Drosophila transcriptional repressor protein that binds specifically to negative control elements in fat body enhancers

Expression of the Drosophila melanogaster Adh gene in adults requires a fat body-specific enhancer called the Adh adult enhancer (AAE). We have identified a protein in Drosophila nuclear extracts that binds specifically to a site within the AAE (adult enhancer factor 1 [AEF-1]). In addition, we have shown that AEF-1 binds specifically to two other Drosophila fat body enhancers. Base substitutions in the AEF-1 binding site that disrupt AEF-1 binding in vitro result in a significant increase in the level of Adh expression in vivo. Thus, the AEF-1 binding site is a negative regulatory element within the AAE. A cDNA encoding the AEF-1 protein was isolated and shown to act as a repressor of the AAE in cotransfection studies. The AEF-1 protein contains four zinc fingers and an alanine-rich sequence. The latter motif is found in other eukaryotic proteins known to be transcriptional repressors.

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The ecdysone response enhancer of the Fbp1 gene of Drosophila melanogaster is a direct target for the EcR/USP nuclear receptor.

Molecular and Cellular Biology ◽

10.1128/mcb.14.7.4465 ◽

1994 ◽

Vol 14 (7) ◽

pp. 4465-4474 ◽

Cited By ~ 47

Author(s):

C Antoniewski ◽

M Laval ◽

A Dahan ◽

J A Lepesant

Keyword(s):

Drosophila Melanogaster ◽

Binding Site ◽

Fat Body ◽

Sequence Similarity ◽

High Concentration ◽

Direct Role ◽

Palindromic Structure ◽

Fbp1 Gene

The transcription of the Drosophila melanogaster Fbp1 gene is induced by the steroid hormone 20-hydroxyecdysone and restricted to the late-third-instar fat body tissue. In a previous study we showed that the -68 to -138 region relative to the transcription start site acts as an ecdysone-dependent third-instar fat body-specific enhancer in a transgenic assay. Here we report that seven nucleoprotein complexes are formed in vitro on this enhancer when a nuclear extract from late-third-instar fat body is used in a gel shift assay. Accurate mapping of the binding sites of the complexes revealed a remarkably symmetrical organization. Using specific antibodies, one of the complexes was identified as a heterodimer consisting of the ecdysone receptor (EcR) and Ultraspiracle (USP) proteins. The binding site of the heterodimer as defined by mutagenesis and methylation interference experiments bears strong sequence similarity to the canonical hsp27 ecdysone response element, including an imperfect palindromic structure. The two elements diverge at three positions in both half-sites, indicating that the structure of an active EcR/USP binding site allows considerable sequence variations. In vivo footprinting experiments using ligation-mediated PCR and wild-type or ecdysteroid-deficient larvae show that occupancy of the Fbp1 EcR/USP binding site and adjacent region is dependent on a high concentration of ecdysteroids. These results provide strong evidence for a direct role of the EcR/USP heterodimer in driving gene expression in response to changes of the ecdysteroid titer during Drosophila larval development.

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Transcription Termination and 3′-End Processing of the Spliced Leader RNA in Kinetoplastids

Molecular and Cellular Biology ◽

10.1128/mcb.19.2.1595 ◽

1999 ◽

Vol 19 (2) ◽

pp. 1595-1604 ◽

Cited By ~ 51

Author(s):

Nancy R. Sturm ◽

Michael C. Yu ◽

David A. Campbell

Keyword(s):

Binding Site ◽

Transcription Termination ◽

Small Nuclear Rna ◽

Stem Loop ◽

Spliced Leader ◽

Nuclear Extracts ◽

Sequence Elements ◽

Rna Genes

ABSTRACT Addition of a 39-nucleotide (nt) spliced leader (SL) bytrans splicing is a basic requirement for all trypanosome nuclear mRNAs. The SL RNA in Leishmania tarentolae is a 96-nt precursor transcript synthesized by a polymerase that resembles polymerase II most closely. To analyze SL RNA genesis, we mutated SL RNA intron structures and sequence elements: stem-loops II and III, the Sm-binding site, and the downstream T tract. Using an exon-tagged SL RNA gene, we examined the phenotypes produced by a second-site 10-bp linker scan mutagenic series and directed mutagenesis. Here we report that transcription is terminated by the T tract, which is common to the 3′ end of all kinetoplastid SL RNA genes, and that more than six T’s are required for efficient termination in vivo. We describe mutants whose SL RNAs end in the T tract or appear to lack efficient termination but can generate wild-type 3′ ends. Transcriptionally active nuclear extracts show staggered products in the T tract, directed by eight or more T’s. The in vivo and in vitro data suggest that SL RNA transcription termination is staggered in the T tract and is followed by nucleolytic processing to generate the mature 3′ end. We show that the Sm-binding site and stem-loop III structures are necessary for correct 3′-end formation. Thus, we have defined the transcription termination element for the SL RNA gene. The termination mechanism differs from that of vertebrate small nuclear RNA genes and the SL RNA homologue in Ascaris.

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Identification of a critical regulatory site in the human interleukin-3 promoter by in vivo footprinting

Blood ◽

10.1182/blood.v83.10.2851.2851 ◽

1994 ◽

Vol 83 (10) ◽

pp. 2851-2859 ◽

Cited By ~ 44

Author(s):

S Cameron ◽

DS Taylor ◽

EC TePas ◽

NA Speck ◽

B Mathey-Prevot

Keyword(s):

T Cell ◽

Regulatory Element ◽

Cell Receptor ◽

Interleukin 3 ◽

Core Sequence ◽

Nuclear Extracts ◽

Specific Regulation ◽

In Vivo Footprinting

Abstract Interleukin-3 (IL-3) is involved in proliferation and differentiation of hematopoietic progenitor cells. Its expression is subject to precise, tissue-specific regulation, and has been studied extensively in the gibbon T-cell line MLA 144 by a combination of functional assays and DNA binding experiments. To extend these studies, the gibbon IL-3 promoter was cloned and in vivo footprinting of the gibbon and human IL- 3 proximal promoters was performed. The gibbon IL-3 promoter was found to be highly homologous to its human counterpart and both promoters yielded identical in vivo footprints after induction of IL-3 synthesis. In particular, we observed specific protection of three guanines over a core sequence TGTGGTTT (IF-1IL3) that had not been recognized in previous studies. IF-1IL3 is not found in other cytokine promoters, but it is conserved in the IL-3 promoter of several species and is similar to a recurring motif in viral and T-cell-specific cellular enhancers. IF-1IL3 binds a specific complex in MLA 144 and Jurkat nuclear extracts in vitro, which shares the same specificity as the complex bound by the polyoma virus and T-cell receptor delta enhancers. Mutation of the three guanines in IF-1IL3 core sequence disrupts binding in vitro and abrogates the ability of the IL-3 promoter to mediate inducible expression in T cells. Although IF-1IL3 is necessary for IL-3 expression, it is not sufficient: a truncated IL-3 promoter with an intact IF-1IL3 site but no other activator sites is transcriptionally silent. These studies describe a new regulatory element within the IL-3 promoter that is essential for expression and conserved between species.

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Regulation of Drosophila yolk protein genes by an ovary-specific GATA factor.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.6943 ◽

1995 ◽

Vol 15 (12) ◽

pp. 6943-6952 ◽

Cited By ~ 24

Author(s):

M Lossky ◽

P C Wensink

Keyword(s):

Fat Body ◽

Regulatory Element ◽

Ovarian Follicle ◽

In Vitro Transcription ◽

Yolk Protein ◽

Follicle Cells ◽

Gata Factor ◽

Yolk Protein Genes

The divergently transcribed yolk protein genes (Yp1 and Yp2) of Drosophila melanogaster are expressed only in adult females, in fat body tissue and in ovarian follicle cells. Using an in vitro transcription assay, we have identified a single 12-bp DNA element that activates transcription from the promoters of both Yp genes. In vivo, this regulatory element is tissue specific: it activates transcription of Yp1 and Yp2 reporter genes in follicle cells but has no detectable effect in fat body or other tissues. The sequence of the element consists of two recognition sites for the GATA family of transcription factors. We show that among the Drosophila genes known to encode GATA factors, only dGATAb is expressed in ovaries. The single transcript that we detect in ovaries is alternatively spliced or initiated to produce an ovary-specific isoform of the protein. Bacterially expressed dGATAb binds to the 12-bp element; a similar binding activity is also present in the Kc0 nuclear extracts used for in vitro transcription assays. These in vitro and in vivo results lead us to propose that dGATAb makes several developmentally regulated products, one of which is a follicle cell-specific protein activating transcription of Yp1 and Yp2 from a known regulatory element.

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Two Roles for the DNA Recognition Site of theKlebsiella aerogenes Nitrogen Assimilation Control Protein

Journal of Bacteriology ◽

10.1128/jb.180.3.578-585.1998 ◽

1998 ◽

Vol 180 (3) ◽

pp. 578-585 ◽

Cited By ~ 19

Author(s):

Pablo J. Pomposiello ◽

Brian K. Janes ◽

Robert A. Bender

Keyword(s):

Binding Site ◽

Transcriptional Activation ◽

Nitrogen Assimilation ◽

Proximal Half ◽

Weak Binding ◽

A Site ◽

Control Protein ◽

Half Site

ABSTRACT The nitrogen assimilation control protein (NAC) binds to a site within the promoter region of the histidine utilization operon (hutUH) of Klebsiella aerogenes, and NAC bound at this site activates transcription of hutUH. This NAC-binding site was characterized by a combination of random and directed DNA mutagenesis. Mutations that abolished or diminished in vivo transcriptional activation by NAC were found to lie within a 15-bp region contained within the 26-bp region protected by NAC from DNase I digestion. This 15-bp core has the palindromic ends ATA and TAT, and it matches the consensus for LysR family transcriptional regulators. Protein-binding experiments showed that transcriptional activation in vivo decreased with decreasing binding in vitro. In contrast to the NAC-binding site from hutUH, the NAC-binding site from thegdhA promoter failed to activate transcription from a semisynthetic promoter, and this failure was not due to weak binding or greatly distorted protein-DNA structure. Mutations in the promoter-proximal half-site of the NAC-binding site fromgdhA allowed this site to activate transcription. Similar studies using the NAC-binding site from hut showed that two mutations in the promoter proximal half-site increased binding but abolished transcriptional activation. Interestingly, for symmetric mutations in the promoter-distal half-site, loss of transcriptional activation was always correlated with a decrease in binding. We conclude from these observations that if the binding in vitro reflects the binding in vivo, then binding of NAC to DNA is not sufficient for transcriptional activation and that the NAC-binding site can be functionally divided in two half-sites, with related but different functions.

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Salicylate, Sulphinpyrazone, Indomeihacin And Aspirin May Interact With Platelet Cyclooxygenase At A Site Different From The Substrate Binding Site

10.1055/s-0038-1652019 ◽

1981 ◽

Author(s):

C Cerletti ◽

M Livio ◽

G Rajtar ◽

G de Gaetano

Keyword(s):

Binding Site ◽

Platelet Rich Plasma ◽

Hydrolysis Product ◽

Inhibitory Effect ◽

Dose Ratio ◽

Inflammatory Drugs ◽

A Site ◽

Dose Dependent

In vitro studies have shown that salicylate(SA), inactive by itself, prevents the inhibitory effect of aspirin (ASA)on platelet prostaglandin production. The purpose of this study was to verify this interaction in vivo and the ability of other non-steroidal anti-inflammatory drugs to counter ASA effect on platelet cyclooxygenase. Groups of male CD-COBS rats received one of the following drugs : SA (25-200 mg/kg i.p.),sulphinpyrazone(200 mg/kg p.o.), indomethacin (0.5 mg/kg i.p.) or the corresponding vehicles, followed after 30 min by ASA(1-50 mg/kg i.p.)as its soluble lysine salt. One or 24 hours later platelet-rich plasma was prepared and MDA (spec- trophotanetric assay) or TxB2 (RIA) generated on stimulation with 0.4-1 mM Na arachidonate were measured. All 3 compounds blunted ASA inhibitory action. In particular: 1. The preverting effect of SA was dose-dependent and could be overcome by increasing the dose of ASA. At a dose ratio (SA:ASA) of 20 the effect of ASA was reduced by about 50%, but no interaction was apparent at a ratio below 5. 2. Sulphinpyrazone had a greater preventing effect when it was administered 6 h (100%) than 3 h or 30 min (60%) before ASA (5 mg). 3. Indo-methacin reduced by 50% the effect of 5 mg of ASA but not of 10 mg. Tests were made 24 h after ASA administration, when the inhibitory effect of indcmethacin was no longer detectable. 4. 200 mg/kg SA reduced by 50% the inhibitory effect of indcmethacin.This data suggests that the 4 drugs interact with the same enzyme and that competition does not necessarily involve the substrate arachidonic acid. This supports the existence of a supplementary binding site which regulates cyclooxygenase activity, but is not directly involved with the substrate site. The clinical implications of these results include the interference of ASA with its hydrolysis product SA and a reappraisal of the pharmacological basis for the association of sulphinpyrazone and ASA in thrombosis prevention trials.

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The ecdysone response enhancer of the Fbp1 gene of Drosophila melanogaster is a direct target for the EcR/USP nuclear receptor

Molecular and Cellular Biology ◽

10.1128/mcb.14.7.4465-4474.1994 ◽

1994 ◽

Vol 14 (7) ◽

pp. 4465-4474

Author(s):

C Antoniewski ◽

M Laval ◽

A Dahan ◽

J A Lepesant

Keyword(s):

Drosophila Melanogaster ◽

Binding Site ◽

Fat Body ◽

Sequence Similarity ◽

High Concentration ◽

Direct Role ◽

Palindromic Structure ◽

Fbp1 Gene

The transcription of the Drosophila melanogaster Fbp1 gene is induced by the steroid hormone 20-hydroxyecdysone and restricted to the late-third-instar fat body tissue. In a previous study we showed that the -68 to -138 region relative to the transcription start site acts as an ecdysone-dependent third-instar fat body-specific enhancer in a transgenic assay. Here we report that seven nucleoprotein complexes are formed in vitro on this enhancer when a nuclear extract from late-third-instar fat body is used in a gel shift assay. Accurate mapping of the binding sites of the complexes revealed a remarkably symmetrical organization. Using specific antibodies, one of the complexes was identified as a heterodimer consisting of the ecdysone receptor (EcR) and Ultraspiracle (USP) proteins. The binding site of the heterodimer as defined by mutagenesis and methylation interference experiments bears strong sequence similarity to the canonical hsp27 ecdysone response element, including an imperfect palindromic structure. The two elements diverge at three positions in both half-sites, indicating that the structure of an active EcR/USP binding site allows considerable sequence variations. In vivo footprinting experiments using ligation-mediated PCR and wild-type or ecdysteroid-deficient larvae show that occupancy of the Fbp1 EcR/USP binding site and adjacent region is dependent on a high concentration of ecdysteroids. These results provide strong evidence for a direct role of the EcR/USP heterodimer in driving gene expression in response to changes of the ecdysteroid titer during Drosophila larval development.

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A Binding Site for Multiple Transcriptional Activators in the fushi tarazu Proximal Enhancer Is Essential for Gene Expression In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.18.6.3384 ◽

1998 ◽

Vol 18 (6) ◽

pp. 3384-3394 ◽

Cited By ~ 14

Author(s):

Wei Han ◽

Yan Yu ◽

Kai Su ◽

Ronald A. Kohanski ◽

Leslie Pick

Keyword(s):

Gene Expression ◽

Binding Site ◽

Binding Sites ◽

Zinc Finger Protein ◽

Regulatory Element ◽

Site Directed Mutagenesis ◽

Nuclear Proteins ◽

Fushi Tarazu

ABSTRACT The Drosophila homeobox gene fushi tarazu(ftz) is expressed in a highly dynamic striped pattern in early embryos. A key regulatory element that controls theftz pattern is the ftz proximal enhancer, which mediates positive autoregulation via multiple binding sites for the Ftz protein. In addition, the enhancer is necessary for stripe establishment prior to the onset of autoregulation. We previously identified nine binding sites for multiple Drosophilanuclear proteins in a core 323-bp region of the enhancer. Three of these nine sites interact with the same cohort of nuclear proteins in vitro. We showed previously that the nuclear receptor Ftz-F1 interacts with this repeated module. Here we purified additional proteins interacting with this module from Drosophila nuclear extracts. Peptide sequences of the zinc finger protein Ttk and the transcription factor Adf-1 were obtained. While Ttk is thought to be a repressor of ftz stripes, we have shown that both Adf-1 and Ftz-F1 activate transcription in a binding site-dependent fashion. These two proteins are expressed ubiquitously at the timeftz is expressed in stripes, suggesting that either may activate striped expression alone or in combination with the Ftz protein. The roles of the nine nuclear factor binding sites were tested in vivo, by site-directed mutagenesis of individual and multiple sites. The three Ftz-F1–Adf-1–Ttk binding sites were found to be functionally redundant and essential for stripe expression in transgenic embryos. Thus, a biochemical analysis identifiedcis-acting regulatory modules that are required for gene expression in vivo. The finding of repeated binding sites for multiple nuclear proteins underscores the high degree of redundancy built into embryonic gene regulatory networks.

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Function of NF-kappa B/Rel binding sites in the major histocompatibility complex class II invariant chain promoter is dependent on cell-specific binding of different NF-kappa B/Rel subunits.

Molecular and Cellular Biology ◽

10.1128/mcb.14.5.2926 ◽

1994 ◽

Vol 14 (5) ◽

pp. 2926-2935 ◽

Cited By ~ 47

Author(s):

A M Brown ◽

M W Linhoff ◽

B Stein ◽

K L Wright ◽

A S Baldwin ◽

...

Keyword(s):

Cell Lines ◽

Binding Sites ◽

Specific Binding ◽

Regulatory Element ◽

Invariant Chain ◽

Nf Kappa B ◽

Histocompatibility Complex ◽

A Site

The promoter of the human major histocompatibility complex class II-associated invariant-chain gene (Ii) contains two NF-kappa B/Rel binding sites located at -109 to -118 (Ii kappa B-1) and -163 to -172 (Ii kappa B-2) from the transcription start site. We report here that the differential function of each of these NF-kappa B/Rel sites in several distinct cell types depends on cell-specific binding of NF-kappa B/Rel transcription factors. Ii kappa B-1 is a positive regulatory element in B-cell lines and in the Ii-expressing T-cell line, H9, but acts as a negative regulatory element in myelomonocytic and glia cell lines. In vivo protein-DNA contacts are detectable at Ii kappa B-1 in cell lines in which this site is functional as either a positive or negative regulator. Electrophoretic mobility supershift assays determine that members of the NF-kappa B/Rel family of transcription factors can bind to this site in vitro and that DNA-binding complexes that contain p50, p52, p65, and cRel correlate with positive regulation whereas the presence of p50 correlates with negative regulation. Ii kappa B-2 is a site of positive regulation in B-cell lines and a site of negative regulation in H9 T cells, myelomonocytic, and glial cell lines. In vivo occupancy of this site is observed only in the H9 T-cell line. Again, in vitro supershift studies indicate that the presence of p50, p52, p65, and cRel correlates with positive function whereas the presence of only p50 and p52 correlates with negative function. This differential binding of specific NF-kappa B/Rel subunits is likely to mediate the disparate functions of these two NF-kappa B/Rel binding sites.

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