Pdgfrα and Flk1 are direct target genes of Mixl1 in differentiating embryonic stem cells.
ABSTRACT The Mixl1 homeodomain protein plays a key role in mesendoderm patterning during embryogenesis, but its target genes remain to be identified. We compared gene expression in differentiating heterozygous Mixl1(GFP/w) and homozygous null Mixl1(GFP/Hygro) mouse embryonic stem cells to identify potential downstream transcriptional targets of Mixl1. Candidate Mixl1 regulated genes whose expression was reduced in GFP+ cells isolated from differentiating Mixl1(GFP/Hygro) embryoid bodies included Pdgfrα and Flk1. Mixl1 bound to ATTA sequences located in the Pdgfrα and Flk1 promoters and chromatin immunoprecipitation assays confirmed Mixl1 occupancy of these promoters in vivo. Furthermore, Mixl1 transactivated the Pdgfrα and Flk1 promoters through ATTA sequences in a DNA binding dependent manner. These data support the hypothesis that Mixl1 directly regulates Pdgfrα and Flk1 gene expression and strengthens the position of Mixl1 as a key regulator of mesendoderm development during mammalian gastrulation.
Article: Brachyury and related Tbx proteins interact with the Mixl1 homeodomain protein and negatively regulate Mixl1 transcriptional activity.[show abstract] [hide abstract]
ABSTRACT: Mixl1 is a homeodomain transcription factor required for mesoderm and endoderm patterning during mammalian embryogenesis. Despite its crucial function in development, co-factors that modulate the activity of Mixl1 remain poorly defined. Here we report that Mixl1 interacts physically and functionally with the T-box protein Brachyury and related members of the T-box family of transcription factors. Transcriptional and protein analyses demonstrated overlapping expression of Mixl1 and Brachyury during embryonic stem cell differentiation. In vitro protein interaction studies showed that the Mixl1 with Brachyury associated via their DNA-binding domains and gel shift assays revealed that the Brachyury T-box domain bound to Mixl1-DNA complexes. Furthermore, luciferase reporter experiments indicated that association of Mixl1 with Brachyury and related T-box factors inhibited the transactivating potential of Mixl1 on the Gsc and Pdgfrα promoters. Our results indicate that the activity of Mixl1 can be modulated by protein-protein interactions and that T-box factors can function as negative regulators of Mixl1 activity.PLoS ONE 01/2011; 6(12):e28394. · 4.09 Impact Factor