scholarly journals Activation and repression by the C-terminal domain of Dorsal

Development ◽  
2001 ◽  
Vol 128 (10) ◽  
pp. 1869-1879 ◽  
Author(s):  
R.D. Flores-Saaib ◽  
S. Jia ◽  
A.J. Courey
Keyword(s):  
Fusion Protein ◽  
Concentration Gradient ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Fusion Proteins ◽  
Drosophila Embryo ◽  
Terminal Domain ◽  
Localization Signal ◽  
Pattern Forming ◽  
Rel Homology Domain

In the Drosophila embryo, Dorsal, a maternally expressed Rel family transcription factor, regulates dorsoventral pattern formation by activating and repressing zygotically active fate-determining genes. Dorsal is distributed in a ventral-to-dorsal nuclear concentration gradient in the embryo, the formation of which depends upon the spatially regulated inhibition of Dorsal nuclear uptake by Cactus. Using maternally expressed Gal4/Dorsal fusion proteins, we have explored the mechanism of activation and repression by Dorsal. We find that a fusion protein containing the Gal4 DNA-binding domain fused to full-length Dorsal is distributed in a nuclear concentration gradient that is similar to that of endogenous Dorsal, despite the presence of a constitutively active nuclear localization signal in the Gal4 domain. Whether this fusion protein activates or represses reporter genes depends upon the context of the Gal4-binding sites in the reporter. A Gal4/Dorsal fusion protein lacking the conserved Rel homology domain of Dorsal, but containing the non-conserved C-terminal domain also mediates both activation and repression, depending upon Gal4-binding site context. A region close to the C-terminal end of the C-terminal domain has homology to a repression motif in Engrailed - the eh1 motif. Deletion analysis indicates that this region mediates transcriptional repression and binding to Groucho, a co-repressor known to be required for Dorsal-mediated repression. As has previously been shown for repression by Dorsal, we find that activation by Dorsal, in particular by the C-terminal domain, is modulated by the maternal terminal pattern-forming system.

scholarly journals Transcriptional repression by Drosophila and mammalian Polycomb group proteins in transfected mammalian cells.

1994 ◽  
Vol 14 (3) ◽  
pp. 1721-1732 ◽  
Author(s):  
C A Bunker ◽  
R E Kingston
Keyword(s):  
Binding Sites ◽  
Transcriptional Repression ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Polycomb Group ◽  
Polycomb Group Proteins ◽  
Related Gene ◽  
Activation Domains ◽  
Sex Combs ◽  
Significant Homology

The Polycomb group (Pc-G) genes are essential for maintaining the proper spatially restricted expression pattern of the homeotic loci during Drosophila development. The Pc-G proteins appear to function at target loci to maintain a state of transcriptional repression. The murine oncogene bmi-1 has significant homology to the Pc-G gene Posterior sex combs (Psc) and a highly related gene, Suppressor two of zeste [Su(z)2]. We show here that the proteins encoded by bmi-1 and the Pc-G genes Polycomb (Pc) and Psc as well as Su(z)2 mediate repression in mammalian cells when targeted to a promoter by LexA in a cotransfection system. These fusion proteins repress activator function by as much as 30-fold, and the effect on different activation domains is distinct for each Pc-G protein. Repression is observed when the LexA fusion proteins are bound directly adjacent to activator binding sites and also when bound 1,700 bases from the promoter. These data demonstrate that the products of the Pc-G genes can significantly repress activator function on transiently introduced DNA. We suggest that this function contributes to the stable repression of targeted loci during development.

scholarly journals AP-2/Eps15 Interaction Is Required for Receptor-mediated Endocytosis

1998 ◽  
Vol 140 (5) ◽  
pp. 1055-1062 ◽  
Author(s):  
Alexandre Benmerah ◽  
Christophe Lamaze ◽  
Bernadette Bègue ◽  
Sandra L. Schmid ◽  
Alice Dautry-Varsat ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fusion Protein ◽  
Binding Sites ◽  
Fluorescent Protein ◽  
Coated Vesicle ◽  
Mutant Form ◽  
A431 Cells ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Terminal Domain

We have previously shown that the protein Eps15 is constitutively associated with the plasma membrane adaptor complex, AP-2, suggesting its possible role in endocytosis. To explore the role of Eps15 and the function of AP-2/Eps15 association in endocytosis, the Eps15 binding domain for AP-2 was precisely delineated. The entire COOH-terminal domain of Eps15 or a mutant form lacking all the AP-2–binding sites was fused to the green fluorescent protein (GFP), and these constructs were transiently transfected in HeLa cells. Overexpression of the fusion protein containing the entire COOH-terminal domain of Eps15 strongly inhibited endocytosis of transferrin, whereas the fusion protein in which the AP-2–binding sites had been deleted had no effect. These results were confirmed in a cell-free assay that uses perforated A431 cells to follow the first steps of coated vesicle formation at the plasma membrane. Addition of Eps15-derived glutathione-S-transferase fusion proteins containing the AP-2–binding site in this assay inhibited not only constitutive endocytosis of transferrin but also ligand-induced endocytosis of epidermal growth factor. This inhibition could be ascribed to a competition between the fusion protein and endogenous Eps15 for AP-2 binding. Altogether, these results show that interaction of Eps15 with AP-2 is required for efficient receptor-mediated endocytosis and thus provide the first evidence that Eps15 is involved in the function of plasma membrane–coated pits.

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 Both TEL and AML-1 Contribute Repression Domains to the t(12;21) Fusion Protein

1999 ◽  
Vol 19 (10) ◽  
pp. 6566-6574 ◽  
Author(s):  
Randy Fenrick ◽  
Joseph M. Amann ◽  
Bart Lutterbach ◽  
Lilin Wang ◽  
Jennifer J. Westendorf ◽  
...  
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Wild Type ◽  
Deletion Mutagenesis ◽  
Binding Domains ◽  
Active Mechanism ◽  
Dna Binding Sites ◽  
Interaction Domains

ABSTRACT t(12;21) is the most frequent translocation found in pediatric B-cell acute lymphoblastic leukemias. This translocation fuses a putative repressor domain from the TEL DNA-binding protein to nearly all of the AML-1B transcription factor. Here, we demonstrate that fusion of the TEL pointed domain to the GAL4 DNA-binding domain resulted in sequence-specific transcriptional repression, indicating that the pointed domain is a portable repression motif. The TEL pointed domain functioned equally well when the GAL4 DNA-binding sites were moved 600 bp from the promoter, suggesting an active mechanism of repression. This lead us to demonstrate that wild-type TEL and the t(12;21) fusion protein bind the mSin3A corepressor. In the fusion protein, both TEL and AML-1B contribute mSin3 interaction domains. Deletion mutagenesis indicated that both the TEL and AML-1B mSin3-binding domains contribute to repression by the fusion protein. While both TEL and AML-1B associate with mSin3A, TEL/AML-1B appears to bind this corepressor much more stably than either wild-type protein, suggesting a mode of action for the t(12;21) fusion protein.

scholarly journals A Role for Groucho Tetramerization in Transcriptional Repression

1998 ◽  
Vol 18 (12) ◽  
pp. 7259-7268 ◽  
Author(s):  
Guoqing Chen ◽  
Pierre H. Nguyen ◽  
Albert J. Courey
Keyword(s):  
Transcriptional Repression ◽  
Fusion Proteins ◽  
Leucine Zipper ◽  
Point Mutations ◽  
Cross Linking ◽  
Transcriptional Repressors ◽  
Terminal Domain ◽  
Tetramerization Domain ◽  
Repeat Domain ◽  
Eukaryotic Organisms

ABSTRACT The Drosophila Groucho (Gro) protein is a corepressor required by a number of DNA-binding transcriptional repressors. Comparison of Gro with its homologues in other eukaryotic organisms reveals that Gro contains, in addition to a conserved C-terminal WD repeat domain, a conserved N-terminal domain, which has previously been implicated in transcriptional repression. We determined, via a variety of hydrodynamic measurements as well as protein cross-linking, that native Gro is a tetramer in solution and that tetramerization is mediated by two putative amphipathic α-helices (termed leucine zipper-like motifs) found in the N-terminal region. Point mutations in the leucine zipper-like motifs that block tetramerization also block repression by Gro, as assayed in cultured Drosophila cells with Gal4-Gro fusion proteins. Furthermore, the heterologous tetramerization domain from p53 fully substitutes for the Gro tetramerization domain in transcriptional repression. These findings suggest that oligomerization is essential for Gro-mediated repression and that the primary function of the conserved N-terminal domain is to mediate this oligomerization.

scholarly journals Targeting to Transcriptionally Active Loci by the Hydrophilic N-Terminal Domain of Drosophila DNA Topoisomerase I

1998 ◽  
Vol 18 (7) ◽  
pp. 4358-4367 ◽  
Author(s):  
Wen-Ling Shaiu ◽  
Tao-shih Hsieh
Keyword(s):  
Heat Shock ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Topoisomerase I ◽  
Polytene Chromosomes ◽  
Dna Topoisomerase I ◽  
Dna Topoisomerase ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Terminal Domain

ABSTRACT DNA topoisomerase I (topo I) from Drosophila melanogaster contains a nonconserved, hydrophilic N-terminal domain of about 430 residues upstream of the conserved core domains. Deletion of this N terminus did not affect the catalytic activity of topo I, while further removal of sequences into the conserved regions inactivated its enzymatic activity. We have investigated the cellular function of the Drosophila topo I N-terminal domain withtop1-lacZ transgenes. There was at least one putative nuclear localization signal within the first 315 residues of the N-terminal domain that allows efficient import of the large chimeric proteins into Drosophila nuclei. The top1-lacZfusion proteins colocalized with RNA polymerase II (pol II) at developmental puffs on the polytene chromosomes. Either topo I or thetop1-lacZ fusion protein was colocalized with RNA pol II in some but not all of the nonpuff, interband loci. However, the fusion proteins as well as RNA pol II were recruited to heat shock puffs during heat treatment, and they returned to the developmental puffs after recovery from heat shock. By immunoprecipitation, we showed that two of the largest subunits of RNA pol II coprecipitated with the N-terminal 315-residue fusion protein by using antibodies against β-galactosidase. These data suggest that the topo I fusion protein can be localized to the transcriptional complex on chromatin and that the N-terminal 315 residues were sufficient to respond to cellular processes, especially during the reprogramming of gene expression.

scholarly journals Transcriptional repression by Drosophila and mammalian Polycomb group proteins in transfected mammalian cells

1994 ◽  
Vol 14 (3) ◽  
pp. 1721-1732
Author(s):  
C A Bunker ◽  
R E Kingston
Keyword(s):  
Binding Sites ◽  
Transcriptional Repression ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Polycomb Group ◽  
Polycomb Group Proteins ◽  
Related Gene ◽  
Activation Domains ◽  
Sex Combs ◽  
Significant Homology

The Polycomb group (Pc-G) genes are essential for maintaining the proper spatially restricted expression pattern of the homeotic loci during Drosophila development. The Pc-G proteins appear to function at target loci to maintain a state of transcriptional repression. The murine oncogene bmi-1 has significant homology to the Pc-G gene Posterior sex combs (Psc) and a highly related gene, Suppressor two of zeste [Su(z)2]. We show here that the proteins encoded by bmi-1 and the Pc-G genes Polycomb (Pc) and Psc as well as Su(z)2 mediate repression in mammalian cells when targeted to a promoter by LexA in a cotransfection system. These fusion proteins repress activator function by as much as 30-fold, and the effect on different activation domains is distinct for each Pc-G protein. Repression is observed when the LexA fusion proteins are bound directly adjacent to activator binding sites and also when bound 1,700 bases from the promoter. These data demonstrate that the products of the Pc-G genes can significantly repress activator function on transiently introduced DNA. We suggest that this function contributes to the stable repression of targeted loci during development.

scholarly journals Roles for the Saccharomyces cerevisiae SDS3, CBK1 and HYM1 Genes in Transcriptional Repression by SIN3

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 573-586 ◽  
Author(s):  
Scott Dorland ◽  
Michelle L Deegenaars ◽  
David J Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Analysis ◽  
Fusion Protein ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Cell Separation ◽  
Multiprotein Complex ◽  
Genetic Pathway ◽  
Lexa Binding ◽  
Sin3 Complex

Abstract The Saccharomyces cerevisiae Sin3 transcriptional repressor is part of a large multiprotein complex that includes the Rpd3 histone deacetylase. A LexA-Sin3 fusion protein represses transcription of promoters with LexA binding sites. To identify genes involved in repression by Sin3, we conducted a screen for mutations that reduce repression by LexA-Sin3. One of the mutations identified that reduces LexA-Sin3 repression is in the RPD3 gene, consistent with the known roles of Rpd3 in transcriptional repression. Mutations in CBK1 and HYM1 reduce repression by LexA-Sin3 and also cause defects in cell separation and altered colony morphology. cbk1 and hym1 mutations affect some but not all genes regulated by SIN3 and RPD3, but the effect on transcription is much weaker. Genetic analysis suggests that CBK1 and HYM1 function in the same pathway, but this genetic pathway is separable from that of SIN3 and RPD3. The remaining gene from this screen described in this report is SDS3, previously identified in a screen for mutations that increase silencing at HML, HMR, and telomere-linked genes, a phenotype also seen in sin3 and rpd3 mutants. Genetic analysis demonstrates that SDS3 functions in the same genetic pathway as SIN3 and RPD3, and coimmunoprecipitation experiments show that Sds3 is physically present in the Sin3 complex.

scholarly journals A direct contact between the dorsal rel homology domain and Twist may mediate transcriptional synergy.

1997 ◽  
Vol 17 (6) ◽  
pp. 3345-3355 ◽  
Author(s):  
J M Shirokawa ◽  
A J Courey
Keyword(s):  
Transcriptional Activation ◽  
Binding Sites ◽  
Drosophila Embryo ◽  
Binding Interaction ◽  
Protein Protein Interaction ◽  
Helix Loop Helix ◽  
Early Drosophila Embryo ◽  
Sufficient Strength ◽  
Rel Homology Domain

The establishment of mesoderm and neuroectoderm in the early Drosophila embryo relies on interactions between the Dorsal morphogen and basic-helix-loop-helix (bHLH) activators. Here we show that Dorsal and the bHLH activator Twist synergistically activate transcription in cell culture and in vitro from a promoter containing binding sites for both factors. Somewhat surprisingly, a region of Twist outside the conserved bHLH domain is required for the synergy. In Dorsal, the rel homology domain appears to be sufficient for synergy. Protein-protein interaction assays show that Twist and Dorsal bind to one another in vitro. However, this interaction does not appear to be of sufficient strength to yield cooperative binding to DNA. Nonetheless, the regions of Twist and Dorsal required for the binding interaction are also required for synergistic transcriptional activation.

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.

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