in F9 cells.
- To determine if distinct trans-acting factors control IFN expression in undifferentiated versus differentiated F9 cells.
- To analyze the role of specific regulatory elements in the human beta-IFN gene promoter in this developmental control.
Main Methods:
- Construction of a DNA probe (-104 to -39) from the human beta-IFN upstream regulatory region.
- Gel mobility shift assays (EMSA) using nuclear extracts from induced and uninduced F9 cells and induced L929 cells.
- Competition analysis with oligonucleotides representing positive regulatory domains PRDI and PRDII.
Main Results:
- Nuclear extracts from induced L929 cells showed two shifted bands, while F9 cells (induced or uninduced) showed only one.
- Competition analysis revealed differential binding affinities: PRDII competed strongly in F9 cells, whereas PRDI competed strongly in L929 cells.
- These differences suggest the presence of distinct, developmentally regulated transcription factors interacting with the beta-IFN promoter.
Conclusions:
- The differential expression of beta-interferon in F9 cells is regulated at the transcriptional level.
- Distinct DNA-binding protein complexes interact with the human beta-IFN promoter in F9 cells compared to IFN-producing cells.
- These findings indicate the involvement of uncharacterized, developmentally regulated transcriptional factors controlling IFN gene expression.