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The transcription factor PDX-1 plays a crucial role during pancreatic development and in the function of insulin-producing beta cells. Disruption of the pdx-1 gene in these cells induces overt diabetes in mice, and this gene is modified in several type 2 diabetic families. It is thus crucial to determine the molecular mechanisms involved in the regulation of PDX-1 expression and/or activation. We identified new proteins associated with PDX-1 by mass spectrometry. These proteins, Ku70 and Ku80, are regulatory subunits of DNA-dependent protein kinase (DNA-PK). We determined that the interaction between PDX-1 and Ku70 or Ku80 is dependent on the homeodomain of PDX-1. Most interestingly, we demonstrated in vitro that the DNA-PK phosphorylates PDX-1 on threonine 11. Although this residue is located in the transactivation domain, this phosphorylation does not seem to be implicated in the transcriptional activation of PDX-1. However, in response to radiation, which activates DNA-PK, a second form of the PDX-1 protein appears rapidly. This form is phosphorylated on threonine and seems to drive PDX-1 degradation by the proteosome. In correlation with this degradation, we observed a subsequent reduction in the activation of the insulin promoter and a decrease in PDX-1-mediated gene expression, i.e. glut2 and glucokinase. Our study demonstrates that radiation, through the activation of DNA-PK, may regulate PDX-1 protein expression.

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 Xvent family of homeobox transcription factors is essential for the establishment of the dorsal-ventral body axis during Xenopus embryogenesis. In contrast to Xvent-2B and other members of the Xvent-2 subfamily, Xvent-1B is not a direct response gene of bone morphogenetic protein-4 signaling. Xvent-1B is activated by Xvent-2, but CHX experiments revealed the requirement of additional factors. In this study, we report on the cooperative effect of Xvent-2 and the zinc finger transcription factor GATA-2 on the promoter of the Xvent-1B gene. We show that GATA-2 is a direct target gene of bone morphogenetic protein-4 and that GATA-2 interacts with Xvent-2 to activate transcription of Xvent-1B. Both transcription factors bind to distinct elements within the Xvent-1B promoter, and GATA-2 physically interacts with the C-terminal domain of Xvent-2. Promoter/reporter studies in Xenopus embryos revealed that full activation of Xvent-1B requires both Xvent-2 and GATA-2. Moreover, the two factors are sufficient to direct transcription of Xvent-1B in the presence of CHX at the ventral side of the embryo. The failure of both factors to activate Xvent-1B on the dorsal side suggests the existence of a dorsal inhibitor. This inhibitor is likely a component of the dorsal Wnt signaling pathway because nuclear translocation of beta-catenin before midblastula transition results in a suppression of Xvent-1B 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.

Homeodomain proteins specify developmental pathways and cell-specific gene transcription whereby proteins of the PBC subclass can direct target gene specificity of Hox proteins. Proteins encoded by nonclustered homeobox genes have been shown to be essential for cell lineage differentiation and gene expression in pancreatic islets. Using specific antiserum in an electrophoretic mobility shift assay and in vitro transcribed/translated proteins, the nuclear proteins binding domain B of the G3 enhancer-like element of the glucagon gene were identified in the present study as heterodimers consisting of the ubiquitously expressed homeodomain protein Prep1 and the also widely expressed PBC homeoprotein Pbx (isoform 1a, 1b, or 2). These heterodimeric complexes were found to bind also to the glucagon cAMP response element and to a newly identified element termed G5 (from -169 to -140). Whereas the expression of Prep1 or Pbx forms alone had no effect, coexpression of Pbx1a/1b-Prep1 inhibited the glucagon promoter when activated by cotransfected Pax6 or another transcription factor in non-glucagon-producing cells. In contrast, in glucagon-producing pancreatic islet cells, Pbx-Prep1 had no effect on GAL4-Pax6-induced mutant glucagon promoter activity or on Pax6-dependent wild-type glucagon promoter activity. Furthermore, 5'-deletion of G5 enhanced glucagon promoter activity in a non-glucagon-producing cell line but not in glucagon-producing islet cells. This study thus identifies a novel target and Hox-independent function of Pbx-Prep1 heterodimers that, through repression of glucagon gene transcription in non-glucagon-producing cells, may help to establish islet cell-specific expression of the glucagon gene.

Transcription factors of the ONECUT class, whose prototype is HNF-6, contain a single cut domain and a divergent homeodomain characterized by a phenylalanine at position 48 and a methionine at position 50. The cut domain is required for DNA binding. The homeodomain is required either for DNA binding or for transcriptional stimulation, depending on the target gene. Transcriptional stimulation by the homeodomain involves the F48M50 dyad. We investigate here how HNF-6 stimulates transcription. We identify transcriptionally active domains of HNF-6 that are conserved among members of the ONECUT class and show that the cut domain of HNF-6 participates to DNA binding and, via a LXXLL motif, to transcriptional stimulation. We also demonstrate that, on a target gene to which HNF-6 binds without requirement for the homeodomain, transcriptional stimulation involves an interaction of HNF-6 with the coactivator CREB-binding protein (CBP). This interaction depends both on the LXXLL motif of the cut domain and on the F48M50 dyad of the homeodomain. On a target gene for which the homeodomain is required for DNA binding, but not for transcriptional stimulation, HNF-6 interacts with the coactivator p300/CBP-associated factor but not with CBP. These data show that a transcription factor can act via different, sequence-specific, mechanisms that combine distinct modes of DNA binding with the use of different coactivators.

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.

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.

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.

Homeodomain proteins play important roles in various developmental processes, and their functions are modulated by polypeptide cofactors. Here we report that both in vitro and in vivo, 14-3-3eta is associated with the TLX-2 homeodomain transcription factor that is required for mouse embryogenesis. Expression of 14-3-3eta shifts the predominant localization of TLX-2 in COS cells from the cytoplasm to the nucleus. Tlx-2 and 14-3-3eta are expressed in the developing peripheral nervous system with spatially and temporally overlapping patterns, and they are also coexpressed in PC12 cells. Increased expression of either gene by transfection considerably inhibited nerve growth factor-induced neurite outgrowth of PC12 cells, and cotransfection of both genes led to a synergistic effect of suppression. These findings define 14-3-3eta as a functional modulator of the TLX-2 homeodomain transcription factor and suggest that the in vivo function of TLX-2 in neural differentiation is likely regulated by signaling mediated by 14-3-3eta.

Rieger syndrome is an autosomal-dominant developmental disorder that includes glaucoma and mild craniofacial dysmorphism in humans. Mutations in the Pitx2 homeobox gene have been linked to Rieger syndrome. We have characterized wild type and mutant Pitx2 activities using electrophoretic mobility shift assays, protein binding, and transient transfection assays. Pitx2 preferentially binds the bicoid homeodomain binding site and transactivates reporter genes containing this site. The combination of Pitx2 and another homeodomain protein, Pit-1, yielded a synergistic 55-fold activation of the prolactin promoter in transfection assays. Addition of Pit-1 increased Pitx2 binding to the bicoidelement in electrophoretic mobility shift assays. Furthermore, we demonstrate specific binding of Pit-1 to Pitx2 in vitro. Thus, wild type Pitx2 DNA binding activity is modulated by protein-protein interactions. We next studied two Rieger mutants. A threonine to proline mutation (T68P) in the second helix of the homeodomain retained DNA binding activity with the same apparentKD and only about a 2-fold reduction in theBmax. However, this mutant did not transactivate reporter genes containing the bicoid site. The mutant Pitx2 protein binds Pit-1, but there was no detectable synergism on the prolactin promoter. A second mutation (L54Q) in a highly conserved residue in helix 1 of the homeodomain yielded an unstable protein. Our results provide insights into the potential mechanisms underlying the developmental defects in Rieger syndrome.

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.

The commitment of cells to specific lineages during development is determined in large part by the relative expression of various homeodomain (HOX) selector proteins, which mediate the activation of distinct genetic programs. But the mechanisms by which individual HOX genes are themselves targeted for expression in different cell types remain largely uncharacterized. Here, we demonstrate that STF-1, a homeodomain protein that functions in pancreatic morphogenesis and in glucose homeostasis is encoded by an “orphan” homeobox gene on mouse chromosome 5. When fused to a β-galactosidase reporter gene, a 6.5-kilobase genomic fragment of 5′-flanking sequence from the STF-1 gene shows pancreatic islet specific activity in transgenic mice. Two distinct elements within the STF-1 promoter are required for islet-restricted expression: a distal enhancer sequence located between −3 and −6.5 kilobases and a proximal E-box sequence located at −104, which is recognized primarily by the helix loop helix/leucine zipper nuclear factor USF. As point mutations within the −104 E-box that disrupt USF binding correspondingly impair STF-1 promoter activity, our results demonstrate that USF is an important component of the regulatory apparatus which directs STF-1 expression to pancreatic islet cells.

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.