Fibroblast growth factor-8 expression is regulated by intronic engrailed and Pbx1-binding sites

Area of Science:

• Developmental Biology
• Gene Regulation
• Molecular Biology

Background:

• Fibroblast growth factor-8 (FGF8) is crucial for vertebrate development, with precise spatiotemporal expression patterns.
• Understanding the transcriptional regulation of FGF8 is vital for deciphering developmental processes.

Purpose of the Study:

• To identify regulatory regions of the Fgf8 gene responsible for its transcriptional control.
• To investigate the role of specific transcription factors in Fgf8 gene regulation using mouse embryonic stem cells.

Main Methods:

• Stable transfection of mouse embryonic stem (ES) cells with a human growth hormone reporter gene.
• Analysis of promoter and enhancer activities using Fgf8 gene fragments.
• Identification of transcription factor binding sites (AP2, Pbx1, Engrailed) within the Fgf8 intron.
• Gel shift and Western blot assays to detect transcription factor presence.
• In vitro mutagenesis to assess the functional impact of binding sites.

Main Results:

• A 2-kilobase 5' region of Fgf8 demonstrated promoter activity.
• A 0.8-kilobase intronic fragment enhanced reporter gene expression 3-4 fold in an orientation-dependent manner.
• The intronic fragment contains binding sites for AP2, Pbx1, and Engrailed transcription factors, which were detected in ES cell nuclear extracts.
• Mutagenesis of Engrailed or Pbx1 sites affected intronic fragment activity, suggesting their modulatory role.
• Further mutagenesis indicated that unidentified sites also contribute to the observed transcriptional enhancement.

Conclusions:

• Specific regulatory elements within the Fgf8 gene, including a potent intronic enhancer, control its transcription.
• AP2, Pbx1, and Engrailed transcription factors play a role in modulating Fgf8 gene expression.
• The precise mechanisms of Fgf8 transcriptional regulation involve multiple, potentially unidentified, regulatory elements and factors.