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At least 19 recordsLinked to original sources
PubMed1992

A novel B cell-derived coactivator potentiates the activation of immunoglobulin promoters by octamer-binding transcription factors.

A novel B cell-restricted activity, required for high levels of octamer/Oct-dependent transcription from an immunoglobulin heavy chain (IgH) promoter, was detected in an in vitro system consisting of HeLa cell-derived extracts complemented with fractionated B cell nuclear proteins. The factor responsible for this activity was designated Oct coactivator from B cells (OCA-B). OCA-B stimulates the transcription from an IgH promoter in conjunction with either Oct-1 or Oct-2 but shows no significant effect on the octamer/Oct-dependent transcription of the ubiquitously expressed histone H2B promoter and the transcription of USF- and Sp1-regulated promoters. Taken together, our results suggest that OCA-B is a tissue-, promoter-, and factor-specific coactivator and that OCA-B may be a major determinant for B cell-specific activation of immunoglobulin promoters. In light of the evidence showing physical and functional interactions between Oct factors and OCA-B, we propose a mechanism of action for OCA-B and discuss the implications of OCA-B for the transcriptional regulation of other tissue-specific promoters.

B-Lymphocytes↗
PubMed1992

Recognition of the surface of a homeo domain protein.

Homeo domain proteins exhibit distinct biological functions with specificities that cannot be predicted by their sequence specificities for binding DNA. Recognition of the surface of the Oct-1 POU homeo domain provides a general model for the contribution of selective protein-protein interactions to the functional specificity of the homeo domain family of factors. The assembly of Oct-1 into a multiprotein complex on the herpes simplex virus alpha/IE enhancer is specified by the interactions of its homeo domain with ancillary factors. This complex (C1 complex) is composed of the viral alpha TIF protein (VP16), Oct-1, and one additional cellular component, the C1 factor. Variants of the Oct-1 POU homeo domain were generated by site-directed mutagenesis, which altered the residues predicted to form the exposed surface of the domain-DNA complex. Proteins with single amino acid substitutions on the surface of either helix 1 or 2 of the Oct-1 POU homeo domain had decreased abilities to form the C1 complex. The behavior of these mutants in a cooperative DNA-binding assay with alpha TIF suggested that the Oct-1 POU homeo domain is principally recognized by alpha TIF in the C1 complex. The preferential recognition of Oct-1 over the closely related Oct-2 protein is critically influenced by a single residue on the surface of helix 1 because the introduction of this residue into the Oct-2 POU homeo domain significantly enhanced its ability to form a C1 complex.

Amino Acid Sequence↗
PubMed1992

A single amino acid exchange transfers VP16-induced positive control from the Oct-1 to the Oct-2 homeo domain.

The selective association of the herpesvirus trans-activator VP16 with the human Oct-1 homeo domain is a model for differential positive transcriptional control by homeo domains. VP16 discriminates between the closely related homeo domains of Oct-1 and Oct-2 by distinguishing among their seven amino-acid differences; these differences lie on the surface that is thought to be accessible when the homeo domain is bound to DNA. Only two of these seven differences are recognized by VP16, one in each of the first two alpha-helices of the tri-alpha-helical homeo domain. The major determinant for selective association with VP16 in vitro and VP16-induced positive control in vivo is a single glutamic acid residue at position 22 in the first alpha-helix of the Oct-1 homeo domain, but the acidic properties of this residue are not critical for association with VP16 in vitro or in vivo, because it can be replaced by glutamine with little or no deleterious effect. Mere replacement of the single corresponding alanine residue in the Oct-2 homeo domain with the key glutamic acid residue is sufficient to confer on the Oct-2 homeo domain the ability to associate with VP16 in vitro and respond to VP16-induced positive control in vivo. Thus, the specificity of homeo domain positive control can be conferred by a single amino acid difference.

Amino Acid Sequence↗
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↗
PubMed1992

Identification of sites for distinct DNA binding proteins including Oct-1 and Oct-2 in the Cr2 gene.

The murine Cr2 gene produces two distinct products in a variety of murine cell types. Both of these transcripts appear to initiate from the same position within the gene but vary from one another via an alternative splicing event within the coding exons. An analysis of those gene sequences that might control the cell specific expression of the Cr2 gene has identified a region of Cr2 5' of the transcription start site that is conserved in both the murine Cr2 and human CR2 genes. When this region was examined using the gel shift assay with nuclear extracts from cells expressing Cr2 (B cells) and those that do not (T cells and fibroblasts), at least four distinct proteins were identified that bound to at least three distinct sites. The DNA sequence recognized by two of these proteins is the octamer sequence recognized by a family of transcriptional regulators including the B cell specific Oct-2 protein. During an acute bacterial infection, the levels of Oct-2 and Cr2 mRNA are both depressed. This suggests that the Oct-2 protein directly controls the transcriptional activity of the Cr2 gene and that during such an infection, the levels of Ag receptors on B cells (Ig and complement receptors) are diminished.

Animals↗
PubMed1992

Characterization of a high affinity octamer transcription factor binding site in the human lipoprotein lipase promoter.

A high affinity octamer transcription factor (OTF-1) binding site has been identified and characterized at position--46 base pairs (bp) in the proximal human lipoprotein lipase (LPL) promoter. The affinity of the LPL OTF-1 binding site was approximately 15-fold greater than a consensus octamer sequence, ATTTGCAT, present at position--66 bp in the mouse Vk T1 promoter, and approximately 5-fold greater than the OTF-1 site present at position--49 bp in the human histone H2B promoter. Diethylpyrocarbonate interference assays have identified both 5' and 3' adenine nucleotides, which flank the core LPL ATTTGCAT sequence and interfere with OTF-1 binding when chemically modified. Introduction of mutations in either 5' or 3' flanking AT-rich sequences lowered the affinity of OTF-1 binding below the level observed with the wild-type LPL octamer oligomer. A double mutation in both flanking AT regions, however, greatly reduced the affinity of this site to levels similar to that observed with the mouse Vk T1 OTF site. An additional nuclear transcription factor, NF-Y, has been shown to bind to a functional CCAAT box motif located at -65 bp in the LPL promoter using specific alpha-NF-Y antisera. The observation of high affinity OTF-1 and NF-Y binding sites in a region of the proximal LPL promoter which is necessary for high levels of LPL transcription suggests that these sites with their associated proteins play important functional roles in the transcriptional activation of the LPL promoter during adipocyte differentiation.

3T3 Cells↗
PubMed1992

Protein-DNA interactions in the 5'-flanking region of the bovine pancreatic ribonuclease gene.

In the 5'-flanking region of the bovine pancreatic ribonuclease gene a sequence has been identified which specifically binds one or more factors present in nuclear protein extracts prepared from bovine pancreas. The binding site, as delineated by footprinting analysis, is located in a region extending from positions -113 to -146 relative to the transcription initiation site of the ribonuclease gene. This region contains consensus sequences for known control transcriptional elements. The observed pattern of protein-DNA interactions is likely to be pancreas-specific as it could not be detected with nuclear extracts prepared from HeLa or bovine aorta endothelium cells.

Animals↗
PubMed1992

Determination of transcription factor isoelectric point by two-dimensional native isoelectric focusing and electrophoretic mobility shift analysis.

A rapid, sensitive, and inexpensive assay is described for the determination of isoelectric points of native transcription factors derived from cell nuclei. This assay depends on a transcription factors' ability to bind DNA with high specificity and obviates the need for specific antisera or additional detection methods in identifying a particular protein following isoelectric focusing. This method has been applied to two ubiquitous proteins, the octamer transcription factor, Oct-1, and the multisubunit CCAAT box factor, NF-Y. Isoelectric points have been determined under native conditions using conventional isoelectric focusing in the first dimension, followed by binding to a specific oligonucleotide DNA probe, and separation of specific DNA/protein complexes from unbound DNA in the second dimension using native polyacrylamide gel electrophoresis. Oct-1 from HeLa cell nuclei was shown to have a pI of 9.6, while NF-Y was shown to have a pI of 4.5. This method is applicable to any transcription factor which binds DNA specifically and may be used to identify changes in surface charge characteristics which occur as a result of alternative splicing events and/or following transcription factor post-translational modification(s).

Base Sequence↗
PubMed1992

Candidate proto-oncogene bcl-3 encodes a subunit-specific inhibitor of transcription factor NF-kappa B.

The NF-kappa B subunits p50 and p65 and the product of the rel proto-oncogene are members of a growing class of transcription factors with a unique DNA-binding and dimerization domain. Nuclear transfer of each of these factors is controlled by cytoplasmic inhibitors, and regulated by specific stimuli. The inhibitors I kappa B-alpha and -beta and pp40 recognize either p65 or the c-rel protein. We show here that the proto-oncogene bcl-3, believed to be involved in certain human B-cell leukaemias, encodes a protein that functions as an I kappa B-like molecule for native NF-kappa B but is specific for the p50 subunit. The ankyrin repeat domain of the bcl-3 product is shown to mediate complex formation with NF-kappa B dimers by contracting the conserved dimerization domain of NF-kappa B.

B-Cell Lymphoma 3 Protein↗
PubMed1992

Ethidium bromide provides a simple tool for identifying genuine DNA-independent protein associations.

DNA-dependent and DNA-independent associations of DNA-binding proteins are important in transcriptional regulation. The analysis of DNA-independent associations frequently relies on assaying protein interaction in the absence of target DNA sequences. We have found that contaminating DNA in protein preparations can stabilize DNA-dependent associations that may appear DNA-independent. Three cellular proteins of 70, 85, and 110 kDa coimmunoprecipitated with the octamer motif-binding protein Oct-2 because of the presence of contaminating DNA in the cell extracts. In addition, heterodimer formation between Oct-1 (or Oct-2) and Pit-1 during protein-affinity chromatography was stabilized by the contaminating DNA. In both instances, these DNA-dependent protein associations were selectively inhibited by ethidium bromide in the precipitation reaction without any evident effect on DNA-independent protein associations. Thus, ethidium bromide may serve as a simple and general indicator of DNA-dependent and DNA-independent protein associations.

Animals↗
PubMed1992

In vivo footprint analysis of the HLA-DRA gene promoter: cell-specific interaction at the octamer site and up-regulation of X box binding by interferon gamma.

Analysis of the major histocompatibility complex class II gene promoter DRA has previously identified at least five cis-acting regions required for maximal expression. We have examined the DRA promoter for protein-DNA interactions in the intact cell, which may mediate transcriptional activation. Using in vivo genomic footprinting we identified interactions in B-cell lines at the octamer site and the Y, X1, and X2 boxes. Class II antigen expressing T-cell lines maintained contacts identical to B-cell lines, while class II-negative T-cell lines exhibited no interactions. In lymphoid cell lines, the octamer site is occupied and required for maximal expression. This is most likely due to the presence of the lymphoid-specific OTF-2 factor. In contrast, the class II-positive nonlymphoid glioblastoma cell line does not exhibit interactions at the octamer site despite the presence of the ubiquitous OTF-1 factor and an open binding site. Thus, the DRA promoter discriminates against OTF-1 activation at the level of DNA binding in the glioblastoma line. Interferon gamma induces class II expression in this glioblastoma cell line and, in parallel, up-regulates X1 and X2 box protein-DNA interactions, while all other interactions remain unchanged. These results suggest that interferon gamma functions on a poised promoter by altering weak, nonproductive interactions at the X boxes to strong interactions. These findings provide direct in vivo evidence to strongly suggest that the modulation of X1 and X2 interactions is an important constituent of the interferon gamma induction pathway.

B-Lymphocytes↗
PubMed1991

The constitutively expressed octamer binding protein OTF-1 and a novel octamer binding protein expressed specifically in cervical cells bind to an octamer-related sequence in the human papillomavirus 16 enhancer.

A novel octamer binding protein expressed specifically in cervical cells but not in other cell types has been identified. This protein differs in size and sequence specificity from the constitutively expressed octamer binding protein OTF-1. In particular it binds with higher affinity to a sequence in the human papillomavirus 16 (HPV) upstream regulatory region which has a seven out of eight base pair match compared to the consensus octamer motif. This is the first example of a tissue specific protein which has been observed to bind to the papillomavirus enhancer. The possible role of this protein in producing the observed tissue specific activity of the enhancer and in cervical carcinogenesis induced by HPV is discussed.

Base Sequence↗
PubMed1992

Multiple octamer binding sites in the promoter region of the bovine alpha s2-casein gene.

Using a set of overlapping oligonucleotides from the promoter region of the bovine alpha s2-casein gene we have identified two nuclear factors which probably are involved in expression of this gene and the related calcium sensitive alpha s1- and beta-casein genes. One of these factors which was present in extracts of all tissues that have been tested including Hela cells turned out to be the octamer binding protein OCT-1. Oct-1 binds with different affinity to 4 sites at positions centred around -480, -260, -210 and -50. The strongest of these 4 binding sites, the one around position -50, is highly conserved in all calcium sensitive caseins of mouse, rat, rabbit and cattle. The other nuclear factor (MGF, mammary gland factor) which is specifically expressed in the mammary gland, binds to a site around position -90. This binding site is also highly conserved in all calcium sensitive caseins of mouse, rat, rabbit and cattle.

Animals↗
PubMed1992

Evidence that nucleosomes on the mouse mammary tumor virus promoter adopt specific translational positions.

We have previously demonstrated that an array of six nucleosomes are phased on the mouse mammary tumor virus (MMTV) long terminal repeat (1,2). In this study, we devised a new assay to measure the translational positions of specific nucleosomes on the MMTV promoter. Nucleosome core particles were purified and shown to contain A and B nucleosomal DNA by Taq polymerase primer extension with nucleosome-specific primers. The 5' and 3' boundaries of A and B nucleosomes were measured by extending to the end of the core DNA with internal primers. This approach yielded results consistent with major translational positions of -23 to +123 and -221 to -75 for A and B nucleosomes, respectively. The micrococcal nuclease cleavage patterns of A and B nucleosome regions in isolated nuclei are conserved at base-pair resolution in multiple murine cell lines containing either stable MMTV-reporter chimeras or endogenous proviruses. As the refined nucleosome positions place important transcription factor binding sites at the 3' edge of the B nucleosome and in the nucleosome A/B linker, we propose that linker histone depletion and chromatin unfolding may be required to expose these cis-elements during steroid hormone-induced transcription initiation.

Animals↗
PubMed1991

Repression of the human papillomavirus type 18 enhancer by the cellular transcription factor Oct-1.

The role of cellular factors involved in the transcriptional regulation of the cancer-associated human papillomavirus type 18 (HPV18) is yet poorly understood. The presence of an Oct-1-binding site within the HPV18 upstream regulatory region led us to investigate the influence of Oct-1 on viral transcription. Cotransfection of Oct-1 expression plasmids together with luciferase reporter constructs containing HPV18 regulatory sequences indicated that Oct-1 can transcriptionally repress the HPV18 upstream regulatory region. In contrast, heterologous control regions were not affected by Oct-1. HPV18 cis elements that can be repressed by Oct-1 mapped to a 135-bp subregion of the viral constitutive enhancer. Analysis of an Oct-1 mutant defective in DNA binding suggested that HPV18 down-modulation does not require direct binding of Oct-1 to DNA. These results make Oct-1 a candidate factor involved in the intracellular surveillance of HPV18 transcription and support the notion of a host cell mechanism that can specifically repress HPV E6-E7 transforming gene expression.

DNA-Binding Proteins↗
PubMed1991

trans activation of the simian virus 40 late promoter by large T antigen requires binding sites for the cellular transcription factor TEF-1.

Simian virus 40 (SV40) T antigen stimulates the level of transcription from several RNA polymerase II promoters, including the SV40 late promoter. The mechanism of trans activation appears to be indirect since binding of T antigen to specific DNA sequences is not required. However, specific promoter elements that respond to T antigen have not previously been defined. We identified DNA sequences from the SV40 late promoter whose ability to stimulate transcription is induced by the expression of T antigen. In particular, the Sph I + II motifs of the SV40 enhancer can confer T-antigen inducibility to the normally uninducible herpes simplex virus thymidine kinase gene promoter when multiple copies of the sequence are inserted 5' of the transcription initiation site and TATA sequence. Binding sites for the cellular transcription factor TEF-1 and octamer binding proteins are contained within the Sph I + II motifs, as well as at other positions in the SV40 promoter. To study the role of individual protein-binding sites in trans activation by T antigen, mutations were constructed in various TEF-1 and octamer protein-binding sites of the SV40 late promoter. These mutations did not significantly affect basal promoter activity. However, mutation of all three TEF-1 sites prevented detectable activation by T antigen. DNase I footprinting of the mutated promoters with purified proteins demonstrated that inducibility by T antigen correlated with binding affinity of TEF-1 for the DNA and not with binding affinity of an octamer binding protein.

Animals↗