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PubMed2000

Domains of Brn-2 that mediate homodimerization and interaction with general and melanocytic transcription factors.

The class III POU gene brn-2, encoding the Brn-2/N-Oct-3 transcription factor, is widely expressed in the developing mammalian central nervous system. Brn-2 has also been found to regulate the melanocytic phenotype with N-Oct-3 DNA binding activity elevated in malignant melanoma, however, its mode of action is yet to be defined. The functional role of the Brn-2 transcription factor has been investigated through the analysis of protein-protein interactions it forms with a number of basal and melanocytic transcriptional regulatory proteins. In vivo interactions were tested by gene-cotransfection using the mammalian GAL4-Herpes Simplex viral protein 16 (VP16) two hybrid formation and direct protein binding by in vitro glutathione S-transferase (GST)-pull down assay. The Brn-2 protein was found to homodimerize in vivo with high affinity, using Brn-2 deletion constructs dimer complex formation was found to be dependent on the presence of both the homeodomain and linker regions of the POU-domain. However, the POU-homoedomain was dispensable for the formation of the dimerization interface in one of the partner molecules but not both, when the POU-linker region was removed the ability to interact was lost irrespective of the presence of the homeodomain. Dimerization of Brn-2/N-Oct-3 was also found to occur in DNA binding assays using melanoma cell line nuclear extracts and a recently reported dimer target sequence probe, which may have significant consequences for gene regulation in melanocytic tumours. Low affinity Brn-2 protein contacts have also been found with the basal transcription complex, including TATA binding protein (TBP) and the transcriptional coactivator p300, and with the Sox-10 and Pax-3 transcription factors that are known to play an important role in melanocyte cell formation.

Binding Sites↗
PubMed1997

Pituitary dwarfism in the R271W Pit-1 gene mutation.

UNLABELLED: The Pit-1 gene encodes the POU-domain transcription factor Pit-1 which is important for the differentiation of the anterior pituitary and regulation of the PRL, GH and TSH genes. As a member of the POU domain transcription factors, Pit-1 contains a DNA-binding region, consisting of a POU-specific domain and a POU homeodomain. Mutation of the Pit-1 gene causes hypoplasia of the pituitary gland and deficiencies of GH, PRL and TSH. In a DNA sample from a 3-month-old girl with severe growth deficiency from birth, single stranded conformational polymorphism analysis of the Pit-1 gene identified a gel shift in exon 6. DNA-sequencing disclosed a single base mutation in codon 271 (CGG to TGG) that changes arginine to tryptophan (R271W) in the POU homeodomain. The patient presented distinct facial features with prominent forehead, marked mid-facial hypoplasia with depressed nasal bridge, deep-set eyes and a short nose with anteverted nostrils. MRI examination showed a hypoplastic pituitary gland. Low serum GH did not respond to insulin-arginine provocation or GHRH tests. PRL levels below the detection limit did not increase in response to a TRH test. T4 and free T4 was below detection limit (< 20 nmol/l and < 4 pmol/l). TSH was 2.0 mU/l and showed a blunt response to 6.0 mU/l following TRH test. TBG was normal. In spite of inappropriately low TSH and very low T4, T3 was in the low normal range (1.4-1.6 nmol/l) and she was clinically euthyroid. The thyroid function tests are consistent with increased monodeiodination activity and increased conversion of T4 to T3, possibly related to the Pit-1 gene mutation. GH and T4 treatment resulted in catch-up growth continued during 5 years of therapy. CONCLUSION: Reports of nine other cases of R271W mutations of different populations as well as the present Norwegian patient suggest codon 271 of exon 6 to be a "hot spot" for Pit-1 mutations. To enable rapid and simple detection of this type of de novo mutation we have designed a specific amplification-created-restriction-site assay to check for the R271W mutation in patients suspected to have this rare form of genetic defect in growth hormone production.

Arginine↗
PubMed1997

Transcriptional regulatory elements in the upstream and intron of the fibroin gene bind three specific factors POU-M1, Bm Fkh and FMBP-1.

The transcriptional modulator in the fibroin gene intron is composed of multiple octamer-like AT-rich elements, to which several specific DNA-binding proteins named fibroin-modulator-binding proteins (FMBPs) bind. Three major FMBPs in the silk gland were characterized. Two of them (FMBP-2 and -3) were identified as a Fork head homologue (Bm Fkh) and a POU-domain protein (POU-M1) respectively. These factors were expressed in the silk gland with distinct temporal- and spatial-specificities during late larval development as well as during embryogenesis, and did not correlate directly with fibroin gene expression. The other (FMBP-1) appeared to correlate with the expression of the fibroin gene for temporal- and spatial-specificity. These FMBPs also bind to the elements in the upstream modulator. Transcriptional enhancement by both modulators was inhibited by binding competition for these factors with oligonucleotides. These results suggest that expression of the fibroin gene is controlled by co-ordination of these factors with distinct specificities during silk-gland development.

Animals↗
PubMed1996

Similar DNA recognition properties of alternatively spliced Drosophila POU factors.

The POU-IV or "Brn-3" class of POU-domain transcription factors is represented in Drosophila by I-POU and twin-of-I-POU, alternative splice products of the I-POU gene. I-POU has been previously reported to inhibit DNA binding by the POU-III class factor drifter/Cf1a via the formation of heterodimeric complexes. Here we report that expression of the I-POU/tI-POU message is maximal late in the embryonic phase of Drosophila development, and I-POU is the preferred splice variant. Although I-POU lacks two basic amino acid residues in the POU-homeodomain found in tI-POU and Brn-3.0, these three POU-IV class proteins exhibit very similar DNA-binding specificity. In contrast to previously published reports, the results presented here show no effect of I-POU on DNA binding by drifter, and no evidence for I-POU/drifter dimerization. These results suggest that the I-POU/tI-POU gene products function by transcriptional mechanisms similar to those of the homologous POU-IV class factors expressed in other species, not by unique inhibitory mechanism.

Alternative Splicing↗
PubMed1995

High mobility group protein 2 functionally interacts with the POU domains of octamer transcription factors.

The octamer transcription factors Oct1 and Oct2 are involved in the transcriptional regulation of both lymphoid-specific and ubiquitously expressed genes. Their activity depends critically on their interaction with distinct cellular cofactors. Therefore, we have isolated cDNAs encoding proteins that physically interact with Oct2. Here we describe the analysis of one such clone, representing the murine homologue of high mobility group (HMG) protein 2. We have mapped the interaction domains for both proteins and have shown that HMG2 and Oct2 interact via their HMG domains and POU homeodomains, respectively. This interaction is not restricted to Oct2, as other members of the octamer transcription factor family like Oct1 and Oct6 also interact with HMG2. The interaction with HMG2 results in a marked increase in the sequence-specific DNA binding activity of the Oct proteins. Interestingly, the HMG2 protein is not present in the protein-DNA complex detected by an electrophoretic mobility shift assay. The Oct and HMG2 proteins also interact in vivo. A chimeric protein, in which the strong transactivation domain of VP16 was fused directly to the HMG domains of HMG2, stimulated the activity of an octamer-dependent reporter construct upon cotransfection. Furthermore, the expression of antisense RNA for HMG2 specifically reduces octamer-dependent transcription. These results suggest that one of the functions of HMG2 is to support the octamer transcription factors in their role as transcriptional activators.

Amino Acid Sequence↗
PubMed2004

GATA3 and NeuroD distinguish auditory and vestibular neurons during development of the mammalian inner ear.

The function of the zinc finger transcription factor GATA3 was studied in a newly established, conditionally immortal cell line derived to represent auditory sensory neuroblasts migrating from the mouse otic vesicle at embryonic day E10.5. The cell line, US/VOT-33, expressed GATA3, the bHLH transcription factor NeuroD and the POU-domain transcription factor Brn3a, as do auditory neuroblasts in vivo. When GATA3 was knocked down reversibly with antisense oligonucleotides, NeuroD was reversibly down-regulated. Auditory and vestibular neurons form from neuroblasts that express NeuroD and that migrate from the antero-ventral, otic epithelium at E9.5-10.5. On the medial side, neuroblasts and epithelial cells express GATA3 but on the lateral side they do not. At E13.5 most auditory neurons express GATA3 but no longer express NeuroD, whereas vestibular neurons express NeuroD but not GATA3. Neuroblasts expressing NeuroD and GATA3 were located in the ventral, otic epithelium, the adjacent mesenchyme and the developing auditory ganglion. The results suggest that auditory and vestibular neurons arise from different, otic epithelial domains and that they gain their identity prior to migration. In auditory neuroblasts, NeuroD appears to be dependent on the expression of GATA3.

Animals↗
PubMed1993

Neurons regulate Schwann cell genes by diffusible molecules.

Successful peripheral nerve regeneration and functional recovery require the reestablishment of the neuron-Schwann cell relationship in the regenerating rat sciatic nerve, neurons differentially regulate Schwann cell genes. The message for the low-affinity NGF receptor, p75NGFR, is induced in Schwann cells distal to the injury and is repressed as regenerating axons make contact with these cells. The inverse is true for mRNA of the myelin gene P0; expression decreases distal to injury and increases as new axons contact Schwann cells and a program of myelination is initiated. Using an in vitro co-culture paradigm in which primary neurons and adult Schwann cells are separated by a microporous membrane, we show that axon contact is not an absolute requirement for neuronal regulation of Schwann cell genes. In this system neurons but not other cell types, repress the expression of Schwann cell p75NGFR while inducing the expression of the POU domain transcription factor, suppressed cAMP inducible POU, and myelin P0. These results demonstrate that regenerating axons can direct the Schwann cell genetic program from a distance through diffusible molecules.

Animals↗
PubMed2001

DNA recognition by the herpes simplex virus transactivator VP16: a novel DNA-binding structure.

Upon infection, the herpes simplex virus (HSV) transcriptional activator VP16 directs the formation of a multiprotein-DNA complex-the VP16-induced complex-with two cellular proteins, the host cell factor HCF-1 and the POU domain transcription factor Oct-1, on TAATGARAT-containing sequences found in the promoters of HSV immediate-early genes. HSV VP16 contains carboxy-terminal sequences important for transcriptional activation and a central conserved core that is important for VP16-induced complex assembly. On its own, VP16 displays little, if any, sequence-specific DNA-binding activity. We show here that, within the VP16-induced complex, however, the VP16 core has an important role in DNA binding. Mutation of basic residues on the surface of the VP16 core reveals a novel DNA-binding surface with essential residues which are conserved among VP16 orthologs. These results illuminate how, through association with DNA, VP16 is able to interpret cis-regulatory signals in the DNA to direct the assembly of a multiprotein-DNA transcriptional regulatory complex.

Amino Acid Substitution↗
PubMed2000

Protein interaction surface of the POU transcription factor UNC-86 selectively used in touch neurons.

The Caenorhabditis elegans POU protein UNC-86 specifies the HSN motor neurons, which are required for egg-laying, and six mechanosensory neurons. To investigate how UNC-86 controls neuronal specification, we characterized two unc-86 mutants that do not respond to touch but show wild-type egg-laying behavior. Residues P145 and L195, which are altered by these mutations, are located in the POU-specific domain and abolish the physical interaction of UNC-86 with the LIM homeodomain protein, MEC-3. This results in a failure to maintain mec-3 expression and in loss of expression of the mechanosensory neuron-specific gene, mec-2. unc-86-dependent expression of genes in other neurons is not impaired. We conclude that distinct residues in the POU domain of UNC-86 are involved in modulating UNC-86 activity during its specification of different neurons. A structural model of the UNC-86 POU domain, including base pairs and amino acid residues required for MEC-3 interaction, revealed that P145 and L195 are part of a hydrophobic pocket which is similar to the OCA-B-binding domain of the mammalian POU protein, Oct-1.

Amino Acid Sequence↗
PubMed2006

Characterization of two POU transcription factor family members from the urochordate Oikopleura dioica.

Three POU domain containing transcription factors have been cloned from the urochordate Oikopleura dioica. Phylogenetic analysis showed that two of these (OctA1 and OctA2) are closely related members of the class II POU domain family, and one (OctB) is a member of the class III POU domain family. All three transcription factors contained a highly conserved bipartite DNA-binding POU domain with POU specific and POU homeodomains, separated by a linker region. All three proteins were shown to bind specifically to the canonical octamer motif, ATGCAAAT. The ability of these factors to drive transcription from an octamer-containing reporter construct was assessed in vertebrate B lymphocyte cell lines. Both OctA1 and OctA2 drove transcription in murine and catfish B cell lines, however, OctB did not increase the level of transcription above background levels. It is concluded that Oct transcription factors capable of functioning in a similar fashion to vertebrate Oct1/2 were present at the phylogenetic level of the urochordates.

Amino Acid Motifs↗
PubMed1994

The POU domains of the Oct1 and Oct2 transcription factors mediate specific interaction with TBP.

We had previously shown that the ubiquitous Oct1 and the lymphoid-specific Oct2 transcription factors stimulate transcription at the level of stable preinitiation complex formation. We have therefore investigated whether the octamer binding proteins might physically interact with TBP, the TATA box binding protein component of the TFIID factor. By using several different experimental systems we show that TBP efficiently associates with Oct1 and Oct2. The interaction is direct and does not depend on the presence of DNA or additional proteins. N- and C-terminal deletions of the different proteins were used to localize the domains involved in the interaction. We show that the POU homeodomain of Oct2 and the evolutionarily conserved C-terminal core domain of TBP are both required and sufficient for the interaction. The Oct1 POU domain, which is highly homologous to the Oct2 POU domain, likewise mediates interaction with TBP. The interaction can also be observed in vivo, as TBP can be co-precipitated with Oct2 from co-transfected Cos1 cells and TBP co-immunoprecipitates with the endogenous Oct1 from HeLa cells. Co-transfection of human TBP and Oct2 expression vectors into B cells resulted in a synergistic activation of an octamer motif containing promoter.

Binding Sites↗
PubMed1997

Repression of gonadotropin-releasing hormone promoter activity by the POU homeodomain transcription factor SCIP/Oct-6/Tst-1: a regulatory mechanism of phenotype expression?

POU domain transcription factors are required for neuropeptide expression in selected subsets of hypothalamic neuroendocrine neurons. We now report that expression of the gonadotropin-releasing hormone (GnRH) gene, which controls sexual development, is regulated by the POU protein SCIP/Oct-6/Tst-1. Reverse transcriptase PCR cloning and RNase protection assays demonstrated the presence of SCIP/Oct-6/Tst-1 mRNA in the GnRH-producing neuronal cell line GT1-7. The physiological relevance of this regulatory activity was suggested by the detection of SCIP/Oct-6/Tst-1 mRNA in a subset of GnRH neurons in the hypothalamus of prepubertal female rats. Coexpression of SCIP/Oct-6/Tst-1 in neuronal cells inhibited rat GnRH (rGnRH) promoter activity via three regions of the proximal rGnRH promoter containing SCIP/Oct-6/Tst-1 binding sites. DNase I footprinting, gel shift assays, and DNA and protein mutagenesis studies indicated that both direct DNA binding and protein-protein interactions are required for SCIP/Oct-6/Tst-1 modulation of GnRH gene expression. Activation of SCIP/Oct-6/Tst-1 expression in terminally differentiated GnRH neurons may be a factor determining the ratio of phenotypically "inactive" versus "active" GnRH neurons during postnatal life.

Animals↗
PubMed1997

Expression of the POU transcription factor Brn-5 inhibits proliferation of NG108-15 cells.

The POU domain transcription factors are a subgroup of homeodomain proteins that appears to control cellular phenotypes. The expression of the POU protein Brn-5 occurs selectively in postmitotic CNS neurons. Ectopic expression of Brn-5 in dividing NG108-15 cells reduces the level of RNA encoding the proliferating cell nuclear antigen (PCNA). This ectopic expression also inhibits DNA synthesis as measured by the incorporation of bromodeoxyuridine (BrdU). Thus, Brn-5 may inhibit the continued proliferation of these cells. A potential function of Brn-5 may be to suppress the action of proliferative signals in postmitotic neurons and thus prevents them from reentering the cell cycle.

Cell Differentiation↗
PubMed1997

OCA-B is a functional analog of VP16 but targets a separate surface of the Oct-1 POU domain.

OCA-B is a B-cell-specific coregulator of the broadly expressed POU domain transcription factor Oct-1. OCA-B associates with the Oct-1 POU domain, a bipartite DNA-binding structure containing a POU-specific (POU[S]) domain joined by a flexible linker to a POU homeodomain (POU[H]). Here, we show that OCA-B alters the activity of Oct-1 in two ways. It provides a transcriptional activation domain which, unlike Oct-1, activates an mRNA-type promoter effectively, and it stabilizes Oct-1 on the Oct-1-responsive octamer sequence ATGCAAAT. These properties of OCA-B parallel those displayed by the herpes simplex virus Oct-1 coregulator VP16. OCA-B, however, interacts with a different surface of the DNA-bound Oct-1 POU domain, interacting with both the POU(S) and POU(H) domains and the center of the ATGCAAAT octamer sequence. The OCA-B and VP16 interactions with the Oct-1 POU domain are sufficiently different to permit OCA-B and VP16 to bind the Oct-1 POU domain simultaneously. These results emphasize the structural versatility of the Oct-1 POU domain in its interaction with coregulators.

Amino Acid Sequence↗
PubMed1997

Structure of Pit-1 POU domain bound to DNA as a dimer: unexpected arrangement and flexibility.

Pit-1, a member of the POU domain family of transcription factors, characterized by a bipartite DNA-binding domain, serves critical developmental functions based on binding to diverse DNA elements in its target genes. Here we report a high resolution X-ray analysis of the Pit-1 POU domain bound to a DNA element as a homodimer. This analysis reveals that Pit-1 subdomains bind to perpendicular faces of the DNA, rather than opposite faces of the DNA as in Oct-1. This is accomplished by different spacing and orientation of the POU-specific domain. Contrary to previous predictions, the dimerization interface involves the carboxyl terminus of the DNA recognition helix of the homeodomain, which in an extended conformation interacts with specific residues at the amino terminus of helix alpha1 and in the loop between helices alpha3 and alpha4 of the POU-specific domain of the symmetry related monomer. These features suggest the molecular basis of disease-causing mutations in Pit-1 and provide potential basis for the flexible allostery between protein domains and DNA sites in the activation of target genes.

Amino Acid Sequence↗
PubMed2001

Drosophila neuroblasts sequentially express transcription factors which specify the temporal identity of their neuronal progeny.

Neural precursors often generate distinct cell types in a specific order, but the intrinsic or extrinsic cues regulating the timing of cell fate specification are poorly understood. Here we show that Drosophila neural precursors (neuroblasts) sequentially express the transcription factors Hunchback --> Krüppel --> Pdm --> Castor, with differentiated progeny maintaining the transcription factor profile present at their birth. Hunchback is necessary and sufficient for first-born cell fates, whereas Krüppel is necessary and sufficient for second-born cell fates; this is observed in multiple lineages and is independent of the cell type involved. We propose that Hunchback and Krüppel control early-born temporal identity in neuroblast cell lineages.

Animals↗
PubMed1998

New POU dimer configuration mediates antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and Sox-2.

The POU transcription factor Oct-4 is expressed specifically in the germ line, pluripotent cells of the pregastrulation embryo and stem cell lines derived from the early embryo. Osteopontin (OPN) is a protein secreted by cells of the preimplantation embryo and contains a GRGDS motif that can bind to specific integrin subtypes and modulate cell adhesion/migration. We show that Oct-4 and OPN are coexpressed in the preimplantation mouse embryo and during differentiation of embryonal cell lines. Immunoprecipitation of the first intron of OPN (i-opn) from covalently fixed chromatin of embryonal stem cells by Oct-4-specific antibodies indicates that Oct-4 binds to this fragment in vivo. The i-opn fragment functions as an enhancer in cell lines that resemble cells of the preimplantation embryo. Furthermore, it contains a novel palindromic Oct factor recognition element (PORE) that is composed of an inverted pair of homeodomain-binding sites separated by exactly 5 bp (ATTTG +5 CAAAT). POU proteins can homo- and heterodimerize on the PORE in a configuration that has not been described previously. Strong transcriptional activation of the OPN element requires an intact PORE. In contrast, the canonical octamer overlapping with the downstream half of the PORE is not essential. Sox-2 is a transcription factor that contains an HMG box and is coexpressed with Oct-4 in the early mouse embryo. Sox-2 represses Oct-4 mediated activation of i-opn by way of a canonical Sox element that is located close to the PORE. Repression depends on a carboxy-terminal region of Sox-2 that is outside of the HMG box. Expression, DNA binding, and transactivation data are consistent with the hypothesis that OPN expression is regulated by Oct-4 and Sox-2 in preimplantation development.

Amino Acid Sequence↗
PubMed2006

The WNT/Beta-catenin pathway in melanoma.

The Wnt/beta-catenin pathway is involved in various cellular activities--including determination, proliferation, migration and differentiation--in embryonic development and adult homeostasis. The deregulation or constitutive activation of the Wnt/beta-catenin pathway may lead to cancer formation. This review focuses on the role of the Wnt/beta-catenin canonical signaling pathway in the melanocyte lineage, and more specifically, in melanoma. Several components of the Wnt/beta-catenin pathway, such as APC, ICAT, LEF1 and beta-catenin are modified in melanoma tumors and cell lines, leading to activation of this signaling. A hallmark of the activation of this pathway is the presence of beta-catenin in the nucleus. Indeed, beta-catenin is found in about 30% of human melanoma nuclei, indicating a potentially specific role for this signaling pathway in this aggressive type of cancer. Beta-catenin can induce ubiquitous genes such as myc or cyclinD1, cell lineage-restricted genes such as Brn2 and melanocyte-specific genes such as Mitf-M and Dct. The Mitf-M and Brn-2 genes encode transcription factors. Mitf plays a critical role in melanocyte survival, proliferation and differentiation. Brn-2 is involved in melanoma proliferation. Determining how the Wnt/beta-catenin signaling pathway, alone or with other pathways, orchestrates the induction of target genes involved in a diverse range of activities represents a major challenge in research into melanoma formation and tumor progression.

Cell Adhesion Molecules↗