The mechanisms controlling growth and patterning along the proximal-distal axis of the vertebrate limb are yet to be understood. We show that restriction of expression of the homeobox gene Meis2 to proximal regions of the limb bud is essential for limb development, since ectopic Meis2 severely disrupts limb outgrowth. We also uncover an antagonistic relationship between the secreted factors Gremlin and BMPs required to maintain the Shh/FGF loop that regulates distal outgrowth. These proximal and distal factors have coordinated activities: Meis2 can repress distal genes, and Bmps and Hoxd genes restrict Meis2 expression to the proximal limb bud. Moreover, combinations of BMPs and AER factors are sufficient to distalize proximal limb cells. Our results unveil a novel set of proximal-distal regulatory interactions that establish and maintain outgrowth of the vertebrate limb.
Vertebrate limb development occurs along three cardinal axes -proximodistal, anteroposterior and dorsoventral -that are established via the organization of signaling centers, such as the zone of polarizing activity (ZPA). Distal limb development, in turn, requires a molecular feedback loop between the ZPA expression of sonic hedgehog (Shh) and the apical ectodermal ridge. The TALE homeoprotein Pbx1 has been shown to be essential for proximal limb development. In this study, we first uncover that Pbx1 and Pbx2 are co-expressed in the lateral plate and early limb field mesoderm. Later, Pbx2 is expressed throughout the limb, unlike Pbx1, which is expressed only in the proximal bud. By exploiting a Pbx1/Pbx2 loss-of-function mouse model, we demonstrate that, despite the lack of limb abnormalities in Pbx2-deficient (Pbx2 embryos lack limbs altogether. Furthermore, we establish that, unlike in flies, where the leg develops independently of Hox and where the Pbx ortholog Exd is required for specification of proximal (but not distal) limbs, in vertebrates, distal limb patterning is Pbx1/Pbx2 dependent. Indeed, we demonstrate that Pbx genetic requirement is mediated, at least in part, through their hierarchical control of Hox spatial distribution and Shh expression. Overall, we establish that, by controlling the spatial expression of Hox genes in the posterior limb and regulating ZPA function, Pbx1/Pbx2 exert a primary hierarchical function on Hox genes, rather than behaving merely as Hox ancillary factors.
Milano, Italy 4Corresponding authorHigh mobility group protein 1 (HMG1) is a nonhistone, chromatin-associated nuclear protein with a proposed role in the regulation of eukaryotic gene expression. We show that HMG1 interacts with proteins encoded by the HOX gene family by establishing protein-protein contacts between the HMG box domains and the HOX homeodomain. The functional role of these interactions was studied using the transcriptional activity of the human HOXD9 protein as a model. HMG1 enhances, in a dose-dependent fashion, the sequence-specific DNA binding activity in vitro, and the transcriptional activation in a co-transfection assay in vivo, of the HOXD9 protein. Functional interaction between HMG1 and HOXD9 is dependent on the DNA binding activity of the homeodomain, and requires the HOXD9 transcriptional activation domain. HMG1 enhances activation by HOXD9, but not by HOXD8, of the HOXD9-controlled element. Specific target recognition and functional interaction with HMG1 can be transferred to HOXD8 by homeodomain swapping. We propose that HMG1-like proteins might be general co-factors in HOX-mediated transcriptional activation, which facilitate access of HOX proteins to specific DNA targets, and/or introduce architectural constraints in the assembly of HOX-containing transcriptional complexes.
Nuclear localization of the Extradenticle (EXD) and PBX1 proteins is regionally restricted during Drosophila and mammalian development. We studied the subcellular localization of EXD, PBX, and their partners Homothorax (HTH) and PREP1, in different cell contexts. HTH and PREP1 are cytoplasmic and require association with EXD/PBX for nuclear localization. EXD and PBX1 are nuclear in murine fibroblasts but not in Drosophila Schneider cells, in which they are actively exported to the cytoplasm. Coexpression of EXD/PBX with HTH/PREP1 causes nuclear localization of their heterodimers in both cell contexts. We propose that heterodimerization with HTH/PREP induces nuclear translocation of EXD and PBX1 in specific cell contexts by blocking their nuclear export.
The human transcription factor, UEF3, is important in regulating the activity of the urokinase plasminogen activator (uPA) gene enhancer. The UEF3 DNA target site is a regulatory element in the promoters of several growth factor and protease genes. We reported previously that purified UEF3 is a complex of several subunits. In this paper we report the cloning of the cDNA of one of the subunits which encodes for a novel human homeodomain protein, which we have termed Prep1. The Prep1 homeodomain belongs to the TALE class of homeodomains, is most closely related to those of the TGIF and Meis1 proteins, and like these, recognizes a TGACAG motif. We further identify the other UEF3 subunit as a member of the Pbx protein family. Unlike other proteins known to interact with Pbx, Prep1 forms a stable complex with Pbx independent of DNA binding. Heterodimerization of Prep1 and Pbx results in a strong DNA binding affinity towards the TGACAG target site of the uPA promoter. Overall, these data indicate that Prep1 is a stable intracellular partner of Pbx in vivo.
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