Transcriptional repression by the Drosophila giant protein: cis element positioning provides an alternative means of interpreting an effector gradient

Development ◽  
1999 ◽  
Vol 126 (6) ◽  
pp. 1201-1210 ◽  
Author(s):  
G.F. Hewitt ◽  
B.S. Strunk ◽  
C. Margulies ◽  
T. Priputin ◽  
X.D. Wang ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Short Range ◽  
Initiation Site ◽  
Step Function ◽  
Drosophila Embryo ◽  
Transcriptional Initiation ◽  
Cis Element ◽  
Alternative Means

Early developmental patterning of the Drosophila embryo is driven by the activities of a diverse set of maternally and zygotically derived transcription factors, including repressors encoded by gap genes such as Kruppel, knirps, giant and the mesoderm-specific snail. The mechanism of repression by gap transcription factors is not well understood at a molecular level. Initial characterization of these transcription factors suggests that they act as short-range repressors, interfering with the activity of enhancer or promoter elements 50 to 100 bp away. To better understand the molecular mechanism of short-range repression, we have investigated the properties of the Giant gap protein. We tested the ability of endogenous Giant to repress when bound close to the transcriptional initiation site and found that Giant effectively represses a heterologous promoter when binding sites are located at −55 bp with respect to the start of transcription. Consistent with its role as a short-range repressor, as the binding sites are moved to more distal locations, repression is diminished. Rather than exhibiting a sharp ‘step-function’ drop-off in activity, however, repression is progressively restricted to areas of highest Giant concentration. Less than a two-fold difference in Giant protein concentration is sufficient to determine a change in transcriptional status of a target gene. This effect demonstrates that Giant protein gradients can be differentially interpreted by target promoters, depending on the exact location of the Giant binding sites within the gene. Thus, in addition to binding site affinity and number, cis element positioning within a promoter can affect the response of a gene to a repressor gradient. We also demonstrate that a chimeric Gal4-Giant protein lacking the basic/zipper domain can specifically repress reporter genes, suggesting that the Giant effector domain is an autonomous repression domain.

scholarly journals Differential Regulation of the Bordetella bipA Gene: Distinct Roles for Different BvgA Binding Sites

2002 ◽  
Vol 184 (24) ◽  
pp. 6942-6951 ◽  
Author(s):  
Rajendar Deora
Keyword(s):  
Binding Sites ◽  
Transcriptional Repression ◽  
Intermediate Phase ◽  
Initiation Site ◽  
Transcriptional Level ◽  
Transcriptional Initiation ◽  
Reporter Enzyme ◽  
High Concentrations ◽  
Mutant Promoter

ABSTRACT The BvgAS signal transduction system of Bordetella controls an entire spectrum of gene expression states in response to differences in environmental conditions. In particular, the Bordetella Bvg-intermediate-phase gene bipA displays a complex regulatory pattern in response to various concentrations of modulators. Expression of bipA is low in the absence of modulating signals, maximal at intermediate concentrations of modulators, and near background levels at high concentrations of modulators. bipA is regulated at the transcriptional level, and the bipA promoter contains multiple BvgA binding sites present both upstream and downstream of the transcriptional initiation site. In vivo transcriptional analyses, utilizing several mutant promoter fusions to the reporter enzyme β-galactosidase, suggest that the upstream binding site IR1 is essential for expression and that the downstream binding sites IR2 and IR3 are involved in transcriptional repression. Mutations of IR2 or IR3 convert the expression profile of bipA from that of a Bvg-intermediate-specific-phase gene to that of a Bvg+-phase gene. To gain insight into the mechanism responsible for differential bipA regulation, DNase I protection studies were conducted with various mutant promoters. These analyses suggest that IR1 and IR2 function as core binding sites and are the primary determinants for the phosphorylation-induced oligomerization of BvgA to the adjacent regions.

scholarly journals CtBP-Independent Repression in the Drosophila Embryo

2003 ◽  
Vol 23 (11) ◽  
pp. 3990-3999 ◽  
Author(s):  
Yutaka Nibu ◽  
Kate Senger ◽  
Michael Levine
Keyword(s):  
Drosophila Melanogaster ◽  
Binding Site ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Short Range ◽  
Core Promoter ◽  
Drosophila Embryo ◽  
The Third ◽  
The Core ◽  
Dna Binding Site

ABSTRACT There are three mechanisms of transcriptional repression in eukaryotes. The first is quenching, whereby repressors and activators co-occupy closely linked sites and then the repressor inhibits adjacent activators. The second is direct repression, in which repressors block the function of the core transcription complex. The third is competition, in which repressors compete with activators for a common DNA-binding site. Previous studies have shown that the Drosophila melanogaster CtBP corepressor (dCtBP) is essential for the quenching activity of three short-range sequence-specific repressors in the early Drosophila embryo: Krüppel, Knirps, and Snail. Here we demonstrate that dCtBP is dispensable for target enhancers that contain overlapping activator and repressor binding sites. However, it is essential when Krüppel and Knirps repressor sites do not overlap activator sites but are instead located adjacent to either activators or the core promoter. These findings provide evidence that competition is distinct from quenching and direct repression. Quenching and direct repression depend on dCtBP, whereas competition does not.

scholarly journals Genes encoding components of the olfactory signal transduction cascade contain a DNA binding site that may direct neuronal expression.

1993 ◽  
Vol 13 (9) ◽  
pp. 5805-5813 ◽  
Author(s):  
M M Wang ◽  
R Y Tsai ◽  
K A Schrader ◽  
R R Reed
Keyword(s):  
Signal Transduction ◽  
Dna Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Initiation Site ◽  
Olfactory Neuron ◽  
Promoter Regions ◽  
Transcriptional Initiation ◽  
Genes Encoding

Genes which mediate odorant signal transduction are expressed at high levels in neurons of the olfactory epithelium. The molecular mechanism governing the restricted expression of these genes likely involves tissue-specific DNA binding proteins which coordinately activate transcription through sequence-specific interactions with olfactory promoter regions. We have identified binding sites for the olfactory neuron-specific transcription factor, Olf-1, in the sequences surrounding the transcriptional initiation site of five olfactory neuron-specific genes. The Olf-1 binding sites described define the consensus sequence YTCCCYRGGGAR. In addition, we have identified a second binding site, the U site, in the olfactory cyclic nucleotide gated channel and type III cyclase promoters, which binds factors present in all tissue examined. These experiments support a model in which expression of Olf-1 in the sensory neurons coordinately activates a set of olfactory neuron-specific genes. Furthermore, expression of a subset of these genes may be modulated by additional binding factors.

scholarly journals The histone variant H2A.Z and chromatin remodeler BRAHMA act coordinately and antagonistically to regulate transcription and nucleosome dynamics in Arabidopsis

2018 ◽  
Author(s):  
E. Shannon Torres ◽  
Roger B. Deal
Keyword(s):  
Transcriptional Regulation ◽  
Transcription Factors ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Nucleosome Positioning ◽  
Chromatin Organization ◽  
Histone H2a ◽  
Nucleosome Dynamics ◽  
Environmentally Responsive ◽  
Context Specific

ABSTRACTPlants adapt to changes in their environment by regulating transcription and chromatin organization. The histone H2A variant H2A.Z and the SWI2/SNF2 ATPase BRAHMA have overlapping roles in positively and negatively regulating environmentally responsive genes in Arabidopsis, but the extent of this overlap was uncharacterized. Both have been associated with various changes in nucleosome positioning and stability in different contexts, but their specific roles in transcriptional regulation and chromatin organization need further characterization. We show that H2A.Z and BRM act both cooperatively and antagonistically to contribute directly to transcriptional repression and activation of genes involved in development and response to environmental stimuli. We identified 8 classes of genes that show distinct relationships between H2A.Z and BRM and their roles in transcription. We found that H2A.Z contributes to a range of different nucleosome properties, while BRM stabilizes nucleosomes where it binds and destabilizes and/or repositions flanking nucleosomes. H2A.Z and BRM contribute to +1 nucleosome destabilization, especially where they coordinately regulate transcription. We also found that at genes regulated by both BRM and H2A.Z, both factors overlap with the binding sites of light-regulated transcription factors PIF4, PIF5, and FRS9, and that some of the FRS9 binding sites are dependent on H2A.Z and BRM for accessibility. Collectively, we comprehensively characterized the antagonistic and cooperative contributions of H2A.Z and BRM to transcriptional regulation, and illuminated their interrelated roles in chromatin organization. The variability observed in their individual functions implies that both BRM and H2A.Z have more context-specific roles within diverse chromatin environments than previously assumed.

Transcriptional repression in the Drosophila embryo

1995 ◽  
Vol 349 (1329) ◽  
pp. 257-262 ◽  
Keyword(s):  
Long Range ◽  
Transcriptional Repression ◽  
Short Range ◽  
Boundary Elements ◽  
Early Embryo ◽  
Tissue Differentiation ◽  
Drosophila Embryo ◽  
Quenching Mechanism ◽  
Chromatin Boundary

Transcriptional repression is essential for the conversion of crude maternal gradients into sharp territories of tissue differentiation in the Drosophila embryo. Evidence will be presented suggesting that some of the embryonic repressors function through a short-range ‘quenching’ mechanism, whereby a repressor works over short distances ( ca. 50 b.p.) to block neighbouring activators within a target enhancer. This type of repression can explain how different enhancers work autonomously within complex modular promoters. However, at least one of the repressors operating in the early embryo works through a long-range, or silencing, mechanism. The binding of a silencer to a given enhancer leads to the inactivation of all enhancers within a complex promoter. The analysis of chromatin boundary elements suggest that silencers and enhancers might work through distinct mechanisms. We speculate that silencers constrain the evolution of complex promoters.

Isolation and Characterization of Human ADAMTS13 Gene Promoter Region - Control of ADAMTS13 Gene Expression by Hepatic Transcription Factors and Inflammatory Cytokines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1603-1603 ◽  
Author(s):  
Xingwu Zheng ◽  
Masami Niiya ◽  
X. Long Zheng ◽  
Eleanor S. Pollak
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Inflammatory Cytokines ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Promoter Activity ◽  
Initiation Site ◽  
Luciferase Reporter ◽  
Tnf Alpha

Abstract ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats-13) controls von Willebrand factor multimer sizes by cleaving the Tyr1605-Met1606 bond in the central A2 domain. Deficiency of plasma ADAMTS13 activity can result in a lethal syndrome, thrombotic thrombocytopenic purpura (TTP). ADAMTS13 is primarily synthesized in hepatic stellate cells (HSCs), endothelial cells and megakaryocytes. We determined the transcription initiation site, the core region for promoter activity, the putative transcription factor binding sites as well as the influence of inflammatory cytokines on ADAMTS13 promoter activity. To explore the transcriptional control of ADAMTS13 gene expression, we constructed reporter genes containing 991 base pairs (bp) of the ADAMTS13 5′ untranslated (UT) region. We showed by deletion mutagenesis and luciferase reporter expression that the proximal-most 197 bp region was required for maximal luciferase activity in transfected cells in the human hepatic stellate cell line (LX-2) and in the human hepatocyte-like cell line (HepG2); the major transcription initiation site determined by 5′ - RACE was found at 77 bp upstream from the translation start site (ATG). However, the minimal sequences that were required for the promoter activity varied depending on the cells, with required sequences of approximately 147 and 127 bp in LX-2 and HepG2 cells, respectively. The proximal ADAMTS13 promoter region is evolutionally conserved between humans, mice and rats. This region is rich in GC content (72%) and contains putative binding sites for the transcription factors heat shock factor-2 (HSF2), FOXa2 [also named hepatocyte nuclear factor 3beta (HNF-3b)] and AP-1. A footprint assay demonstrated that the region between −116 and −126, containing the putative FOXa2 binding site, was largely protected by Dnase I digestion. The luciferase reporter activity was suppressed in cells transfected with the plasmid containing the proximal 314 bp human 5′ UT ADAMTS13 sequence in parallel with the inflammatory cytokines found to be elevated in patients with TTP: IL-4, TNF-alpha and INF-gamma. These inflammatory cytokines inhibited the Adamts13 mRNA and protein expression in rat primary HSCs in culture in a dose dependent manner. Approximately 70%, 71% and 80% of Adamts13 mRNA (by real time RT-PCR) and 77%, 78% and 92% of Adamts13 proteolytic activity (by FRETS-VWF73) were suppressed at 48 hours by IL-4 (10 ng/ml), TNF-alpha (10 ng/ml) and INF-gamma (100 ng/ml), respectively. We conclude that under physiological conditions ADAMTS13 synthesis may be strictly maintained at relatively low levels by binding transcription factors, whereas under pathological conditions inflammatory cytokines, released due to systemic inflammation, may further suppress ADAMTS13 gene expression, which may result in thrombotic complications. However, the mechanism regarding how the inflammatory cytokines negatively regulate ADAMTS13 (or Adamts13) synthesis remains to be determined.

scholarly journals cis-Regulatory Logic of Short-Range Transcriptional Repression in Drosophila melanogaster

2005 ◽  
Vol 25 (9) ◽  
pp. 3411-3420 ◽  
Author(s):  
Meghana M. Kulkarni ◽  
David N. Arnosti
Keyword(s):  
Binding Sites ◽  
Transcriptional Repression ◽  
Short Range ◽  
Transcriptional Control ◽  
Regulatory Sequences ◽  
Transcriptional Repressors ◽  
Quantitative Parameters ◽  
Transcriptional Regulatory ◽  
Distance Dependence ◽  
Enhancer Identification

ABSTRACT Bioinformatics analysis of transcriptional control is guided by knowledge of the characteristics of cis-regulatory regions or enhancers. Features such as clustering of binding sites and co-occurrence of binding sites have aided enhancer identification, but quantitative predictions of enhancer function are not yet generally feasible. To facilitate the analysis of regulatory sequences in Drosophila melanogaster, we identified quantitative parameters that affect the activity of short-range transcriptional repressors, proteins that play key roles in development. In addition to the previously noted distance dependence, repression is strongly influenced by the stoichiometry, affinity, spacing, and arrangement of activator binding sites. Repression is insensitive to the type of activation domain, suggesting that short-range repression may primarily affect activators at the level of DNA binding. The activity of several short-range, but not long-range, repressors is circumscribed by the same quantitative parameters. This cis-regulatory “grammar” may aid the identification of enhancers regulated by short-range repressors and facilitate bioinformatic prediction of the functional output of transcriptional regulatory sequences.

Cooperative activation of transcription by bovine papillomavirus type 1 E2 can occur over a large distance

1990 ◽  
Vol 10 (8) ◽  
pp. 4431-4437
Author(s):  
F Thierry ◽  
N Dostatni ◽  
F Arnos ◽  
M Yaniv
Keyword(s):  
Binding Sites ◽  
Initiation Site ◽  
Dna Looping ◽  
Binding Motif ◽  
Bovine Papillomavirus ◽  
Kinase Gene ◽  
Transcriptional Initiation ◽  
Reading Frame ◽  
Simplex Virus

The viral transcriptional factors encoded by the E2 open reading frame bind to the specific DNA sequence elements ACCGNNNNCGGT, allowing activation or repression of transcription. We have analyzed bovine papillomavirus type 1 E2 transactivation using recombinant genes containing E2-binding sites inserted at either 3' or 5' positions relative to the heterologous transcriptional initiation site of the herpes simplex virus thymidine kinase gene. In these hybrid plasmids, strong transactivation required the presence of a minimum of two E2-binding sites in close proximity to the promoter or five binding sites at a distance. The presence of a single E2-binding motif 5', close to the initiation site, increased the efficiency of E2 transactivation from a distance in a more-than-additive manner. Since each E2-binding site bound a dimer of the E2 protein, these experiments suggest that transactivation by E2 requires the interaction between several E2 dimers with other essential transcription factors. This interaction may be facilitated by DNA looping, which would bring E2 molecules close to the promoter.

scholarly journals The promoter of the human interleukin-2 gene contains two octamer-binding sites and is partially activated by the expression of Oct-2.

1990 ◽  
Vol 10 (10) ◽  
pp. 5464-5472 ◽  
Author(s):  
M P Kamps ◽  
L Corcoran ◽  
J H LeBowitz ◽  
D Baltimore
Keyword(s):  
T Cells ◽  
Binding Sites ◽  
Interleukin 2 ◽  
Point Mutations ◽  
Transcriptional Response ◽  
Initiation Site ◽  
Gene Encoding ◽  
Transcriptional Initiation

The gene encoding interleukin-2 (IL-2) contains a sequence 52 to 326 nucleotides upstream of its transcriptional initiation site that promotes transcription in T cells that have been activated by costimulation with tetradecanoyl phorbol myristyl acetate (TPA) and phytohemagglutinin (PHA). We found that the ubiquitous transcription factor, Oct-1, bound to two previously identified motifs within the human IL-2 enhancer, centered at nucleotides -74 and -251. Each site in the IL-2 enhancer that bound Oct-1 in vitro was also required to achieve a maximal transcriptional response to TPA plus PHA in vivo. Point mutations within either the proximal or distal octamer sequences reduced the response of the enhancer to activation by 54 and 34%, respectively. Because the murine T-cell line EL4 constitutively expresses Oct-2 and requires only TPA to induce transcription of the IL-2 gene, the effect of Oct-2 expression on activation of the IL-2 promoter in Jurkat T cells was determined. Expression of Oct-2 potentiated transcription 13-fold in response to TPA plus PHA and permitted the enhancer to respond to the single stimulus of TPA. Therefore, both the signal requirements and the magnitude of the transcription response of the IL-2 promoter can be modulated by Oct-2.

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