scholarly journals Phosphorylation represses Ets-1 DNA binding by reinforcing autoinhibition

2000 ◽  
Vol 14 (3) ◽  
pp. 366-376 ◽  
Author(s):  
Dale O. Cowley ◽  
Barbara J. Graves
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Inhibitory Effect ◽  
Dna Binding Domain ◽  
Structural Elements ◽  
Binding Assays ◽  
Control Of Gene Expression ◽  
Structural Mechanism ◽  
Calcium Dependent ◽  
Dependent Inhibition

Phosphorylation of transcription factors is a key link between cell signaling and the control of gene expression. Here we report that phosphorylation regulates DNA binding of the Ets-1 transcription factor by reinforcing an autoinhibitory mechanism. Quantitative DNA-binding assays show that calcium-dependent phosphorylation inhibits Ets-1 DNA binding 50-fold. The four serines that mediate this inhibitory effect are distant from the DNA-binding domain but near structural elements required for autoinhibition. Mutational analyses demonstrate that an intact inhibitory module is required for phosphorylation-dependent regulation. Partial proteolysis studies indicate that phosphorylation stabilizes an inhibitory conformation. These findings provide a structural mechanism for phosphorylation-dependent inhibition of Ets-1 DNA binding and demonstrate a new function for inhibitory modules as structural mediators of negative signaling events.

Solution Structure of the B-Myb DNA-Binding Domain:  A Possible Role for Conformational Instability of the Protein in DNA Binding and Control of Gene Expression†,‡

Biochemistry ◽  
1998 ◽  
Vol 37 (27) ◽  
pp. 9619-9629 ◽  
Author(s):  
Pauline B. McIntosh ◽  
Tom A. Frenkiel ◽  
Ute Wollborn ◽  
John E. McCormick ◽  
Karl-Heinz Klempnauer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Solution Structure ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Control Of Gene Expression ◽  
And Control

Steroid receptor-mediated inhibition of rat prolactin gene expression does not require the receptor DNA-binding domain

Cell ◽  
1988 ◽  
Vol 52 (5) ◽  
pp. 685-695 ◽  
Author(s):  
Stuart Adler ◽  
Marian L. Waterman ◽  
Xi He ◽  
Michael G. Rosenfeld
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Steroid Receptor ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Prolactin Gene

scholarly journals Adaptive evolution of transcription factor binding affinities

2017 ◽  
Author(s):  
Jungeui Hong ◽  
Nathan Brandt ◽  
Ally Yang ◽  
Tim Hughes ◽  
David Gresham
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Dna Binding ◽  
Adaptive Evolution ◽  
Consensus Sequence ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Missense Mutations ◽  
Binding Affinities ◽  
Synonymous Mutations

Understanding the molecular basis of gene expression evolution is a central problem in evolutionary biology. However, connecting changes in gene expression to increased fitness, and identifying the functional basis of those changes, remains challenging. To study adaptive evolution of gene expression in real time, we performed long term experimental evolution (LTEE) of Saccharomyces cerevisiae (budding yeast) in ammonium-limited chemostats. Following several hundred generations of continuous selection we found significant divergence of nitrogen-responsive gene expression in lineages with increased fitness. In multiple independent lineages we found repeated selection for non-synonymous mutations in the zinc finger DNA binding domain of the activating transcription factor (TF), GAT1, that operates within incoherent feedforward loops to control expression of the nitrogen catabolite repression (NCR) regulon. Missense mutations in the DNA binding domain of GAT1 reduce its binding affinity for the GATAA consensus sequence in a promoter-specific manner, resulting in increased expression of ammonium permease genes via both direct and indirect effects, thereby conferring increased fitness. We find that altered transcriptional output of the NCR regulon results in antagonistic pleiotropy in alternate environments and that the DNA binding domain of GAT1 is subject to purifying selection in natural populations. Our study shows that adaptive evolution of gene expression can entail tuning expression output by quantitative changes in TF binding affinities while maintaining the overall topology of a gene regulatory network.

scholarly journals AguR Is Required for Induction of the Streptococcus mutans Agmatine Deiminase System by Low pH and Agmatine

2009 ◽  
Vol 75 (9) ◽  
pp. 2629-2637 ◽  
Author(s):  
Yaling Liu ◽  
Lin Zeng ◽  
Robert A. Burne
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Streptococcus Mutans ◽  
Low Ph ◽  
Binding Domain ◽  
Negative Regulator ◽  
Dna Binding Domain ◽  
Mutant Proteins ◽  
Acidic Conditions ◽  
Agmatine Deiminase

ABSTRACT Acidic conditions and the presence of exogenous agmatine are required to achieve maximal expression of the agmatine deiminase system (AgDS) of Streptococcus mutans. Here we demonstrate that the transcriptional activator of the AgDS, AguR, is required for the responses to agmatine and to low pH. Linker scanning mutagenesis was used to create a panel of mutated aguR genes that were utilized to complement an aguR deletion mutant of S. mutans. The level of production of the mutant proteins was shown to be comparable to that of the wild-type AguR protein. Mutations in the predicted DNA binding domain of AguR eliminated activation of the agu operon. Insertions into the region connecting the DNA binding domain to the predicted extracellular and transmembrane domains were well tolerated. In contrast, a variety of mutants were isolated that had a diminished capacity to respond to low pH but retained the ability to activate AgDS gene expression in response to agmatine, and vice versa. Also, a number of mutants were unable to respond to either agmatine or low pH. AguD, which is a predicted agmatine-putrescine antiporter, was found to be a negative regulator of AgDS gene expression in the absence of exogenous agmatine but was not required for low-pH induction of the AgDS genes. This study reveals that the control of AgDS gene expression by both agmatine and low pH is coordinated through the AguR protein and begins to identify domains of the protein involved in sensing and signaling.

The snail repressor establishes a muscle/notochord boundary in the Ciona embryo

Development ◽  
1998 ◽  
Vol 125 (13) ◽  
pp. 2511-2520 ◽  
Author(s):  
S. Fujiwara ◽  
J.C. Corbo ◽  
M. Levine
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Sites ◽  
Promoter Region ◽  
Muscle Cells ◽  
Specific Pattern ◽  
Binding Assays ◽  
Heterologous Promoter ◽  
Present Evidence ◽  
Tail Muscle

Previous studies have identified a minimal 434 bp enhancer from the promoter region of the Ciona Brachyury gene (Ci-Bra), which is sufficient to direct a notochord-specific pattern of gene expression. Here we present evidence that a Ciona homolog of snail (Ci-sna) encodes a repressor of the Ci-Bra enhancer in the tail muscles. DNA-binding assays identified four Ci-Sna-binding sites in the Ci-Bra enhancer, and mutations in these sites cause otherwise normal Ci-Bra/lacZ transgenes to be misexpressed in ectopic tissues, particularly the tail muscles. Selective misexpression of Ci-sna using a heterologous promoter results in the repression of Ci-Bra/lacZ transgenes in the notochord. Moreover, the conversion of the Ci-Sna repressor into an activator results in the ectopic induction of Ci-Bra/lacZ transgenes in the muscles, and also causes an intermixing of notochord and muscle cells during tail morphogenesis. These results suggest that Ci-Sna functions as a boundary repressor, which subdivides the mesoderm into separate notochord and tail muscle lineages.

scholarly journals RstA Is a Major Regulator ofClostridioides difficileToxin Production and Motility

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Adrianne N. Edwards ◽  
Brandon R. Anjuwon-Foster ◽  
Shonna M. McBride
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Toxin Production ◽  
Toxin Gene ◽  
Dna Binding Domain ◽  
Content Type ◽  
Toxin Genes ◽  
Clostridioides Difficile ◽  
Species Specific ◽  
Toxin Gene Expression

ABSTRACTClostridioides difficileinfection (CDI) is a toxin-mediated diarrheal disease. Several factors have been identified that influence the production of the two majorC. difficiletoxins, TcdA and TcdB, but prior published evidence suggested that additional unknown factors were involved in toxin regulation. Previously, we identified aC. difficileregulator, RstA, that promotes sporulation and represses motility and toxin production. We observed that the predicted DNA-binding domain of RstA was required for RstA-dependent repression of toxin genes, motility genes, andrstAtranscription. In this study, we further investigated the regulation of toxin and motility gene expression by RstA. DNA pulldown assays confirmed that RstA directly binds therstApromoter via the predicted DNA-binding domain. Through mutational analysis of therstApromoter, we identified several nucleotides that are important for RstA-dependent transcriptional regulation. Further, we observed that RstA directly binds and regulates the promoters of the toxin genestcdAandtcdB, as well as the promoters for thesigDandtcdRgenes, which encode regulators of toxin gene expression. Complementation analyses with theClostridium perfringensRstA ortholog and a multispecies chimeric RstA protein revealed that theC. difficileC-terminal domain is required for RstA DNA-binding activity, suggesting that species-specific signaling controls RstA function. Our data demonstrate that RstA is a transcriptional repressor that autoregulates its own expression and directly inhibits transcription of the two toxin genes and two positive toxin regulators, thereby acting at multiple regulatory points to control toxin production.IMPORTANCEClostridioides difficileis an anaerobic, gastrointestinal pathogen of humans and other mammals.C. difficileproduces two major toxins, TcdA and TcdB, which cause the symptoms of the disease, and forms dormant endospores to survive the aerobic environment outside the host. A recently discovered regulatory factor, RstA, inhibits toxin production and positively influences spore formation. Herein, we determine that RstA directly binds its own promoter DNA to repress its own gene transcription. In addition, our data demonstrate that RstA directly represses toxin gene expression and gene expression of two toxin gene activators, TcdR and SigD, creating a complex regulatory network to tightly control toxin production. This study provides a novel regulatory link betweenC. difficilesporulation and toxin production. Further, our data suggest thatC. difficiletoxin production is regulated through a direct, species-specific sensing mechanism.

scholarly journals An IFN regulatory factor-2 DNA-binding domain dominant negative mutant exhibits altered cell growth and gene expression

Oncogene ◽  
2000 ◽  
Vol 19 (11) ◽  
pp. 1411-1418 ◽  
Author(s):  
Y R Rubinstein ◽  
P H Driggers ◽  
V V Ogryzko ◽  
A M Thornton ◽  
K Ozato ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Growth ◽  
Dna Binding ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dna Binding Domain ◽  
Regulatory Factor ◽  
Altered Cell ◽  
Negative Mutant

The discovery of zinc fingers and their development for practical applications in gene regulation and genome manipulation

2010 ◽  
Vol 43 (1) ◽  
pp. 1-21 ◽  
Author(s):  
Aaron Klug
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Zinc Finger ◽  
Binding Proteins ◽  
Zinc Fingers ◽  
Dna Binding Proteins ◽  
Functional Domains ◽  
Control Of Gene Expression ◽  
Activation Domains ◽  
Finger Peptides

AbstractA long-standing goal of molecular biologists has been to construct DNA-binding proteins for the control of gene expression. The classical Cys2His2 (C2H2) zinc finger design is ideally suited for such purposes. Discriminating between closely related DNA sequences both in vitro and in vivo, this naturally occurring design was adopted for engineering zinc finger proteins (ZFPs) to target genes specifically.Zinc fingers were discovered in 1985, arising from the interpretation of our biochemical studies on the interaction of the Xenopus protein transcription factor IIIA (TFIIIA) with 5S RNA. Subsequent structural studies revealed its three-dimensional structure and its interaction with DNA. Each finger constitutes a self-contained domain stabilized by a zinc (Zn) ion ligated to a pair of cysteines and a pair of histidines and also by an inner structural hydrophobic core. This discovery showed not only a new protein fold but also a novel principle of DNA recognition. Whereas other DNA-binding proteins generally make use of the 2-fold symmetry of the double helix, functioning as homo- or heterodimers, zinc fingers can be linked linearly in tandem to recognize nucleic acid sequences of varying lengths. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA (or RNA). It is therefore not surprising that the zinc finger is found widespread in nature, including 3% of the genes of the human genome.The zinc finger design can be used to construct DNA-binding proteins for specific intervention in gene expression. By fusing selected zinc finger peptides to repression or activation domains, genes can be selectively switched off or on by targeting the peptide to the desired gene target. It was also suggested that by combining an appropriate zinc finger peptide with other effector or functional domains, e.g. from nucleases or integrases to form chimaeric proteins, genomes could be modified or manipulated.The first example of the power of the method was published in 1994 when a three-finger protein was constructed to block the expression of a human oncogene transformed into a mouse cell line. The same paper also described how a reporter gene was activated by targeting an inserted 9-base pair (bp) sequence, which acts as the promoter. Thus, by fusing zinc finger peptides to repression or activation domains, genes can be selectively switched off or on. It was also suggested that, by combining zinc fingers with other effector or functional domains, e.g. from nucleases or integrases, to form chimaeric proteins, genomes could be manipulated or modified.Several applications of such engineered ZFPs are described here, including some of therapeutic importance, and also their adaptation for breeding improved crop plants.

Deletion and overexpression of the Aspergillus nidulans GATA factor AreB reveals unexpected pleiotropy

Microbiology ◽  
2009 ◽  
Vol 155 (12) ◽  
pp. 3868-3880 ◽  
Author(s):  
Koon Ho Wong ◽  
Michael J. Hynes ◽  
Richard B. Todd ◽  
Meryl A. Davis
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Aspergillus Nidulans ◽  
Zinc Finger ◽  
Leucine Zipper ◽  
Binding Domain ◽  
Negative Regulator ◽  
Dna Binding Domain ◽  
Gata Factor ◽  
Nitrogen Acquisition

The Aspergillus nidulans transcription factor AreA is a key regulator of nitrogen metabolic gene expression. AreA contains a C-terminal GATA zinc finger DNA-binding domain and activates expression of genes necessary for nitrogen acquisition. Previous studies identified AreB as a potential negative regulator of nitrogen catabolism showing similarity with Penicillium chrysogenum NreB and Neurospora crassa ASD4. The areB gene encodes multiple products containing an N-terminal GATA zinc finger and a leucine zipper motif. We deleted the areB gene and now show that AreB negatively regulates AreA-dependent nitrogen catabolic gene expression under nitrogen-limiting or nitrogen-starvation conditions. AreB also acts pleiotropically, with functions in growth, conidial germination and asexual development, though not in sexual development. AreB overexpression results in severe growth inhibition, aberrant cell morphology and reduced AreA-dependent gene expression. Deletion of either the DNA-binding domain or the leucine zipper domain results in loss of both nitrogen and developmental phenotypes.

scholarly journals The PAX3 and 7 homeodomains have evolved unique determinants that influence DNA-binding, structure and communication with the paired domain

2019 ◽  
Author(s):  
Gareth N. Corry ◽  
Brian D. Sykes ◽  
D. Alan Underhill
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Ternary Complex ◽  
Phylogenetic Analyses ◽  
Inhibitory Effect ◽  
Binding Assays ◽  
Paired Domain ◽  
Functional Diversification ◽  
H Nmr

ABSTRACTThe PAX (paired box) family is a collection of metazoan transcription factors defined by the paired domain, which confers sequence-specific DNA-binding. Ancestral PAX proteins also contained a homeodomain, which can communicate with the paired domain to modulate DNA-binding. In the present study, we sought to identify determinants of this functional interaction using the paralogous PAX3 and 7 proteins. First, we evaluated a group of heterologous paired domains and homeodomains for the ability to bind DNA cooperatively through formation of a ternary complex (paired domain:homeodomain:DNA). This revealed that capacity for ternary complex formation was unique to the PAX3 and 7 homeodomains and therefore not simply a consequence of DNA-binding. We also found PAX3 and 7 were distinguished by an extended region of conservation N-terminal to the homeodomain (NTE). Phylogenetic analyses established the NTE was restricted to PAX3/7 orthologs of segmented metazoans, indicating it arose in a bilaterian precursor prior to separation of deuterostomes and protostomes. In DNA-binding assays, presence of the NTE caused a decrease in monomeric binding by the PAX3 homeodomain that reflected a lack of secondary structure in 1D-1H-NMR. Nevertheless, this inhibitory effect could be overcome by homeodomain dimerization or cooperative binding with the paired domain, establishing that protein interactions could induce homeodomain folding in the presence of the NTE. Strikingly, the PAX7 counterpart did not impair homeodomain binding, revealing inherent differences that could account for its distinct target profile in vivo. Collectively, these findings identify critical determinants of PAX3 and 7 activity, which contribute to their functional diversification.

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