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A Belayew

Publications and source records attributed to A Belayew.

At least 19 recordsLinked to original sources
PubMed1998

Characterization of a double homeodomain protein (DUX1) encoded by a cDNA homologous to 3.3 kb dispersed repeated elements.

Target genes for the helicase-like transcription factor (HLTF), a member of the SNF/SWI family, were immunoprecipitated from HeLa chromatin fragments with an anti-HLTF antibody. A 182 bp fragment ( HEFT1 ) presented 87% sequence identity with 3.3 kb dispersed repeats from the 4q35 D4Z4 locus linked to facioscapulohumeral muscular dystrophy (FSHD). The HEFT1 loci were, however, not genetically linked to FSHD. Transfection and in vitro binding studies identified within HEFT1 a promoter whose basal activity required a GC box activated by Sp1 or Sp3. A 4.4 kb homologous transcript was found mostly in human skeletal muscle and heart. A 1.2 kb cDNA fragment was cloned that encoded a 170 amino acid protein (DUX1) with two paired-type homeodomains. In vitro translated DUX1 specifically interacted in electrophoretic mobility shift assay (EMSA) with a P5 oligonucleotide (5'-GATCTGAGTCTAATTGAGAATTACTGTAC-3'). DUX1 co-expression activated up to 5-fold transient expression in insect cells of a minimal promoter-luciferase construct fused to P5. The presence of 20 kDa DUX1 in vivo in rhabdomyosarcoma TE671 cell extracts was shown by western blotting with a rabbit antiserum raised against a DUX1 peptide. This antiserum suppressed a TE671 protein-P5 complex in EMSA with identical migration as the in vitro translated DUX1-P5 complex. Genomic PCR experiments could not identify a gene fragment linking the HEFT1 and DUX1 sequences, which present one mismatch in their overlapping region. However, a similar gene was found in another 3.3 kb element comprising the HEFT1 promoter and a DUX1 -like open reading frame. In addition, homologous gene sequences were identified in 3.3 kb elements of the D4Z4/FSHD locus, considered until now 'junk' DNA.

Amino Acid Sequence↗
PubMed1997

Nonisotopic quantitative analysis of protein-DNA interactions at equilibrium.

Two versions of an enzyme-linked immunosorbent assay-type method to quantify protein-DNA interactions at equilibrium were developed. The first variant comprised immobilization of DNA-binding protein on microtiter plates, incubation with biotinylated DNA, and tagging of bound DNA with streptavidin- and biotin-substituted horseradish peroxidase. In the second version, biotinylated DNA was immobilized on streptavidin-substituted microtiter plates, incubated with DNA-binding protein, and bound protein was quantified with specific antibodies. To illustrate the method, the interaction of a fusion protein between glutathione-S-transferase and the DNA-binding domain of the helicase-like transcription factor with its cis-element (the B box of the plasminogen activator inhibitor-1 promoter) was determined with both versions: a 1:1 stoichiometric interaction with an equilibrium dissociation constant (Kd) of 1 nM was found, which is similar to the value determined by electrophoretic mobility shift assay, demonstrating the validity of the assays.

Bacterial Proteins↗
PubMed1997

Identification of a multihormone responsive enhancer far upstream from the human tissue-type plasminogen activator gene.

A 2.4-kilobase (kb) DNA fragment, located 7.1 kb upstream from the human tissue-type plasminogen activator (t-PA) gene (t-PA2.4), acts as an enhancer which is activated by glucocorticoids, progesterone, androgens, and mineralocorticoids. Transient expression of t-PA-chloramphenicol acetyltransferase reporter constructs in HT1080 human fibrosarcoma cells identified a glucocorticoid responsive unit with four functional binding sites for the glucocorticoid receptor, located between bp -7,501 and -7,974. The region from bp -7,145 to -9,578 (t-PA2.4) was found to confer a cooperative induction by dexamethasone and all-trans-retinoic acid (RA) to its homologous and a heterologous promoter, irrespective of its orientation. The minimal enhancer, defined by progressive deletion analysis, comprised the region from -7.1 to -8.0 kb (t-PA0.9) and encompassed the glucocorticoid responsive unit and the previously identified RA-responsive element located at -7.3 kb (Bulens, F., Ibañez-Tallon, I., Van Acker, P., De Vriese, A., Nelles, L., Belayew, A., and Collen, D. (1995) J. Biol. Chem. 270, 7167-7175). The amplitude of the synergistic response to dexamethasone and RA increased by reducing the distance between the enhancer and the proximal t-PA promoter. The synergistic interaction was also observed between the aldosterone and the RA receptors. It is postulated that the t-PA0.9 enhancer might play a role in the hormonal regulation of the expression of human t-PA in vivo.

Cell Line↗
PubMed1997

Characterization and targeting of the murine alpha2-antiplasmin gene.

Alpha2-Antiplasmin (alpha2-AP) is the main physiological plasmin inhibitor in mammalian plasma. As a first step toward the generation of alpha2-AP deficient mice, the murine alpha2-AP gene was characterized and a targeting vector for homologous recombination in embryonic stem (ES) cells constructed. Alignment of nucleotide sequences obtained from genomic subclones allowed location of exons 2 through 10 of the alpha2-AP gene, but failed to identify the 5' boundary of exon 1. Compared to the human gene, exons 2 through 9 in the murine gene have identical size and intron-exon boundaries obeying the GT/AG rule. The 5' boundary of exon 10 is identical in both genes while the 3' non-coding region is 64 bp longer in the human gene. Introns 2, 3, 6 and 8 have similar sizes in the mouse and human genes; intron 1 is 6-fold smaller, introns 5, 7 and 9 are 2- to 3-fold smaller, whereas intron 4 is about 2-fold larger in the mouse gene. Compared to the human 5' flanking sequence, an insertion of a simple repeat region with sequence (TGG)n has occurred. The open reading frame of the mouse alpha2-AP gene encodes a 491-amino-acid protein comprising the experimentally determined NH2-terminus of the mature protein Val-Asp-Leu-Pro-Gly-. A targeting vector, pPNT.alpha2-AP, was constructed by introducing a homologous sequence of 8.3 kb in total in the parental pPNT vector. In pPNT.alpha2-AP, the neomycin resistance expression cassette replaces a 7 kb genomic fragment comprising exon 2 through part of exon 10 (including the stop codon), which represents the entire sequence encoding the mature protein, including the fibrin-binding domain, the reactive site peptide bond and the plasmin(ogen)-binding region. Electroporation of 129R1 embryonic stem (ES) cells with the linearized vector pPNT.alpha2-AP yielded three targeted clones with correct homologous recombination at the 5'- and 3'-ends, as confirmed by Southern blot analysis of purified genomic DNA with appropriate restriction enzymes and probes. These targeted clones will be used to generate alpha2-AP deficient mice.

Amino Acid Sequence↗
PubMed1996

Characterization of a helicase-like transcription factor involved in the expression of the human plasminogen activator inhibitor-1 gene.

A 5.4-kb cDNA encoding the protein that binds to the B Box of the plasminogen activator inhibitor-1 (PAI-1) gene was isolated and sequenced. The protein, named helicase-like transcription factor (HLTF), contains a DNA-binding domain, a RING finger domain, and seven helicase domains and is homologous to SWI/SNF proteins. Two HLTF mRNAs of 5.5 and 4.5 kb were detected in most human tissues, a single gene was located on chromosome 3q24-25, and the protein was located in the nucleoplasm. Two HLTF proteins differing in translation start site (Met-1 or Met-123) were obtained by in vitro translation in reticulocyte lysate or by immunoprecipitation from HeLa cell nuclear extracts. In vitro transcription from the PAI-1 promoter in HeLa cell extracts was inhibited by HLTF antibodies and by the HLTF DNA binding domain. Over-expression of HLTF or HLTFMet123 produced a three-fold induction of PAI-1-LUC transient expression in HeLa cells. Mutation of the PAI-1 B Box led to an eight-fold reduction of basal PAI-1-LUC expression in these cell lines, but did not affect the four- to six-fold induction by phorbol esters.

Amino Acid Sequence↗
PubMed1996

The tilapia prolactin I gene: evolutionary conservation of the regulatory elements directing pituitary-specific expression.

To study the elements involved in the pituitary specific transcriptional regulation of the tilapia prolactin I gene (tiPRL I), we have cloned and entirely sequenced a 3.4-kb genomic fragment immediately upstream from the first exon. In footprinting experiments, three tilapia sequences are protected from DNase I digestion by rat pituitary extracts (base pair coordinates -643 to -593, -160 to -111, and -73 to -46). Computer analysis of the nucleotide sequence reveals significant homology to mammalian binding sites for Pit-1, a transcription factor that is known to mediate pituitary-specific expression of the PRL genes in mammals. The tiPRL I 5'-flanking sequences can direct transient expression of a linked luciferase reporter gene in transfected rat pituitary cell lines and tilapia pituitary primary cell cultures. Transient expression experiments with 5'-deletion mutants reveal three regulatory regions. Two have a stimulatory effect on transcription and one an inhibitory effect. Electrophoretic mobility-shift assays (EMSA) demonstrate that the rat Pit-1 factor specifically binds to tilapia DNA sequences. Several such tilapia Pit-1 binding sites mediate activation of a linked heterologous promoter in transfected rat and tilapia pituitary cells. As evidenced by EMSA, a Pit-1-like protein is present in tilapia pituitary extracts. All these data point to a high conservation of the molecular mechanisms involved in pituitary-specific expression of the PRL genes in vertebrates.

Animals↗
PubMed1996

The enhancers of the human placental lactogen B, A, and L genes: progressive activation during in vitro trophoblast differentiation and importance of the DF-3 element in determining their respective activities.

The hCS-A and hCS-B genes encoding human chorionic somatomammotropin and the related hCS-L gene are very similar in their coding and flanking sequences. For each of these genes, downstream enhancers, varying in strength, have been identified with the help of cytotrophoblast-derived JEG-3 cells, which do not express the hCS genes. Here we study the activity of the hCS enhancers in human syncytiotrophoblast in primary culture, which naturally expresses the hCS genes. We show that the activity of the hCS-B gene enhancer is mediated by two elements, DF-3 and DF-4, whereas the hCS-L and hCS-A gene enhancers display weaker activity due to mutations in their respective DF-3 sites. Replacement of the hCS-B DF-3 site with the homologous hCS-A sequence causes hCS-B enhancer activity to decrease. Primary cytotrophoblasts differentiate in culture to form the syncytiotrophoblast. We show that during this process the production of hCS progressively increases and that concomitantly all three hCS enhancers are progressively activated. A targeted mutation in the 3' part of the DF-4 element abolishes the binding of a protein present only in syncytiotrophoblast extracts and inactivates the DF-4 element. Thus, a direct correlation exists between the appearance of this syncytiotrophoblast-specific protein and hCS enhancer activity. This primary culture model proves useful in studying the regulation of the hCS genes.

Base Sequence↗
PubMed1995

Retinoic acid induction of human tissue-type plasminogen activator gene expression via a direct repeat element (DR5) located at -7 kilobases.

All-trans-retinoic acid (RA) and retinoids induce synthesis of tissue-type plasminogen activator (t-PA) in endothelial and neuroblastoma cells in vitro and in rats in vivo. In HT1080 fibrosarcoma cells, induction of t-PA-related antigen secretion and t-PA mRNA steady state levels by RA were found to depend on de novo protein and mRNA synthesis. Fragments derived from the 5'-flanking region of the t-PA gene (+197 to -9578 base pairs (bp)) were linked to the chloramphenicol acetyltransferase gene. Transfection studies demonstrated that the region spanning bp -7145 to -9578 mediated induction by RA. A functional retinoic acid response element (RARE), consisting of a direct repeat of the GGGTCA motif spaced by 5 nucleotides (t-PA/DR5), was localized at -7.3 kilobases. The t-PA/DR5 element interacted with the heterodimer composed of retinoic acid receptor alpha and retinoid X receptor alpha in vitro, whereas its mutation abolished induction by RA in transient expression. In human EA.hy926 hybrid endothelial and in SK-N-SH neuroblastoma cells, the activity of t-PA/DR5 was found to be independent of the intervening sequence (-632 to -7144 bp) and of its distance from the transcription initiation site. Staurosporine, an inhibitor of protein kinase activity, inhibited induction by RA, suggesting that it required protein phosphorylation.

Alkaloids↗
PubMed1995

Rapamycin, FK506 and cyclosporin A inhibit human prolactin gene expression.

In this work we demonstrate that transcription of the human prolactin gene is inhibited by the immunosuppressants FK506 (IC50 = 25 nM), cyclosporin A (IC50 = 190 nM) and rapamycin (IC50 = 25 nM). Whereas the effect of FK506 and cyclosporin A is specific for prolactin gene transcription, rapamycin has a more general effect on transcription and/or translation in pituitary cells. In view of recent work demonstrating the immunoactivating role of prolactin, these results suggest that inhibition of prolactin gene expression in the pituitary may contribute to the mechanism of action of immunosuppressants.

Animals↗
PubMed1995

Cyclosporin A, rapamycin and FK506 decrease prolactin release from rat pituitary cells in primary culture.

It is at present well established that prolactin exerts a non-specific immunoactivating function. In this work we tested whether the immunosuppressant drugs cyclosporin A, FK506 and rapamycin influence prolactin release from rat pituitary cells in primary culture. The tested drugs had no effect on the prolactin release measured during a 2h incubation period, indicating that they do not influence the secretion of prolactin from intracellular stores into the culture medium. During longer incubation times (48h), however, prolactin release was diminished to 56% +/- 18 (10 microM cyclosporin A), 64% +/- 14 (1 microM rapamycin) or 64% +/- 7 (1 microM FK506), suggesting an effect on prolactin production. At these drug concentrations no toxic effects were observed. The data indicate that inhibition of pituitary prolactin synthesis might contribute to the immunosuppressant action of cyclosporin A, rapamycin and FK506.

Animals↗
PubMed1994

A TEF-1 binding motif that interacts with a placental protein is important for the transcriptional activity of the hCS-B enhancer.

The transcriptional activity of the human placental lactogen genes (choriosomatomammotropic hormone, hCS) is controlled by tissue-specific enhancers located 4 kb downstream from their respective origins of transcription. The hCS-B enhancer is the strongest; its activity is mediated by synergism between two protein-binding sites (DF-3 and DF-4). The DF-4 site possesses a potential binding sequence for TEF-1, a known transcription factor. In this paper, we show by electrophoretic mobility-shift assays and antibody supershift experiments that TEF-1 does not bind to site DF-4. Mutations in the TEF-1-like binding motif of site DF-4 prevent formation of the DNA-protein complex, called complex f, in the presence of placental JEG-3 cell extracts. When HeLa cell extracts are used, another complex (complex c) is also affected. In transient expression experiments, TKCAT constructs linked to this mutated DF-4 site exhibit greatly reduced transcriptional activity when introduced into JEG-3 cells. Some cell lines contain both protein c and protein f (the proteins forming complexes c and f); when transfected, these lines display reduced DF-4-driven activity, suggesting that the two proteins could compete for the same DF-4 sequence. We conclude that protein f is important for the placenta-specific activity of the hCS-B enhancer. By UV cross-linking, we show that protein f is actually three polypeptides ranging in size from about 12 to 21 kD.

Base Sequence↗
PubMed1994

Characterization of a single strong tissue-specific enhancer downstream from the three human genes encoding placental lactogen.

The human genes coding for growth hormone (hGH) and placental lactogen (choriosomatomammotropic hormone [hCS]) are clustered on chromosome 17 in the following order: 5' hGH-N hCS-L hCS-A hGH-V hCS-B 3'. So far, a single placenta-specific enhancer has been identified in the locus, 2 kb downstream from the hCS-B gene, and shown to comprise one in vitro binding site for a nuclear protein. We here provide evidence that the hCS-B enhancer is more complex: (i) protection against DNase I digestion in the 3' flanking region of the hCS-B gene reveals four binding sites (DF-1, DF-2, DF-3, and DF-4) for nuclear proteins from either placental or HeLa cells, and (ii) placenta-specific enhancer activity can be fully exerted in transient expression experiments by a 126-bp fragment comprising the DF-3 and DF-4 protein-binding sites. By dissecting this region, we show that enhancer activity is mediated by a synergy between DF-3 and DF-4. Competitions with various oligonucleotides in footprinting and gel retardation experiments indicate that the same protein or set of proteins, different in HeLa and placenta cell nuclei, interacts with sites DF-2, DF-3, and DF-4. We also studied the regions of the hCS-L and hCS-A genes which are highly similar to the hCS-B enhancer. Although they each present the same four protein-binding sites, they exhibit only minor enhancer activity.

Base Sequence↗
PubMed1994

Replication, expression, and fate of foreign DNA during embryonic and larval development of the African catfish (Clarias gariepinus).

The transfer of exogenous DNA in fish represents a powerful strategy to study the regulation of gene expression in vivo. The African catfish (Clarias gariepinus) was chosen for this study because of its scientific and economic importance due to its easy husbandry, its short developmental period, and its value as a protein source in Africa and Asia. Fertilized eggs (1- and 2-cell stage) were cytoplasmatically injected with either supercoiled or linearized plasmids harboring the fusion genes encoding beta-galactosidase (lacZ) or luciferase (Luc) without a promoter or fused to the promoter/enhancer of human cytomegalovirus (CMV). Replication of the exogenous DNA peaked at 4 hours (early gastrula) and again at 2 days (which corresponds to the developmental stage of yolksac resorption). Foreign DNA persisted during embryogenesis, and it was still detectable 8 months after injection. In vivo transient expression of both CMV fusion genes was mosaic and peaked within 24 hours after DNA injection. Transient expression of the luciferase reporter gene could be detected with a much higher sensitivity than the lacZ gene. These data establish African catfish as a suitable in vivo assay system, and they confirm the luciferase reporter gene as a high quality reporter gene in fish.

Animals↗
PubMed1993

Efficient lipofection of human trophoblast cells in primary cultures.

Human choriosomatomammotropic hormone, also known as placental lactogen, is expressed in syncytiotrophoblast cells of the placenta. Studying transcriptional regulation of the genes coding for this hormone, we became interested in transfecting primary cultures of these trophoblast cells. In this study, we show that it is possible to transfect, by the lipofection method, these giant cells in an efficient and reproducible manner. We show the presence of an enhancer region downstream from the hCS-B gene, functionally active in these cells; furthermore, we demonstrate the placenta-specific characteristic of this enhancer, previously identified in a human choriocarcinoma cell line.

Cells, Cultured↗
PubMed1993

The transcriptional regulation of the growth hormone gene is conserved in vertebrate evolution.

Growth hormone (GH) gene expression in mammals is regulated by the interaction of the transcription factor Pit-1 with two binding sites within the proximal promoter. Four sequences, homologous to the mammalian Pit-1 motif occur in the rainbow trout (Oncorhynchus mykiss) GHII (rtGH) gene promoter, two of which partly overlap. The three regions containing these putative Pit-1 binding sequences were protected from deoxyribonuclease I digestion by nuclear extracts of GC cells, a rat pituitary tumor cell line producing Pit-1. In gel shift assays, nuclear proteins from GC cells and from trout pituitaries were found to interact specifically with one of these protected sites. Transfection experiments showed that the rtGH promoter is transcriptionally active in GC cells, the response being strongly enhanced in the presence of a cAMP analogue. The results demonstrate that rat Pit-1 binds to and activates the rtGH promoter, indicating that the basic mechanisms regulating GH gene transcription have been conserved between fish and mammals.

Animals↗
PubMed1993

Thyrotropin-releasing hormone and epidermal growth factor induce human prolactin expression via identical multiple cis elements.

Pituitary GH3 cells were transfected with different deletion mutants of the human prolactin (hPRL) promoter fused to the CAT reporter gene. The proximal region (-250 to -42) was sufficient to confer stimulation by both thyrotropin-releasing hormone (TRH) and epidermal growth factor (EGF). Further deletion analyses demonstrated the importance of the three proximal Pit-1 binding sites in this response. However, Pit-1 binding oligonucleotides confer neither TRH nor EGF induction to a linked neutral promoter, suggesting that other elements might be involved. We have previously shown that sequence A (-115 to -85) is needed together with Pit-1 binding sites for full cyclic AMP response of hPRL-CAT. Mutation of this sequence strongly affects TRH and EGF induction. On the other hand, three copies of sequence A confer both TRH and EGF response to a linked neutral promoter. In conclusion, although TRH and EGF activate mostly different intracellular pathways, they mediate transcriptional induction of the hPRL promoter via identical cis elements.

Animals↗
PubMed1993

Okadaic acid, a protein phosphatase inhibitor, enhances transcription of a receptor gene containing sequence A of the human prolactin promoter.

Human PRL (hPRL) gene expression is controlled by cAMP and Ca2+. This control is mediated by two cis-elements: a Pit-1 binding site (-62 to -35) and sequence A (-110 to -85), present in the hPRL promoter. We have investigated whether protein phosphatases could be involved in this regulation. GC-type rat pituitary tumor cells were transfected with sequence -138 to -35 of the hPRL gene promoter, upstream from a thymidine kinase promoter and a chloramphenicol acetyltransferase (CAT) reporter gene. Addition of okadaic acid (OA), a specific inhibitor of protein phosphatases 1 and 2A, stimulates transient expression of the CAT gene. The dose-response curve shows a maximal effect at 25 nM OA (2.2-fold stimulation above controls). The OA effect is also observed with a natural 4500-base pair hPRL promoter. A single copy of the hPRL promoter sequence -115 to -85 (sequence A) confers to a thymidine kinase-CAT construct an identical response to OA, whereas a single copy of the proximal Pit-1 binding site does not. Synergism is observed between cAMP and OA in activating PRL gene transcription. This synergism is also observed with a single copy of sequence A. The effect of cAMP is not mediated by an L-type Ca2+ channel, since addition of the Ca2+ channel antagonist verapamil does not decrease it, nor does complexing extracellular Ca2+ significantly reduce it. Furthermore, OA and the Ca2+ channel opener BAY K8644 exert additive effects.

Calcium↗