Regulatory role for a conserved motif adjacent to the homeodomain of Hox10 proteins

Abstract

SUMMARYDevelopment of the vertebrate axial skeleton requires the concerted activity of several Hox genes. Among them, Hox genes belonging to the paralog group 10 are essential for the formation of the lumbar region of the vertebral column, owing to their capacity to block rib formation. In this work, we explored the basis for the rib-repressing activity of Hox10 proteins. Because genetic experiments in mice demonstrated that Hox10 proteins are strongly redundant in this function, we first searched for common motifs among the group members. We identified the presence of two small sequences flanking the homeodomain that are phylogenetically conserved among Hox10 proteins and that seem to be specific for this group. We show here that one of these motifs is required but not sufficient for the rib-repressing activity of Hox10 proteins. This motif includes two potential phosphorylation sites, which are essential for protein activity as their mutation to alanines resulted in a total loss of rib-repressing properties. Our data indicates that this motif has a significant regulatory function, modulating interactions with more N-terminal parts of the Hox protein, eventually triggering the rib-repressing program. In addition, this motif might also regulate protein activity by alteration of the protein's DNA-binding affinity through changes in the phosphorylation state of two conserved tyrosine residues within the homeodomain.

The regulation of Hox gene expression during animal development

“…Interestingly, alternatively spliced mRNAs are also produced from the Hoxa10 locus, and encode a predicted protein that is essentially the homeodomain without any significant N-terminal attached to it (Benson et al, 1995). The role of such a molecule is still unclear, as functional analysis in transgenic mice indicate that it is not able to activate the typical Hoxa10 patterning programme in the axial skeleton or interfere with it (Guerreiro et al, 2012). Hoxb3 and Hoxa5 also produce different mRNA isoforms through a combination of alternative splicing and alternative promoter usage; interestingly, the resulting isoforms are expressed in different domains during embryonic development (Chan et al, 2010;Coulombe et al, 2010;Sham et al, 1992), once again suggesting that the resulting isoforms might carry out specific functions.…”

DeepCAGE transcriptomics identify HOXD10 as a transcription factor regulating lymphatic endothelial responses to VEGF-C

“…Combining gene expression data and transcription factor activity prediction based on the presence of known TFBS in the promoters of differentially expressed genes, we found that HOXD10 is not induced by VEGFR-3 stimulation on the mRNA level, but is activated as early as 15 min after the stimulus. The mechanism by which activated VEGFR-3 triggers HOXD10 activity is entirely unknown, but it has been reported that the transcription factors of the HOX cluster 10, to which HOXD10 belongs, contain several putative tyrosine phosphorylation sites in the homeodomain flanking regions (Guerreiro et al, 2012). Thus, the trigger of HOXD10 activity downstream of VEGFR-3 stimulation might well be a phosphorylation event.…”

Hoxb6 can interfere with somitogenesis in the posterior embryo through a mechanism independent of its rib-promoting activity

“…A similar tag was also present in Pax3 (Guerreiro et al, 2013). Deletion and swapping mutants were produced using a PCR-based mutagenesis strategy (Guerreiro et al, 2012) and their sequences verified.…”

“…Electrophoretic mobility shift assays were performed as previously described (Guerreiro et al, 2012) using proteins produced in 293T cells. The DNA probe corresponding to the H1 enhancer has been described previously (Guerreiro et al, 2012).…”

“…It is clear that Hox functional specificity is largely encoded in non-HD parts of the protein Potter, 2001, 2002;Guerreiro et al, 2012), either in the form of intrinsic functional activities or by their ability to recruit additional factors (Mann et al, 2009;Casaca et al, 2014). Understanding the patterning activity of these Hox proteins thus requires the identification and characterization of the molecular signatures responsible for their functional specificity.…”