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p48ZnF is a C3H1 zinc finger domain-containing protein that is involved in the control of gene transcription and translation. In the present study a novel transgenic p48ZnF mouse model is described that is useful for in vivo brain imaging using luciferase as bioluminescence-mediating reporter gene. Yeast two-hybrid screening and western blot analyses revealed Drg1 (developmentally regulated GTP binding protein 1) and Pcbp1 (poly (rC)-binding protein 1) as p48ZnF-associated proteins. Interestingly, p48ZnF' cellular location of action depends on the cell's differentiation status: nuclear in proliferating cells and cytoplasmic in differentiated neurons.

Abstract 2629 Hodgkin/Reed-Sternberg (HRS) cells represent the malignant fraction of infiltrated lymph nodes in Hodgkin lymphoma (HL). Although HRS cells display multiple chromosomal aberrations, few are recurrent and the targeted genes unknown. However, understanding the pathology of HL and developing rational therapies may well require identification and characterization of these genes and their deregulating mechanisms. Recently, we have identified a novel chromosomal rearrangement in HL cell line L-1236, t(4;8)(q27;q24), which targets ZHX2 at the recurrent breakpoint 8q24. ZHX2 encodes a transcription factor of the homeobox gene family and possesses tumour suppressor activity connected with a worse prognosis in HL-related multiple myeloma. Comparative expression analysis in HL cell lines and primary hematopoietic cells indicated aberrant downregulation in HL as well. In L-1236 the translocation breakpoint at 8q24 deletes the regulative far upstream region of ZHX2, indicating loss of activating elements. Therefore, we have looked for potential binding sites within this deleted region to analyze deregulation of this B-cell tumour suppressor gene. Accordingly, we identified two transcription factors, homeodomain protein MSX1 and bZIP protein XBP1, regulating ZHX2 expression. SiRNA-mediated knockdown and reporter gene analyses indicated that both factors activated transcription of ZHX2 directly via their corresponding binding sites. Furthermore, MSX1-cofactor histone H1C mediated repression of ZHX2 and showed enhanced expression levels in L-1236. As analyzed by fluorescence in situ hybridization (FISH) and genomic arrays, the gene loci of MSX1 at 4p16 and H1C at 6p21 were rearranged in several HL cell lines, correlating with their altered expression activity. As compared to primary hematopoietic cells, XBP1 expression was reduced in 6/7 HL cell lines while one cell line showed elevated levels, corresponding to a chromosomal rearrangement therein. Interestingly, the neighbouring pro-apoptotic genes EDEM1 and BHLHE40 were removed by del(3p26) in this cell line and activated by XBP1 in other HL cells. Taken together, our results demonstrate multiple mechanisms decreasing expression of tumour suppressor gene ZHX2 in HL cells: loss of enhancing binding sites, reduced expression of activators MSX1 and XBP1, and overexpression of MSX1-corepressor H1C. Moreover, chromosomal deregulations of genes involved in this regulative network highlight their role in development and malignancy of B-cells. Disclosures: No relevant conflicts of interest to declare.

Zinc finger-homeodomain proteins (ZF-HDs) have been identified in many plant species. In soybean (Glycine max), GmZF-HD1 functions as a transcription factor that activates the soybean calmodulin isoform-4 (GmCaM-4) gene in response to pathogens. Recently, we reported specific binding of GmZF-HD1 to a 30-nt A/T-rich cis-element which constitutes two repeats of a conserved homeodomain binding site, ATTA, within -1207 to -1128bp of the GmCaM-4 promoter. Herein, homeodomain sequences of the GmZF-HD1 protein were compared to those of other homeodomain proteins and characterized the specificity of DNA sequences in the interaction of the GmCaM-4 promoter with GmZF-HD1 protein. Considering the conservation of homeodomains in plants, the AG sequence within a 30-nt A/T-rich cis-element is required for binding of the GmZF-HD1 protein. Approximately 25-bp of A/T-rich DNA sequences containing an AG sequence is necessary for effective binding to the GmZF-HD1 protein. Taken together, the results support the notion that the GmZF-HD1 protein specifically functions in plant stress signalling by interacting with the promoter of GmCaM-4.

Homeobox genes encode transcription factors whose expression organizes programs of development. A number of homeobox genes expressed in reproductive tissues have been identified recently, including a colinear cluster on the X chromosome in mice. This has led to an increased interest in understanding the role(s) of homeobox genes in regulating development of reproductive tissues including the testis, ovary, and placenta. Here we report the identification and characterization of a novel homeobox gene of the paired-like class on the X chromosome distal to the reproductive homeobox (Rhox) cluster in mice. Transcripts are found in the testis and ovary as early as 13.5 days post-coitum (dpc). Transcription ceases in the ovary by 3 days post-partum (dpp), but continues in the testis through adulthood. The Rhox13 gene encodes a 25.3 kDa protein expressed in the adult testis in germ cells at the basal aspect of the seminiferous epithelium.

A set of 96 homeobox transcription factors was identified in the Strongylocentrotus purpuratus genome using permissive blast searches with a large collection of authentic homeodomain sequences from mouse, human and fly. A phylogenetic tree was constructed to compare the sea urchin homeobox gene family to those of vertebrates, with the result that with the only a few exceptions, orthologs of all vertebrate homeodomain genes were uncovered by our search. QPCR time course measurements revealed that 65% of these genes are expressed within the first 48 h of development (late gastrula). For genes displaying sufficiently high levels of transcript during the first 24 h of development (late blastula), whole mount in situ hybridization was carried out up to 48 h to determine spatial patterns of expression. The results demonstrate that homeodomain transcription factors participate in multiple and diverse developmental functions, in that they are used at a range of time points and in every territory of the developing embryo.

The Drosophila melanogaster genes, ladybird early (lbe) and ladybird late (lbl), encode transcriptional regulators, which play an important role in neurogenesis, myogenesis and cardiogenesis. Here, we report an isolation of a novel ladybird family homeobox gene (lbx3) and its expression during chick development. The open reading frame of lbx3 encodes a predicted protein of 213 amino acids including a homeodomain, a PST motif and a nuclear localization signal. The homeodomain of lbx3 protein has 80% identities with the chick and mouse lbx1 homeodomains, and 75% identity with the mouse lbx2 homeodomain. Both lbx1 and lbx3 are expressed in prospective hypaxial myoblasts at cervical and limb level. In addition, lbx3 transcripts are detected in the medial dermomyotomal lips of somites of all axial levels at stage 23, but not detected in the neural tube.

Although HOXB6 and other HOX genes have previously been associated with hematopoiesis and leukemias, the precise mechanism of action of their protein products remains unclear. Here we use a biological model in which HOXB6 represses alpha- and gamma-globin mRNA levels to perform a structure/function analysis for this homeodomain protein. HOXB6 protein represses globin transcript levels in stably transfected K562 cells in a DNA-binding dependent fashion. However, the capacity to form cooperative DNA-binding complexes with the PBX co-factor protein is not required for HOXB6 biological activity. Neither the conserved extreme N-terminal region, a polyglutamic acid region at the protein C terminus, nor the Ser(214) CKII phosphorylation site was required for DNA binding or activity in this model. We have previously reported that HOX proteins can inhibit CREB-binding protein (CBP)-histone acetyltransferase-mediated potentiation of reporter gene transcription. We now show that endogenous CBP is co-precipitated with exogenous HOXB6 from nuclear and cytoplasmic compartments of transfected K562 cells. Furthermore, endogenous CBP co-precipitates with endogenous HOXB6 in day 14.5 murine fetal liver cells during active globin gene expression in this tissue. The CBP interaction motif was localized to the homeodomain but does not require the highly conserved helix 3. Our data suggest that the homeodomain contains most or all of the important structures required for HOXB6 activity in blood cells.

Certain cases of familial Alzheimer's disease are caused by mutants of amyloid-beta precursor protein (AbetaPP), including V642I-AbetaPP, K595N/M596L-AbetaPP (NL-AbetaPP), A617G-AbetaPP, and L648P-AbetaPP. By using an unbiased functional screening with transfection and expression of a human brain cDNA library, we searched for genes that protect neuronal cells from toxicity by V642I-AbetaPP. One protective clone was identical to the human GTX, a neuronal homeobox gene. Human Gtx (hGtx) inhibited caspase inhibitor-sensitive neuronal cell death not only by V642I-AbetaPP but also by L648P-, NL-, A617G-AbetaPP, apolipoprotein E4, and Abeta. The region of hGtx responsible for this rescue function was specified to be its homeodomain (Lys148-His207). The rescue function was shared by DLX4, a distal-less family gene with a homeodomain only 38.3% homologous to that of hGtx, suggesting that this function would be generally shared by homeodomains. The neuroprotective function of hGtx was attributable to hGtx-stimulated production and secretion of insulin-like growth factor-I. This study provides molecular clues to understand how neuronal cells developmentally regulate themselves against cell death as well as to develop reagents effective in curative therapeutics of Alzheimer's disease.

The sponge homeobox gene EmH-3 had not been attributed to any homeobox family. Comparative promoter and homeodomain sequence analyses suggest that it is related to the Hox11 gene, which belongs to the Tlx homeobox family. Hox11 is highly expressed in proliferating progenitor cells, but expression is downregulated during cell differentiation. Using reporter gene methodology, we monitored function of the sponge EmH-3 promoter transfected into human erythroleukemia K562 cells. These cells express the Tlx/Hox11 gene constitutively, and downregulate its expression upon differentiation. The same pattern of expression and downregulation was observed for the sponge reporter construct. We propose that Tlx/Hox11 genes have structural and functional homologies conserved in phylogenetically distant groups, that represent a deep homology in the regulation of cell proliferation, commitment and differentiation.

The proline-rich homeodomain protein (PRH), also known as Hex, is a transcriptional repressor expressed in a variety of cell types. The PRH protein contains a proline-rich N-terminal domain that can repress transcription when attached to a heterologous DNA binding domain, a central homeodomain that mediates sequence-specific DNA binding, and an acidic C-terminal domain of unknown function. Although individual domains of PRH have been expressed in bacterial cells as GST- and histidine-tagged fusion proteins, attempts to express and purify the full-length protein have met with little success. Here we describe the purification of a histidine-tagged full-length PRH fusion protein. The protein described here will allow us to determine the mechanisms whereby PRH represses transcription.

Homeodomain proteins are a highly conserved class of DNA-binding proteins that are found in virtually every eukaryotic organism. The conserved mechanism that these proteins use to bind DNA suggests that there may be at least a partial DNA recognition code for this class of proteins. To test this idea, we have investigated the sequence-specific requirements for DNA binding and repression by the yeast alpha2 homeodomain protein in association with its cofactors, Mcm1 and Mata1. We have determined the contribution for each residue in the alpha2 homeodomain that contacts the DNA in the co-crystal structures of the protein. We have also engineered mutants in the alpha2 homeodomain to alter the DNA-binding specificity of the protein. Although we were unable to change the specificity of alpha2 by making substitutions at residues 47, 54, and 55, we were able to alter the DNA-binding specificity by making substitutions at residue 50 in the homeodomain. Since other homeodomain proteins show similar changes in specificity with substitutions at residue 50, this suggests that there is at least a partial DNA recognition code at this position.

A novel human homeobox gene related to the mouse Esx1 homeobox gene, which we have designated ESXR1, has been identified. ESXR1 and Esx1 share 65% identity within their homeodomains and have glutamic acid-rich and proline-rich N- and C-terminal regions, respectively. Unlike Esx1, ESXR1 contains 12 repeats of a unique nine amino acid motif, PPMAP(V/L)PPG, located C-terminal to the homeodomain. The general exon–intron structures of ESXR1 and Esx1 appear to be conserved. ESXR1 has been localized to human Xq22.1–q22.3, the same region of synteny shared by the map position of Esx1. ESXR1 expression appears to be restricted to the placenta and testis, the tissues in which Esx1 is also expressed. These data suggest that ESXR1 may be the orthologue of Esx1. The findings that there are similarities between ESXR1 and Esx1, yet differences between their encoded products, are consistent with the idea that placental genes evolve rapidly between mammalian species.

HIPK2 (homeodomain-interacting protein kinase 2) is a CD95 binding partner in yeast. Its primary amino acid sequence is highly conserved between human and mouse. The highest HIPK2 mRNA expression is found in neuronal tissue. The HIPK2 gene is located on human chromosome 7q33-35 and the protein is mainly localized in the nucleus. HIPK2 has been described to play a role as a co-repressor for homeodomain transcription factors.

The human gene for the fourth member of the protein 4.1 family, KIAA0987, was recently identified by comprehensive cDNA analysis. To further characterize the corresponding gene and its product in rats, we cloned and sequenced rat KIAA0987 cDNA. RNA blot analyses revealed that the rat KIAA0987 gene was abundantly expressed only in the brain, kidney, and testis. Although we have previously reported that the third member of the protein 4.1 family, the KIAA0338 gene product, is predominantly expressed in rat brain, and thus was named brain 4.1, quantitative RNA blot analyses indicated that KIAA0987 should be called something other than brain 4.1 because the level of KIAA0987 mRNA was found to be of the same order as that of KIAA0338 mRNA. Our quantitative immunoblot analysis showed that the most predominant member of the protein 4.1 family at the protein level was the product of the KIAA0987 gene, not that of the KIAA0338 gene. Taking these results together, we consider it reasonable to name the KIAA0338 and KIAA0987 gene products 'type I brain 4.1' and 'type II brain 4.1,' respectively, because these two products were found to be more prominently produced in rat brain than the other two members of the protein 4.1 family, erythroid 4.1 and 4.1G.

In Drosophila, the tinman homeobox gene is absolutely required for heart development. In the vertebrates, a small family of tinman-related genes, the cardiac NK-2 genes, appear to play a similar role in the formation of the vertebrate heart. However, targeted gene ablation of one of these genes, Nkx2-5, results in defects in only the late stages of cardiac development suggesting the presence of a rescuing gene function early in development. Here, we report the characterization of a novel tinman-related gene, XNkx2-10, which is expressed during early heart development in Xenopus. Using in vitro assays, we show that XNkx2-10 is capable of transactivating expression from promoters previously shown to be activated by other tinman-related genes, including Nkx2-5. Furthermore, Xenopus Nkx2-10 can synergize with the GATA-4 and SRF transcription factors to activate reporter gene expression.

We report the isolation and characterization of the cDNAs encoded by the murine and human homeobox genes, Irx4 (Iroquois homeobox gene 4). Mouse and human Irx4 proteins are highly conserved (83%) and their 63-aa homeodomain is more than 93% identical to that of the Drosophila Iroquois patterning genes. Human IRX4 maps to chromosome 5p15.3, which is syntenic to murine chromosome 13. Irx4 transcripts are present in the developing central nervous system, skin, and vibrissae, but are predominantly expressed in the cardiac ventricles. In mice at embryonic day (E) 7.5, Irx4 transcripts are found in the chorion and at low levels in a discrete anterior domain of the cardiac primordia. During the formation of the linear heart tube and its subsequent looping (E8.0–8.5), Irx4 expression is restricted to the ventricular segment and is absent from both the posterior (eventual atrial) and the anterior (eventual outflow tract) segments of the heart. Throughout all subsequent stages in which the chambers of the heart become morphologically distinct (E8.5–11) and into adulthood, cardiac Irx4 expression is found exclusively in the ventricular myocardium. Irx4 gene expression was also assessed in embryos with aberrant cardiac development: mice lacking RXRα or MEF2c have normal Irx4 expression, but mice lacking the homeobox transcription factor Nkx2-5 (Csx) have markedly reduced levels of Irx4 transcripts. dHand-null embryos initiate Irx4 expression, but cannot maintain normal levels. These data indicate that the homeobox gene Irx4 is likely to be an important mediator of ventricular differentiation during cardiac development, which is downstream of Nkx2-5 and dHand.

The homeodomain is a DNA-binding motif within transcription factor proteins. These transcription factors may be involved in cell differentiation and control of cell growth, as well as patterning of diverse organisms. Given their strong evolutionary conservation, it has been suggested that homeodomain proteins have been fundamental to the evolution of animal species. We have previously shown that the major classes of animal homeodomains originated early in the evolution of metazoans. Here, I investigate the evolutionary relationships of homeodomain sequences in plants. Using distance matrix approaches with unweighted or weighted character state transitions, the overall results support the same conclusion: in plants, as in animals, multiple independent classes of divergent homeodomains can be distinguished, again suggesting an early evolutionary origin. These studies advance the hypothesis that the homeodomain represents a fundamental motif of gene regulation in multicellular organisms.

The differentiation and maintenance of a neurotransmitter phenotype is guided by the interaction of exogenous cues with intrinsic genetic machinery. For the noradrenergic phenotype, these influences combine to activate the expression of the catecholaminergic biosynthetic enzymes tyrosine hydroxylase and dopamine beta-hydroxylase (DBH). In this study, we evaluate the molecular mechanisms by which the transcription factor Arix/Phox2a contributes to DBH gene transcription. We have evaluated the contribution of individual homeodomain binding sites in the rat DBH promoter region and find that all are essential for both basal and cAMP-dependent protein kinase A (PKA)-stimulated transcription. Using mammalian one-hybrid and two-hybrid systems, we demonstrate that recruitment of Arix to the positions of homeodomain core recognition sites 1 and 2 at -153 to -166 of the DBH gene restores complete responsiveness of the promoter to PKA in SHSY-5Y neuroblastoma and HepG2 hepatoma cells. Intracellular Arix-Arix interactions are evident and may contribute to the interdependence of homeodomain binding sites. Analysis of functional domains of Arix reveals an N-terminal activation domain and a C-terminal repression domain. The N terminus of Arix contains an amino acid motif similar to a region in Brachyury and Pax9 transcription factors. The N-terminal activation domain of Arix interacts with the transcriptional co-activator, cAMP-response element-binding protein-binding protein, which potentiates transcription from the DBH promoter in a PKA-dependent manner. The present study supports the hypothesis that the paired-like homeodomain protein, Arix, acts as a critical phenotype-specific regulator of the DBH promoter by serving as an integrator of signal-dependent transcription activators within the network of the general transcription machinery.

Higher vertebrates appear to possess six genes encoding a homeodomain of the distal-less type. We report the cloning and expression pattern of the chicken DLX3 gene, a homeobox gene highly related to the DLX5 gene with regard to both the encoded protein structure and the expression pattern. DLX3 RNA was observed during the development of the olfactory and otic placodes, in the distal portion of the first and second visceral arch mesenchyme, in the growing limb buds, and in the tail tip. No expression occurs in the central nervous system.

Alx4 is a paired class homeodomain protein involved in defining anterior/posterior polarity in the developing limb bud. The paired class of homeodomain proteins cooperatively bind palindromic DNA elements as homodimers or as heterodimers with other paired homeodomain proteins. Previous characterization demonstrates that the strength of the cooperativity as well as the preference for targets is dictated largely by the identity of amino acid 50 of the homeodomain. Here we compare and contrast the DNA binding properties of a glutamine 50 paired homeodomain protein, Alx4, and a lysine 50 paired homeodomain protein, Goosecoid. We demonstrate that Alx4 homodimers, Gsc homodimers, and Alx4/Gsc heterodimers each have distinct DNA binding properties, and each can discriminate between highly related palindromic elements. Using reporter gene assays, we show that Alx4 activates transcription in a site-specific manner, and that Gsc is capable of antagonizing Alx4-mediated activation only from promoter elements that support heterodimer formation. These data demonstrate that paired homeodomain proteins with different DNA binding properties are able to form heterodimeric complexes with unique DNA binding and transcriptional activities. Thus, heterodimerization regulates the DNA binding specificity of these transcription factors and may partially explain how paired homeodomain proteins direct specific developmental functions.

Homeodomain proteins play essential roles in basic processes during embryogenesis and development. NBPhox, a vertebrate paired-like homeodomain protein specific to catecholaminergic cells, may have a fundamental role in determining and maintaining the catecholaminergic phenotype. Here we describe the human and mouse genomic structures and the functional characterization of NBPhox. The genomic structure of NBPhox is highly conserved at the nucleotide level between human and mouse and is also quite similar to that of other paired-like homeodomain proteins, Arix/Phox2a, CRX, OTX1, and OTX2. The human NBPhox gene (HGMW-approved symbol PMX2B) maps to chromosomal region 5p12-p13. When the NBPhox expression plasmid was introduced into PC12h cells, the transcription from the promoter of the dopamine beta-hydroxylase (DBH) gene was slightly stimulated. However, NBPhox substantially enhances second messenger-mediated activation of the DBH promoter by forskolin and/or phorbol ester. Furthermore, we found that NBPhox can also enhance second messenger-mediated activation of the c-fos promoter and several enhancers, including cyclic AMP-response element, the binding site for activator protein 1, and serum-response element. Our findings provide strong evidence that a homeodomain protein is involved in the activation of several genetic regulatory elements responsive to second messenger-mediated signals. These data suggest that this family of proteins may be involved in determining and maintaining a cell-specific phenotype through regulation of certain genes responsive to second messenger-mediated signals.

The recently described Hmx family of homeodomain proteins is predominately expressed in discrete regions of developing sensory tissues. In this report, we have identified the preferred DNA-binding site of the murine Hmx3 homeodomain protein by the selection and amplification binding (SAAB) technique. The consensus Hmx-binding site contained the sequence 5′-CAAGTG-3′, which differs from the 5′-TAAT-3′ motif commonly associated with homeodomain proteins. Instead, the Hmx consensus is similar to the 5′-CAAGTG-3′-binding sites of Nkx2.1 and Nkx2.5 homeodomain proteins. Based on mutation studies, both the 5′-CAAG-3′ core and the 3′-TG dinucleotide are required for high affinity binding by Hmx3 and the homologous Hmx1 protein. A critical determinant of this specificity is the glutamine at position 50 in the third helix of the Hmx homeodomain. Hmx1 binds to the 5′-CAAGTG-3′ element with an apparent dissociation constant of 20 nm. Unexpectedly, the human Hmx1 protein specifically repressed transcription from a luciferase reporter gene containing 3 copies of the 5′-CAAGTG-3′ sequence. In contrast, the Nkx2.5 protein transactivated this luciferase reporter. Interestingly, co-expression of Hmx1 and Nkx2.5 attenuated each others activity, suggesting that genes containing the CAAGTG element can integrate signals from these proteins. Therefore, Hmx1 and Nkx2.5 proteins bind a unique DNA sequence and act as transcriptional antagonists.

The Xvent homeobox multigene family is essential for the patterning of the ventral mesoderm in Xenopus embryos. We have identified two novel members of this family, Xvent-1B and Xvent-2B, and have characterized their genomic structures. These two genes show a clustered organization and have probably arisen by gene duplication with subsequent inversion. Cis-regulatory elements within the promoters of both genes have been identified which contribute to their spatial activation. Xvent-2B is activated by BMP-2/4 in the absence of de novo protein synthesis, suggesting that this gene is a direct target of BMP-signalling. In contrast, Xvent-1B does not directly respond to BMP-2/4, but is activated by Xvent-2B. This activation is documented by Xvent-1B promoter/reporter studies, Xvent-2B overexpression and loss-of-function analysis using a dominant-negative Xvent-2 mutant. However, cycloheximide experiments reveal that Xvent-2B by itself is not sufficient to activate transcription of the Xvent-1B gene, but that there is a requirement for additional factor(s) being synthesized after midblastula transition.

Previous studies in both Xenopus and zebrafish have shown that goosecoid is one of the first genes to be transcribed at the onset of gastrulation. Goosecoid transcription still initiates when embryos are treated with protein synthesis inhibitors, indicating that it is mediated by preexisting factors and suggesting that goosecoid transcription is immediately downstream of the maternal mesoderm-inducing signal. However, goosecoid transcription continues long after this maternal signal has ceased to be active, indicating that there are mechanisms to maintain activin-induced transcription. Our study has focused on understanding the factors required to maintain this transcription. We have defined an element within the zebrafish goosecoid promoter that is sufficient for activin inducibility in both Xenopus and zebrafish embryos. This element, the goosecoid activin element, interacts with two developmentally regulated proteins from Xenopus embryos. A maternal protein interacts through cleavage stages until the midblastula transition, and a second protein binds from the onset of gastrulation. The second protein is zygotically expressed, and its binding is required for activin inducibility in our assay system. We suggest that the zygotic protein we have identified is a good candidate to be involved in the maintenance of goosecoid transcription. Furthermore, this zygotic protein is likely to contain a paired class homeodomain since a consensus binding site for such proteins is present within the goosecoid activin element and is essential for its function.

The HOXC6 homeodomain-containing proteins act as transcription factors in the genetic control of multiple genes involved in development and cell differentiation. Two HOXC6 polypeptides are encoded by a single homeobox ('HOX') gene described as 'master gene' for the crucial role it plays in the patterning and axial morphogenesis of multiple species. Transcription of the HOXC6 gene is initiated from two promoters and generates two proteins that share the same DNA-binding domain but harbor a distinct N-terminal region. Recent studies have demonstrated that both HOXC6 products can activate or repress transcription, depending on the cellular context. Functional in vivo specificity of HOXC6 proteins may be achieved through combinatorial interactions with other members of the HOX family as well as with co-factors whose identities are largely unknown. Disruption of this 'HOX code' may lead to pathology such as developmental defects.

Homeodomain containing transcription factors serve important functions in patterning the embryo during vertebrate development. We have isolated cDNA clones encoding a novel protein, named Alx-4, that contains a paired-type homeodomain. Analysis of the homeodomain sequence shows that Alx-4 belongs to a family of genes that are related to the Drosophila gene aristaless, and includes the mammalian genes Alx3, Cart-1, MHox, and S8. We have analyzed the expression of Alx-4 during development by Northern blot and whole mount in situ hybridization. In addition, we have generated antibodies to recombinant Alx-4 protein and identified Alx-4 protein in nuclear extracts prepared from mouse embryos. The expression pattern of Alx-4 suggests that it may play a role in the patterning of structures derived from craniofacial mesenchyme, the first branchial arch, and the limb bud. Our results provide a starting point for the analysis of a new member of the family of paired type homeodomain proteins.

Pbx1 is a homeodomain transcription factor involved in cAMP-dependent transcriptional regulation of the bovine CYP17 gene. In this study, we have investigated the involvement of Pbx1 in the transcriptional regulation of the human CYP17 gene. Although a sequence identical to previously determined Pbx-binding sites is not present in the promoter region of the human CYP17 gene, three putative Pbx-binding sites are identified by sequence similarity analysis. Coexpression of Pbx1 and a catalytic subunit of protein kinase A (PKA) greatly enhances reporter gene transcription via the 5′-flanking region of the human CYP17 gene. Upon gel shift analysis utilizing nuclear extracts from human adrenal H295R cells, one of the three putative Pbx1-binding sites, −250/−241 bp, shows the typical intense doublet observed with other Pbx-binding sites. 5′-Deletion analyses of the reporter construct containing this Pbx-binding site showed approximately sixfold induction by coexpression of Pbx1 and PKA compared to the basal transcription, suggesting that Pbx1 binds the −250/−241 bp sequence and participates in cAMP-dependent regulation of the human CYP17 gene.

Spx1, a novel mouse homeobox gene, encodes a homeodomain characteristic of the paired-like class of homeobox genes and has been mapped to the distal end of the X chromosome. Northern blot hybridization of adult tissues detected high levels of a single Spx1 transcript in the testis. Further analysis by in situ hybridization revealed predominant Spx1 expression within the spermatogonia/preleptotene spermatocytes and round spermatids of spermatogenic stages IV-VII. These expression data suggest SPX1 may play a role in the regulation of spermatogenesis.

Pbx1 is a DNA-binding homeodomain protein originally discovered in the t(1;19) chromosomal translocation associated with pediatric pre-B acute lymphoblastic leukemia. Previously we reported a cAMP-regulatory sequence (CRS1) in the promoter region of the bovine CYP17 gene encoding steroid 17alpha-hydroxylase cytochrome P450 (P450c17) to be the first endogenous Pbx1 binding site and that overexpression of Pbx1 in mouse adrenal Y1 tumor cells enhances cAMP-dependent transcription mediated by this element. Here we report further characterization of Pbx1 binding site in CRS1 and role of Pbx1 in cAMP-dependent, CRS1-mediated transcription. By gel shift analysis utilizing nuclear extracts from Y1 cells, a high-affinity Pbx-binding sequence has been determined to be TTGAT(T/G)GA(T/C)A which represents the 5' portion of CRS1. An artificial Pbx-binding sequence (PRS), previously determined by random PCR analysis, is similar to the Pbx1-binding sequence in CRS1 and by both gel shift analysis and transfection studies shows characteristics very similar to CRS1. Upon overexpression, Pbx1 is found capable of enhancing CRS1-mediated transcription in both steroidogenic (Y1, JEG3) and nonsteroidogenic (HepG2 and S194) cells when coexpressed with the catalytic subunit of cAMP-dependent protein kinase A. Thus even though Pbx1 has been found to be involved only in cAMP-dependent transcription of a gene involved in steroidogenesis (CYP17), Pbx1 is capable of participating in cAMP-dependent transcription of target genes without complex formation with steroidogenic tissue-specific nuclear factors.

We have determined the genomic structure of the humanLHX1 gene, a member of the LIM/homeobox (Lhx) gene family. The transcript is assembled from five exons, which are separated by introns ranging in size from 93 nt to 2.3 kb. The two LIM domains are entirely contained in the first and second exons, respectively, while the homeodomain is split into exons three and four. This structure closely parallels the organization of other mouse and human Lhx genes whose genomic structure is known. An exception is the mouse and humanisl1 genes, whose homeodomain does not contain introns. An intron at the same position also occurs in theXlim1 gene as well as in other homeobox genes, such asevx1 andevx2, suggesting that this intron insertion represents an ancestral event, from which homeobox genes of different families originated. In this context, evolution of theLhxgene family probably involved the shuffling of this intron-containing homeobox in the proximity of a LIM-only gene, whileIsletgenes were formed either by the shuffling of an intronless homeobox to the same LIM domain or, alternatively, by intron loss during their evolution.

The cut locus of Drosophila encodes a diverged homeodomain-containing protein that is required for the development of external sensory (es) organs and other tissues. A homologous gene (Cux-1) that encodes a transcriptional repressor has been identified in the mouse and other mammals. We have identified a second murine homeobox-containing gene (designated Cux-2) that is structurally related to Drosophila cut. The murine Cux-2 homeobox was similar to Drosophila cut and encoded a homeodomain that contained a characteristic histidine residue at position 50. The predicted Cux-2 protein contained 1426 amino acids and included three internal 60-amino acid repeats (Cut repeats) that were previously found in Drosophila Cut and murine Cux-1. Unlike Cux-1, expression of Cux-2 was restricted to neural tissue. In the adult brain, Cux-2 was prominently expressed in neurons in the thalamus and limbic system. In embryos, Cux-2 was expressed in the developing central and peripheral nervous systems, including the telencephalon and peripheral ganglia of the trigeminal and glossopharyngeal nerves. A glutathione S-transferase fusion protein containing the carboxyl-terminal Cut repeat and homeodomain of Cux-2 exhibited sequence-specific binding to oligonucleotides derived from the promoter of the Ncam gene. Using an interspecific backcross panel, Cux-1 and Cux-2 were mapped to distinct loci that were genetically linked on distal chromosome 5. These results demonstrate that a family of homeobox genes related to Drosophila cut is located on chromosome 5 in the mouse. Cux-2 is expressed exclusively in the central and peripheral nervous systems, and the Cux-2 gene product binds to DNA in a sequence-specific manner. Cux-2 may encode a transcription factor that is involved in neural specification in mammals.

Homeodomain-containing proteins play a crucial role as transcriptional regulators in the process of cell differentiation. The homeodomain performs a dual function in this regard, acting as a DNA binding domain and participating in protein-protein interactions that enhance DNA binding specificity or regulatory activity. Here we describe a homeodomain class-specific interaction with the beta subunit of the general transcription factor TFIIE. We show that the Antennapedia and Abdominal-B homeodomains bind to TFIIEbeta, but the even-skipped homeodomain does not. Using a two-hybrid assay performed in cultured cells, we demonstrate that the homeodomain-TFIIEbeta interaction occurs in vivo. The Abdominal-B homeodomain is shown to activate transcription in vitro, and this activation can be blocked with anti-TFIIEbeta antibody without affecting basal transcription levels. Together with published data demonstrating an interaction between proteins containing even-skipped class homeodomains and the TATA-binding protein (Um, M., Li, C., and Manley, J. L. (1995) Mol. Cell. Biol. 15, 5007-5016; Zhang, H., Catron, K. M., and Abate-Shen, C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1764-1769), these results suggest various homeodomain containing proteins interact with different general transcription factors, an observation that may have important implications for transcriptional regulation.

A hallmark of homeobox genes is their high degree of sequence conservation in distantly related species. Here, we report the chromosomal localization, sequence, and expression pattern of an orphan homeobox gene, Pem, that encodes a homeodomain (HD) that has undergone a surprisingly high rate of evolutionary change. The N-terminal portion of the Pem HD, which includes the first two alpha-helices, exhibits only 44% sequence identity between rat Pem (r.Pem) and mouse Pem (m.Pem). This N-terminal subdomain exhibited an extremely high frequency of nonsynonymous substitutions, severalfold higher than other regions of the Pem protein. In contrast, the third helix, which is known to confer most of the base-specific contacts of HDs with DNA, was almost identical in r. Pem and m.Pem. Several lines of evidence suggested that the rat and mouse genes that we identified as Pem genes are true homologues: (1) the r.Pem and m.Pem genes both reside on the X chromosome; (2) they possess identical exon/intron splice junctions; (3) they both encode a distinctive motif upstream of the HD that is unique to Pem; and (4) the only m.Pem-like gene we were able to identify in the rat genome other than r.Pem was a pseudogene, r.Pem-ps, whose sequence and chromosomal localization indicated that it was derived by reverse transcription and reinsertion into the genome. The functional r.Pem gene is selectively expressed in placenta, testis, epididymis, and ovary. This expression pattern is of interest since other genes transcribed in reproductive tissue have also been shown to undergo high rates of sequence divergence. The high rate of amino acid substitutions in the N-terminal region of the Pem HD suggests the possibility of species-specific directional selection.

Msx2 is a mammalian homeodomain protein that is expressed during craniofacial development. A proline-to-histidine substitution at residue 148 of human Msx2 results in an autosomal dominant form of craniosynostosis. In this study, both wild-type and mutant Msx2 were shown to specifically bind to a DNA sequence previously identified as a high-affinity binding site for the related homeodomain protein Msx1. In co-transfection assays both wild-type and mutant Msx2 repressed reporter gene transcription in a dose-dependent but binding-site-independent manner. These results provide evidence that Msx2 is a transcriptional repressor and suggest that the mutant form of Msx2 may exert its pathophysiologic effects on craniofacial development by a gain-of-function mechanism.

We have isolated and characterized cDNA clones encoding a novel human homeobox gene, MOX2, the homologue of the murine mox-2 gene. The MOX2 protein contains all of the characteristic features of Mox-2 proteins of other vertebrate species, namely the homeobox, the polyhistidine stretch, and a number of potential serine/threonine phosphorylation sites. The homeodomain of MOX2 protein is identical to all other vertebrate species reported so far (rodents and amphibians). Outside the homeodomain, Mox-2 proteins share a high degree of identity, except for a few amino acid differences encountered between the human and the rodent polypeptides. A polyhistidine stretch of 12 amino acids in the N terminal region of the protein is also conserved among humans, rodents, and (only partly) amphibians. The chromosomal position of MOX2 was assigned to 7p22.1-p21.3. 31 refs., 3 figs.

Genes encoding transcription factors are fundamental to developmental processes because their DNA-binding proteins can control expression of a multitude of genes. Therefore, activation of a single transcription factor gene can act as a genetic switch that controls the developmental destinies of groups of cells. Results of expression studies and mutational analyses in mice and Drosophila suggest an essential role in heart development for the homeobox class of transcription factor genes. Understanding the genetic circuitry of cardiogenesis will facilitate the identification of individuals with inherited cardiac diseases, and the possible prevention of these diseases with drug therapy or lifestyle modification. It may also allow for genetic counseling of affected individuals and family members and enable gene therapy aimed at correcting these defects.

Extensive analyses of homeobox gene expression and function during murine embryogenesis have demonstrated that homeobox gene products are key components in the establishment of pattern formation and regional identity during development. In this paper we report the molecular characterization and expression of a novel murine homeobox sequence, Hesx1, isolated from pluripotent embryonic stem cells. Hesx1 is expressed as two transcripts of 1.0 and 1.2 kilobases which encode an identical 185 amino acid open reading frame. The transcripts differ in the 3'-untranslated region due to the differential utilization of a weak splice donor site located immediately downstream of the translation termination codon. The Hesx1 homeodomain shared 80% identity with the Xenopus homeoprotein XANF-1 and was less than 50% related to other homeodomain sequences. Hesx1 and XANF-1 therefore constitute the founder members of a new homeodomain class. Hesx1 expression was down-regulated during embryonic stem cell differentiation and was detected in tissue-specific RNA samples derived from the embryonic liver, and at lower levels in viscera, amnion, and yolk sac. Expression in adult mice was not detected. These sites of expression are consistent with a role for Hesx1 in the regulation of developmental decisions in the early mouse embryo and during fetal hematopoiesis.

Homeobox genes from the urochordates Styela clava (AHox2) and S. plicata (AHox3) were cloned and analyzed. The two genes are homologous and Antennapedia-like. The homeobox regions have 87% identity at the nucleotide level and are identical at the amino-acid level. No introns are present in the homeobox region of AHoxB, and AHox3 is represented at a low copy number per haploid genome. AHox2 and AHoxB represent the second type of homeobox gene found in this evolutionarily and developmentally important group of organisms.

Homeobox genes are a major group of genes involved in regulating embryogenesis. Here we describe the identification of GH6, a novel chicken homeobox-containing gene and its spatial and temporal expression pattern in the developing chick embryo. Identity comparisons of the GH6 homeodomain suggest that it is closely related to the human homeobox gene H6 , with 93% amino acid conservation. Temporally, GH6 expression is highest between embryonic stages 23 and 26; however, some expression is also detectable as early as stage 13. In situ hybridization of stage 23 embryos indicates that GH6 expression occurs at high levels in discrete craniofacial regions including the second branchial arch, the neural retina, the lens epithelium, the optic nerve, and the infundibulum. GH6 expression was also seen in the developing ventricular myocardium, representing the first report of homeobox gene expression in the developing ventricle. GH6 is also expressed in sensory spinal and cranial ganglia, suggesting that GH6 plays several roles not only in the development of craniofacial structures such as the eye and ear, but also in formation of functionally defined ganglia and myocardial structures.

Two of the most common DNA-binding motifs found in eukaryotic transcriptional regulatory proteins are the homeodomain and the C2-H2 zinc finger. In Drosophila, homeodomain and zinc-finger proteins have been implicated in a wide variety of developmental processes. Until now, no proteins have been described in which both these DNA-binding motifs are present. We report here the isolation of genes encoding two such Drosophila proteins from a cDNA expression library. The product of the zfh-1 gene (zinc-finger homeodomain protein 1) contains one homeodomain and nine C2-H2 zinc fingers. The product of the zfh-2 gene possesses three homeodomains and sixteen C2-H2 zinc fingers. For zfh-1, antisera raised against nonoverlapping regions of the gene product all recognize a 145 kDa protein on protein immunoblots, suggesting that the different DNA-binding motifs are actually all present in the mature gene product. The novel arrangement of interspersed homeodomain and zinc-finger motifs in the primary sequences of the zfh-1 and zfh-2 gene products may signify an unusual mechanism of transcriptional regulation by these proteins.

The mammalian homeobox genes are a family of genes that are expressed at high levels in the nervous system and encode sequence-specific DNA-binding proteins and transcriptional factors. To understand the expression and function of the homeobox genes in the nervous system, we have studied the expression of the rat homeobox genes in the RT4 cell lines, which mimic specific properties of the neuronal, glial, and their bipotential progenitor cell types. Northern blot analyses indicate that the rat homeobox R5 and Rlb genes are expressed in all cell types in the RT4 cell family. The sizes and levels of RNA transcripts are comparable to those observed in vivo in rat spinal cord and rat embryos. In contrast, another rat homeobox gene Rla, which is expressed in vivo and is closely linked to the R1b gene, does not express detectable RNA species in the RT4 cell family. Thus, the rat homeobox genes are differentially regulated in the RT4 cell family. In protein blotting analyses, anti-R5 peptide antisera react with a 60-kDa protein in cell extracts from all the RT4 cell lines. Immunofluorescence experiments demonstrate that the homeobox R5 gene product is present in all cell lines and that the protein is predominantly localized in the nucleus. This suggests that the rat homeodomain proteins may function as transcriptional regulators in the RT4 cells.

The homeotic selector genes of Drosophila melanogaster encode transcriptional regulatory proteins that control the determination of different segmental fates. Binding of selector proteins to regulatory DNA sequences is mediated by an evolutionary conserved protein domain, the homeodomain. Although homeodomains encoded by the selector genes are very similar in their amino acid sequence and in vitro DNA-binding properties, here we provide additional evidence that the homeodomain is responsinle for most of the regulatory specificity of the entire protein. A heat-shock promoter/selectro gene was constructed that encodes a Deformed / Abdominal-B chimera in which the Abdominal-B homeodomain is substituted for that of Deformed. Expression of this chimeric protein throughout the embryo causes morphological transformations of anterior segments toward more posterior identities. A number of other homeotic selector genes, all normally repressed by Abdominal-B, are ectopically activated by the chimeric protein. These results support the hypothesis that the target specificity of similar homeodomain proteins is largely determined by the amino acid sequence of the homeodomain.

By nuclease protection and mobility shift assays, we have shown that the 5' flanking sequence of the Bombyx mori fibroin gene, which is known to be required for a maximal level of transcription in vitro, contains five regions (A to E) that bind protein factors from the posterior silk gland extract. A silk gland-specific factor (SGF-1) and a ubiquitous factor (FBF-A1) were found to interact with the proximal A region, while the related B region can only bind SGF-1. The three distal regions (C, D and E) bind one posterior silk gland-specific factor (SGF-2) and two ubiquitous factors (SGF-3 and -4). SGF-1 might play an important role in the expression of silk protein genes because it also binds to a similar site in the sericin-1 gene and potential SGF-1 binding sites can be found in two other silk protein genes of Bombyx mori and the fibroin gene of Antheraea yamamai. The three distal regions for SGF-2, -3 and -4 contain ten base-pairs of A + T-rich repeats that resemble the consensus binding site (TCAATTAAAT) of a number of homeodomain proteins. The TAAT motif in the core of these regions is shown to be important for the binding of these three proteins and, as described elsewhere, two Drosophila homeodomain proteins, ZEN (zerknüllt) and EVE (even-skipped). Interestingly, SGF-3 appears to be a Bombyx octamer binding protein and may also be involved in the regulation of the sericin-1 gene. The possibility that the fibroin gene and other silk protein genes may be a group of target genes for some members of the homeobox gene family is discussed.

Multiple A + T-rich stretches in the 5' flanking region of the Bombyx mori fibroin light-chain gene have been shown to bind two Drosophila homeodomain proteins, EVE (even-skipped) and ZEN (zerknüllt), with high affinities. Some of these sites fall into a class that has the established consensus sequence of the binding sites (TCAATTAAAT) for a diverse group of Drosophila homeodomain proteins, while others are quite heterogenous except that they all possess a core TAAT motif. Since clusters of homeodomain binding sites can also be found in the promoters of other silk protein genes, the fibroin gene and the sericin-1 gene, these observations suggest a possible involvement of some homeobox genes in the regulation of a group of silk protein genes.

Oct-1 is a sequence-specific transcription and DNA replication factor that recognizes the octameric sequence ATGCAAAT. This protein shares an extended region of sequence similarity, called the POU domain, with the lymphoid-specific transcription factor Oct-2, the pituitary-specific transcription factor Pit-1, and the Caenorhabditis elegans cell lineage gene product unc-86. Two subdomains, POU-related homeobox and POU-specific box, lie within the POU domain. Unlike other POU or homeodomain proteins, Oct-1 is ubiquitously expressed. A ubiquitous 95- to 100-kDa protein that has the same DNA-binding properties and is variously referred to as OTF-1, NFIII, or OBP100 is probably identical to Oct-1. The human gene was mapped by Southern blot analyses of human x rodent hybrid cell lines to chromosome 1, region cen-q32. In Chinese hamster x mouse hybrid cell lines the mouse locus was also mapped to chromosome 1. With the BXD (from progenitor strains C57BL/6J and DBA/2J) set of recombinant inbred strains of mice, the murine gene was localized on the linkage map of mouse chromosome 1. There were no recombinants among 26 strains with Ly-22 and 1 recombinant among 24 strains with Ltw-4. The gene symbol is OTF1 for humans and Oct-1 for mouse.

The interactions of one homeodomain protein with another may be a general way in which their specificity is enhanced.