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Results for “POU Domain Factors”

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PubMed1998

Non-cell autonomous induction of apoptosis and loss of posterior structures by activation domain-specific interactions of Oct-1 in the Xenopus embryo.

Oct-1, a member of the POU family of transcription factors, is expressed at relatively high levels in ectodermal and mesodermal cell lineages during early Xenopus embryogenesis (Veenstra et al, 1995). Here we show that overexpression of Oct-1 induces programmed cell death concomitant with the loss of the posterior part of the body axis. Truncated Oct-1 variants, missing either the C-terminal or N-terminal trans-activation domain, exhibit a different capacity to cause such developmental defects. Oct-1-induced cell death is rescued in unilaterally injected embryos by non-injected cells, indicative of the non-cell autonomous character of the developmental effects of Oct-1. This was confirmed by marker gene analysis, which showed a significant decrease in brachyury expression, suggesting that Oct-1 interferes with an FGF-type signalling pathway.

Animals↗

PubMed1990

A two-base change in a POU factor-binding site switches pituitary-specific to lymphoid-specific gene expression.

The structurally related POU homeo domain proteins Pit-1 and Oct-2 activate pituitary- and lymphoid-specific transcription, respectively, by binding to similar AT-rich motifs in their target genes. In this study we identify bases critical for recognition and activation by Pit-1 and examine how small differences in Pit-1 and Oct-2-binding sites can impart differential transcriptional responses in pituitary and B-lymphoid cells. Scanning mutagenesis of Pit-1 response elements in both the rat prolactin and growth hormone genes reveals a critical binding motif recognized in an identical manner by the native Pit-1 protein and cloned Pit-1 gene product. This motif, ATTATTCCAT, differs by only two bases from the octamer element, ATTTGCAT, required for Oct-2-dependent activation of immunoglobulin genes. Cross recognition of Pit-1 and Oct-2 sites by both factors can be demonstrated in competitive binding assays, in which an oligometric Pit-1 site from the prolactin gene is converted to an Oct-2 site by a double point mutation. In contrast to the binding data, no cross activation of transcription is detectable in cultured cell lines. When inserted immediately 5' to a prolactin TATA box, the wild-type prolactin element enhances transcription strongly in pituitary cells but is inactive in B cells, whereas the octamer variant of the prolactin site activates expression in B cells but is silent in pituitary lines. Both elements are nonfunctional in heterologous cell lines that lack Pit-1 and Oct-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Solution structure of a POU-specific homeodomain: 3D-NMR studies of human B-cell transcription factor Oct-2.

The POU DNA-binding motif defines a conserved family of eukaryotic transcription factors involved in regulation of gene expression. This bipartite motif consists of an N-terminal POU-specific domain (POUs), a flexible linker, and a C-terminal POU-specific homeodomain (POUHD). Here we describe the solution structure of a POU-specific homeodomain. An NMR model is obtained from Oct-2, a human B-cell specific transcription factor which participates in the regulation of immunoglobulin genes. A fragment of Oct-2 containing POUHD and an adjoining linker was expressed in Escherichia coli and characterized by three-dimensional nuclear magnetic resonance (3D-NMR) spectroscopy. Complete 1H and 15N resonance assignment of the POUHD moiety is presented. The POUHD solution structure, as calculated by distance geometry and simulated annealing (DG/SA), is similar to that of canonical homeodomains. A salient difference between solution and crystal structures is observed in the C-terminal segment of alpha-helix 3 (the HTH recognition helix), which is not well ordered in solution. Because this segment presumably folds upon specific DNA binding, its flexibility in solution may reduce the intrinsic DNA affinity of POUHD in the absence of POUs.

Amino Acid Sequence↗

PubMed1988

The POU domain is a bipartite DNA-binding structure.

The POU domain (pronounced 'pow') is a highly charged 155-162-amino-acid (aa) region of sequence similarity contained within three mammalian transcription factors. Pt-1 (ref. 2), Oct-1 (ref. 3) and Oct-2 (ref. 4), and the product of the nematode gene unc-86 (ref. 5) which is involved in determining neural cell lineage. This domain consists of two subdomains, a C-terminal homoeo domain and an N-terminal POU-specific region separated by a short nonconserved linker; the sequence relationship shows that the POU homoeo domains form a distinct POU-related family. In the ubiquitous and lymphoid-specific octamer-motif binding proteins Oct-1 and Oct-2, the POU domain is sufficient for sequence-specific DNA binding. Homoeobox domains contain a helix-turn-helix DNA-binding motif, first identified in bacterial repressors. The helix-turn-helix region of the POU domain is important for DNA binding and, in other classes of homoeo-containing proteins, the entire homoeo domain is sufficient for DNA binding; thus the new POU-specific region could be involved in other functions such as protein-protein interactions. Nevertheless, we show here that in fact the POU domain is a novel bipartite DNA-binding structure in which the POU homoeo and POU-specific regions form two subdomains that are both required for DNA binding but are held together by a flexible linker.

Base Sequence↗

PubMed2000

The Pit-1 homeodomain and beta-domain interact with Ets-1 and modulate synergistic activation of the rat prolactin promoter.

Pit-1/GHF-1 is a pituitary-specific, POU homeodomain transcription factor required for development of somatotroph, lactotroph, and thyrotroph cell lineages and regulation of the temporal and spatial expression of the growth hormone, prolactin (PRL), and thyrotropin-beta genes. Synergistic interaction of Pit-1 with a member of the Ets family of transcription factors, Ets-1, has been shown to be an important mechanism regulating basal and Ras-induced lactotroph-specific rat (r) PRL promoter activity. Pit-1beta/GHF-2, an alternatively spliced isoform containing a 26-amino acid insert (beta-domain) within its transcription-activation domain, physically interacts with Ets-1 but fails to synergize. By using a series of Pit-1 internal-deletion constructs in a transient transfection protocol to reconstitute rPRL promoter activity in HeLa cells, we have determined that the functional and physical interaction of Pit-1 and Ets-1 is mediated via the POU homeodomain, which is common to both Pit-1 and Pit-1beta. Although the Pit-1 homeodomain is both necessary and sufficient for direct binding to Ets-1 in a DNA-independent manner, an additional interaction surface was mapped to the beta-domain, specific to the Pit-1beta isoform. Thus, the unique transcriptional properties of Pit-1 and Pit-1beta on the rPRL promoter may be due to the formation of functionally distinct complexes of these two Pit-1 isoforms with Ets-1.

Animals↗

PubMed2000

Phosphorylation of Oct-2 at sites located in the POU domain induces differential down-regulation of Oct-2 DNA-binding ability.

We compared the effects of phosphorylation of Oct-2 protein on its binding to the consensus octamer sequence (ATGCAAAT) and two non-canonical sequences present in human (AAGCAAAT) and murine (AAACAAAT) promoters of the BLR1 (Burkitts' lymphoma receptor 1) gene encoding chemokine receptor CXCR5 (CXC-chemokine receptor 5). The latter cis-acting elements represent low-affinity recognition sequences for the octamer transcription factors. Okadaic acid was found to induce hyperphosphorylation of Oct-2 specifically in cells of lymphoid lineage. Potentially phosphorylated amino acid residues localized to the POU-specific domain of Oct-2. Whereas binding of Oct-2 to the octamer site from the human BLR1 promoter or to the consensus octamer sequence was unaffected by phosphorylation of this factor, a strong reduction of Oct-2 binding to the octamer site from the murine BLR1 promoter was observed. This finding correlates well with the down-regulation of expression of the BLR1 gene in murine splenic cells but not in lymphoid cells of human origin treated with okadaic acid. These data support the hypothesis that phosphorylation of Oct-2 may be a mechanism by which activities of the promoters containing non-canonical octamer sequences are differentially regulated in response to extracellular stimuli.

Amino Acid Sequence↗

PubMed2000

Modulation of the activity of multiple transcriptional activation domains by the DNA binding domains mediates the synergistic action of Sox2 and Oct-3 on the fibroblast growth factor-4 enhancer.

Fibroblast growth factor (FGF)-4 gene expression in the inner cell mass of the blastocyst and in EC cells requires the combined activity of two transcriptional regulators, Sox2 and Oct-3, which bind to adjacent sites on the FGF-4 enhancer DNA and synergistically activate transcription. Sox2 and Oct-3 bind cooperatively to the enhancer DNA through their DNA-binding, high mobility group and POU domains, respectively. These two domains, however, are not sufficient to activate transcription. We have analyzed a number of Sox2 and Oct-3 deletion mutants to identify the domains within each protein that contribute to the activity of the Sox2 x Oct-3 complex. Within Oct-3, we have identified two activation domains, the N-terminal AD1 and the C-terminal AD2, that play a role in the activity of the Sox2 x Oct-3 complex. AD1 also displays transcriptional activation functions in the absence of Sox2 while AD2 function was only detected within the Sox2 x Oct-3 complex. In Sox2, we have identified three activation domains within its C terminus: R1, R2, and R3. R1 and R2 can potentiate weak activation by Sox2 in the absence of Oct-3 but their deletion has no effect on the Sox2 x Oct-3 complex. In contrast, R3 function is only observed when Sox2 is complexed with Oct-3. In addition, analysis of Oct-1/Oct-3 chimeras indicates that the Oct-3 homeodomain also plays a critical role in the formation of a functional Sox2 x Oct-3 complex. Our results are consistent with a model in which the synergistic action of Sox2 and Oct-3 results from two major processes. Cooperative binding of the factors to the enhancer DNA, mediated by their binding domains, stably tethers each factor to DNA and increases the activity of intrinsic activation domains within each protein. Protein-protein and protein-DNA interactions then may lead to reciprocal conformational changes that expose latent activation domains within each protein. These findings define a mechanism that may also be utilized by other Sox x POU protein complexes in gene activation.

Base Sequence↗

PubMed1999

Heritable disorders of pituitary development.

Basic and translational research achievements over the past 2 decades have disclosed the molecular mechanisms underlying several genetic forms of hypopituitarism. Disorders that are limited to the hypothalamic, pituitary, GH axis are caused by mutations in individual components of that axis. Disorders involving GH and one or more additional pituitary hormones are caused by mutations in the homeodomain transcription factors that direct embryological development of the anterior pituitary gland. Pit-1 has a POU-specific and a POU-homeo DNA-binding domain. The phenotype produced by mutations in the PIT1 gene involves deficiencies of GH, PRL, and TSH. Pituitary glands are either small or normally sized. The PROP1 gene encodes a transcription factor with a single paired-like DNA-binding domain. Persons with inactivating mutations in PROP1 have deficiencies of LH and FSH, as well as GH, PRL, and TSH. Their pituitary glands may be small, normally sized, or extremely large and show suprasellar extension. Pituitary degeneration may produce acquired deficiency of ACTH. Expression of the HESX1 gene precedes expression of PROP1 and PIT1, and it is much more widespread. The protein has a paired-like domain, and it competes with the product of PROP1 for DNA-binding. Homozygosity for inactivating mutations of HESX1 produces a complex phenotype that resembles septo-optic dysplasia. Much more needs to be learned about the role of HESX1 mutations in other forms of hypopituitarism.

Basic Helix-Loop-Helix Proteins↗

PubMed1996

Two variants of the pituitary specific transcription factor Pit-1 in Atlantic salmon.

We have cloned two 1.6 kb cDNAs encoding variants of the POU-type pituitary-specific transcription factor Pit-1 from Atlantic salmon. Sequence comparison with mammalian Pit-1 revealed that the POU domain was highly similar while flanking regions were less conserved. The N-terminal region contained three insertions relative to mammalian Pit-1, one of these corresponded to the insertion found in the alternatively spliced Pit-1a isoform. While two different salmon Pit-1 transcripts were expressed, alternative splicing in the 5'-region did not appear to contribute to further transcript diversity. Both salmon Pit-1 cDNAs encoded 39.5 kDa proteins that specifically bind a consensus Pit-1 recognition sequence in vitro. The salmon Pit-1 proteins also recognized the classical octamer motif; however, a point mutation in the POU homeodomain abolished this interaction.

Alternative Splicing↗

PubMed2004

Herpes simplex virus infections are arrested in Oct-1-deficient cells.

Expression of the herpes simplex virus (HSV) immediate early (IE) genes is regulated by a multiprotein complex that is assembled on the TAATGARAT enhancer core element. The complex contains the cellular POU domain protein Oct-1, the viral transactivator VP16, and the cellular cofactor host cell factor 1. The current model suggests that the assembly depends on recognition of the core element by Oct-1. Here, HSV infection of Oct-1-deficient mouse embryonic fibroblast cells demonstrates that Oct-1 is critical for IE gene expression at low multiplicities of infection (moi). However, the protein is not essential for IE gene expression at high moi, indicating that VP16-mediated transcriptional induction through other IE regulatory elements is also important. This induction depends, at least in part, on the GA-binding protein binding elements that are present in each IE enhancer domain. Surprisingly, whereas the viral IE genes are expressed after high moi infection of Oct-1-deficient cells, the assembly of viral replication factories is severely impaired, revealing a second critical role for Oct-1 in HSV replication. The results have implications for both the HSV lytic and latency-reactivation cycles.

3T3 Cells↗

PubMed2001

Optimal Oct-2 affinity for an extended DNA site and the effect of GST fusion on site preference.

The regulator of immunoglobulin expression Oct-2 and the related widely expressed transcription factor Oct-1 have been shown to interact with DNA sequences containing an "octamer" motif, ATGC(A/T)AAT. To better understand Oct-2 function we have used random oligonucleotide selection and competition assays to define the optimal recognition site for this protein. The selected site contains an extended sequence that is remarkably similar to octamer-heptamer sequences found in immunoglobulin heavy-chain regulatory sequences, and the affinity of Oct-2 for this site is at least 50-fold greater than for sites containing the octamer motif alone. Fusion to glutathione S-transferase, a widely used model for protein-DNA and protein-protein interaction, does not alter the optimal Oct-2 recognition site, but inhibits Oct-2 POU-domain dimerization, slows the dissociation rate of the GST-Oct-2/DNA complex, and increases the relative importance of the heptamer domain for Oct-2 binding. These data advance our ability to identify in vivo targets of POU-factor regulation and also suggest that GST-fusion proteins should be used with caution in DNA-binding studies.

Amino Acid Sequence↗

PubMed2002

Analysis of associations of PIT1 genotypes with growth, meat quality and carcass composition traits in pigs.

PIT1 was chosen as a candidate gene to investigate its associations with growth, meat quality and carcass composition traits in the pig. PIT1 is known as the pituitary-specific activator of the growth hormone in several mammals. Furthermore, PIT1 is a positive regulatory factor of prolactin and thyroid-stimulating hormone beta. PIT1 is a member of the POU-domain family of genes and is located on porcine chromosome 13. Two informative three-generation families of the University of Hohenheim were used for the presented investigations. The families were based on crosses of the European Wild boar (W) x Pietrain (P) and Meishan (M) x Pietrain (P). Each family included 310 F(2) animals. A RsaI (PCR) RFLP described by Yu et al. (1994) was used for genotyping the animals. Altogether over 50 parameters of growth, meat quality, carcass composition and stress susceptibility were evaluated concerning their associations with RsaI PCR-RFLP. The statistical model of association analyses was used including fixed effects of sex, family, PIT1 genotypes and covariate age at slaughter. Taking the significance level of p < 0.05 as the basis, fourteen traits of growth and carcass composition were associated with PIT1 genotypes in family W x P. Results from this study suggest that there are contributions of PIT1 gene to variations in the analysed performance traits in pigs. The influence of PIT1 genotypes could not be confirmed under the supposition of a genome-wide test limit.

Animals↗

PubMed1999

Assembly requirements of PU.1-Pip (IRF-4) activator complexes: inhibiting function in vivo using fused dimers.

Gene expression in higher eukaryotes appears to be regulated by specific combinations of transcription factors binding to regulatory sequences. The Ets factor PU.1 and the IRF protein Pip (IRF-4) represent a pair of interacting transcription factors implicated in regulating B cell-specific gene expression. Pip is recruited to its binding site on DNA by phosphorylated PU.1. PU.1-Pip interaction is shown to be template directed and involves two distinct protein-protein interaction surfaces: (i) the ets and IRF DNA-binding domains; and (ii) the phosphorylated PEST region of PU.1 and a lysine-requiring putative alpha-helix in Pip. Thus, a coordinated set of protein-protein and protein-DNA contacts are essential for PU.1-Pip ternary complex assembly. To analyze the function of these factors in vivo, we engineered chimeric repressors containing the ets and IRF DNA-binding domains connected by a flexible POU domain linker. When stably expressed, the wild-type fused dimer strongly repressed the expression of a rearranged immunoglobulin lambda gene, thereby establishing the functional importance of PU.1-Pip complexes in B cell gene expression. Comparative analysis of the wild-type dimer with a series of mutant dimers distinguished a gene regulated by PU.1 and Pip from one regulated by PU.1 alone. This strategy should prove generally useful in analyzing the function of interacting transcription factors in vivo, and for identifying novel genes regulated by such complexes.

Amino Acid Sequence↗

PubMed1990

New type of POU domain in germ line-specific protein Oct-4.

Members of a family of murine octamer-binding proteins interact specifically with the octamer motif, a transcription regulatory element found in the promoter and enhancer regions of many genes. Oct-4 is a maternally expressed protein that is also present in the pre-implantation mouse embryo. Although many regulatory proteins are expressed in post-implantation embryos, transcription factors regulating pre-implantation processes have remained elusive. The Oct-4 gene is therefore a prime candidate for an early developmental control gene. Here we report the complementary DNA cloning of the mouse Oct-4 gene, and the characterization of the encoded protein(s) by sequential in vitro transcription, translation, DNA-binding and protease-clipping analysis. Deletion analysis shows that the DNA-binding activity is mediated by a POU domain encoded in an open reading frame corresponding to a 324-amino-acid protein. Sequence comparison with known POU domains reveals that Oct-4 is a novel member of the POU-family.

Animals↗

PubMed2003

Functions of HNF1 family members in differentiation of the visceral endoderm cell lineage.

The two members of the hepatocyte nuclear factor 1 (HNF1) transcription factor family, HNF1 and variant HNF1 (vHNF1), show a strong homology in their atypical POU-homeodomain and dimerization domain but differ in their transactivation domains. Moreover, two vHNF1 isoforms generated by alternative splicing are present in all tissues expressing this gene. vHnf1-deficient mouse embryos die soon after implantation due to defective visceral endoderm formation, an extraembryonic tissue essential for development and survival of the embryo proper. In contrast, invalidation of Hnf1, which is expressed at later developmental stages than vHnf1, does not lead to embryonic lethality or developmental defects. To examine the specific or potential equivalent functions of vHNF1 isoforms and HNF1 during the process of visceral endoderm differentiation, we stably reexpressed these factors in vHnf1-deficient embryonic stem cells. Analysis of these embryonic stem cells upon differentiation into embryoid bodies shows that vHNF1 isoforms exhibit specific behaviors depending on particular target genes and cooperate in the establishment of a functional visceral endoderm. Furthermore, forced expression of HNF1 in vHnf1-deficient embryonic stem cells fully restores the formation of a mature visceral endoderm with the correct expression profile of early and late markers of this lineage. Thus, in this context, HNF1 functionally replaces both vHNF1 isoforms, suggesting that the different developmental functions of these transcription factors are mainly due to the acquisition of novel expression patterns.

Alternative Splicing↗

PubMed1994

Identification of comS, a gene of the srfA operon that regulates the establishment of genetic competence in Bacillus subtilis.

Genetic competence (the ability to internalize exogenous DNA) in Bacillus subtilis is dependent on a regulatory pathway that activates the expression of a battery of competence-specific genes. The srfA operon, encoding the subunits of surfactin synthetase, which catalyzes the nonribosomal synthesis of the peptide antibiotic surfactin, also functions in the competence regulatory pathway. The DNA encoding only one of the seven amino acid-activating domains of surfactin synthetase, the valine-activating domain (srfAB1), is necessary for competence. Deletion analysis revealed that a 569-bp fragment of srfAB1, fused to the srfA promoter, complements a srfA deletion mutation (delta srfA) with respect to competence. This fragment contains an open reading frame consisting of 46 amino acids (orf46), which is out of frame with srfAB1. A frameshift mutation in srfAB upstream of orf46 has no effect on competence but a frameshift and nonsense mutation in orf46 resulted in failure to complement the delt srfA mutation. These results indicate that orf46 encodes the srfA-associated competence regulatory factor. Computer-aided analysis of the putative orf46 product (ComS) shows similarity to the homeodomain of the POU domain class of eukaryotic transcriptional regulators.

Amino Acid Sequence↗

PubMed1993

The octamer binding protein Oct-2 inhibits transactivation of the herpes simplex virus immediate-early genes by the virion protein Vmw65.

Transactivation by a complex of the cellular transcription factor Oct-1 and the virion protein Vmw65 is necessary for the high-level activity of the HSV immediate-early promoters during lytic infection. We show that this trans-activation can be inhibited by two forms of the Oct-2 transcription factor which are expressed at high levels in neuronal cells as well as by the isolated DNA binding, POU domain of Oct-2. The inhibition of Oct-1-Vmw65 DNA binding by these neuronal forms of Oct-2 is likely to play a critical role in the nonpermissivity of neuronal cells for the HSV lytic cycle and therefore in the establishment of latent infections.

Alternative Splicing↗

PubMed1999

Defective retinoic acid regulation of the Pit-1 gene enhancer: a novel mechanism of combined pituitary hormone deficiency.

Pit-1 is a pituitary-specific transcription factor responsible for pituitary development and hormone expression in mammals. Pit-1 contains two protein domains, termed POU-specific and POU-homeo, which are both necessary for DNA binding and activation of the GH and PRL genes and regulation of the PRL, TSH-beta subunit (TSH-beta), and Pit-1 genes. Pit-1 is also necessary for retinoic acid induction of its own gene during development through a Pit-1-dependent enhancer. Combined pituitary hormone deficiency is caused by defective transactivation of target genes in the anterior pituitary. In the present report, we provide in vivo evidence that retinoic acid induction of the Pit-1 gene can be impaired by a Pit-1 gene mutation, suggesting a new molecular mechanism for combined pituitary hormone deficiency in man.

Animals↗