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Results for “POU Domain Factors”

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PubMed1995

A novel octamer regulatory element in the VH11 leader exon of B-1 cells.

B-1 cells (CD5 B cells) represent an initial fetal wave of B cell lymphopoiesis. B-1 cells have fundamental properties that are unique from conventional B cells, including a restricted Ab repertoire. We investigated the mechanism for the overrepresentation of one such Ig H chain variable-region gene, VH11, by murine B-1 cells. We postulated that a cis-regulatory element contributed to the use of VH11. We observed that the DNA encoding the leader peptide of VH11 was atypically A/T rich and thus was a candidate for nuclear protein binding. By electrophoretic mobility shift analysis, we found that the VH11 leader DNA specifically bound to three protein complexes present in the nucleus of the B-1 cell line AJ9. Of these bands, one was ubiquitous for all cells examined (lymphoid and nonlymphoid); another band was present only in B cells, and the third band was specific for B-1 cells that expressed VH11 or VH12. In addition to its binding properties, the VH11 leader sequence also displayed modest tissue-specific enhancer activity. By DNA footprint analysis, all three protein complexes were found to bind to an octamer motif embedded within the VH11 leader DNA. To identify the octamer-binding proteins, a panel of octamer-specific Abs was used. We found that the ubiquitous band was Oct-1, and the B cell-specific band was Oct-2. The B-1 cell-specific nuclear binding protein was neither Oct-1 nor Oct-2, but may be a novel POU domain protein. We hypothesize that the VH11 leader octamer site may target this gene for preferential rearrangement and/or expression and therefore would be a contributing factor in the increased use of this gene by B-1 cells.

Animals↗

PubMed1997

Luman, a new member of the CREB/ATF family, binds to herpes simplex virus VP16-associated host cellular factor.

The human host cell factor (HCF) is expressed in a variety of adult and fetal tissues, and its gene is conserved in animals as diverse as mammals and insects. However, its only known function is to stabilize the herpes simplex virus virion transactivator VP16 in a complex with the cellular POU domain protein Oct-1 and cis-acting regulatory elements in promoters of immediate-early viral genes. To identify a cellular function for HCF, we used the yeast two-hybrid system to identify a cellular ligand for HCF. This protein, Luman, appears to be a cyclic AMP response element (CRE)-binding protein/activating transcription factor 1 protein of the basic leucine zipper superfamily. It binds CREs in vitro and activates CRE-containing promoters when transfected into COS7 cells. This activation of transcription was synergistically enhanced by the presence of CCAAT/enhancer-binding protein elements and inhibited by AP-1 elements in the promoter. In addition to a basic DNA binding domain, Luman possesses an unusually long leucine zipper and an acidic amino-terminal activation domain. These features in Luman are also present in what appear to be homologs in the mouse, Drosophila melanogaster, and Caenorhabditis elegans. Luman and VP16 appear to have similar mechanisms for binding HCF, as in vitro each competitively inhibited the binding of the other to HCF. In transfected cells, however, while VP16 strongly inhibited the ability of GAL-Luman to activate transcription from a GAL4 upstream activation sequence-containing promoter, Luman was unable to inhibit the activity of GAL-VP16. Luman appears to be a ubiquitous transcription factor, and its mRNA was detected in all human adult and fetal tissues examined. The possible role of HCF in regulating the function of this ubiquitous transcription factor is discussed.

Adult↗

PubMed2002

Oct-1 potentiates CREB-driven cyclin D1 promoter activation via a phospho-CREB- and CREB binding protein-independent mechanism.

Cyclin D1, the regulatory subunit for mid-G(1) cyclin-dependent kinases, controls the expression of numerous cell cycle genes. A cyclic AMP-responsive element (CRE), located upstream of the cyclin D1 mRNA start site, integrates mitogenic signals that target the CRE-binding factor CREB, which can recruit the transcriptional coactivator CREB-binding protein (CBP). We describe an alternative mechanism for CREB-driven cyclin D1 induction that involves the ubiquitous POU domain protein Oct-1. In the breast cancer cell line MCF-7, overexpression of Oct-1 or its POU domain strongly increases transcriptional activation of cyclin D1 and GAL4 reporter genes that is specifically dependent upon CREB but independent of Oct-1 DNA binding. Gel retardation and chromatin immunoprecipitation assays confirm that POU forms a complex with CREB bound to the cyclin D1 CRE. In solution, CREB interaction with POU requires the CREB Q2 domain and, notably, occurs with CREB that is not phosphorylated on Ser 133. Accordingly, Oct-1 also potently enhances transcriptional activation mediated by a Ser133Ala CREB mutant. Oct-1/CREB synergy is not diminished by the adenovirus E1A 12S protein, a repressor of CBP coactivator function. In contrast, E1A strongly represses CBP-enhanced transactivation by CREB phosphorylated on Ser 133. Our observation that Oct-1 potentiates CREB-dependent cyclin D1 transcriptional activity independently of Ser 133 phosphorylation and E1A-sensitive coactivator function offers a new paradigm for the regulation of cyclin D1 induction by proliferative signals.

Breast Neoplasms↗

PubMed2006

Regulation of the immunoglobulin heavy chain locus expression at the phylogenetic level of a bony fish: transcription factor interaction with two variant octamer motifs.

Transcriptional control of the IGH locus in teleosts is not fully understood, but evidence from catfish and zebrafish indicates major roles for octamer-binding (Oct) and E-protein transcription factors. A pair of variant octamer motifs in the Emu3' enhancer of the catfish has been shown to be particularly important in driving expression, justifying detailed study of their function. These octamer motifs were examined to determine if they bound Oct2 POU domains in monomeric or dimeric (PORE and MORE) configurations. While catfish Oct2 was shown to be capable of binding PORE and MORE motifs in dimeric conformation, the two octamer motifs in Emu3' bound Oct2 POU domains only in monomeric configuration. Catfish Oct2, when bound in this monomeric conformation, was shown to bend the DNA helix. The relative position of the two octamer motifs in Emu3' affected the activity of the enhancer, and moving the octamer motifs closer together by 5 bp greatly reduced the activity of the enhancer. This effect was not due to steric hindrance preventing the binding of Oct transcription factors to the two motifs, but rather was shown to be due to the disruption of an additional transcription factor binding site lying between the two octamer motifs.

Animals↗

PubMed1996

Identification of new genes expressed in a human erythroleukemia cell line.

We cloned novel cDNAs from MB02 human erythroleukemia cells using PCR based approaches: a) Differential display by means of RT-PCR using one 5' primer CTTGATTGCC and four different 3' primers (T12AA, T12CA, T12GA, and T12AT). Ninety-three percent of the differential clones which were reamplified and sequenced were cDNAs of previously unidentified genes. b) Cloning using degenerate TFIIIA-like zinc finger domain specific oligonucleotide. Of the 54 clones sequenced, 20 contained two or more zinc finger sequences. Ten of these clones were new zinc finger cDNAs and one belonged to a known zinc finger gene (ZFP7). c) Cloning using degenerate tyrosine kinase(TK) domain-specific oligonucleotides corresponding to the highly conserved amino acid sequences IHRDLAA and DVWSFG. Of the 28 cDNA clones sequenced, 7 were JAK1 TK, one was atk TK, one was tec TK. The remaining sequences belonged to new ESTs or to ribosomal genes. d) Cloning using degenerate POU domain-specific oligonucleotides corresponding to sequence FK(QV)RRIK of the POU-specific domain and to sequence VWFCN(RQ)R of the POU-homeodomain. Sixteen clones were sequenced and all but one were identical with the Oct-1 transcriptional factor. Differential display RT-PCR and PCR-based cDNA cloning using degenerate primers for zinc finger motifs yielded the largest number of new genes expressed in MBO2 cells.

Amino Acid Sequence↗

PubMed1995

Comparative analysis of mouse NotI linking clones with mouse and human genomic sequences and transcripts.

NotI cleavage sites are frequently associated with CpG islands that identify the 5' regulatory sites of functional genes in the genome. Therefore we analyzed a sample of 22 NotI linking clones prepared from mouse brain DNA, to determine whether these mouse NotI site associated clones could be used for comparative analysis of mouse and human genomes by cross-reaction with both mouse and human genomic DNA and RNA in Southern and Northern hybridization. We further examined whether we could establish the identity of these clones with known genes by comparing the nucleotide sequences surrounding the NotI site with the GenBank database. We observed that 70% of the clones cross-hybridized with human DNA and that 4 of 11 tested clones (36%) detected a transcript in human HeLa cells RNA whereas 73% clones (8/11) detected transcripts in mouse RNAs from one or more organs. Single pass sequence analysis was successful on 16 of 19 clones. The GC content in these sequence was very high (48.8% to 73.8%) suggesting that 12 of 16 sequenced clones contained a CpG island. Three out of 19 clones showed significant similarity with previously analyzed mouse gene sequences in GenBank, including the mouse rRNA gene family, cathepsin and the scip POU-domain genes. In addition, two sequences showed significant similarity to the human and rabbit protein phosphatase 2A-beta subunit and the human transforming growth factor-beta. Thus, 5 of 16 clones showed homology with identified genes. These results and the recent work of using RLGS methods for genetic mapping indicate that NotI linking clones can be used to efficiently cross reference a comparative analysis of the mouse and human genomic maps.

Animals↗

PubMed1997

The Brn-3a transcription factor induces neuronal process outgrowth and the coordinate expression of genes encoding synaptic proteins.

The Brn-3a POU family transcription factor is expressed only in posmitotic neurons in the central nervous system and identifies the first differentiated neurons to appear in the midbrain, hindbrain, and spinal cord during development. This factor is also induced when undifferentiated proliferating ND7 cells cease dividing and differentiate to a mature neuronal-like phenotype bearing numerous neurite processes. We show that overexpression of Brn-3a in undifferentiated ND7 cells induces a mature neuronal phenotype characterized by process outgrowth and the induction of genes encoding synaptic proteins, although the cells continue to proliferate. In contrast, the closely related factors Brn-3b and Brn-3c do not have this effect. Although the N-terminal activation domain of Brn-3a is required for maximum induction of neurite outgrowth and gene expression, these effects are primarily dependent on the DNA binding POU domain, which also acts as an activation domain. Overexpression of the isolated POU domain of Brn-3a is sufficient to induce neurite outgrowth, while the ability of full-length Brn-3a to do so is abolished by mutating a single amino acid in the Brn-3a POU homeodomain to its equivalent in Brn-3b. Thus, Brn-3a appears to play a critical role in the specification of the mature neuronal phenotype, acting by stimulating the expression of genes whose products are required for process outgrowth and synapse formation.

Animals↗

PubMed1994

Two independent pathways for transcription from the MMTV promoter.

The influence of progesterone receptor (PR) and glucocorticoid receptor (GR) on transcription from the mouse mammary tumour virus (MMTV) promoter was analyzed using cell-free transcription of DNA templates with a G-free cassette. Preincubation of the templates with either PR or GR stimulates the rate of transcription initiation 10-50 fold, whereas the recombinant DNA binding domain of GR is inactive. Mutations that inactivate the nuclear factor I (NFI) binding site, or NFI depletion of the nuclear extract, decrease basal transcription without influencing receptor-dependent induction. Recombinant NFI, but not its DNA-binding domain, restores efficient basal transcription of the depleted extract. Recombinant OTF1 or OTF2, but not the POU domain of OTF1, enhance MMTV transcription independently of NF1. In agreement with this finding, NFI and OTF1 do not cooperate, but rather compete for binding to the wild type MMTV promoter, though they have the potential to bind simultaneously to properly oriented sites. Our results imply the existence of two independent pathways for MMTV transcription: one initiated by NFI and the other dependent on octamer transcription factors. Only the second pathway is stimulated by steroid hormone receptors in vitro.

Base Sequence↗

PubMed1993

Specific transcriptional activation in vitro by the herpes simplex virus protein VP16.

The herpes simplex virus protein VP16 interacts with cellular factors, including the protein Oct-1, to activate viral immediate early (IE) gene transcription. We have reproduced this effect by addition of purified, full-length VP16 and the DNA-binding 'POU' domain of Oct-1 (Oct-1/POU) to a HeLa cell in vitro transcription system. Stimulation of transcription was dependent on the IE-specific element, TAATGARAT. In agreement with earlier observations from electrophoretic mobility shift assays, activation was not observed when Oct-2/POU, the DNA-binding domain from the Oct-2 protein, was substituted for Oct-1/POU. Single round transcription assays revealed that, together, VP16 and Oct-1/POU facilitate the assembly of pre-initiation complexes at target gene promoters.

Base Sequence↗

PubMed2001

Combined pituitary hormone deficiency due to the F135C human Pit-1 (pituitary-specific factor 1) gene mutation: functional and structural correlates.

The pituitary-specific transcription factor Pit-1 (pituitary-specific factor 1) is known to play a key role in the differentiation of PRL-, GH-, and TSH-secreting cells, and in the regulation of expression of the corresponding genes. In recent years, 12 distinct mutations of the Pit-1 gene have been shown to be responsible for a phenotype of multiple congenital pituitary hormone deficiency involving PRL, GH, and TSH. We had previously identified, in four siblings with GH, PRL, and TSH deficiencies, a mutation (F135C) resulting in a single amino acid change within the POU-specific binding domain of the Pit-1 molecule. In the present report, we have explored the functional effect of the F135C mutation. In vitro activity tests performed by transfection in human HeLa cells showed decreased transactivation capacity on the PRL, GH, and Pit-1 genes. The DNA binding experiments performed by gel shift showed that the F135C mutation generated a protein capable of binding to DNA response elements. To analyze how the F135C mutation might affect functionality of the transcription factor despite a normal DNA binding, we used a structure modelization approach and also analyzed two other Pit-1 mutant proteins (F135A and F135Y). The loss of functionality in these two mutants was similar to that of F135C. This finding was in keeping with our molecular modeling studies. According to structural data derived from the crystallographic analysis of the DNA/Pit-1 POU domain complex, the conformation of the first helix of the F135C-mutated POU-specific domain could be perturbed to such an extent that any interaction with other transcription cofactors might be definitively prevented.

Amino Acid Sequence↗

PubMed1995

OBF-1, a novel B cell-specific coactivator that stimulates immunoglobulin promoter activity through association with octamer-binding proteins.

Recent biochemical and genetic studies indicate that in addition to the octamer-binding proteins Oct-1 and Oct-2, other B cell components are required for lymphoid-restricted, octamer site-mediated immunoglobulin gene promoter activity. Using a genetic screen in yeast, we have isolated B cell-derived cDNAs encoding Oct-binding factor 1 (OBF-1), a novel protein that specifically associates with Oct-1 and Oct-2. Biochemical studies demonstrate that OBF-1 has no intrinsic DNA-binding activity and recognizes the POU domains of Oct-1 and Oct-2, but not those of Oct-4 and Oct-6. The OBF-1 mRNA is expressed in a highly cell-specific manner, being most abundant in B cells and essentially absent in most of the other cells or tissues tested. Furthermore, expression of OBF-1 in HeLa cells selectively stimulates the activity of a natural immunoglobulin promoter in an octamer site-dependent manner. Thus, OBF-1 has all the properties expected for a B cell-specific transcriptional coactivator protein.

Amino Acid Sequence↗

PubMed1992

Mutations in the Pit-1 gene in children with combined pituitary hormone deficiency.

Pit-1 is a pituitary-specific transcription factor that binds to and transactivates promoters of growth hormone and prolactin genes. In three unrelated Japanese children with combined pituitary hormone deficiency, we identified three point mutations in the Pit-1 gene, Pro24Leu, Arg143Gln, and Arg271Trp, located on the major transactivation region, POU-specific domain, and POU-homeodomain, respectively.

Amino Acid Sequence↗

PubMed2001

Atypical binding of the neuronal POU protein N-Oct3 to noncanonical DNA targets. Implications for heterodimerization with HNF-3 beta.

The capacity of POU proteins to recognize different DNA sequences and to bind target DNA in the form of monomers, cooperative dimers or heterodimers is important in relation to their transcriptional regulatory properties. The N-Oct3 neuron-specific protein binds to an octamer-like sequence (AAATAATGC) within the (-102/-72) neuronal promoter region of the human aromatic L-amino acid decarboxylase (AADC) gene. In this atypical case the POUh and POUs tetrameric subsites are spaced one nucleotide apart and in switched order as compared with the consensus octamer. Moreover this POU binding motif overlaps the hepatocyte nuclear factor HNF-3 beta binding site (TGCTCAGTAAA) which itself contains a heptamer-like sequence (CTCAGTA). Using the isolated DNA binding domains (DBD) of the two proteins, it is shown that, when binding to this unusual recognition sequence, N-Oct3 either exhibits noncooperative homodimerization or allows the simultaneous binding of the second transcription activator HNF-3 beta. CD studies indicate that the binding of N-Oct3 monomers/dimers and N-Oct3-HNF-3 beta heterodimers to the DNA induces conformational changes of both protein and DNA. Partial proteolysis/MALDI-MS was used in conjunction with molecular modelling to show that the protein conformational change resulting from binary N-Oct3/DNA complex formation occurs within the linker peptide joining the POUs and POUh subdomains. Furthermore, modelling the N-Oct3/HNF-3 beta/DNA ternary complex predicts a nucleotide rearrangement in the overlap region and an interaction between both transcription factors. In the light of our findings, which illustrate both site-dependent and site-independent protein and DNA conformational changes, general implications for the allosteric function of DNA response elements in transcriptional regulation are discussed.

Allosteric Site↗

PubMed1993

Developmental regulation and tissue distribution of the liver transcription factor LFB1 (HNF1) in Xenopus laevis.

The transcription factor LFB1 (HNF1) was initially identified as a regulator of liver-specific gene expression in mammals. It interacts with the promoter element HP1, which is functionally conserved between mammals and amphibians, suggesting that a homologous factor, XLFB1, also exists in Xenopus laevis. To study the role of LFB1 in early development, we isolated two groups of cDNAs coding for this factor from a Xenopus liver cDNA library by using a rat LFB1 cDNA probe. A comparison of the primary structures of the Xenopus and mammalian proteins shows that the myosin-like dimerization helix, the POU-A-related domain, the homeo-domain-related region, and the serine/threonine-rich activation domain are conserved between X. laevis and mammals, suggesting that all these features typical for LFB1 are essential for function. Using monoclonal antibodies, we demonstrate that XLFB1 is present not only in the liver but also in the stomach, intestine, colon, and kidney. In an analysis of the expression of XLFB1 in the developing Xenopus embryo, XLFB1 transcripts appear at the gastrula stage. The XLFB1 protein can be identified in regions of the embryo in which the liver diverticulum, stomach, gut, and pronephros are localized. The early appearance of XLFB1 expression during embryogenesis suggests that the tissue-specific transcription factor XLFB1 is involved in the determination and/or differentiation of specific cell types during organogenesis.

Amino Acid Sequence↗

PubMed2005

EWSR1 is fused to POU5F1 in a bone tumor with translocation t(6;22)(p21;q12).

POU5F1(OCT3/4) is a sequence-specific transcription factor that is essential for keeping germ cells and embryonic stem cells in an immature and pluripotent status. In this article, we report that POU5F1 was fused to EWSR1 in a case of undifferentiated sarcoma derived from pelvic bone with chromosomal translocation t(6;22)(p21;q12). The EWSR1-POU5F1 chimera consists of exons 1-6 of EWSR1 and exons 2-5 and a part of exon 1 of POU5F1. The predicted amino acid sequence indicates that the chimera is composed of the N-terminal QSY domain of EWS that functions as a transcriptional activation domain and the C-terminal POU DNA-binding domains derived from POU5F1. The t(6;22) tumor does not belong to any known categories of bone and soft-tissue tumors (BSTs). It is suggested that EWS-POU5F1 may act as an oncogenic transcription factor and that its expression may contribute to undifferentiated and immature phenotypes of BST.

Artificial Gene Fusion↗

PubMed2005

Solution structure and DNA-binding mode of the matrix attachment region-binding domain of the transcription factor SATB1 that regulates the T-cell maturation.

SATB1 is a transcriptional regulator controlling the gene expression that is essential in the maturation of the immune T-cell. SATB1 binds to the nuclear matrix attachment regions of DNA, where it recruits histone deacetylase and represses transcription through a local chromatin remodeling. Here we determined the solution structure of the matrix attachment region-binding domain, possessing similarity to the CUT DNA-binding domain, of human SATB1 by NMR spectroscopy. The structure consists of five alpha-helices, in which the N-terminal four are arranged similarly to the four-helix structure of the CUT domain of hepatocyte nuclear factor 6alpha. By an NMR chemical shift perturbation analysis and by surface plasmon resonance analyses of SATB1 mutant proteins, an interface for DNA binding was revealed to be located at the third helix and the surrounding regions. Surface plasmon resonance experiments using groove-specific binding drugs and methylated DNAs indicated that the domain recognizes DNA from the major groove side. These observations suggested that SATB1 possesses a DNA-binding mode similar to that of the POU-specific DNA-binding domain, which is known to share structural similarity to the four-helix CUT domain.

Amino Acid Sequence↗

PubMed1992

Trans-dominant inhibition of transcription activator LFB1.

Liver-enriched factor LFB1 (also named HNF1) is a dimeric transcription activator which is essential for the expression of many hepatocyte-specific genes. Here we demonstrate that LFB1 mutants in the POU A-like or in the homeo domains inhibit wild-type DNA binding by forming inactive heterodimeric complexes. Co-transfection of one of these mutants with wild-type LFB1 in HeLa cells eliminated LFB1 DNA binding and transcriptional activities through a trans-dominant mechanism. Expression of the same dominant negative mutant in human hepatoma HepG2 cells only partially inhibited endogenous LFB1 activity, due to stabilization of LFB1 dimers in these cells. Dimer stabilization in hepatoma cells is mediated by a heat-labile association with an 11kD polypeptide, analogous to the DCoH cofactor identified in rat liver by Mendel et al. (1). The property of stabilizing LFB1 dimers is also shared by HeLa cells which produce a HeLa homolog of DCoH. These results demonstrate that LFB1 dimer stabilization as well as the synthesis of 'stabilizing factors' are not restricted to cells expressing LFB1 or other members of its family.

Binding Sites↗

PubMed1999

HCF-dependent nuclear import of VP16.

Transactivation by VP16 requires the formation of a multicomponent complex, the TAATGAAAT recognition factor complex (TRF.C), that contains in addition to VP16, two cellular proteins, Oct-1 and HCF. HCF binds directly to VP16 and this promotes subsequent interaction of the VP16-HCF complex with the POU DNA-binding domain of Oct-1 and selective assembly onto target sites. Here we demonstrate a novel role of HCF in the intracellular compartmentalization of VP16. We show that while VP16 does not contain a consensus nuclear localization signal (NLS) and is largely cytoplasmic, co-expression with HCF resulted in VP16 nuclear accumulation. A candidate NLS within the C-terminus of HCF was identified and insertion of this motif into green fluorescent protein (GFP) promoted nuclear accumulation. Conversely, removal of this signal from HCF (HCFDeltaNLS) resulted in its cytoplasmic accumulation. Co-expression of HCFDeltaNLS with wild-type (wt) VP16, or of wt HCF with VP16 mutants lacking HCF-binding activity failed to promote the nuclear enrichment of VP16. These results indicate that in addition to its role in stabilizing TRF.C, HCF acts as a nuclear import factor for VP16.

Amino Acid Sequence↗