The Hematopoietic Regulator Mouse Mix Functions as a Transcriptional Activator in Embryonic Stem Cells.

Abstract During mammalian embryogenesis, hemangioblastic and hematopoietic progenitors are differentiated from mesoderm, but the underlying mechanisms are poorly understood. Mouse Mixl (also known as Mixl1 or Mml) is a member of the Mix/Bix family of Paired-class homeobox genes regulated by Nodal/activin/BMP signaling. Targeted disruption of mMix results in mesodermal and endodermal defects in the embryo and deficient hematopoiesis in embryonic stem (ES) cell-derived embryoid bodies (EBs). Conditional induction of mMix (i-Mix) in EBs results in accelerated mesodermal development and increased numbers of mesodermal, hemangioblastic, and hematopoietic progenitors, suggesting that mMix promotes the recruitment and/or expansion of mesodermal progenitors to these lineages (Willey et al., Blood 107:3122, 2006). Conditional activation of mMix in differentiating i-Mix ES cells led to rapid upregulation of goosecoid (gsc). In the frog, gsc is a direct target of XMix.1 and represses transcription of the Brachuyry/T homologue, Xbra. XMix.1 is, therefore, thought to function indirectly in the negative regulation of Xbra. Genetic ablation of mMix in the mouse resulted in an expanded domain of T expression, also suggesting direct or indirect suppression. However, induction of mMix in differentiating i-Mix EBs was followed by early activation of T. Like all known vertebrate members of the Mix/Bix family, mMix contains a highly conserved C-terminal region of polar and acidic residues that have the potential to form an amphipathic helix (a characteristic of many transcriptional activators) and is part of a larger region that can activate transcription in yeast two-hybrid assays. Therefore, mMix may function as an activator or a repressor depending on the cellular context, e.g. through differential recruitment of coactivators and corepressors. Indeed, microarray analysis of induced and uninduced i-Mix EBs revealed dynamic changes in gene expression (both up- and down-regulation) within 24 hr of addition of DOX (unpublished data). To understand the molecular mechanisms by which mMix regulates mesodermal differentiation and hematopoiesis, we have identified the optimal target DNA sequence of the mouse Mix protein in vitro using a PCR-assisted binding site selection assay and a GST-mMix homeodomain fusion protein. The consensus target sequence consists of two 4 base pair half sites separated by a 3 base pair spacer. This consensus DNA recognition sequence is different from the consensus target sequence identified for the Drosophila Paired protein, the founder member of the Paired-class homeodomain protein family. We found that mMix bound to the bipartite target sequence primarily as a dimer. Importantly, mMix protein activates the transcription of a luciferase reporter gene linked to multimerized copies of the target sequence in both NIH3T3 cells and in ES cells in which mMix is conditionally expressed. Mutations of critical DNA contacting residues in the homeodomain of mMix abolish transcriptional activation, suggesting that transactivation by mMix is dependent on homeodomain-DNA interactions. In summary, these findings indicate that mMix can function as a sequence-specific transcriptional activator. We propose that mMix may regulate mesodermal and hematopoietic development, at least in part, through activation of essential target genes. To test this hypothesis, we are currently identifying potential mMix target genes using microarray analysis and chromatin immunoprecipitation assays.