Background: Hox proteins are transcription factors involved in crucial processes during animal development. Their mode of action remains scantily documented. While other families of transcription factors, like Smad or Stat, are known cell signaling transducers, such a function has never been squarely addressed for Hox proteins. Results: To investigate the mode of action of mammalian Hoxa1, we characterized its interactome by a systematic yeast two-hybrid screening against ~12,200 ORF-derived polypeptides. Fifty nine interactors were identified of which 45 could be confirmed by affinity co-purification in animal cell lines. Many Hoxa1 interactors are proteins involved in cell-signaling transduction, cell adhesion and vesicular trafficking. Forty-one interactions were detectable in live cells by Bimolecular Fluorescence Complementation which revealed distinctive intracellular patterns for these interactions consistent with the selective recruitment of Hoxa1 by subgroups of partner proteins at vesicular, cytoplasmic or nuclear compartments. Conclusions: The characterization of the Hoxa1 interactome presented here suggests unexplored roles for Hox proteins in cell-to-cell communication and cell physiology.

Recent advances in single-cell transcriptomics techniques have opened the door to the study of gene regulatory networks (GRNs) at the single-cell level. Here, we studied the GRNs controlling the emergence of hematopoietic stem and progenitor cells from mouse embryonic endothelium using a combination of single-cell transcriptome assays. We found that a heptad of transcription factors (Runx1, Gata2, Tal1, Fli1, Lyl1, Erg and Lmo2) is specifically co-expressed in an intermediate population expressing both endothelial and hematopoietic markers. Within the heptad, we identified two sets of factors of opposing functions: one (Erg/Fli1) promoting the endothelial cell fate, the other (Runx1/Gata2) promoting the hematopoietic fate. Surprisingly, our data suggest that even though Fli1 initially supports the endothelial cell fate, it acquires a pro-hematopoietic role when co-expressed with Runx1. This work demonstrates the power of single-cell RNA-sequencing for characterizing complex transcription factor dynamics.

Hox proteins are conserved homeodomain transcription factors known to be crucial regulators of animal development. As transcription factors, the functions and modes of action (co-factors, target genes) of Hox proteins have been very well studied in a multitude of animal models. However, a handful of reports established that Hox proteins may display molecular activities distinct from gene transcription regulation. Here, we reveal that Hoxa2 interacts with 20S proteasome subunits and RCHY1 (also known as PIRH2), an E3 ubiquitin ligase that targets p53 for degradation. We further show that Hoxa2 promotes proteasome-dependent degradation of RCHY1 in an ubiquitin-independent manner. Correlatively, Hoxa2 alters the RCHY1-mediated ubiquitination of p53 and promotes p53 stabilization. Together, our data establish that Hoxa2 can regulate the proteasomal degradation of RCHY1 and stabilization of p53.

The homeodomain transcription factor Hoxa2 interacts with the RING-finger type E3 ubiquitin ligase RCHY1 and induces its proteasomal degradation. In this work, we dissected this non-transcriptional activity of Hoxa2 at the molecular level. The Hoxa2-mediated decay of RCHY1 involves both the 19S and 20S proteasome complexes. It relies on both the Hoxa2 homeodomain and C-terminal moiety although no single deletion in the Hoxa2 sequence could disrupt the RCHY1 interaction. That the Hoxa2 homeodomain alone could mediate RCHY1 binding is consistent with the shared ability all the Hox proteins we tested to interact with RCHY1. Nonetheless, the ability to induce RCHY1 degradation although critically relying on the homeodomain is not common to all Hox proteins. This identifies the homeodomain as necessary but not sufficient for what appears to be an almost generic Hox protein activity. Finally we provide evidence that the Hoxa2-induced degradation of RCHY1 is evolutionarily conserved among vertebrates. These data therefore support the hypothesis that the molecular and functional interaction between Hox proteins and RCHY1 is an ancestral Hox property.