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

Development 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 Xenopus laevis Hox 2.1 homeodomain protein is expressed in a narrow band of the hindbrain

The expression pattern of the Xenopus homeodomain protein Hox 2.1 during development was determined using an affinity-purified antibody directed against a carboxyterminal peptide. Nuclear staining was detected in a very narrow band of the hindbrain. This pattern was compared to that of the previously described Xenopus gene XIHbox 1 in serial sections and found to be more anterior than the XIHbox 1 long protein expression but overlapping with that of the short protein. Xenopus Hox 2.1 protein expression is restricted to a much narrower antero-posterior band than that reported for mouse Hox 2.1 RNA expression by in situ hybridization.     

Cell cycle regulation of the c-Myc transcriptional activation domain.

The product of the c-myc gene (c-Myc) is a sequence-specific DNA-binding protein that has previously been demonstrated to be required for cell cycle progression. Here we report that the c-Myc DNA binding site confers cell cycle regulation to a reporter gene in Chinese hamster ovary cells. The observed transactivation was biphasic with a small increase in G1 and a marked increase during the S-to-G2/M transition of the cell cycle. This cell cycle regulation of transactivation potential is accounted for, in part, by regulatory phosphorylation of the c-Myc transactivation domain. Together, these data demonstrate that c-Myc may have an important role in the progression of cells through both the G1 and G2 phases of the cell cycle.     

Hox homeodomain proteins exhibit selective complex stabilities with Pbx and DNA.

Eight of the nine homeobox genes of the Hoxb locus encode proteins which contain a conserved hexapeptide motif upstream from the homeodomain. All eight proteins (Hoxb-1-Hoxb-8) bind to a target oligonucleotide in the presence of Pbx1a under conditions where minimal or no binding is detected for the Hox or Pbx1a proteins alone. The stabilities of the Hox-Pbx1a-DNA complexes vary >100-fold, with the proteins from the middle of the locus (Hoxb-5 and Hoxb-6) forming very stable complexes, while Hoxb-4, Hoxb-7 and Hoxb-8 form complexes of intermediate stability and proteins at the 3'-side of the locus (Hoxb-1-Hoxb-3) form complexes which are very unstable. Although Hox-b proteins containing longer linker sequences between the hexapeptide and homeodomains formed unstable complexes, shortening the linker did not confer complex stability. Homeodomain swapping experiments revealed that this motif does not independently determine complex stability. Naturally occurring variations within the hexapeptides of specific Hox proteins also do not explain complex stability differences. However, two core amino acids (tryptophan and methionine) which are absolutely conserved within the hexapeptide domains appear to be required for complex formation. Removal of N- and C-terminal flanking regions did not influence complex stability and the members of paralog group 4 (Hoxa-4, b-4, c-4 and d-4), which share highly conserved hexapeptides, linkers and homeodomains but different flanking regions, form complexes of similar stability. These data suggest that the structural features of Hox proteins which determine Hox-Pbx1a-DNA complex stability reside within the precise structural relationships between the homeodomain, hexapeptide and linker regions.     

Direct regulation of the muscle-identity gene apterous by a Hox protein in the somatic mesoderm

Hox genes control segment identity in the mesoderm as well as in other tissues. Most evidence indicates that Hox genes act cell-autonomously in muscle development, although this remains a controversial issue. We show that apterous expression in the somatic mesoderm is under direct Hox control. We have identified a small enhancer element of apterous (apME680) that regulates reporter gene expression in the LT1-4 muscle progenitors. We show that the product of the Hox gene Antennapedia is present in the somatic mesoderm of the second and third thoracic segments. Through complementary alterations in the Antennapedia protein and in its binding sites on apME680, we show that Antennapedia positively regulates apterous in a direct manner, demonstrating unambiguously its cell-autonomous role in muscle development. Finally, we determine that LT1-4 muscles contain more nuclei in the thorax than in the abdomen and we propose that one of the segmental differences under Hox control is the number of myoblasts allocated to the formation of specific muscles in different segments.