Mouse embryo Hox gene enhancers assayed in cell culture: Hoxb4, b8 and a7 are activated by Cdx1 protein

Mouse Hox gene enhancer elements have typically been identified and characterized using Hox/lacZ transgenic mouse embryos. Such studies have, for example, identified Cdx responsive binding motifs in the enhancers of Hoxb8 and Hoxa7. Production of transgenic mouse embryos involves issues of cost, welfare, and considerable technical skill. It would be of benefit if these studies could be performed, or advanced, in cell culture. It is shown here that Cdx1 activation of mouse Hoxb4, b8 and a7 embryo-active enhancers can be detected using a HepG2 cell culture model system. The technique employed uses co-transfection of an inducible Cdx1 expression construct together with a Hox enhancer/luciferase reporter construct. Cultures to be compared receive identical DNAs and differ only in whether or not they also receive inducer (doxycycline). Response of all three Hox enhancers to Cdx1 protein is inhibited by mutation of Cdx binding motifs which are conserved in sequence from fish or Xenopus to mammals. The magnitude of transfected chick Hoxa7 activation by Cdx1 is increased by multiple copies of its enhancer, but for maximum effect these must contain intact Cdx binding motifs. Cdx1 protein was found not to activate Hoxb4, b8 or a7 enhancers in P19 mouse pluripotential cells.