Abstract
Members of the Mix/Bix family of paired class homeobox genes play important roles in the development of vertebrate mesoderm and endoderm. The single Mix/Bix family member identified in the mouse, Mix-like 1 (Mixl1), is required for mesendoderm patterning during gastrulation and promotes mesoderm formation and hematopoiesis in embryonic stem cell (ESC)-derived embryoid bodies. Despite its crucial functions the transcriptional activity and targets of Mixl1 have not been well described. To investigate the molecular mechanisms of Mixl1-mediated transcriptional regulation, we have characterized the DNA-binding specificity and transcriptional properties of this homeodomain protein in differentiating ESCs. Mixl1 binds preferentially as a dimer to an 11-base pair (bp) Mixl1 binding sequence (MBS) that contains two inverted repeats separated by a 3-bp spacer. The MBS mediates transcriptional activation by Mixl1 in both NIH 3T3 cells and in a new application of an inducible ESC differentiation system. Consistent with our previous observation that early induction of Mixl1 expression in ESCs results in premature activation of Goosecoid (Gsc), we have found that Mixl1 occupies two variant MBSs within and activates transcription from the Gsc promoter in vitro and in vivo. These results strongly suggest that Gsc is a direct target gene of Mixl1 during embryogenesis. STEM CELLS 2009;27:2884-2895.