The molecular function of the Mixl1 transcription factor during early embryonic stem cell development

The Mixl1 gene encodes a homeodomain transcription factor that is required for normal mesoderm and endoderm development in the mouse. We have examined the consequences of enforced Mixl1 expression during mouse ESC differentiation. We found that in serum containing media differentiation to endoderm was promoted at the expense of ventral mesoderm. Luciferase reporter experiments indicate that Mixl1 can transactivate the Gsc, Sox17 and E-Cad promoters, supporting the hypothesis that Mixl1 has a direct role in definitive endoderm formation. To identify additional downstream transcriptional targets of Mixl1, we generated heterozygous Mixl1GFP/WT and homozygous null Mixl1GFP/Hygro ESCs. Candidate Mixl1 regulated genes with reduced expression in GFP+ cells isolated from differentiating Mixl1GFP/Hygro embryoid bodies included Pdgfrα and Flk1. Mixl1 bound to ATTA sequences located in Pdgfrα and Flk1 promoters in vivo. Mixl1 transactivated through these sequences in a DNA binding dependent manner. Using in vitro protein interactions studies Mixl1’s DNA-binding domain was shown to associate with T-box protein Brachyury. Gene expression and immunofluorescence analysis demonstrated overlapping expression of Mixl1 and the Brachyury during embryonic stem cell differentiation. Gel shift assays revealed that the Brachyury T-box domain could bind to Mixl1-DNA complexes. Furthermore, luciferase reporter experiments indicated that association with Mixl1 by Brachyury and related T-box factors inhibited the transactivation potential of Mixl1 on Gsc and Pdgfrα promoters. Taken together our results strengthen the position of Mixl1 as a key regulator of mesendoderm development during mammalian gastrulation.