Role of the N-terminal arm of Hox genes in interacting with PBX proteins

The labial group protein HOXA-1 has intrinsically weak DNA-binding activity due to residues in the N-terminal arm of its homeodomain. This observation, among others, suggests that HOX and HOM proteins require cofactors for stable interactions with DNA. A putative HOX cofactor, PBX1A, participates in cooperative DNA binding with HOXA-1 and the Deformed group protein HOXD-4. Three Abdominal-B class HOX proteins fail to cooperate with PBX1A. The interacting domain of HOXD-4 maps to the YPWMK pentapeptide motif, a conserved sequence found N terminal to the homeodomain of HOXA-1 and many other homeoproteins, but one that is absent from the Abdominal-B class. The naturally occurring fusion of the transcriptional activation domain of E2A with PBX1 creates an oncoprotein implicated in human pre-B-cell leukemias. A pentapeptide mutation that abolished cooperative interaction with PBX1A in vitro also abrogates synergistic transcriptional activation with the E2A/PBX oncoprotein. The direct contact of PBX family members by the HOX pentapeptide is likely to play an important role in developmental and oncogenic processes (Phelan, 1995).

The highly conserved pentapeptide motif F/Y-P-W-M-R/K, which occurs in numerous Hox proteins and is positioned 8 to 50 amino acids N terminal to the homeodomain, is essential for cooperative DNA binding with Pbx1 and the oncogenic E2A-Pbx1. Point mutational analysis demonstrates that the tryptophan and methionine residues within the core of this motif are critical for cooperative DNA binding. A peptide containing the wild-type pentapeptide sequence, but not one in which phenylalanine is substituted for tryptophan, blocks the ability of Hox proteins to bind cooperatively with Pbx1 or E2A-Pbx1, suggesting that the pentapeptide itself provides at least one surface through which Hox proteins bind Pbx1. The same peptide, but not the mutant peptide, stimulates DNA binding by Pbx1, suggesting that interaction of Hox proteins with Pbx1 through the pentapeptide motif raises the DNA-binding ability of Pbx1 (Knoepfler, 1995).

Specific recognition of target sites in DNA is partly achieved through cooperative interaction with the extradenticle/pre-B-cell transformation-related gene (EXD/PBX) family of homeodomain-containing proteins. This interaction is mediated by the YPWM motif present N-terminal to the homeodomain in HOX proteins. YPWM peptides were used to confirm the importance of this motif for mediating HOX/PBX interactions. A novel monoclonal antibody directed against the YPWM was used to show that occlusion of this motif abrogates cooperativity with PBX. Residues flanking the YPWM, both N-terminally and C-terminally, stabilize the HOX.PBX cooperative complex. Because these flanking residues are also conserved among paralogs, they are likely to help distinguish the specificity of HOX/PBX interactions. These data further show that the relative importance of individual residues within and flanking the YPWM depends on the identity of position 6 of the cooperative binding site (TGATTNATGG). These results suggest that interactions between PBX and the YPWM motif are modified by a base pair predicted to contact the N-terminal arm of the HOX homeodomain (Shanmugam, 1997).

Autoregulation of vertebrate homeobox cluster genes requires the vertebrate homolog of extradenticle. Hoxb-1 has a labial class homeodomain, and its expression was studied in transgenic mice and Drosophila embryos. Comparison of Hoxb-1 regulatory regions from different vertebrates identified three related sequence motifs critical for rhombomere 4 (r4) expression in the hindbrain. The conserved elements are involved in a positive autoregulatory loop dependent on Labial family members. Binding of HOXB-1 to these elements in vitro requires cofactors, and the motifs closely resemble the consensus binding site for PBX1, a homolog of the Drosophila Extradenticle. In vitro EXD/PBX serves as a Hoxb-1 cofactor in cooperative binding and in Drosophila expression mediated by the r4 enhancer is dependent on both lab and exd. This provides in vivo and in vitro evidence that r4 expression involves direct autoregulation dependent on cooperative interactions of HOXB-1 with EXD/PBX proteins as cofactors (Popperl, 1995).

HOX proteins are dependent on cofactors of the PBX family for the specificity of their DNA binding. Two regions that have been implicated in HOX/PBX cooperative interactions are the YPWM motif, found N-terminal to the HOX homeodomain, and the GKFQ domain (also known as the Hox cooperativity motif) immediately C-terminal to the PBX homeodomain. Using derivatives of the E2A-PBX oncoprotein, it was found that the GKFQ domain is not essential for cooperative interaction with HOXA1 but contributes to the stability of the complex. In contrast, the YPWM motif is strictly required for cooperative interactions in vitro and in vivo, even with mutants of E2A-PBX that lack the GKFQ domain. Using truncated PBX proteins, it was shown that the YPWM motif contacts the PBX homeodomain. The presence of the GKFQ domain increases monomer binding by the PBX homeodomain 5-fold, and the stability of the HOXA1.E2A-PBX complex 2-fold. These data suggest that the GKFQ domain acts mainly to increase DNA binding by PBX, rather than providing a primary contact site for the YPWM motif of HOXA1. Two residues, Glu-301 and Tyr-305, have been identified that are required for GKFQ function and it is suggested that this is dependent on alpha-helical character (Green, 1998).

A hydrophobic pocket in Pbx proteins

Specific residues located within the Pbx homeodomain are essential for cooperative DNA binding with Hox and Engrailed gene products. Within the N-terminal region of the Pbx homeodomain, a residue has been identified that is required for cooperative DNA binding with three Antennapedia class Hox gene products (Hoxb-7, Hoxb-8 and Hoxc-6) but not for cooperativity with Engrailed-2 (En-2) (See Drosophila Engrailed). There are similarities between heterodimeric interactions involving the yeast mating type homeodomain proteins MATa1 and MATalpha2, and those that allow the formation of Pbx/Hox and Pbx/En-2 heterodimers. Specifically, residues located in the a1 homeodomain that form a hydrophobic pocket allowing the alpha2 C-terminal tail to bind, are also required for Pbx/Hox and Pbx/En-2 cooperativity (Peltenburg, 1997).

Three residues located at another site, in the turn between helix 1 and helix 2 are characteristic of many atypical homeodomain proteins. These residues, present in Pbx type homeodomains, are required for cooperative DNA binding involving both Hox and En-2. Replacement of the three residues located in the turn between helix 1 and helix 2 of the Pbx homeodomain with those of the atypical homeo-domain proteins controlling cell fate in the basidiomycete Ustilago maydis, bE5 and bE6, allows cooperative DNA binding with three Hox members but abolishes interactions with En-2. The data suggest that the molecular mechanism of homeodomain protein interactions that control cell fate in Saccharomyces cerevisiae and in the basidiomycetes may well be conserved in part in multicellular organisms. While a number of structural determinates, such as the hydrophobic pocket, are required for cooperativity involving both Hox and Engrailed, others, such as the three amino acid insert, are clearly more specific (Peltenburg, 1997).

Transcriptional targets of Pbx proteins

Members of the Hox family of homeoproteins and their cofactors play a central role in pattern formation of all germ layers. During postembryonic development of C. elegans, non-gonadal mesoderm arises from a single mesoblast cell M. Starting in the first larval stage, M divides to produce 14 striated muscles, 16 non-striated muscles, and two non-muscle cells (coelomocytes). The role of the C. elegans Hox cluster and of the exd ortholog ceh-20 in patterning of the postembryonic mesoderm has been investigated. By examining the M lineage and its differentiation products in different Hox mutant combinations, an essential but overlapping role was found for two of the Hox cluster genes, lin-39 (Scr homolog) and mab-5 (Antp homolog), in diversification of the postembryonic mesoderm. This role of the two Hox gene products requires the CEH-20 cofactor. One target of these two Hox genes is the C. elegans twist ortholog hlh-8. Using both in vitro and in vivo assays, it has been demonstrated that twist is a direct target of Hox activation. Evidence from mutant phenotypes is presented that twist is not the only target for Hox genes in the M lineage: in particular lin-39 mab-5 double mutants exhibit a more severe M lineage defect than the hlh-8 null mutant (Liu, 2000).

The C. elegans twist ortholog hlh-8 is a direct and critical target of Hox genes and ceh-20 in the postembryonic M lineage. A critical site has been identified in the hlh-8 promoter that is a binding site for the LIN-39/CEH-20 protein complex. The similarity between core binding sequences for Drosophila Antp and Dfd proteins in vitro, and the functional equivalence of mab-5 and lin-39 in activating hlh-8 expression in the M lineage, strongly suggest that this site is also a binding site for MAB-5/CEH-20. Although hlh-8 is a target for Hox/CEH-20 function in the M lineage, it is not the only such target. Several indirect observations demonstrate the existence of additional targets. One line of evidence comes from the observation that forced expression of hlh-8 in lin-39 mab-5 mutants fails to rescue the M lineage defects. An independent line of evidence comes from a comparison of mutant phenotypes: lin-39 mab-5 mutants show a more severe patterning defect in the M lineage than null hlh-8 mutants: (1) while lin-39 mab-5 animals lack both M-derived coelomocytes, the majority of hlh-8 mutants contain normal numbers of M-derived coelomocytes; (2) while lin-39 mab-5 mutants lack all M-derived bodywall muscle, hlh-8 mutants produce variable number of these cells; (3) sex muscles can be produced in hlh-8 mutants, although they are not fully differentiated. The identity of other Hox targets in the M lineage is not known (Liu, 2000).

Fibroblast growth factor-8 (Fgf8) plays a critical role in vertebrate development and is expressed normally in temporally and spatially restricted regions of the vertebrate embryo. This study reports the identification of regions of Fgf8 important for its transcriptional regulation in murine ES cell-derived embryoid bodies. Stable transfection of ES cells, using a human growth hormone reporter gene, was employed to identify regions of the Fgf8 gene with promoter/enhancer activity. A 2-kilobase 5' region of Fgf8 contains promoter activity. A 0.8-kilobase fragment derived from the large intron of Fgf8 enhances 3-4 fold the human growth hormone expressed from the Fgf8 promoter, in an orientation dependent manner. The intronic fragment contains DNA-binding sites for the AP2, Pbx1, and Engrailed transcription factors. Gel shift and Western blot experiments document the presence of these transcription factors in nuclear extracts from ES cell embryoid bodies. In vitro mutagenesis of the Engrailed or Pbx1 site demonstrate that these sites modulate the activity of the intronic fragment. In addition, in vitro mutagenesis of both Engrailed and Pbx1 sites indicates that other unidentified sites are responsible for the transcriptional enhancement observed with the intronic fragment (Gemel, 1999).

Pbx homeodomain proteins direct Myod activity to promote fast-muscle differentiation

The basic helix-loop-helix (bHLH) transcription factor Myod directly regulates gene expression throughout the program of skeletal muscle differentiation. It is not known how a Myod-driven myogenic program is modulated to achieve muscle fiber-type-specific gene expression. Pbx homeodomain proteins mark promoters of a subset of Myod target genes, including myogenin (Myog); thus, Pbx proteins might modulate the program of myogenesis driven by Myod. By inhibiting Pbx function in zebrafish embryos, this study has shown that Pbx proteins are required in order for Myod to induce the expression of a subset of muscle genes in the somites. In the absence of Pbx function, expression of myog and of fast-muscle genes is inhibited, whereas slow-muscle gene expression appears normal. By knocking down Pbx or Myod function in combination with another bHLH myogenic factor, Myf5, it was shown that Pbx is required for Myod to regulate fast-muscle, but not slow-muscle, development. Furthermore, this study shows that Sonic hedgehog requires Myod in order to induce both fast- and slow-muscle markers but requires Pbx only to induce fast-muscle markers. These results reveal that Pbx proteins modulate Myod activity to drive fast-muscle gene expression, thus showing that homeodomain proteins can direct bHLH proteins to establish a specific cell-type identity (Maves, 2007).

Pbx1 functions in distinct regulatory networks to pattern the great arteries and cardiac outflow tract

The patterning of the cardiovascular system into systemic and pulmonic circulations is a complex morphogenetic process, the failure of which results in clinically important congenital defects. This process involves extensive vascular remodeling and coordinated division of the cardiac outflow tract (OFT). The homeodomain transcription factor Pbx1 orchestrates separate transcriptional pathways to control great-artery patterning and cardiac OFT septation in mice. Pbx1-null embryos display anomalous great arteries owing to a failure to establish the initial complement of branchial arch arteries in the caudal pharyngeal region. Pbx1 deficiency also results in the failure of cardiac OFT septation. Pbx1-null embryos lose a transient burst of Pax3 expression in premigratory cardiac neural crest cells (NCCs) that ultimately specifies cardiac NCC function for OFT development, but does not regulate NCC migration to the heart. Pbx1 directly activates Pax3, leading to repression of its target gene Msx2 in NCCs. Compound Msx2/Pbx1-null embryos display significant rescue of cardiac septation, demonstrating that disruption of this Pbx1-Pax3-Msx2 regulatory pathway partially underlies the OFT defects in Pbx1-null mice. Conversely, the great-artery anomalies of compound Msx2/Pbx1-null embryos remain within the same spectrum as those of Pbx1-null embryos. Thus, Pbx1 makes a crucial contribution to distinct regulatory pathways in cardiovascular development (Chang, 2008).

In vitro studies were conducted to further assess the potential role of Pbx1 in the transcriptional regulation of Pax3, which contains Pbx1 binding sites in its promoter. Electrophoretic mobility shift assays (EMSA) confirmed that Site A, which contains a consensus Pbx1/Meis1 binding sequence (5'-TGACAGTT-3'), supported robust cooperative binding by Pbx1 and Meis1, but not binding by either protein alone. By contrast, Pbx1 did not form binding complexes with several representative Hox proteins (HoxB2, HoxB4 or HoxB7) on Site A. Site B, which is located 1.1 kb upstream of the Pax3 transcriptional start site, was bound robustly by HoxB4 or Meis1 in the presence of Pbx1. DNA binding by HoxB2 and HoxB7 on Site B was also dependent on Pbx1. Pbx1-Meis1-Hox trimeric complexes did not form on either isolated Site A or Site B. The requirement for Pbx1 in regulating Pax3 promoter activity was assessed using a reporter gene containing the 1.6 kb Pax3 promoter fragment in PC12 pheochromocytoma cells, which are derivatives of NCCs. Whereas HoxB4 alone produced a modest increase in Pax3 promoter activity, co-transfection of Pbx1 and HoxB4 strongly activated transcription. Consistent with the binding studies, adding Meis1 to the transfection mixture did not further enhance the Pax3 transcriptional response. Thus, Pbx1 partners with Meis and Hox proteins to directly activate expression of Pax3 through its proximal promoter elements. Taken together, these results show that Pbx1 is essential for Pax3 proximal promoter activity and for transient premigratory cardiac NCC expression of Pax3. It is proposed that activity of the Pax3 1.6 kb proximal promoter in vivo partially reflects Pbx1-dependent Pax3 expression in premigratory cardiac NCCs. The broader and sustained dorsal neural tube expression of Pax3 is likely to require additional regulatory elements outside the 1.6 kb region that are not under Pbx1 control (Chang, 2008).

Pbx proteins and spleen development

The genetic control of cell fate specification, morphogenesis and expansion of the spleen, a crucial lymphoid organ, is poorly understood. Recent studies of mutant mice implicate various transcription factors in spleen development, but the hierarchical relationships between these factors have not been explored. This report establishes a genetic network that regulates spleen ontogeny, by analyzing asplenic mice mutant for the transcription factors Pbx1, Hox11 (Tlx1), Nkx3.2 (Bapx1) and Pod1 (capsulin, Tcf21). Hox11 and Nkx2.5, among the earliest known markers for splenic progenitor cells, are absent in the splenic anlage of Pbx1 homozygous mutant (-/-) embryos, implicating the TALE homeoprotein Pbx1 in splenic cell specification. Pbx1 and Hox11 genetically interact in spleen formation and loss of either is associated with a similar reduction of progenitor cell proliferation and failed expansion of the splenic anlage. Chromatin immunoprecipitation assays show that Pbx1 binds to the Hox11 promoter in spleen mesenchymal cells, which co-express Pbx1 and Hox11. Furthermore, Hox11 binds its own promoter in vivo and acts synergistically with TALE proteins to activate transcription, supporting its role in an auto-regulatory circuit. These studies establish a Pbx1-Hox11-dependent genetic and transcriptional pathway in spleen ontogeny. Additionally, it is demonstrated that while Nkx3.2 and Pod1 control spleen development via separate pathways, Pbx1 genetically regulates key players in both pathways, and thus emerges as a central hierarchical co-regulator in spleen genesis (Brendolan, 2005).

Scapula development is governed by genetic interactions of Pbx1 with its family members and with Emx2 via their cooperative control of Alx1

The genetic pathways underlying shoulder blade development are largely unknown, as gene networks controlling limb morphogenesis have limited influence on scapula formation. Analysis of mouse mutants for Pbx and Emx2 genes has suggested their potential roles in girdle development. In this study, by generating compound mutant mice, the genetic control of scapula development by Pbx genes and their functional relationship with Emx2 were examined. Analyses of Pbx and Pbx1;Emx2 compound mutants revealed that Pbx genes share overlapping functions in shoulder development and that Pbx1 genetically interacts with Emx2 in this process. A biochemical basis for Pbx1;Emx2 genetic interaction is provided by showing that Pbx1 and Emx2 can bind specific DNA sequences as heterodimers. Moreover, the expression of genes crucial for scapula development is altered in these mutants, indicating that Pbx genes act upstream of essential pathways for scapula formation. In particular, expression of Alx1, an effector of scapula blade patterning, is absent in all compound mutants. Pbx1 and Emx2 bind in vivo to a conserved sequence upstream of Alx1 and cooperatively activate its transcription via this potential regulatory element. These results establish an essential role for Pbx1 in genetic interactions with its family members and with Emx2 and delineate novel regulatory networks in shoulder girdle development (Capellini, 2010).

Pbx proteins, developmental biology and 0ncogenesis

Retinoic acid (RA) upregulates Pbx protein abundance coincident with transcriptional activation of Hox genes in P19 embryonal carcinoma cells undergoing neuronal differentiation. However, in contrast to Hox induction, Pbx upregulation is predominantly a result of post-transcriptional mechanisms. Interestingly, Pbx1, Pbx2, and Pbx3 exhibit different profiles of upregulation, suggesting possible functional divergence. The parallel upregulation of Pbx and Hox proteins in this model suggests an important role for transcriptional control by Pbx-Hox heterodimers during neurogenesis, and argues for precise control by RA. The distinctive patterns of RA-induced upregulation of both Hox and Pbx proteins coincident with RA-induced neuronal differentiation suggests that Pbx-binding motifs will be occupied by different Pbx-containing heterodimers at different stages of differentation (Knoepfler, 1997a).

Recently, a new family of homeodomain proteins has emerged, that includes Extradenticle, ceh-20, Pbx1, Pbx2 and Pbx3. The Pbx family has been shown to modulate the biological activities of the Hox proteins. Pbx1 transcripts are present in many embryonic tissues. Highest levels of Pbx1 expression in the developing embryo, from 12 to 20 days post coitum, are found in neuronal tissues, including brain, spinal cord and ganglia. In addition, Pbx1 transcripts are also detectable in the gut, lung, olfactory epithelium and kidney. The expression pattern of Pbx1 overlaps with that of many of the Hox gene products and is consistent with them acting in parallel to regulate common target genes (Roberts, 1995).

Many Hox proteins are thought to require Pbx and Meis co-factors to specify cell identity during embryogenesis. Meis3 synergizes with Pbx4 and Hoxb1b in promoting hindbrain fates in the zebrafish. Hoxb1b and Pbx4 act together to induce ectopic hoxb1a expression in rhombomere 2 of the hindbrain. In contrast, Hoxb1b and Pbx4 acting together with Meis3 induce hoxb1a, hoxb2, krox20 and valentino expression rostrally and cause extensive transformation of forebrain and midbrain fates to hindbrain fates, including differentiation of excess rhombomere 4-specific Mauthner neurons. This synergistic effect requires that Hoxb1b and Meis3 have intact Pbx-interaction domains, suggesting that their in vivo activity is dependent on binding to Pbx4. In the case of Meis3, binding to Pbx4 is also required for nuclear access. These results are consistent with Hoxb1b and Meis3 interacting with Pbx4 to form complexes that regulate hindbrain development during zebrafish embryogenesis (Vlachakis, 2001).

Notably, these experiments do not indicate the composition of Hoxb1b-, Pbx4- and Meis3-containing complexes, and several issues remain to be resolved. (1) It is not clear if all complexes contain a Meis family member. Ectopic expression of Hoxb1b by itself induces hoxb1a expression in r2. To perform this function, Hoxb1b needs to interact with an endogenous Pbx protein (most likely Pbx4 as this is the predominant Pbx protein at this stage), but does not require exogenous Meis3. While this is consistent with Hoxb1b and Pbx4 acting in the absence of a Meis protein, it leaves open the possibility that an endogenous Meis protein is involved. Indeed, the zebrafish prep1 gene appears to be ubiquitously expressed and endogenous Prep1 may interact with Hoxb1b and Pbx4 in these experiments. However, while the Pbx and Hox binding sites appear to be required for in vivo expression of most Hox-dependent genes, a Meis/Prep1 binding site is only required for some genes. Meis proteins may therefore not always be required (at least not as a DNA binding component) for Pbx and Hox proteins to function in vivo (Vlachakis, 2001).

(2) Different Hox proteins have different effects in vivo, but it is not clear if different Meis family members differ functionally. For instance, if Hoxb1b and Pbx4 require Prep1 to activate hoxb1a expression, the synergistic effect seen following co-expression of Meis3 could be due to Prep1 being limiting in vivo. In this scenario, Prep1 and Meis3 would be functionally equivalent. An alternative explanation to the synergistic effect is that exogenously supplied Meis3 provides a unique function, perhaps by replacing Prep1, thus playing an instructive role. The latter model is favored, primarily because published experiments suggest that Prep1 cannot substitute for Homothorax in Drosophila (Vlachakis, 2001 and references therein).

(3) Although both Hoxb1b and Meis3 appear to require Pbx4 interaction to be functional in vivo, it is not known whether Hoxb1b and Meis3 interact with the same Pbx4 molecule to form a trimeric complex, or whether they interact with separate Pbx4 molecules to form a pair of dimers. However, several pieces of data indicate the formation of trimeric complexes: (1) both the hoxb1 and hoxb2 r4 enhancers contain adjacent Hox/Pbx binding sites and a more distant Meis site, but there is no Pbx site near the Meis site; consistent with this, DNA fragments containing these sequences support formation of trimeric Hox/Pbx/Meis complexes, but not of a pair of dimers, in vitro; (2) a DNA-binding mutant Prep1 forms dimers with Pbx that bind DNA only very weakly. Therefore, if Meis3, Pbx4 and Hoxb1b act as a pair of dimers, a DNA-binding mutant of Meis3 (MutMeis3) should not be able to form a functional dimer with Pbx4 and should not have any in vivo activity. However, MutMeis3 still functions in vivo, as does a DNA-binding mutant Hth (Vlachakis, 2001).

(4) Misexpression of Xenopus Meis3 by itself has a minimal effect on krox20 and hoxb1 expression, but nevertheless mediates anterior deletions in Xenopus. This is in contrast with the analysis reported here, where zebrafish Meis3 requires Pbx4 and Hoxb1b for the transformation of anterior fates. Since lineage labeling was not utilized to analyze the deletions in Xenopus, it is not known if a distinct mechanism is at work, or if some Meis family members may be able to function independently of Pbx and Hox (Vlachakis, 2001 and references therein).

The effects mediated by Hoxb1b, Pbx4 and Meis3 co-expression are likely to be causally related and to occur in sequence. Since murine hoxb1 and hoxb2 have Pbx, Hox and Meis binding sites in their enhancers it is likely that zebrafish hoxb1a and hoxb2 are directly induced by Hoxb1b, Pbx4 and Meis3. Ectopic expression of hoxb2 induces krox20 and valentino expression in zebrafish, suggesting that these genes may be activated subsequent to hoxb2. Thus, expression of Hoxb1b, Pbx4 and Meis3 is sufficient to promote the differentiation of hindbrain fates, particularly r4 fates, and it is speculated that they normally perform this function within the caudal hindbrain during zebrafish embryogenesis (Vlachakis, 2001).

The vertebrate branchiomotor neurons are organized in a pattern that corresponds with the segments, or rhombomeres, of the developing hindbrain and have identities and behaviors associated with their position along the anterior/posterior axis. These neurons undergo characteristic migrations in the hindbrain and project from stereotyped exit points. lazarus/pbx4, which encodes an essential Hox DNA-binding partner in zebrafish, is required for facial (VIIth cranial nerve) motor neuron migration and for axon pathfinding of trigeminal (Vth cranial nerve) motor axons. lzr/pbx4 is required for Hox paralog group 1 and 2 function, suggesting that Pbx interacts with these proteins. Consistent with this, lzr/pbx4 interacts genetically with hoxb1a to control facial motor neuron migration. Using genetic mosaic analysis, it has been shown that lzr/pbx4 and hoxb1a are primarily required cell-autonomously within the facial motor neurons; however, analysis of a subtle non-cell-autonomous effect indicates that facial motor neuron migration is promoted by interactions among the migrating neurons. At the same time, lzr/pbx4 is required non-cell-autonomously to control the pathfinding of trigeminal motor axons. Thus, Pbx/Hox can function both cell-autonomously and non-cell-autonomously to direct different aspects of hindbrain motor neuron behavior (Cooper, 2003).

Homeodomain-containing Hox proteins regulate segmental identity in Drosophila in concert with two partners known as Extradenticle (Exd) and Homothorax (Hth). These partners are themselves DNA-binding, homeodomain proteins, and probably function by revealing the intrinsic specificity of Hox proteins. Vertebrate orthologs of Exd and Hth, known as Pbx and Meis (named for a myeloid ecotropic leukemia virus integration site), respectively, are encoded by multigene families and are present in multimeric complexes together with vertebrate Hox proteins. The zygotically encoded Pbx4/Lazarus (Lzr) protein is required for segmentation of the zebrafish hindbrain and proper expression and function of Hox genes. Meis functions in the same pathway as Pbx in zebrafish hindbrain development, since expression of a dominant-negative mutant Meis results in phenotypes that are remarkably similar to those of lzr mutants. Surprisingly, expression of Meis protein partially rescues the lzr- phenotype. Lzr protein levels are increased in embryos overexpressing Meis and are reduced for lzr mutants that cannot bind to Meis. This implies a mechanism whereby Meis rescues lzr mutants by stabilizing maternally encoded Lzr. These results define two functions of Meis during zebrafish hindbrain segmentation: that of a DNA-binding partner of Pbx proteins, and that of a post-transcriptional regulator of Pbx protein levels (Waskiewicz, 2001).

Pbx1 and a subset of homeodomain proteins collaboratively bind DNA as higher-order molecular complexes with unknown consequences for mammalian development. Pbx1 contributions were investigated through characterization of Pbx1-deficient mice. Pbx1 mutants die at embryonic day 15/16 with severe hypoplasia or aplasia of multiple organs and widespread patterning defects of the axial and appendicular skeleton. An obligatory role for Pbx1 in limb axis patterning is apparent from malformations of proximal skeletal elements, but distal structures are unaffected. In addition to multiple rib and vertebral malformations, neural crest cell-derived skeletal structures of the second branchial arch are morphologically transformed into elements reminiscent of first arch-derived cartilages. Although the skeletal malformations do not phenocopy single or compound Hox gene defects, they are restricted to domains specified by Hox proteins bearing Pbx dimerization motifs and unaccompanied by alterations in Hox gene expression. In affected domains of limbs and ribs, chondrocyte proliferation is markedly diminished and there is a notable increase of hypertrophic chondrocytes, accompanied by premature ossification of bone. The pattern of expression of genes known to regulate chondrocyte differentiation is not perturbed in Pbx1-deficient cartilage at early days of embryonic skeletogenesis, however precocious expression of Col1a1, a marker of bone formation, was found. These studies demonstrate a role for Pbx1 in multiple developmental programs and reveal a novel function in co-ordinating the extent and/or timing of proliferation with terminal differentiation. This impacts on the rate of endochondral ossification and bone formation and suggests a mechanistic basis for most of the observed skeletal malformations (Selleri, 2001).

The normal Pbx1 homeodomain protein, as well as its oncogenic derivative, E2A-Pbx1, binds the DNA sequence ATCAATCAA cooperatively with the murine Hox-A5, Hox-B7, Hox-B8, and Hox-C8 homeodomain proteins, which are themselves known oncoproteins, as well as with the Hox-D4 homeodomain protein. Cooperative binding to ATCAATCAA required the homeodomain-dependent DNA-binding activities of both Pbx1 and the Hox partner. In cotransfection assays, Hox-B8 suppresses transactivation by E2A-Pbx1. These results suggest that (1) Pbx1 may participate in the normal regulation of Hox target gene transcription in vivo and thereby contribute to aspects of anterior-posterior patterning and structural development in vertebrates; (2) that E2A-Pbx1 could abrogate normal differentiation by altering the transcriptional regulation of Hox target genes in conjunction with Hox proteins, and (3) that the oncogenic mechanism of certain Hox proteins may require their physical interaction with Pbx1 as a cooperating, DNA-binding partner (Lu, 1995).

A transcriptionally activated version of Pbx1, E2a-Pbx1, is an oncoprotein in human pre-B cell leukemia that strongly suppresses differentiation and retains its ability to heterodimerize with Hox proteins. Because monomeric Hox proteins bind very similar DNA motifs, it is unclear how the proteins activate diverse developmental programs. Heterodimers containing different Hox proteins and a common Pbx1 or E2a-Pbx1 partner bind different DNA motifs. Structural models suggest that the specificity of the Hox protein is altered by a conformation change involving residues in the N-terminal arm of the Hox homeodomain. Mutational analysis also supports the hypothesis that unique sequences in the N-terminal arm of the Hox homeodomain are at least partially responsible for mediating this specificity. In vivo, Hox proteins directed E2a-Pbx1-mediated transactivation with moderate specificity to cognate Hox-Pbx motifs. Thus, the development specificity of individual Hox proteins may be mediated, in part, by differential targeting of cellular genes by Pbx1-Hox complexes. Likewise, through its function as a common heterodimer partner, oncoprotein E2a-Pbx1 may be able to interfere with multiple programs of development that are induced by the sequential or simultaneous expression of Hox proteins during hematopoiesis (Lu, 1997).

Oncogenic mutation of nuclear transcription factors often is associated with altered patterns of subcellular localization that may be of functional importance. The leukemogenic transcription factor gene E2A-PBX1 is created through fusion of the genes E2A (see Drosophila Daughterless) and PBX1 as a result of t(1;19) in acute lymphoblastic leukemia. Subcellular localization patterns of E2A-PBX1 protein were evaluated in transfected cells using immunofluorescence. Full-length E2A-PBX1 is exclusively nuclear and is concentrated in spherical domains termed chimeric-E2A oncoprotein domains (CODs). In contrast, nuclear fluorescence for wild-type E2A or PBX1 proteins is diffuse. Enhanced concentrations of RNA polymerase II within many CODs and the requirement for an E2A-encoded activation domain suggest transcriptional relevance. However, in situ co-detection of nascent transcripts labeled with bromouridine fails to confirm altered transcriptional activity in relation to CODs. CODs also fail to co-localize with foci of DNA replication as well as with other proteins known to occupy functional nuclear compartments, including the transcription factor PML, the spliceosome-associated protein SC-35 and the adenovirus replication factor DBP. Co-transfection of Hoxb7, a homeodomain protein capable of enhancing DNA binding by PBX1, impairs COD formation, suggesting that CODs contain E2A-PBX1 protein not associated with DNA. It is concluded that as a 'gain of function' phenomenon requiring protein elements from both E2A and PBX1, COD formation may be relevant to the biology of E2A-PBX1 in leukemogenesis (LeBrun, 1997).

Hoxa9, Meis1 and Pbx1 encode homeodomain containing proteins implicated in leukemic transformation in both mice and humans. Hoxa9, Meis1 and Pbx1 proteins have been shown to physically interact with each other, as Hoxa9 cooperatively binds consensus DNA sequences with Meis1 and with Pbx1, while Meis1 and Pbx1 form heterodimers in both the presence and absence of DNA. Could Hoxa9 transform hemopoietic cells in collaboration with either Pbx1 or Meis1? Primary bone marrow cells, retrovirally engineered to overexpress Hoxa9 and Meis1a simultaneously, induce growth factor-dependent oligoclonal acute myeloid leukemia in 3 months when transplanted into syngenic mice. In contrast, overexpression of Hoxa9, Meis1a or Pbx1b alone, or the combination of Hoxa9 and Pbx1b fail to transform these cells acutely within 6 months post-transplantation. Similar results were obtained when FDC-P1 cells, engineered to overexpress these genes, are transplanted to syngenic recipients. Thus, these studies demonstrate a selective collaboration between a member of the Hox family and one of its DNA-binding partners in transformation of hemopoietic cells (Kroon, 1998).

The products of PBX homeobox genes, which were initially discovered in reciprocal translocations occurring in human leukemias, have been shown to cooperate in the in vitro DNA binding with HOX proteins. Despite the growing body of data implicating Hox genes in the development of various cancers, little is known about the role of HOX-PBX interactions in the regulation of proliferation and induction of transformation of mammalian cells. Both cellular transformation and proliferation induced by Hoxb4 and Hoxb3 are greatly modulated by the levels of available PBX1 present in these cells. The transforming capacity of these two HOX proteins depends on their conserved tetrapeptide and homeodomain regions, which (respectively) mediate binding to PBX and DNA. Taken together, results of this study demonstrate that cooperation between HOX and PBX proteins modulates cellular proliferation and strongly suggest that cooperative DNA binding by these two groups of proteins represents the basis for Hox-induced cellular transformation (Krosl, 1998).

Human Pbx1 and HOXB1 proteins can cooperatively activate transcription through a genetically characterized Hox target, i.e. an autoregulatory element directing spatially restricted expression of the murine Hoxb-1 gene (b1-ARE) in rhombomere 4 of the developing hindbrain. On the b1-ARE, only a restricted subset of HOX proteins (HOXA1, HOXB1, HOXA2) are able to bind cooperatively with Pbx1 and activate transcription. Selective recognition of the b1-ARE is mediated by the N-terminal region of the HOX homeodomain. The DNA-binding and protein-protein interaction functions of HOXB1 and Pbx1 are all necessary for the assembly of a transcriptionally active complex on the b1-ARE. Functional dissection of the complex shows that the localization of the main activation domain is in the HOXB1 N-terminal region, and that an additional activation domain is located in the C-terminal region of Pbx1, contained in the Pbx1a isoform, but not in the alternatively spliced Pbx1b isoform. These results indicate that Pbx1 acts as a transcriptional co-factor of Hox proteins, allowing selective recognition and cooperative activation of regulatory target sequences (Di Rocco, 1997).

Meis homeodomain proteins function as Hox-cofactors by binding Pbx and Hox proteins to form multimeric complexes that control transcription of genes involved in development and differentiation. It is not known what role Meis proteins play in these complexes, nor is it clear which Hox functions require Meis proteins in vivo. A divergent Meis family member, Prep1, acts as a Hox co-factor in zebrafish. This suggests that all Meis family members have at least one shared function and that this function must be carried out by a conserved domain. The Meinox domain, an N-terminal conserved domain shown to mediate Pbx binding, is sufficient to provide Meis activity to a Pbx/Hox complex. This activity is separable from Pbx binding and resides within the M1 subdomain. This finding also presents a rational strategy for interfering with Meis activity in vivo. This was accomplished by expressing the Pbx4/Lzr N-terminus, which sequesters Meis proteins in the cytoplasm away from the nuclear transcription complexes. Sequestering Meis proteins in the cytoplasm leads to extensive loss of rhombomere (r) 3- and r4-specific gene expression, as well as defective rhombomere boundary formation in this region. These changes in gene expression correlate with impaired neuronal differentiation in r3 and r4, e.g. the loss of r3-specific nV branchiomotor neurons and r4-specific Mauthner neurons. It is conclude that Meis family proteins are essential for the specification of r3 and r4 of the hindbrain (Choe, 2002).

The phenotype observed as a result of interfering with Meis activity is also qualitatively similar to that of the lazarus mutant (which carries a mutation in the pbx4 gene). Particularly, in both cases gene expression is affected primarily in r3 and r4 and less in r1, r2 or r5-r7. This suggests that Pbx and Meis function in the same pathway during hindbrain development. This is consistent with work in Drosophila, where the phenotypes of hth and exd mutants are largely indistinguishable and the genes are thought to act in the same pathway. An explanation for Meis and Pbx acting in the same pathway in the hindbrain probably comes from Meis proteins not interacting directly with Hox proteins expressed in the hindbrain (primarily paralog group 1-4), whereas Pbx proteins do interact directly with Hox proteins. Therefore, Meis proteins can only act as Hox cofactors in the hindbrain by binding to Pbx. The finding that Meis and Pbx loss-of-function give similar hindbrain phenotypes is therefore consistent with all hindbrain Hox functions that require Pbx also requiring Meis. However, although the meis loss-of-function and lazarus phenotypes are qualitatively similar, they differ quantitatively. Surprisingly, both a higher frequency and a more severe effect on hindbrain gene expression is observed in the absence of Meis function than reported for the lazarus mutant. It is speculated that this is unlikely to be a result of Pbx-independent effects of Meis proteins on Hox function, but may instead stem from the presence of maternal pbx4/lzr transcript, as well as additional pbx genes expressed in the lazarus mutant. If this is correct, complete removal of Pbx activity might be required to conclusively define the relative roles of Pbx and Meis in regulating Hox function (Choe, 2002).

Pbx1 encodes a TALE homeodomain transcription factor that regulates developmental gene expression in a variety of tissues. Loss-of-function studies have demonstrated a critical role for Pbx1 in cellular proliferation and patterning and suggest its involvement in numerous regulatory pathways. Examination of metanephric development in Pbx1-/- embryos has been conducted to further elucidate Pbx1-dependent processes during organogenesis. Prior to death at E15.5, Pbx1-/- embryos display kidneys that are reduced in size, axially mispositioned, and in more severe cases, exhibit unilateral agenesis. Analysis with molecular markers revealed the effective induction of tubulogenic mesenchyme; however, Pbx1-/- kidneys contain fewer nephrons and are characterized by expanded regions of mesenchymal condensates in the nephrogenic zone. Decreased branching and elongation of the ureter are also observed, despite the restricted expression of Pbx1 in metanephric mesenchyme, developing nephrons, and stroma. Moreover, heterologous recombination studies with explant cultures verified that Pbx1-/- renal defects arise exclusively from mesenchymal dysfunction. Taken together, these data establish a role for Pbx1 in mesenchymal-epithelial signaling and demonstrate that Pbx1 is an essential regulator of mesenchymal function during renal morphogenesis (Schnabel, 2003).

Relevant mouse models of E2a-PBX1-induced pre-B cell leukemia are still elusive. The translocation t(1;19) occurs in 5% of all childhood acute lymphoblastic leukemias (ALL). Those ALLs are typically classified as pre-B ALLs since they express cytoplasmic but not surface immunoglobin. The specific association of t(1;19) with pediatric pre-B ALL (25% of all cases) makes it an attractive disease to model. As a result of this translocation, two transactivation domains in E2A are fused to the C-terminal portion of PBX1. In gene transfer studies the fusion protein E2a-PBX1 showed transforming activity in several cell types, including fibroblasts and lymphoid and myeloid cells. A pre-B leukemia model has been generated using E2a-PBX1 transgenic mice, which lack mature and precursor T-cells as a result of engineered loss of CD3epsilon expression (CD3epsilon-/-). Using insertional mutagenesis and inverse-PCR, it is shown that B-cell leukemia development in the E2a-PBX1 x D3epsilon-/- compound transgenic animals is significantly accelerated when compared to control littermates, and several known and novel integrations in these tumors have been documented. Of all common integration sites, a small region of 19 kb in the Hoxa gene locus, mostly between Hoxa6 and Hoxa10, represented 18% of all integrations in the E2a-PBX1 B-cell leukemia and was targeted in 86% of these leukemias compared to 17% in control tumors. Q-PCR assessment of expression levels for most Hoxa cluster genes in these tumors revealed an unprecedented impact of the proviral integrations on Hoxa gene expression, with tumors having one to seven different Hoxa genes overexpressed at levels up to 6600-fold above control values. Together these studies set the stage for modeling E2a-PBX1-induced B-cell leukemia and shed new light on the complexity pertaining to Hox gene regulation. In addition, these results show that the Hoxa gene cluster is preferentially targeted in E2a-PBX1-induced tumors, thus suggesting functional collaboration between these oncogenes in pre-B-cell tumors (Bijl, 2005).

Pbx proteins and neural development

Pbx proteins are a family of TALE-class transcription factors that are well characterized as Hox co-factors acting to impart segmental identity to the hindbrain rhombomeres. However, no role for Pbx in establishing more anterior neural compartments has been demonstrated. Studies done in Drosophila show that Engrailed requires Exd (Pbx orthologue) for its biological activity. Evidence is presented that zebrafish Pbx proteins cooperate with Engrailed to compartmentalize the midbrain by regulating the maintenance of the midbrain–hindbrain boundary (MHB) and the diencephalic–mesencephalic boundary (DMB). Embryos lacking Pbx function correctly initiate midbrain patterning, but fail to maintain eng2a, pax2a, fgf8, gbx2, and wnt1 expression at the MHB. Formation of the DMB is also defective as shown by a caudal expansion of diencephalic epha4a and pax6a expression into midbrain territory. These phenotypes are similar to the phenotype of an Engrailed loss-of-function embryo, supporting the hypothesis that Pbx and Engrailed act together on a common genetic pathway. Consistent with this model, it has been demonstrated that zebrafish Engrailed and Pbx interact in vitro and that this interaction is required for both the eng2a overexpression phenotype and Engrailed's role in patterning the MHB. The data support a novel model of midbrain development in which Pbx and Engrailed proteins cooperatively pattern the mesencephalic region of the neural tube (Erickson, 2007).

Parallel Pbx-dependent pathways govern the coalescence and fate of motor columns

The clustering of neurons sharing similar functional properties and connectivity is a common organizational feature of vertebrate nervous systems. Within motor networks, spinal motor neurons (MNs) segregate into longitudinally arrayed subtypes, establishing a central somatotopic map of peripheral target innervation. MN organization and connectivity relies on Hox transcription factors expressed along the rostrocaudal axis; however, the developmental mechanisms governing the orderly arrangement of MNs are largely unknown. This study shows that Pbx genes (see Drosophila Extradenticle), which encode Hox cofactors, are essential for the segregation and clustering of neurons within motor columns. In the absence of Pbx1 and Pbx3 function, Hox-dependent programs are lost and the remaining MN subtypes are unclustered and disordered. Identification of Pbx gene targets revealed an unexpected and apparently Hox-independent role in defining molecular features of dorsally projecting medial motor column (MMC) neurons. These results indicate Pbx genes act in parallel genetic pathways to orchestrate neuronal subtype differentiation, connectivity, and organization (Hanley, 2016).

Pbx and cancer

Homeobox transcription factors Meis1 and Hoxa9 promote hematopoietic progenitor self-renewal and cooperate to cause acute myeloid leukemia (AML). While Hoxa9 alone blocks the differentiation of nonleukemogenic myeloid cell-committed progenitors, coexpression with Meis1 is required for the production of AML-initiating progenitors, which also transcribe a group of hematopoietic stem cell genes, including Cd34 and Flt3 (defined as Meis1-related leukemic signature genes). This study used dominant trans-activating (Vp16 fusion) or trans-repressing (engrailed fusion) forms of Meis1 to define its biochemical functions that contribute to leukemogenesis. Surprisingly, Vp16-Meis1 (but not engrailed-Meis1) functioned as an autonomous oncoprotein that mimicked combined activities of Meis1 plus Hoxa9, immortalizing early progenitors, inducing low-level expression of Meis1-related signature genes, and causing leukemia without coexpression of exogenous or endogenous Hox genes. Vp16-Meis1-mediated transformation required the Meis1 function of binding to Pbx and DNA but not its C-terminal domain (CTD). The absence of endogenous Hox gene expression in Vp16-Meis1-immortalized progenitors allowed investigation of how Hox alters gene expression and cell biology in early hematopoietic progenitors. Strikingly, expression of Hoxa9 or Hoxa7 stimulated both leukemic aggressiveness and transcription of Meis1-related signature genes in Vp16-Meis1 progenitors. Interestingly, while the Hoxa9 N-terminal domain (NTD) is essential for cooperative transformation with wild-type Meis1, it is dispensable in Vp16-Meis1 progenitors. The fact that a dominant transactivation domain fused to Meis1 replaces the essential functions of both the Meis1 CTD and Hoxa9 NTD suggests that Meis-Pbx and Hox-Pbx (or Hox-Pbx-Meis) complexes co-occupy cellular promoters that drive leukemogenesis and that Meis1 CTD and Hox NTD cooperate in gene activation. Chromatin immunoprecipitation confirmed co-occupancy of Hoxa9 and Meis1 on the Flt3 promoter (Wang, 2006 Oncogenic mutations of the MLL histone methyltransferase confer an unusual ability to transform non-self-renewing myeloid progenitors into leukemia stem cells (LSCs) by mechanisms that remain poorly defined. Misregulation of Hox genes is likely to be critical for LSC induction and maintenance but alone it does not recapitulate the phenotype and biology of MLL leukemias, which are clinically heterogeneous -- presumably reflecting differences in LSC biology and/or frequency. TALE (three-amino-acid loop extension) class homeodomain proteins of the Pbx and Meis families are also misexpressed in this context, and thus knockout, knockdown, and dominant-negative genetic techniques were employed to investigate the requirements and contributions of these factors in MLL oncoprotein-induced acute myeloid leukemia. The studies show that induction and maintenance of MLL transformation requires Meis1 and is codependent on the redundant contributions of Pbx2 and Pbx3. Meis1 in particular serves a major role in establishing LSC potential, and determines LSC frequency by quantitatively regulating the extent of self-renewal, differentiation arrest, and cycling, as well as the rate of in vivo LSC generation from myeloid progenitors. Thus, TALE proteins are critical downstream effectors within an essential homeoprotein network that serves a rate-limiting regulatory role in MLL leukemogenesis (Wong, 2007).

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