Synergistic action in P19 pluripotential cells of retinoic acid and Wnt3a on Cdx1 enhancer elements

Abstract

Cdx1 encodes a homeodomain protein that regulates expression of some Hox genes. Cdx1 itself is known to be regulated in the primitive streak/tailbud by both retinoic acid (RA) and Wnt3a. Cdx1 in eutherian mammals has two retinoic acid response elements (RAREs), located upstream and in the first intron, and each is adjacent to structural Lef/Tcf motifs. Upstream Lef/Tcf motifs respond to canonical Wnt signalling to activate Cdx1 synergistically with RA. By combined use of reporter assays, immunofluorescence and flow cytometry in mouse P19 embryonal carcinoma cells we show that the Cdx1 intron Lef/Tcf motif also responds to Wnt3a signalling. Synergy between individual Cdx1 RARE and Lef/Tcf motifs can occur whether they are adjacent or distant in the gene. Part, though not all, of the Cdx1 stimulation by RA (in absence of added Wnt3a) likely depends upon Wnt protein produced by the cells themselves, since it is inhibited by mutation of Lef/Tcf motifs, or by IWP-2, an inhibitor of Wnt production. RA and Wnt3a stimulate Cdx1 by increasing both the proportion of P19 cells that are expressing and also their mean level of expression. The expressing/non-expressing sub-populations do not simply correspond with those that express a marker of pluripotentiality, Nanog. We conclude that RA and Wnt3a activate Cdx1 synergistically by overlapping use of both upstream and intron enhancers, and that mouse embryonal carcinoma cell populations display heterogeneity in their response to these activators.