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Cloning and analysis of the mouse follistatin promoter
E de Groot1, J Veltmaat, A Caricasole
1Hubrecht laboratory, Utrecht, The Netherlands.
Abstract:
Follistatin is a secreted protein, which functions as an antagonist of different members of the TGF-beta superfamily, including activin and bone morphogenetic proteins. Expression of follistatin is tightly regulated during mouse development both spatially and temporally. In order to study the regulation of follistatin expression in the mouse embryo we have cloned and analyzed part of the 5' flanking region of the murine follistatin gene. Primer extension and RNase protection assays demonstrate that the murine follistatin promoter region has at least three distinct transcription initiation sites, which are each preceded by a TATA box. All of the transcription initiation sites are located within the first 500 bp upstream of the translational start site. Sequence analysis of this 500 bp region revealed several consensus binding sites for transcription factors including AP-1, Brachyury-T, CREB, Sp1, AP-2 and Tcf. To test whether the 5' region displays promoter activity, we transfected various 5' flanking region deletion constructs into F9 embryonal carcinoma (EC) cells and into P19 EC cells. In these two cell lines a region of only 262 bp upstream of the translation start site could drivereporter expression in a manner that reflects endogenous mRNA expression.
Insights
Researchers identified the promoter region of the mouse follistatin gene, revealing key regulatory elements. A 262 bp sequence upstream of the translation start site drives reporter expression, mimicking endogenous follistatin mRNA levels.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- Follistatin is a secreted protein acting as a TGF-beta superfamily antagonist.
- Follistatin expression is precisely controlled during mouse embryonic development.
Purpose of the Study:
- To investigate the regulatory mechanisms of murine follistatin gene expression.
- To identify and characterize the promoter region of the follistatin gene in mice.
Main Methods:
- Cloning and analysis of the 5' flanking region of the murine follistatin gene.
- Primer extension and RNase protection assays to identify transcription initiation sites.
- Reporter gene assays in F9 and P19 embryonal carcinoma cells using deletion constructs.
Main Results:
- Identified at least three distinct transcription initiation sites within 500 bp upstream of the translational start site.
- Sequence analysis revealed consensus binding sites for transcription factors (AP-1, Brachyury-T, CREB, Sp1, AP-2, Tcf).
- A 262 bp region upstream of the translation start site demonstrated promoter activity, driving reporter expression consistent with endogenous follistatin mRNA levels in EC cells.
Conclusions:
- The 5' flanking region of the murine follistatin gene contains regulatory elements essential for its expression.
- A minimal promoter region of 262 bp is sufficient to direct follistatin gene expression in embryonal carcinoma cells.