Regulation of Germ Cell Promoter Activity in Xenopus Oocytes

Abstract

Here we evaluate the feasibility of using Xenopus laevis oocytes as a model system for assaying the activity and architecture of a germ cell promoter. To test this approach the promoter from the X. laevis ALF transcription factor gene was cloned and evaluated for transcription start site usage and DNA methylation status. Comparison with human, chimpanzee, macaque, mouse, rat, cow, pig, horse, dog, chicken and X. tropicalis revealed low homology and demonstrated the difficulty in the computer prediction of regulatory elements. However, the activity of ALF promoter‐luciferase reporter constructs assayed after microinjection into oocytes was very high and could be localized to a 63 base pair segment. Interestingly, promoter activity in oocytes and somatic Xenopus XR epithelial cells were very different, indicating that these cell types are not interchangeable as assay systems. The use of site‐specific promoter mutants and deletion constructs identified two distinct functional elements (A and B) in the promoter, both of which were required for activity and recognized by proteins present in oocyte extracts. Rearrangement of the two elements displayed effects on activity ranging from minimal to severe. Overall, the results show that microinjection of a germ cell‐specific promoter into Xenopus oocytes provides a viable model system for dissecting the biochemistry of germ cell gene regulation.