scholarly journals Identification of a New Sea Urchin Ets Protein, SpEts4, by Yeast One-Hybrid Screening with the Hatching Enzyme Promoter

1999 ◽  
Vol 19 (2) ◽  
pp. 1271-1278 ◽  
Author(s):  
Zheng Wei ◽  
Robert C. Angerer ◽  
Lynne M. Angerer
Keyword(s):  
Sea Urchin ◽  
Transcriptional Activation ◽  
Transcriptional Regulator ◽  
Hatching Enzyme ◽  
Nuclear Extracts ◽  
Sea Urchin Embryo ◽  
Ets Family ◽  
High Degree ◽  
Hybrid Screening

ABSTRACT We report the use of a yeast one-hybrid system to isolate a transcriptional regulator of the sea urchin embryo hatching enzyme gene, SpHE. This gene is asymmetrically expressed along the animal-vegetal axis of sea urchin embryos under the cell-autonomous control of maternal regulatory activities and therefore provides an excellent entry point for understanding the mechanism that establishes animal-vegetal developmental polarity. To search for transcriptional regulators, we used a fragment of the SpHE promoter containing several individual elements instead of the conventional bait that contains a multimerized cis element. This screen yielded a number of positive clones that encode a new member of the Ets family, named SpEts4. This protein contains transcriptional activation activity, since expression of reporter genes in yeast does not depend on the presence of the yeast GAL4 activation domain. Sequences in the N-terminal region of SpEts4 mediate the activation activity, as shown by deletion or domain-swapping experiments. The newly identified DNA binding protein binds with a high degree of specificity to aSpHE promoter Ets element and forms a complex with a mobility identical to that obtained with 9-h sea urchin embryo nuclear extracts. SpEts4 positively regulates SpHE transcription, since mutation of the SpEts4 site in SpHE promoter transgenes reduces promoter activity in vivo while SpEts4mRNA coinjection increases its output. As expected for a positiveSpHE transcriptional regulator, the timing ofSpEts4 gene expression precedes the transient expression ofSpHE in the very early sea urchin blastula.

scholarly journals Repression of GCN5 Histone Acetyltransferase Activity via Bromodomain-Mediated Binding and Phosphorylation by the Ku–DNA-Dependent Protein Kinase Complex

1998 ◽  
Vol 18 (3) ◽  
pp. 1349-1358 ◽  
Author(s):  
Nickolai A. Barlev ◽  
Vladimir Poltoratsky ◽  
Tom Owen-Hughes ◽  
Carol Ying ◽  
Lin Liu ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Transcriptional Activation ◽  
Histone Acetyltransferase ◽  
Dependent Protein Kinase ◽  
Nuclear Extracts ◽  
Dependent Protein ◽  
Endogenous Proteins ◽  
Hybrid Screening

ABSTRACT GCN5, a putative transcriptional adapter in humans and yeast, possesses histone acetyltransferase (HAT) activity which has been linked to GCN5’s role in transcriptional activation in yeast. In this report, we demonstrate a functional interaction between human GCN5 (hGCN5) and the DNA-dependent protein kinase (DNA-PK) holoenzyme. Yeast two-hybrid screening detected an interaction between the bromodomain of hGCN5 and the p70 subunit of the human Ku heterodimer (p70-p80), which is the DNA-binding component of DNA-PK. Interaction between intact hGCN5 and Ku70 was shown biochemically using recombinant proteins and by coimmunoprecipitation of endogenous proteins following chromatography of HeLa nuclear extracts. We demonstrate that the catalytic subunit of DNA-PK phosphorylates hGCN5 both in vivo and in vitro and, moreover, that the phosphorylation inhibits the HAT activity of hGCN5. These findings suggest a possible regulatory mechanism of HAT activity.

scholarly journals Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

2015 ◽  
Vol 36 (6) ◽  
pp. 913-922 ◽  
Author(s):  
Nallani Vijay Kumar ◽  
Jianbo Yang ◽  
Jitesh K. Pillai ◽  
Swati Rawat ◽  
Carlos Solano ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Transcriptional Response ◽  
Transcriptional Regulator ◽  
Yeast Protein ◽  
Content Type ◽  
Arsenic Tolerance ◽  
Arsenic Sensing

The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeastSaccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)]in vitroandin vivoand that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.

Comparative in vivo evaluation of polyalkoxy substituted 4H-chromenes and oxa-podophyllotoxins as microtubule destabilizing agents in the phenotypic sea urchin embryo assay

2014 ◽  
Vol 24 (16) ◽  
pp. 3914-3918 ◽  
Author(s):  
Marina N. Semenova ◽  
Dmitry V. Tsyganov ◽  
Oleg R. Malyshev ◽  
Oleg V. Ershov ◽  
Ivan N. Bardasov ◽  
...  
Keyword(s):  
Sea Urchin ◽  
In Vivo Evaluation ◽  
Sea Urchin Embryo

Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription

1990 ◽  
Vol 10 (6) ◽  
pp. 2832-2839
Author(s):  
A S Ponticelli ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Site ◽  
Activator Proteins ◽  
Nuclear Extracts ◽  
Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

Spatial expression of the hatching enzyme gene in the sea urchin embryo

1992 ◽  
Vol 150 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Thierry Lepage ◽  
Christian Sardet ◽  
Christian Gache
Keyword(s):  
Sea Urchin ◽  
Enzyme Gene ◽  
Spatial Expression ◽  
Hatching Enzyme ◽  
Sea Urchin Embryo

scholarly journals The DRIP Complex and SRC-1/p160 Coactivators Share Similar Nuclear Receptor Binding Determinants but Constitute Functionally Distinct Complexes

2000 ◽  
Vol 20 (8) ◽  
pp. 2718-2726 ◽  
Author(s):  
Christophe Rachez ◽  
Matthew Gamble ◽  
Chao-Pei Betty Chang ◽  
G. Brandon Atkins ◽  
Mitchell A. Lazar ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Receptor Binding ◽  
Transcriptional Activation ◽  
Thyroid Hormone Receptor ◽  
Nuclear Receptor Coactivators ◽  
Nuclear Extracts ◽  
Sequence Requirements

ABSTRACT Transcriptional activation requires both access to DNA assembled as chromatin and functional contact with components of the basal transcription machinery. Using the hormone-bound vitamin D3receptor (VDR) ligand binding domain (LBD) as an affinity matrix, we previously identified a novel multisubunit coactivator complex, DRIP (VDR-interacting proteins), required for transcriptional activation by nuclear receptors and several other transcription factors. In this report, we characterize the nuclear receptor binding features of DRIP205, a key subunit of the DRIP complex, that interacts directly with VDR and thyroid hormone receptor in response to ligand and anchors the other DRIP subunits to the nuclear receptor LBD. In common with other nuclear receptor coactivators, DRIP205 interaction occurs through one of two LXXLL motifs and requires the receptor's AF-2 subdomain. Although the second motif of DRIP205 is required only for VDR binding in vitro, both motifs are used in the context of an retinoid X receptor-VDR heterodimer on DNA and in transactivation in vivo. We demonstrate that both endogenous p160 coactivators and DRIP complexes bind to the VDR LBD from nuclear extracts through similar sequence requirements, but they do so as distinct complexes. Moreover, in contrast to the p160 family of coactivators, the DRIP complex is devoid of any histone acetyltransferase activity. The results demonstrate that different coactivator complexes with distinct functions bind to the same transactivation region of nuclear receptors, suggesting that they are both required for transcription activation by nuclear receptors.

scholarly journals Ligand-Controlled Proteolysis of the Escherichia coli Transcriptional Regulator ZntR

2007 ◽  
Vol 189 (8) ◽  
pp. 3017-3025 ◽  
Author(s):  
Mihaela Pruteanu ◽  
Saskia B. Neher ◽  
Tania A. Baker
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcriptional Regulator ◽  
Zinc Homeostasis ◽  
Fine Tuning ◽  
Physical Analysis ◽  
Cellular Environment ◽  
High Zinc

ABSTRACT Proteases play a crucial role in remodeling the bacterial proteome in response to changes in cellular environment. Escherichia coli ZntR, a zinc-responsive transcriptional regulator, was identified by proteomic experiments as a likely ClpXP substrate, suggesting that protein turnover may play a role in regulation of zinc homeostasis. When intracellular zinc levels are high, ZntR activates expression of ZntA, an ATPase essential for zinc export. We find that ZntR is degraded in vivo in a manner dependent on both the ClpXP and Lon proteases. However, ZntR degradation decreases in the presence of high zinc concentrations, the level of ZntR rises, and transcription of the zntA exporter is increased. Mutagenesis experiments reveal that zinc binding does not appear to be solely responsible for the zinc-induced protection from proteolysis. Therefore, we tested whether DNA binding was important in the zinc-induced stabilization of ZntR by mutagenesis of the DNA binding helices. Replacement of a conserved arginine (R19A) in the DNA binding domain both enhances ZntR degradation and abolishes zinc-induced transcriptional activation of zntA. Biochemical and physical analysis of ZntRR19A demonstrates that it is structurally similar to, and binds zinc as well as does, the wild-type protein but is severely defective in binding DNA. Thus, we conclude that two different ligands—zinc and DNA—function together to increase ZntR stability and that ligand-controlled proteolysis of ZntR plays an important role in fine-tuning zinc homeostasis in bacteria.

scholarly journals Elf-1 Contributes to the Function of the Complex Interleukin (IL)-2–responsive Enhancer in the Mouse IL-2 Receptor α Gene

1997 ◽  
Vol 185 (7) ◽  
pp. 1211-1222 ◽  
Author(s):  
Irina Serdobova ◽  
Maria Pla ◽  
Patrick Reichenbach ◽  
Peter Sperisen ◽  
Jacques Ghysdael ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Proximal Region ◽  
Binding Motif ◽  
Nuclear Extracts ◽  
Vitro Binding ◽  
Ets Family ◽  
Cd8 Cell ◽  
Promoter Proximal Region

Lymphocytes regulate their responsiveness to IL-2 through the transcriptional control of the IL-2Rα gene, which encodes a component of the high affinity IL-2 receptor. In the mouse IL-2Rα gene this control is exerted via two regulatable elements, a promoter proximal region, and an IL-2–responsive enhancer (IL-2rE) 1.3 kb upstream. In vitro and in vivo functional analysis of the IL-2rE in the rodent thymic lymphoma-derived, CD4−CD8− cell line PC60 demonstrated that three separate elements, sites I, II, and III, were necessary for IL-2 responsiveness; these three sites demonstrate functional cooperation. Site III contains a consensus binding motif for members of the Ets family of transcription factors. Here we demonstrate that Elf-1, an Ets-like protein, binds to site III and participates in IL-2 responsiveness. In vitro site III forms a complex with a protein constitutively present in nuclear extracts from PC60 cells as well as from normal CD4−CD8− thymocytes. We have identified this molecule as Elf-1 according to a number of criteria. The complex possesses an identical electrophoretic mobility to that formed by recombinant Elf-1 protein and is super-shifted by anti–Elf-1 antibodies. Biotinylated IL-2rE probes precipitate Elf-1 from PC60 extracts provided site III is intact and both recombinant and PC60-derived proteins bind with the same relative affinities to different mutants of site III. In addition, by introducing mutations into the core of the site III Ets-like motif and comparing the corresponding effects on the in vitro binding of Elf-1 and the in vivo IL-2rE activity, we provide strong evidence that Elf-1 is directly involved in IL-2 responsiveness. The nature of the functional cooperativity observed between Elf-1 and the factors binding sites I and II remains unresolved; experiments presented here however suggest that this effect may not require direct interactions between the proteins binding these three elements.

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