Emx2 is a dose-dependent negative regulator of Sox2 telencephalic enhancers.

The transcription factor Sox2 is essential for neural stem cells (NSC) maintenance in the hippocampus and in vitro. The transcription factor Emx2 is also critical for hippocampal development and NSC self-renewal. Searching for 'modifier' genes affecting the Sox2 deficiency phenotype in mouse, we observed that loss of one Emx2 allele substantially increased the telencephalic ß-geo (LacZ) expression of a transgene driven by the 5' or 3' Sox2 enhancer. Reciprocally, Emx2 overexpression in NSC cultures inhibited the activity of the same transgene. In vivo, loss of one Emx2 allele increased Sox2 levels in the medial telencephalic wall, including the hippocampal primordium. In hypomorphic Sox2 mutants, retaining a single 'weak' Sox2 allele, Emx2 deficiency substantially rescued hippocampal radial glia stem cells and neurogenesis, indicating that Emx2 functionally interacts with Sox2 at the stem cell level. Electrophoresis mobility shift assays and transfection indicated that Emx2 represses the activities of both Sox2 enhancers. Emx2 bound to overlapping Emx2/POU-binding sites, preventing binding of the POU transcriptional activator Brn2. Additionally, Emx2 directly interacted with Brn2 without binding to DNA. These data imply that Emx2 may perform part of its functions by negatively modulating Sox2 in specific brain areas, thus controlling important aspects of NSC function in development.

Characterization of PREP2, a paralog of PREP1, which defines a novel sub-family of the MEINOX TALE homeodomain transcription factors.

TALE (three amino acid loop extension) homeodomain proteins include the PBC and the MEINOX sub-families. MEINOX proteins form heterodimer complexes with PBC proteins. Heterodimerization is crucial to DNA binding and for nuclear localization. PBC-MEINOX heterodimers bind DNA also in combination with HOX proteins, thereby modulating their DNA-binding specificity. TALE proteins therefore play crucial roles in multiple developmental and differentiation pathways in vivo. We report the identification and characterization of a novel human gene homologous to PREP1, called PREP2. Sequence comparisons indicate that PREP1 and PREP2 define a novel sub-family of MEINOX proteins, distinct from the MEIS sub-family. PREP2 is expressed in a variety of human adult tissues and displays a more restricted expression pattern than PREP1. PREP2 is capable of heterodimerizing with PBC proteins. Heterodimerization with PBX1 appears to be essential for nuclear localization of both PREP2 and PBX1. A comparison between the functional properties of PREP1 and PREP2 reveals that PREP2-PBX display a faster DNA-dissociation rate than PREP1-PBX heterodimers, suggesting different roles in controlling gene expression. Like PREP1, PREP2-PBX heterodimers are capable of forming ternary complexes with HOXB1. The analysis of some PREP2 in vitro properties suggests a functional diversification among PREP and between PREP and MEIS MEINOX proteins.

The recruitment of SOX/OCT complexes and the differential activity of HOXA1 and HOXB1 modulate the Hoxb1 auto-regulatory enhancer function.

Regionally restricted expression patterns of Hox genes in developing embryos rely on auto-, cross-, and para-regulatory transcriptional elements. One example is the Hoxb1 auto-regulatory element (b1-ARE), which drives expression of Hoxb1 in the fourth rhombomere of the hindbrain. We previously showed that HOXB1 and PBX1 activate transcription from the b1-ARE by binding to sequences required for the expression of a reporter gene in rhombomere 4 in vivo. We now report that in embryonal carcinoma cells, which retain characteristics of primitive neuroectodermal cells, the b1-ARE displays higher basal and HOX/PBX-induced activities than in other cell backgrounds. We have identified a bipartite-binding site for SOX/OCT heterodimers within the b1-ARE that accounts for its cell context-specific activity and is required for maximal transcriptional activity of HOX/PBX complexes in embryonal carcinoma cells. Furthermore, we found that in an embryonal carcinoma cell background, HOXB1 has a significantly higher transcriptional activity than its paralog HOXA1. We map the determinants for this differential activity within the HOXB1 N-terminal transcriptional activation domain. By using analysis in transgenic and HOXA1 mutant mice, we extended these findings on the differential activities of HOXA1 and HOXB1 in vivo, and we demonstrated that they are important for regulating aspects of HOXB1 expression in the hindbrain. We found that mutation of the SOX/OCT site and targeted inactivation of Hoxa1 both impair the response of the b1-ARE to retinoic acid in transgenic mice. Our results show that Hoxa1 is the primary mediator of the response of b1-ARE to retinoic acid in vivo and that this function is dependent on the binding of SOX/OCT heterodimers to the b1-ARE. These results uncover novel functional differences between Hox paralogs and their modulators.

Two murine and human homologs of mab-21, a cell fate determination gene involved in Caenorhabditis elegans neural development.

We report the cloning and genetic characterization of one human and two murine homologs of the mab-21 cell fate specification gene. mab-21 participates in the formation of sensory organs in the male nematode tail, and is essential for other developmental functions elsewhere in the Caenorhabditis elegans embryo. The expanding mab-21 gene family, which is strikingly conserved in evolution, includes two putative Drosophila members. The two mammalian genes, encoding 41 kDa nuclear basic proteins, are expressed in partially overlapping territories in the embryonic brain, eye and limbs, as well as in neural crest derivatives. Recent genetic data implicating mab-21 as a downstream target of TGF-beta signaling, together with the distribution of mab-21 transcripts in the mouse embryo, propose these novel genes as relevant factors in various aspects of vertebrate neural development.

The subcellular localization of PBX1 and EXD proteins depends on nuclear import and export signals and is modulated by association with PREP1 and HTH.

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.

Control of vertebrate limb outgrowth by the proximal factor Meis2 and distal antagonism of BMPs by Gremlin.

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.

Definition of the transcriptional activation domains of three human HOX proteins depends on the DNA-binding context.

Hox proteins control developmental patterns and cell differentiation in vertebrates by acting as positive or negative regulators of still unidentified downstream target genes. The homeodomain and other small accessory sequences encode the DNA-protein and protein-protein interaction functions which ultimately dictate target recognition and functional specificity in vivo. The effector domains responsible for either positive or negative interactions with the cell transcriptional machinery are unknown for most Hox proteins, largely due to a lack of physiological targets on which to carry out functional analysis. We report the identification of the transcriptional activation domains of three human Hox proteins, HOXB1, HOXB3, and HOXD9, which interact in vivo with the autoregulatory and cross-regulatory enhancers of the murine Hoxb-1 and human HOXD9 genes. Activation domains have been defined both in a homologous context, i.e., within a HOX protein binding as a monomer or as a HOX-PBX heterodimer to the specific target, and in a heterologous context, after translocation to the yeast Gal4 DNA-binding domain. Transfection analysis indicates that activation domains can be identified in different regions of the three HOX proteins depending on the context in which they interact with the DNA target. These results suggest that Hox proteins may be multifunctional transcriptional regulators, interacting with different cofactors and/or components of the transcriptional machinery depending on the structure of their target regulatory elements.

Prep1, a novel functional partner of Pbx proteins.

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.

The novel homeoprotein Prep1 modulates Pbx-Hox protein cooperativity.

The products of the mammalian Pbx and Drosophila exd genes are able to interact with Hox proteins specifically and to increase their DNA binding affinity and selectivity. In the accompanying paper we show that Pbx proteins exist as stable heterodimers with a novel homeodomain protein, Prep1. Here we show that Prep1-Pbx interaction presents novel structural features: it is independent of DNA binding and of the integrity of their respective homeodomains, and requires sequences in the N-terminal portions of both proteins. The Prep1-Pbx protein-protein interaction is essential for DNA-binding activity. Prep1-Pbx complexes are present in early mouse embryos at a time when Pbx is also interacting with Hox proteins. The use of different interaction surfaces could allow Pbx to interact with Prep1 and Hox proteins simultaneously. Indeed, we observe the formation of a ternary Prep1-Pbx1-HOXB1 complex on a HOXB1-responsive target in vitro. Interaction with Prep1 enhances the ability of the HOXB1-Pbx1 complex to activate transcription in a cooperative fashion from the same target. Our data suggest that Prep1 is an additional component in the transcriptional regulation by Hox proteins.

Functional dissection of a transcriptionally active, target-specific Hox-Pbx complex.

Hox genes control cell fates and specify regional identities in vertebrate development. Hox proteins show a relaxed DNA-binding selectivity in vitro, suggesting that functional specificity is achieved in vivo through the action of transcriptional co-factors. Pbx proteins are good candidates for such a role, on the basis of both genetic and biochemical evidence. We report that the 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 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 allowed the localization of the main activation domain in the HOXB1 N-terminal region, and of an additional one in the C-terminal region of Pbx1 contained in the Pbx1a but not in the alternatively spliced Pbx1b isoform. Our results indicate that Pbx1 acts as a transcriptional co-factor of Hox proteins, allowing selective recognition and cooperative activation of regulatory target sequences.

HMG1 interacts with HOX proteins and enhances their DNA binding and transcriptional activation.

High mobility group protein 1 (HMG1) is a non-histone, 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.

Inhibition of proliferation and induction of differentiation of pluripotent human embryonal carcinoma cells by osteogenic protein-1 (or bone morphogenetic protein-7).

BACKGROUND: Osteogenic protein-1 (OP-1) is a member of the transforming growth factor-beta super family closely related to the bone morphogenetic proteins and also known as bone morphogenetic protein-7. Other members of this family of growth factors influence cell differentiation as well as cell growth in a number of systems. The Drosophila homolog encoded by the decapentaplegic locus is involved in dorsal-ventral pattern formation during embryogenesis, whereas the expression of several bone morphogenetic proteins including OP-1 is developmentally regulated in mammalian embryos. EXPERIMENTAL DESIGN: The effect of recombinant human OP-1 on the proliferation and differentiation of an established pluripotent human embryonal carcinoma (EC) cell line, NTERA2, and three nullipotent human EC cell lines, 2102Ep, 833KE and TERA-1, was investigated. These cells were grown under reduced serum conditions, and differentiation was monitored by morphology and expression of marker antigens. RESULTS: OP-1 inhibited proliferation of NTERA2 and induced their differentiation, marked by changes in cellular morphology, the loss of EC cell antigens (SSEA3, SSEA4, the liver isozyme of alkaline phosphatase), and the appearance of new antigens, notably SSEA1 and class 1 major histocompatibility complex antigens. These changes were irreversible and did not involve significant cell degeneration or cell death. The OP-1-induced differentiation of NTERA2 appeared distinct from that induced by either retinoic acid or hexamethylene bisacetamide. Nevertheless, OP-1 did induce the homeobox gene, HOXA1. By contrast, OP-1 elicited only a limited and partial response from the nullipotent EC cell lines. CONCLUSIONS: Our results suggest that pluripotent human EC cells differentiate in response to OP-1 and that this factor can modulate the differentiation induced by retinoic acid. Like other members of the transforming growth factor-beta super family, OP-1 might play an inductive role in the early embryo. The results also suggest a possible therapeutic value for OP-1 in the treatment of some germ cell tumors.

Inhibition of retinoic acid-induced activation of 3' human HOXB genes by antisense oligonucleotides affects sequential activation of genes located upstream in the four HOX clusters.

Most homeobox genes belonging to the Hox family are sequentially activated in embryonal carcinoma cells upon treatment with retinoic acid. Genes located at the 3' end of each one of the four Hox clusters are activated first, whereas upstream Hox genes are activated progressively later. This activation has been extensively studied for human HOX genes in the NT2/D1 cell line and shown to take place at the transcriptional level. To understand the molecular mechanisms of sequential HOX gene activation in these cells, we tried to modulate the expression of 3' HOX genes through the use of antisense oligonucleotides added to the culture medium. We chose the HOXB locus. A 5- to 15-fold reduction of the expression of HOXB1 and HOXB3 was sufficient to produce a significant inhibition of the activation of the upstream HOXB genes, as well as of their paralogs in the HOXA, HOXC, and HOXD clusters. Conversely, no effect was detectable on downstream HOX genes. The extent of this inhibition increased for progressively more-5' genes. The stability of the corresponding mRNAs appeared to be unaffected, supporting the idea that the observed effect might be mediated at the transcriptional level. These data suggest a cascade model of progressive activation of Hox genes, with a 3'-to-5' polarity.

Specificity of HOX protein function depends on DNA-protein and protein-protein interactions, both mediated by the homeo domain.

Transcription of human HOX gene promoters in cultured cells is positively and negatively regulated by HOX proteins interacting with specific target sequences. The human HOXD9 protein activates transcription of the HOXD9 promoter by interacting with the HCR sequence and is antagonized by the HOXD8 protein. HOXD8 is not intrinsically a repressor, since it can activate transcription on different targets. Complete or partial HOXD8/HOXD9 homeo domain swapping indicates that the ability to recognize, and activate transcription from, the HCR target in vivo depends on the amino terminus and helix 1 of the homeo domain. The inhibitory activity of HOXD8 is not affected by deletion of the homeo domain helix 2/3 region, whereas it requires the amino terminus/helix 1 region and an additional, effector domain located at the protein amino-terminal end. This activity is therefore DNA-binding independent, and possibly mediated by protein-protein interactions. Affinity chromatography experiments show that the homeo domain amino terminus/helix 1 region is able to mediate direct interactions between HOX proteins in solution. These data indicate that specificity of HOX protein function in vivo depends on both DNA-protein and protein-protein interactions, mediated by the same sub region of the homeo domain.

Comparison of mouse and human HOX-4 complexes defines conserved sequences involved in the regulation of Hox-4.4.

We have cloned and sequenced, in both mouse and human, regions of the HOX-4 complex which contain two Abd-B like genes, Hox-4.4 and Hox-4.5 (HOX4C and HOX4D in human, respectively). The high degree of conservation between the homeoprotein sequences extends to non-coding areas, which suggests that the mechanisms of regulation have been conserved. We show that the Hox-4.5/Hox-4.4 intergenic region can be broadly subdivided into three domains based on DNA conservation between rodents and primates. The presence of all these domains in association with sequences located 3' to the transcription termination site are required to mimick the spatial regulation of Hox-4.4 in transgenic mouse embryos. Several highly conserved short sequences located in this region were studied in gel retardation assays for their binding to potential regulatory factors. One such factor is detected in embryonal carcinoma cells but absent from other differentiated cell lines. This specific binding activity is down regulated upon retinoic acid treatment.

The upstream region of the human homeobox gene HOX3D is a target for regulation by retinoic acid and HOX homeoproteins.

We studied the structure, regulation and expression of HOX3D, a human homeobox gene located in the HOX3 cluster on chromosome 12. HOX3D is developmentally regulated during embryogenesis and is activated by retinoic acid (RA) in cultured embryonal carcinoma (EC) cells. Transfection of HOX3D upstream genomic sequences linked to a reporter gene allowed the functional definition of its promoter, containing a canonical TATA element. This promoter directs the expression of the reporter gene in EC cells after induction with RA, and binds RA-induced nuclear factor(s) through a conserved palindromic sequence located approximately 100 bp upstream of the transcription start site. The HOX3D promoter is transactivated in both human and murine cells when cotransfected with vectors expressing the protein product of the upstream gene HOX3C and the paralogs of further upstream genes in the HOX4 cluster (i.e. HOX4D, HOX4C and the murine Hox 4.3). The HOX3D protein, and those encoded by the downstream gene HOX3E and its paralog HOX4B are instead inactive. HOX4C and HOX4D proteins synthesized in bacteria bind to the same conserved sequence located around position -120, as well as to the TATA box and immediately upstream and downstream nucleotides. These data provide evidence that cross-regulatory interactions between mammalian homeogenes take place in cultured cells, thus raising the possibility that a regulatory network may exist in vivo. The sequences on the HOX3D promoter involved in cross-regulation are different from those binding nuclear factors induced by RA.

HOX4 genes encode transcription factors with potential auto- and cross-regulatory capacities.

We have looked for the binding of several HOX4 complex homeoproteins in the genomic region containing the HOX4C promoter, between the human HOX4C and HOX4D genes. The HOX4C, HOX4D and Hox-4.3 homeoproteins bind to a phylogenetically highly conserved DNA fragment, which is located in the proximal part of this intergenic region and contains multiple binding sites for these HOX4 proteins. Using cotransfection experiments, we show that this endogenous DNA sequence can mediate transactivation by the HOX4D and HOX4C proteins and that this effect requires the presence of TAAT-related binding sites. The Hox-4.3 protein, in contrast, is unable to activate and can repress the activation observed with the two other proteins. These results show that the HOX4D and HOX4C genes are genuine sequence-specific transcription factors and suggest that, as in Drosophila, cross-regulatory interactions between these genes might be essential for their proper expression.

Primary structure and embryonic expression pattern of the mouse Hox-4.3 homeobox gene.

We report the cloning, genomic localization, primary structure and developmental expression pattern of the novel mouse Hox-4.3 gene. This gene is located within the HOX-4(5) complex, at a position which classifies it as a member of the Hox-3.1 and -2.4 subfamily, the DNA and predicted protein sequences further confirmed this classification. Hox-4.3 has a primary structure characteristic of a Hox gene but, in addition, contains several monotonic stretches of amino acids, one of the 'paired'-like type. As expected from its presence and position within the complex. Hox-4.3 is developmentally expressed in structures of either mesodermal or neurectodermal origin located or derived from below a precise craniocaudal level. However, a very important offset between anteroposterior boundaries within neuroectoderm versus mesoderm derivatives is observed. Like other genes of the HOX-4(5) complex, Hox-4.3 is expressed in developing limbs and gonads, suggesting that 'cluster specificity' could be a feature of the HOX network.

Molecular mechanisms underlying the expression of the human HOX-5.1 gene.

The complex mechanisms underlying homeobox genes expression involve regulation at transcriptional, post-transcriptional and translational levels. The multiple transcripts of the human HOX-5.1 gene are expressed differentially in tissue- and stage-specific patterns during embryogenesis, and differentially induced by retinoic acid (RA) in human embryonal carcinoma (EC) NT2/D1 cells. We have sequenced 6.3 Kb of the genomic region containing the HOX-5.1 gene and analyzed its mechanisms of expression. Two alternative promoters underlie the transcription of two classes of HOX-5.1-specific mRNAs. These classes differ in tissue and subcellular distribution, induction by RA, structure of the 5'-UT region and mRNA stability: these features are compatible with a differential function of the two classes of transcripts in embryogenesis.