LAUSR

ABSTRACT The mouse T-box transcription factors T and Tbx6 are co-expressed in the primitive streak and have unique domains of expression; T is expressed in the notochord, while Tbx6 is expressed in the presomitic mesoderm. T-box factors are related through a shared DNA binding domain, the T-domain, and can therefore bind to similar DNA sequences at least in vitro. We investigated the functional similarities and differences of T and Tbx6 DNA binding and transcriptional activity in vitro and their interaction genetically in vivo. We show that at one target, Dll1, the T-domains of T and Tbx6 have different affinities for the binding sites present in the mesoderm enhancer. We further show using in vitro assays that T and Tbx6 differentially affect transcription with Tbx6 activating expression tenfold higher than T, that T and Tbx6 can compete at target gene enhancers, and that this competition requires a functional DNA binding domain. Next, we addressed whether T and Tbx6 can compete in vivo. First, we generated embryos that express Tbx6 at greater than wild-type levels embryos and show that these embryos have short tails, resembling the T heterozygous phenotype. Next, using the dominant-negative TWis allele, we show that Tbx6+/− TWis/+ embryos share similarities with embryos homozygous for the Tbx6 hypomorphic allele rib-vertebrae, specifically fusions of several ribs and malformation of some vertebrae. Finally, we tested whether Tbx6 can functionally replace T using a knockin approach, which resulted in severe T null-like phenotypes in chimeric embryos generated with ES cells heterozygous for a Tbx6 knockin at the T locus. Altogether, our results of differences in affinity for DNA binding sites and transcriptional activity for T and Tbx6 provide a potential mechanism for the failure of Tbx6 to functionally replace T and possible competition phenotypes in vivo. Summary: Mouse Tbx6 fails to compensate for heterozygous loss of T; instead ectopic Tbx6 in the T expression-domain in knockin embryos generates T null-like phenotypes suggestive of competition.