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Results for “POU Domain Factors”

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PubMed1994

[Pituitary specific transcription factor Pit-1/GHF-1 and combined pituitary hormone deficiency].

Pit-1/GHF-1 is a pituitary-specific POU-domain DNA binding factor, which binds to, and trans-activates the promoters of both growth hormone (GH) and prolactin (PRL) genes. It is well conserved among vertebrates and is indispensable for both differentiation and proliferation of the somatotrophs, lactotrophs and thyrotrophs. PIT1 abnormality is defined as a genetic abnormality in the PIT1 gene, and results in various types of combined deficiencies of thyrotropin (TSH), GH, and PRL. PIT1 abnormality was first recognized as an excellent model for 'transcription factor disease'. Recently, we found a novel case whose cannot be explained by the PIT1 gene mutation alone. Such case will give us precious clue to identify modifier factors of Pit-1/GHF-1.

Animals↗

PubMed1992

Oct-1 and Oct-2 potentiate functional interactions of a transcription factor with the proximal sequence element of small nuclear RNA genes.

The promoters of both RNA polymerase II- and RNA polymerase III-transcribed small nuclear RNA (snRNA) genes contain an essential and highly conserved proximal sequence element (PSE) approximately 55 bp upstream from the transcription start site. In addition, the upstream enhancers of all snRNA genes contain binding sites for octamer-binding transcription factors (Octs), and functional studies have indicated that the PSE and octamer elements work cooperatively. The present study has identified and characterized a novel transcription factor (designated PTF) which specifically binds to the PSE sequence of both RNA polymerase II- and RNA polymerase III-transcribed snRNA genes. PTF binding is markedly potentiated by Oct binding to an adjacent octamer site. This potentiation is effected by Oct-1, Oct-2, or the conserved POU domain of these factors. In agreement with these results and despite the independent binding of Octs to the promoter, PTF and Oct-1 enhance transcription from the 7SK promoter in an interdependent manner. Moreover, the POU domain of Oct-1 is sufficient for significant in vitro activity in the presence of PTF. These results suggest that essential activation domains reside in PTF and that the potentiation of PTF binding by Octs plays a key role in the function of octamer-containing snRNA gene enhancers.

Base Sequence↗

PubMed1994

The Oct-1 POU domain stimulates adenovirus DNA replication by a direct interaction between the viral precursor terminal protein-DNA polymerase complex and the POU homeodomain.

The bipartite POU domain of transcription factor Oct-1 stimulates adenovirus DNA replication through an interaction with the octamer sequence present in the auxiliary origin. Employing an immobilized in vitro DNA replication system, we show that the POU domain enhances the formation of a pre-initiation complex composed of the viral precursor terminal protein-DNA polymerase (pTP-pol) complex and the origin. To investigate the mechanism of stimulation we have explored protein-protein interactions between the POU domain and the pTP-pol complex. Such an interaction could be detected using a GST-POU fusion protein bound to glutathione-agarose beads. Binding was also observed with the POU homeodomain (POUHD), albeit weaker than with the intact POU domain, but not with the POU specific subdomain. Four point mutations localized in the POUHD were analyzed for pTP-pol binding. Two of these, E22A and E30A, bound pTP-pol equally as well as the wild-type, while the other two, Q24A and E29A, were able to bind 2- to 4-fold better. These mutations are localized in the same region where the HSV transactivator VP16 binds, but did not coincide with the VP16 contacts. A direct correlation between pTP-pol binding and stimulation of DNA replication in vitro was observed for all mutants, suggesting that stimulation by the POU domain is caused by an interaction with the viral pTP-pol complex.

Adenoviridae↗

PubMed2001

The Brn-3b POU family transcription factor regulates the cellular growth, proliferation, and anchorage dependence of MCF7 human breast cancer cells.

The Brn-3b POU domain containing transcription factor is expressed in the developing sensory nervous system as well as in epithelial cells of the breast, cervix, and testes. Brn-3b functionally interacts with the estrogen receptor (ER) and in association with the ER, regulates transcription from estrogen responsive genes. In addition, Brn-3b expression is elevated in breast tumours compared to levels in normal mammary cells. To explore the role of Brn-3b in breast cancer, we established stable cell lines derived from the MCF7 human breast cancer cell line which had been transfected with Brn-3b sense or anti-sense constructs. The Brn-3b over-expressing cell lines exhibited increased growth rate, reached confluence at a higher saturation density, had higher proliferative activity, and an enhanced ability to form colonies in soft agar when compared to the control empty vector transfected cells. Likewise, the Brn-3b anti-sense cell lines showed reduced cellular growth and proliferation, reached confluence at a lower density, and exhibited a decreased ability to form colonies in soft agar when compared to the vector controls. Five to ten per cent of the Brn-3b over-expressing cells exhibited a severely altered morphology characterized by reduced adherence to tissue culture plastic, increased cell size, and a vacuolar cell shape. These results thus further indicate a role for the Brn-3b transcription factor in regulating mammary cell growth and suggest that its elevation in breast cancer is of functional significance.

Adenocarcinoma↗

PubMed1993

The solution structure of the Oct-1 POU-specific domain reveals a striking similarity to the bacteriophage lambda repressor DNA-binding domain.

The POU-specific (POUs) domain, in association with a POU-type homeodomain, forms the bipartite DNA-binding POU domain. The solution structure of the Oct-1 POUs domain has been determined by multidimensional nuclear magnetic resonance spectroscopy and consists of four alpha helices surrounding a conserved hydrophobic core. The POUs domain is structurally similar to the DNA-binding domains of the bacteriophage lambda and 434 repressors and 434 Cro. These domains exhibit superimposable helix-turn-helix (HTH) motifs, except that in the POUs domain, the first helix and the linker to the second helix of the motif are extended. The conserved structural features have been used to propose a plausible model for DNA binding by the POUs domain. A human dwarfism mutation that affects positive control in the related POU domain protein Pit-1 maps to the same region of the HTH motif as do positive control mutations in lambda repressor.

Amino Acid Sequence↗

PubMed2003

Combination of a zinc finger and homeodomain required for protein-interaction.

The Zinc Finger Homeodomain Enhancer-binding Protein (Zfhep) is involved in skeletal patterning, immune cell, muscle, and brain development, and is necessary for life. Zfhep contains a single central homeodomain (HD) adjacent to an isolated zinc finger, the function of which is unknown. The placement of a zinc finger so close to a homeodomain is novel in nature. The aim of this work was to characterize the Zfhep homeodomain (HD) or the zinc finger homeodomain (ZHD), with respect to DNA-binding and protein-protein interactions. Glutathione-S-transferase (GST) fusion proteins containing either just the HD or both the zinc finger and HD (ZHD) were expressed in E. coli. The GST fusion protein affinity-binding assay demonstrated that Zfhep ZHD interacts specifically with the POU domain of the Oct-1 transcription factor. The adjacent zinc finger is required since Zfhep HD alone does not interact with Oct-1 POU domain. Furthermore, ZHD does not bind to the POU homeodomain lacking the POU specific region. These results demonstrate that the Zfhep zinc finger homeodomain motif functions as a protein-binding domain in vitro, and suggests that Zfhep may modulate the activity of POU domain transcription factors. However, neither the Zfhep ZHD nor the HD bound DNA in EMSA or selected a DNA-binding site from a pool of random oligonucleotides. This is the first demonstration of a function for the HD region of Zfhep, which is the first case of a bi-partite domain requiring both a zinc finger and a HD for binding to protein.

Amino Acid Sequence↗

PubMed1995

Structure of the Oct-3 POU-homeodomain in solution, as determined by triple resonance heteronuclear multidimensional NMR spectroscopy.

The POU-homeodomain (POUH) forms the bipartite DNA-binding POU domain in association with the POU-specific domain. The 1H, 15N, and 13C magnetic resonances of the 67-amino acid long POUH of mouse Oct-3 have almost completely been assigned, mainly through the combined use of three-dimensional triple resonance NMR methods. Based on the distance and dihedral angle constraints derived from the NMR data, the solution structure of the POUH domain has been calculated by the ab initio simulated annealing method. The average RMS deviation for all backbone heavy atoms of the 20 best calculated structures for residues 9-53 of the total 67 amino acid residues is 0.44 A. The POUH domain consists of three alpha-helices (helix-I, 10-20; helix-II, 28-38; and helix-III, 42-53), and helices-II and -III form a helix-turn-helix motif. In comparison with other classical homeodomains, the folding of the three helices is quite similar. However, the length of helix-III is fairly short. In the complex of the Oct-1 POU domain with an octamer site (Klemm JD, et al., 1994, Cell 77:21-32), the corresponding region is involved in helix-III. The structural difference between these two cases will be discussed.

Amino Acid Sequence↗

PubMed1999

SNAP(c): a core promoter factor with a built-in DNA-binding damper that is deactivated by the Oct-1 POU domain.

snRNA gene transcription is activated in part by recruitment of SNAP(c) to the core promoter through protein-protein contacts with the POU domain of the enhancer-binding factor Oct-1. We show that a mini-SNAP(c) consisting of a subset of SNAP(c) subunits is capable of directing both RNA polymerase II (Pol II) and Pol III snRNA gene transcription. Mini-SNAP(c) cannot be recruited by Oct-1, but binds as efficiently to the promoter as SNAP(c) together with Oct-1 and directs activated RNA Pol III transcription. Thus, SNAP(c) represses its own binding to DNA, and repression is relieved by interactions with the Oct-1 POU domain that promote cooperative binding. We have shown previously that TBP also represses its own binding, and in that case repression is relieved by cooperative interactions with SNAP(c). This may represent a general mechanism to ensure that core promoter-binding factors, which have strikingly slow off-rates, are recruited specifically to promoter sequences rather than to cryptic-binding sites in the genome.

DNA-Binding Proteins↗

PubMed2002

Androgen receptor interactions with Oct-1 and Brn-1 are physically and functionally distinct.

POU domain proteins interact positively or negatively with steroid hormone receptors, depending on the precise array of these and other factors assembled on target gene promoters. Octamer transcription factor 1 (Oct-1), a ubiquitous POU factor, is implicated in androgen induction of the mouse sex-limited protein (Slp) gene based on protein-DNA interaction studies. However, direct evidence for a role of Oct-1 in the hormone response has been difficult to obtain. Brain 1 (Brn-1), another POU factor, is more tissue-specific, expressing in brain and also in kidney, which is a major site of Slp synthesis. We compared the interaction of the androgen receptor (AR) with Oct-1 and Brn-1 to reveal the more likely candidate for regulation of Slp. In transfection, addition of either Oct-1 or Brn-1 reduced AR activation, regardless of the presence of an octamer-like sequence in the enhancer, suggesting interference was indirect. However, when the octamer-like element was changed to a consensus octamer site, Brn-1, but not Oct-1, strongly enhanced androgen activation. This correlated with Brn-l's preference for the consensus octamer sequence in DNA binding assays. Direct interaction of AR with glutathione-S-transferase-(GST)-fused Oct-1 was DNA-dependent, while Brn-l-AR association was not. Chimeric Brn-1 and Oct-1 POU domains demonstrated that the DNA-dependent AR interaction relied on the origin of the POU homeodomain. However, in the context of full-length Brn-1 and Oct-1 chimeric proteins, the POU homedomain was not sufficient to confer the distinct behaviors of these factors in vivo, but instead revealed the importance of an N-terminal transactivation domain in Brn-1. These results demonstrate that functional interaction of Oct-1 and Brn-1 with AR is determined by the precise sequence of the octamer binding site, and by differential interaction of the POU factors with AR and other components of the transcriptional machinery.

Animals↗

PubMed2001

NF-Y binding is required for transactivation of neuronal aromatic L-amino acid decarboxylase gene promoter by the POU-domain protein Brn-2.

We have previously characterized binding sites for the NF-Y transcription factor (-71/-52) and Brn-2 POU-domain protein (-92/-71) in the neuronal promoter of the human aromatic L-amino acid decarboxylase gene [Mol. Brain Res. 56 (1998) 227]. We have now explored the functional role of these binding sites in transfected SK-N-BE neuroblastoma cells. Mutations of the NF-Y site that abolish binding depressed expression of a luciferase reporter gene up to 25-fold. The overexpression of a dominant negative mutant of NF-YA subunit depressed expression by 60%. Promoter activity was increased by the overexpression of Brn-2. Mutations or deletion of the binding site of Brn-2 did not suppress transcriptional activation by overexpressed Brn-2, while promoters defective in NF-Y binding were not transactivated by Brn-2. A GST-pulldown experiment showed that recombinant human Brn-2 protein weakly interacts with recombinant NF-Y outside of DNA. Cooperative binding of recombinant NF-Y and GST--Brn-2 proteins on the neuronal promoter was evidenced by an electrophoretic mobility shift assay. The POU-domain of Brn-2 was sufficient for such interaction. The results thus suggest that the activation of the neuronal promoter of the aromatic L-amino acid decarboxylase gene requires a direct interaction between the ubiquitous NF-Y factor and a cell-specific POU-domain protein. The NF-Y, but not the Brn-2 binding site, is essential for the recruitment of the NF-Y/Brn-2 complex on the promoter.

Animals↗

PubMed1996

Sequence-specific DNA binding of the B-cell-specific coactivator OCA-B.

B-cell-specific transcription of immunoglobulin genes is mediated by the interaction of a POU domain containing transcription factor Oct-1 or Oct-2, with the B-cell-specific coactivator OCA-B (Bob-1, OBF-1) and a prototype octamer element. We find that OCA-B binds DNA directly in the major groove between the two subdomains of the POU domain, requiring both an A at the fifth position of the octamer element and contact with the POU domain. An amino-terminal fragment of OCA-B binds the octamer site in the absence of a POU domain with the same sequence specificity. Coactivator OCA-B may undergo a POU-dependent conformational change that exposes its amino terminus, allowing it to recognize specific DNA sequences in the major groove within the binding site for Oct-1 or Oct-2. The recognition of both the POU domain and the octamer sequence by OCA-B provides a mechanism for differential regulation of octamer sites containing genes by the ubiquitous factor Oct-1.

B-Lymphocytes↗

PubMed1995

Functional interaction between the POU domain protein Tst-1/Oct-6 and the high-mobility-group protein HMG-I/Y.

The POU domain protein Tst-1/Oct-6 is a transcriptional activator of human papovavirus JC virus in transient transfections. Because of its endogenous expression in myelinating glia, Tst-1/Oct-6 might also be an important determinant for the glia specificity of JC virus in vivo. Activation of viral early and late genes depends on the ability of Tst-1/Oct-6 to interact with an AT-rich element within the viral regulatory region. Here, we show that this element not only is bound by Tst-1/Oct-6 but, in addition, serves as a binding site for the high-mobility-group protein HMG-I/Y. In the presence of HMG-I/Y, Tst-1/Oct-6 exhibited an increased affinity for this AT-rich element. The specificity of this effect was evident from the fact that no stimulation of Tst-1/Oct-6 binding was observed on a site that did not allow binding of HMG-I/Y. In addition, both proteins interacted with each other in solution. Direct contacts were identified between the POU domain of Tst-1/Oct-6 and a short stretch of 10 amino acids in the central portion of HMG-I/Y. These results point to an accessory role for HMG-I/Y in the activation of JC viral gene expression by the POU domain protein Tst-1/Oct-6. In agreement with such a role, HMG-Y synergistically supported the function of Tst-1/Oct-6 in transient transfections, measured on the early promoter of JC virus or on an artificial promoter consisting of only a TATA box and the common binding element for Tst-1 and HMG-I/Y.

Base Sequence↗

PubMed1999

Selective binding of steroid hormone receptors to octamer transcription factors determines transcriptional synergism at the mouse mammary tumor virus promoter.

Transcriptional synergism between glucocorticoid receptor (GR) and octamer transcription factors 1 and 2 (Oct-1 and Oct-2) in the induction of mouse mammary tumor virus (MMTV) transcription has been proposed to be mediated through directed recruitment of the octamer factors to their binding sites in the viral long terminal repeat. This recruitment correlates with direct binding between the GR DNA binding domain and the POU domain of the octamer factors. In present study, in vitro experiments identified several nuclear hormone receptors to have the potential to bind to the POU domains of Oct-1 and Oct-2 through their DNA binding domains, suggesting that POU domain binding may be a property shared by many nuclear hormone receptors. However, physiologically relevant binding to the POU domain appeared to be a property restricted to only a few nuclear receptors as only GR, progesterone receptor (PR), and androgen receptor (AR), were found to interact physically and functionally with Oct-1 and Oct-2 in transfected cells. Thus GR, PR, and AR efficiently promoted the recruitment of Oct-2 to adjacent octamer motifs in the cell, whereas mineralocorticoid receptor (MR), estrogen receptor alpha, and retinoid X receptor failed to facilitate octamer factor DNA binding. For MMTV, although GR and MR both induced transcription efficiently, mutation of the promoter proximal octamer motifs strongly decreased GR-induced transcription without affecting the total level of reporter gene activity in response to MR. These results suggest that the configuration of the hormone response element within the MMTV long terminal repeat may promote a dependence for the glucocorticoid response upon the recruitment of octamer transcription factors to their response elements within the viral promoter.

Animals↗

PubMed1996

Binding preferences of the POU domain protein Brain-4: implications for autoregulation.

The POU domain-containing transcription factor Brain-4 (Brn-4, RHS-2) was examined for its sites of expression and DNA binding preferences. In the rat, Brn-4 is expressed in 76 and 65% of vasopressin neurons in the paraventricular and supraoptic nuclei, respectively; but in only 10% of corticotropin-releasing factor neurons in the paraventricular nucleus of the hypothalamus. From these data we speculate that genes expressed within vasopressinergic neurons are more likely to be regulated by Brn-4 than those in corticotropin-releasing factor neurons. Random oligonucleotide site selection indicates Brn-4 prefers binding the DNA element CAATATGCTAAT and is inflexible in its spacing requirement between putative CAATAT and TAAT half sites, preferring 2 nucleotides between these elements. Electrophoretic mobility shift and DNase I footprinting analyses show five regions between nucleotides -457 and +22 of the Brn-4 promoter that are bound by Brn-4. Furthermore, Brn-4 can transactivate from this region of the Brn-4 promoter, suggesting that Brn-4 expression may be autoregulated.

Adenine↗

PubMed2004

Germline mutations of the POU6F2 gene in Wilms tumors with loss of heterozygosity on chromosome 7p14.

Wilms tumor (WT) is a kidney malignancy of childhood characterized by highly heterogeneous genetic alterations. We previously reported the molecular and cytogenetic characterization of a WT (Case 30) carrying an interstitial deletion in chromosome 7p14 between markers D7S555 and D7S668. Loss of heterozygosity (LOH) analyses had revealed that this same region was lost in 8 out of 38 examined WTs, suggesting that the identified interval contains a putative tumor suppressor gene. To confirm this hypothesis, in this work, we analyzed an additional 35 WTs, four of which showed LOH in the region of interest. Furthermore, we were able to more accurately define the extension of the deletion in Case 30, mapping it within an interval not exceeding 390 kb, proximally to D7S555. To date, only a single expressed gene, POU6F2 (the POU domain, class 6, transcription factor 2; also known as RPF1), has been recognized in this interval. Sequencing of the gene in the 12 WTs showing LOH and in a corresponding numbers of WT cases without LOH, led to the identification of two germline nucleotide substitutions. The first occurred in the 5'-untranslated region, while the second caused an amino acid change in a glutamine repeat domain. These mutations, whose occurrence was not observed in more than 100 control subjects, were detected in two patients showing the loss of the constitutionally wild-type allele in tumor DNA. Together with the finding of the expression of the POU6F2 mouse homolog in both fetal and adult kidney, our observations suggest that the gene is a tumor suppressor and is involved in hereditary predisposition to WT.

Alleles↗

PubMed1996

POU-domain gene expression during spermatogenesis.

Spermatogenesis is an orderly process whereby male germ cells pass through sequential phases of differentiation to develop into mature spermatozoa. At present, little is known about the factors that regulate this process. The POU domain represents a group of transcription-activator proteins with well-described roles in development. These proteins have been shown to regulate the tissue-specific expression of genes involved in cellular differentiation. The recent identification of Sperm-1, a testis-specific POU-domain protein expressed in primary spermatocytes just prior to the onset of meiosis, suggests that this protein may be involved in the regulation of male germ-cell differentiation. Continued evaluation of Sperm-1 and other related transcriptional activators may shed some light on the regulation of spermatogenesis.

Animals↗

PubMed1999

Reconstitution of the protein kinase A response of the rat prolactin promoter: differential effects of distinct Pit-1 isoforms and functional interaction with Oct-1.

PRL gene transcription is primarily regulated by dopamine, which lowers cAMP levels and inhibits protein kinase A (PKA) activity. Current data indicate that the cAMP/PKA response maps to the most proximal Pit-1/Pit-1beta binding site footprint I (FP I) on the rat PRL (rPRL) promoter. Pit-1, a POU-homeo domain transcription factor, is specifically expressed in the anterior pituitary and is required both for the normal development of anterior pituitary cell types, somatotrophs, lactotrophs, and thyrotrophs, and for the expression of their hormones: GH, PRL, and TSHbeta. Pit-1 has been shown to functionally interact, via FP I, with several transcription factors, including Oct-1, a ubiquitous homeobox protein, and thyrotroph embryonic factor, which is found in lactotrophs, to activate basal rPRL promoter activity. Pit-1beta/GHF-2, a distinct splice isoform of Pit-1, acts to inhibit Ras-activated transcription from the rPRL promoter, which is mediated by a functional interaction between Pit-1 and Ets-1 at the most distal Pit-1 binding site (FP IV). In this manuscript we show 1) that the Pit-1beta isoform not only fails to block PKA activation, but is, in fact, a superior mediator of the PKA response; 2) that the PKA response requires intact POU-specific and POU-homeo domains of Pit-1; and 3) that Oct-1, but not thyrotroph embryonic factor, functions as a Pit-1-interacting factor to mediate an optimal PKA response.

Animals↗

PubMed1991

Role of herpes simplex virus type 1 UL46 and UL47 in alpha TIF-mediated transcriptional induction: characterization of three viral deletion mutants.

The transcriptional induction of the alpha or immediate-early gene class of herpes simplex virus type 1 effected by the alpha trans-induction factor (alpha TIF, ICP25, VP16, Vmw65) requires an alpha-specific cis-acting site. Increased transcription does not result from the direct, independent binding of alpha TIF, but rather from an alpha TIF-dependent formation of a protein-DNA complex containing, in addition to alpha TIF, at least one host cell factor. One of the host factors is a POU domain protein which recognizes an octamer element in the alpha-specific consensus. There is evidence that alpha TIF may drive the formation of multiple protein-DNA complexes containing a POU protein and additional host factors. Previously, the gene products of UL46 and UL47 have been implicated in modulating the alpha TIF-dependent transcriptional induction of alpha genes. Our current studies have extended these analyses from a transient-expression system to a series of viral deletion mutants. In these studies we demonstrate that neither UL46- nor UL47-encoded gene product, either separately or in combination, is required for viral growth in cell culture. The absence of UL47 reduces by up to 80% the ability of the virus to induce an alpha-regulated thymidine kinase reporter gene resident in 143TK- cells. Autoradiograms of [35S]methionine pulse-labeled infected cell proteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, show that deleting UL46 and/or UL47 has no discernable effect on the synthesis of alpha TIF or alpha TIF-containing proteins. Subsequent Western immunoblot analysis, with rabbit anti-alpha TIF antibodies made to an alpha TIF-Staphylococcus aureus protein A fusion, demonstrated that the accumulation and steady-state levels of alpha TIF or alpha TIF-containing proteins was indistinguishable from that of the thymidine kinase-negative isogenic parental virus, R delta 305.

Animals↗