A group 13 homeodomain is neither necessary nor sufficient for posterior prevalence in the mouse limb

Posterior prevalence is the general property attributed to HOX proteins describing the dominant effect of more posterior HOX proteins over the function of anterior orthologs in common areas of expression. To explore the HOX group 13 protein domains required for this property, we used the mouse Prx-1 promoter to drive transgenic expression of Hox constructs throughout the entire limb bud during development. This system allowed us to conclusively demonstrate a hierarchy of Hox function in developing limbs. Furthermore, by substituting the HOXD11 or HOXA9 homeodomain for that of HOXD13, we show that a HOXD13 homeodomain is not necessary for posterior prevalence. Proximal expression of these chimeric proteins unexpectedly caused defects consistent with wild-type HOXD13 mediated posterior prevalence. Moreover, group 13 non-homeodomain residues appear to confer the property as proximal expression of HOXA9 containing the HOXD13 homeodomain did not result in limb reductions characteristic of HOXD13. These data are most compatible with models of posterior prevalence based on protein-protein interactions and support examination of the N-terminal non-homeodomain regions of Hox group 13 proteins as necessary agents for posterior prevalence.     

Range of HOX/TALE superclass associations and protein domain requirements for HOXA13:MEIS interaction

AbdB-like HOX proteins form DNA-binding complexes with the TALE superclass proteins MEIS1A and MEIS1B, and trimeric complexes have been identified in nuclear extracts that include a second TALE protein, PBX. Thus, soluble DNA-independent protein-protein complexes exist in mammals. The extent of HOX/TALE superclass interactions, protein structural requirements, and sites of in vivo cooperative interaction have not been fully explored. We show that Hoxa13 and Hoxd13 expression does not overlap with that of Meis1-3 in the developing limb; however, coexpression occurs in the developing male and female reproductive tracts (FRTs). We demonstrate that both HOXA13 and HOXD13 associate with MEIS1B in mammalian and yeast cells, and that HOXA13 can interact with all MEIS proteins but not more diverged TALE superclass members. In addition, the C-terminal domains (CTDs) of MEIS1A (18 amino acids) and MEIS1B (93 amino acids) are necessary for HOXA13 interaction; for MEIS1B, this domain was also sufficient. We also show by yeast two-hybrid assay that MEIS proteins can interact with anterior HOX proteins, but for some, additional N-terminal MEIS sequences are required for interaction. Using deletion mutants of HOXA13 and HOXD13, we provide evidence for multiple HOX peptide domains interacting with MEIS proteins. These data suggest that HOX:MEIS interactions may extend to non-AbdB-like HOX proteins in solution and that differences may exist in the MEIS peptide domains utilized by different HOX groups. Finally, the capability of multiple HOX domains to interact with MEIS C-terminal sequences implies greater complexity of the HOX:MEIS protein-protein interactions and a larger role for variation of HOX amino-terminal sequences in specificity of function.     

HOXA13 regulates the expression of bone morphogenetic proteins 2 and 7 to control distal limb morphogenesis

In humans and mice, loss of HOXA13 function causes defects in the growth and patterning of the digits and interdigital tissues. Analysis of Hoxa13 expression reveals a pattern of localization overlapping with sites of reduced Bmp2 and Bmp7 expression in Hoxa13 mutant limbs. Biochemical analyses identified a novel series of Bmp2 and Bmp7 enhancer regions that directly interact with the HOXA13 DNA-binding domain and activate gene expression in the presence of HOXA13. Immunoprecipitation of HOXA13-Bmp2 and HOXA13-Bmp7 enhancer complexes from the developing autopod confirm that endogenous HOXA13 associates with these regions. Exogenous application of BMP2 or BMP7 partially rescues the Hoxa13 mutant limb phenotype, suggesting that decreased BMP signaling contributes to the malformations present in these tissues. Together, these results provide conclusive evidence that HOXA13 regulates Bmp2 and Bmp7 expression, providing a mechanistic link between HOXA13, its target genes and the specific developmental processes affected by loss of HOXA13 function.     

Evolution of N-terminal sequences of the vertebrate HOXA13 protein

While the the role of the homeodomain in HOX function has been evaluated extensively, little attention has been given to the non-homeodomain portions of the HOX proteins. To investigate the evolution of the HOXA13 protein and to identify conserved residues in the N-terminal region of the protein with potential functional significance, N-terminal Hoxa13 coding sequences were PCR-amplified from fish, amphibian, reptile, chicken, and marsupial and eutherian mammal genomic DNA. Compared with fish HOXA13, the mammalian protein has increased in size by 35% primarily owing to the accumulation of alanine repeats and flanking segments rich in proline, glycine, or serine within the first 215 amino acids. Certain residues and amino acid motifs were strongly conserved, and several HOXA13 N-terminal domains were also shared in the paralogous HOXB13 and HOXD13 genes; however, other conserved regions appear to be unique to HOXA13. Two domains highly conserved in HOXA13 orthologs are shared with Drosophila AbdB and other vertebrate AbdB-like proteins. Marsupial and eutherian mammalian HOXA13 proteins have three large homopolymeric alanine repeats of 14, 12, and 17–18 residues that are absent in reptiles, birds, and fish. Thus, the repeats arose after the divergence of reptiles from the lineage that would give rise to the mammals. In contrast, other short homopolymeric alanine repeats in mammalian HOXA13 have remained virtually the same length, suggesting that forces driving or limiting repeat expansion are context dependent. Consecutive stretches of identical third-base usage in alanine codons within the large repeats were found, supporting replication slippage as a mechanism for their generation. However, numerous species-specific base substitutions affecting third-base alanine repeat codon positions were observed, particularly in the largest repeat. Therefore, if the large alanine repeats were present prior to eutherian mammal development as is suggested by the opossum data, then a dynamic process of recurring replication slippage and point mutation within alanine repeat codons must be considered to reconcile these observations. This model might also explain why the alanine repeats are flanked by proline, serine, and glycine-rich sequences, and it reveals a biological mechanism that promotes increases in protein size and, potentially, acquisition of new functions. Received: 8 June 1999 / Accepted: 23 September 1999     

PBX1 intracellular localization is independent of MEIS1 in epithelial cells of the developing female genital tract

While studies have highlighted the role of HOXA9-13 and PBX1 homeobox genes during the development of the female genital tract, the molecular mechanisms triggered by these genes are incompletely elucidated. In several developmental pathways, PBX1 binds to MEINOX family members in the cytoplasm to be imported into the nucleus where they associate with HOX proteins to form a higher complex that modulates gene expression. This concept has been challenged by a recent report showing that in some cell cultures, PBX1 nuclear localization might be regulated independently of MEINOX proteins (Kilstrup-Nielsen et al., 2003). Our work gives the first illustration of this alternative mechanism in an organogenesis process. Indeed, we show that PBX1 is mostly cytoplasmic in epithelial endometrial cells of the developing female genital tract despite the nuclear localization of MEIS1. We thus provide evidence for a control of PBX1 intracellular distribution which is independent of MEINOX proteins, but is cell cycle correlated.