The Mixl1 homeodomain transcription factor plays a key role in normal mesoderm and endoderm patterning during mammalian embryogenesis 
. Despite the wealth of information regarding the importance of Mixl1 during gastrulation, little is known about the molecular determinants underlying Mixl1 function. In particular the identity of protein cofactors and their effects on Mixl1 activity remains largely unknown. In this study, we addressed this issue and showed that members of the T-box family of transcription factors physically associated with Mixl1 and repressed its transactivation function on target gene promoters. For pragmatic reasons, we employed differentiating mouse ES cells as a model system to study early post-implantation embryonic development. Whilst there is sufficient evidence that findings in ES cells reflect events occurring in the embryo, we acknowledge that aspects of our in vitro model, especially the kinetics and the lack of complex structural organization within EBs might not accurately indicate events occurring during in vivo
Several sets of observations suggested that Brachyury and Mixl1 might regulate each other during mesoderm and endoderm formation. Firstly, in the early mouse embryo, Mixl1 and Brachyury are both expressed in the primitive streak 
and studies in Xenopus
demonstrated cross repression by Mixl1 and Brachyury 
. Similarly, constitutive expression of Mixl1
during ESC differentiation repressed activin induced Brachyury
expression, whilst analysis of Mixl1
-null ESC lines and mice revealed that loss of Mixl1
resulted in an up-regulation of Brachyury
expression in vitro 
and in vivo 
. These results were also consistent with reports in Xenopus
down regulated Xbra
expression, in part through the activation of Goosecoid 
. In RNAi-mediated knockdown (KD) experiments in ESCs, Mixl1
KDs resulted in enhanced Brachyury expression whilst Mixl1
overexpression suppressed Brachyury expression 
. The results presented in our study extend the scope of this previous work, suggesting that a functional relationship in which Brachyury represses Mixl1 might be based on their physical interaction.
We provide several lines of evidence to support this notion. Firstly, by immunofluorescence analysis, we observed the presence of Mixl1+
cells at d4 of differentiation. Second, through co-immunoprecipitation and GST-pulldown experiments, we showed that Mixl1 physically associated with Brachyury and that this interaction was conserved in several vertebrate species. Others have demonstrated physical associations between homeobox proteins and T-box family members 
. Our mapping studies demonstrated that the T-box domain of Brachyury and the Mixl1 homeodomain were important domains for interaction, consistent with previous findings documenting interactions between the T-box and homeodomains 
While our data suggested important roles for the Mixl1 and Brachyury DNA binding domains, we also provide evidence that other regions may contribute to the interaction, supporting a model where multiple domains within Mixl1 and Brachyury underpin their association. This conclusion mirrors previous studies showing that sequences outside the T-box domain of Tbx5 contributed to its interaction with the homeodomain protein, Nkx2-5 
. An important finding was that Mixl1 mutants defective for DNA binding 
were still able to interact with Brachyury. An exception was the Mixl1 mutant (P126I) that displayed substantially reduced binding to Brachyury. Strikingly we observed that this mutation also impaired the ability of Mixl1 to associate with itself, presumably as a homodimer. These results suggest a number of interesting possibilities. Firstly, since the capacity of Mixl1 to bind DNA is not a pre-requisite for it to interact with Brachyury, part of the pool of Mixl1 and Brachyury within the cell may exist as a pre-formed complex sequestered from DNA. These factors might then be recruited alone or as a complex to promoter DNA in order to regulate gene transcription. Second, our data suggests that helix 3 of the Mixl1 HD is a direct interaction surface for Brachyury or that Brachyury has a preference to associate with Mixl1 homodimers. Further structural analysis of the Mixl1-Brachyury complex may shed further light on the function and dynamics of the Mixl1-Brachyury interaction.
is the founding member of a family comprising at least 18 mammalian T-box genes that are involved in the induction and regional specification of mesoderm 
. We provide evidence to support the notion that in addition to Brachyury, other members of the T-box family might interact with Mixl1. Firstly, we confirmed that the expression of the T-box genes Eomes
significantly overlapped with the transient expression of Mixl1
at days 3 and 4 of ESC differentiation. Furthermore, immunofluorescence analysis of differentiating EBs revealed the presence of a Mixl1+
population of cells during ESC differentiation. The expression pattern of these genes is consistent with their previously reported expression in the primitive streak and emerging mesoderm of gastrulation stage embryos 
. Second, our biochemical analyses demonstrated that Eomes, Tbx2, 3, 6 and 20 were all capable of interacting with Mixl1. Our interaction studies suggested that these same four T-box factors could also interact with the related homeodomain protein, Gsc 
. The finding that multiple members of the T-box family bind Mixl1 or Gsc suggests that the Mixl1-Brachyury interaction reflects a generic propensity for association between paired homeobox proteins and T-box factors. Notably, we were unable to detect an interaction between Brachyury and the POU-homeodomain factors Oct4 or Oct6, suggesting that additional sequence requirements govern Mixl1-T-box interactions and that not all classes of homeodomain proteins associate with T-box factors.
The functional significance of the Mixl1-Brachyury association was demonstrated in luciferase reporter experiments in which Brachyury repressed Mixl1 activation of the Gsc
promoter. A similar effect was observed with Eomes and Tbx6. Whilst it was possible that Brachyury would block Mixl1 binding to DNA, we observed that the T-box domain of Brachyury actually formed a ternary complex with DNA-bound Mixl1. These observations suggested that Brachyury and related T-box factors might be recruited to target genes via the association of their T-box domain with promoter bound Mixl1. Such an arrangement might allow T-box factors to regulate genes to whose promoters they do not directly bind. Our observation that non-DNA binding mutants of Brachyury repressed Mixl1 activity and that Brachyury alone did not transactivate the Gsc
promoter are consistent with this idea. This indirect mode of promoter repression through complex formation with an unrelated transactivating factor has previously been described for basic helix-loop-helix factors such as Hey1 protein and its association with the GATA family of transcriptional activators 
The repressive effect of Brachyury might be mediated through the recruitment of co-repressor complexes to the Gsc
promoter (). While Brachyury has largely been described to function as a transcriptional activator 
, mapping of regulatory domains in the carboxy terminal half of Brachyury has identified repression domains 
. In addition, Tbx6 has been shown to repress gene expression 
. More recently, the T-box proteins Tbx15 and Tbx18 have been suggested to repress promoter activity through the recruitment of Groucho/HDAC or CtBP/HDAC repressor complexes 
while a C terminal motif in Tbx2 and Tbx3 mediates repression by direct association with HDAC1 
. It is noteworthy that in our reporter assays, neither the Brachyury T-box domain nor the carboxy-terminal portion alone repressed Mixl1 transactivation activity. These observations lead us to speculate that the T-box, in this context, might target Tbx proteins to promoter bound homeodomain factors while the carboxy-terminal region participates in the recruitment of co-repressor factors ().
Model for repression of Mixl1 transactivating ability by T.
In summary, these studies represent the first demonstration that the paired-like homeodomain protein Mixl1 can interact with several members of the Tbx protein family. These data raise the interesting possibility that the temporal and/or spatial expression of Tbx factors during development may influence the Tbx partner choice and transcriptional activity of Mixl1. Future experiments aimed at establishing which Mixl1-Tbx interactions take place during the establishment and patterning of the primary germ layers will help shed light on the function of Mixl1-Tbx complexes during early development.