Smooth Muscle-Specific Genes Are Differentially Sensitive to Inhibition by Elk-1

ABSTRACT Understanding the mechanism of smooth muscle cell (SMC) differentiation will provide the foundation for elucidating SMC-related diseases, such as atherosclerosis, restenosis, and asthma. In the current study, overexpression of Elk-1 in SMCs down-regulated expression of several endogenous smooth muscle-restricted proteins, including telokin, SM22α, and smooth muscle α-actin. In contrast, down-regulation of endogenous Elk-1 in smooth muscle cells increased the expression of only telokin and SM22α, suggesting that smooth muscle-specific promoters are differentially sensitive to the inhibitory effects of Elk-1. Consistent with this, overexpression of the DNA binding domain of Elk-1, which acts as a dominant-negative protein by displacing endogenous Elk-1, enhanced the expression of telokin and SM22α without affecting expression of smooth muscle α-actin. Elk-1 suppressed the activity of smooth muscle-restricted promoters, including the telokin promoter that does not contain a consensus Elk-1 binding site, through its ability to block myocardin-induced activation of the promoters. Gel mobility shift and chromatin immunoprecipitation assays revealed that Elk-1 binds to a nonconsensus binding site in the telokin promoter and Elk-1 binding is dependent on serum response factor (SRF) binding to a nearby CArG box. Although overexpression of the SRF-binding B-box domain of Elk-1 is sufficient to repress the myocardin activation of the telokin promoter, this repression is not as complete as that seen with an Elk-1 fragment that includes the DNA binding domain. In addition, reporter gene assays demonstrate that an intact Elk-1 binding site in the telokin promoter is required for Elk-1 to maximally inhibit promoter activity. Together, these data suggest that the differential sensitivity of smooth muscle-specific genes to inhibition by Elk-1 may play a role in the complex changes in smooth muscle-specific protein expression that are observed under pathological conditions.

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Role of the IE62 Consensus Binding Site in Transactivation by the Varicella-Zoster Virus IE62 Protein

Journal of Virology ◽

10.1128/jvi.02522-09 ◽

2010 ◽

Vol 84 (8) ◽

pp. 3767-3779 ◽

Cited By ~ 13

Author(s):

Kris White ◽

Hua Peng ◽

John Hay ◽

William T. Ruyechan

Keyword(s):

Dna Binding ◽

Varicella Zoster Virus ◽

Binding Site ◽

Consensus Sequence ◽

Binding Domain ◽

Dna Binding Domain ◽

Consensus Binding Site ◽

Mobility Shift ◽

Varicella Zoster ◽

Mobility Shift Assay

ABSTRACT The varicella-zoster virus (VZV) IE62 protein is the major transcriptional activator. IE62 is capable of associating with DNA both nonspecifically and in a sequence-specific manner via a consensus binding site (5′-ATCGT-3′). However, the function of the consensus site is poorly understood, since IE62 efficiently transactivates promoter elements lacking this sequence. In the work presented here, sequence analysis of the VZV genome revealed the presence of 245 IE62 consensus sites throughout the genome. Some 54 sites were found to be present within putative VZV promoters. Electrophoretic mobility shift assay (EMSA) experiments using an IE62 fragment containing the IE62 DNA-binding domain and duplex oligonucleotides that did or did not contain the IE62 consensus binding sequence yielded KD (equilibrium dissociation constant) values in the nanomolar range. Further, the IE62 DNA binding domain was shown to have a 5-fold-increased affinity for its consensus site compared to nonconsensus sequences. The effect of consensus site presence and position on IE62-mediated activation of native VZV and model promoters was examined using site-specific mutagenesis and transfection and superinfection reporter assays. In all promoters examined, the consensus sequence functioned as a distance-dependent repressive element. Protein recruitment assays utilizing the VZV gI promoter indicated that the presence of the consensus site increased the recruitment of IE62 but not Sp1. These data suggest a model where the IE62 consensus site functions to down-modulate IE62 activation, and interaction of IE62 with this sequence may result in loss or decrease of the ability of IE62 to recruit cellular factors needed for full promoter activation.

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The DNA-binding domain of HPV-16 E2 protein interaction with the viral enhancer: Protein-induced DNA bending and role of the nonconserved core sequence in binding site affinity

Virology ◽

10.1016/0042-6822(90)90109-5 ◽

1990 ◽

Vol 174 (2) ◽

pp. 557-575 ◽

Cited By ~ 26

Author(s):

Camille L. Bedrosian ◽

Deepak Bastiav

Keyword(s):

Dna Binding ◽

Binding Site ◽

Protein Interaction ◽

Dna Bending ◽

Binding Domain ◽

Dna Binding Domain ◽

Hpv 16 ◽

E2 Protein ◽

Core Sequence

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An Integrated Computational and Experimental Binding Study Identifies the DNA Binding Domain as the Putative Binding Site of Novel Pyrimidinetrione Signal Transducer and Activator of Transcription 3 (STAT3) Inhibitors

Drug Designing Open Access ◽

10.4172/2169-0138.1000142 ◽

2017 ◽

Vol 06 (01) ◽

Cited By ~ 1

Author(s):

Shan Sun ◽

Peibin Yue ◽

Mingzhu He ◽

Xiaolei Zhang ◽

David Paladino ◽

...

Keyword(s):

Dna Binding ◽

Binding Site ◽

Binding Domain ◽

Binding Study ◽

Dna Binding Domain ◽

Signal Transducer ◽

Transcription 3 ◽

Putative Binding Site

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Cell Cycle Localization, Dimerization, and Binding Domain Architecture of the Telomere Protein cPot1

Molecular and Cellular Biology ◽

10.1128/mcb.24.5.2091-2102.2004 ◽

2004 ◽

Vol 24 (5) ◽

pp. 2091-2102 ◽

Cited By ~ 28

Author(s):

Chao Wei ◽

Carolyn M. Price

Keyword(s):

Cell Cycle ◽

Dna Binding ◽

Binding Site ◽

Domain Architecture ◽

Binding Domain ◽

Binding Motif ◽

Dna Binding Domain ◽

Telomere Protein ◽

Ob Fold

ABSTRACT Pot1 is a single-stranded-DNA-binding protein that recognizes telomeric G-strand DNA. It is essential for telomere capping in Saccharomyces pombe and regulates telomere length in humans. Human Pot1 also interacts with proteins that bind the duplex region of the telomeric tract. Thus, like Cdc13 from S. cerevisiae, Pot 1 may have multiple roles at the telomere. We show here that endogenous chicken Pot1 (cPot1) is present at telomeres during periods of the cell cycle when t loops are thought to be present. Since cPot1 can bind internal loops and directly adjacent DNA-binding sites, it is likely to fully coat and protect both G-strand overhangs and the displaced G strand of a t loop. The minimum binding site of cPot1 is double that of the S. pombe DNA-binding domain. Although cPot can self associate, dimerization is not required for DNA binding and hence does not explain the binding-site duplication. Instead, the DNA-binding domain appears to be extended to contain a second binding motif in addition to the conserved oligonucleotide-oligosaccharide (OB) fold present in other G-strand-binding proteins. This second motif could be another OB fold. Although dimerization is inefficient in vitro, it may be regulated in vivo and could promote association with other telomere proteins and/or telomere compaction.

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Dissection of a carboxy-terminal region of the yeast regulatory protein RAP1 with effects on both transcriptional activation and silencing

Molecular and Cellular Biology ◽

10.1128/mcb.12.3.1209-1217.1992 ◽

1992 ◽

Vol 12 (3) ◽

pp. 1209-1217

Author(s):

C F Hardy ◽

D Balderes ◽

D Shore

Keyword(s):

Dna Binding ◽

Binding Site ◽

Transcriptional Activation ◽

Binding Sites ◽

Binding Protein ◽

Binding Domain ◽

Dominant Negative Effect ◽

Dna Binding Domain ◽

Wild Type ◽

Carboxy Terminal

RAP1 is an essential sequence-specific DNA-binding protein in Saccharomyces cerevisiae whose binding sites are found in a large number of promoters, where they function as upstream activation sites, and at the silencer elements of the HMR and HML mating-type loci, where they are important for repression. We have examined the involvement of specific regions of the RAP1 protein in both repression and activation of transcription by studying the properties of a series of hybrid proteins containing RAP1 sequences fused to the DNA-binding domain of the yeast protein GAL4 (amino acids 1 to 147). GAL4 DNA-binding domain/RAP1 hybrids containing only the carboxy-terminal third of the RAP1 protein (which lacks the RAP1 DNA-binding domain) function as transcriptional activators of a reporter gene containing upstream GAL4 binding sites. Expression of some hybrids from the strong ADH1 promoter on multicopy plasmids has a dominant negative effect on silencers, leading to either partial or complete derepression of normally silenced genes. The GAL4/RAP1 hybrids have different effects on wild-type and several mutated but functional silencers. Silencers lacking either an autonomously replicating sequence consensus element or the RAP1 binding site are strongly derepressed, whereas the wild-type silencer or a silencer containing a deletion of the binding site for another silencer-binding protein, ABF1, are only weakly affected by hybrid expression. By examining a series of GAL4 DNA-binding domain/RAP1 hybrids, we have mapped the transcriptional activation and derepression functions to specific parts of the RAP1 carboxy terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

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Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome

Nature ◽

10.1038/nature06096 ◽

2007 ◽

Vol 448 (7157) ◽

pp. 1058-1062 ◽

Cited By ~ 683

Author(s):

Yoshiyuki Minegishi ◽

Masako Saito ◽

Shigeru Tsuchiya ◽

Ikuya Tsuge ◽

Hidetoshi Takada ◽

...

Keyword(s):

Dna Binding ◽

Binding Domain ◽

Dominant Negative ◽

Dna Binding Domain ◽

Hyper Ige Syndrome ◽

Dominant Negative Mutations

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Characterization of a dominant negativeC. elegansTwist mutant protein with implications for human Saethre-Chotzen syndrome

Development ◽

10.1242/dev.129.11.2761 ◽

2002 ◽

Vol 129 (11) ◽

pp. 2761-2772

Author(s):

Ann K. Corsi ◽

Thomas M. Brodigan ◽

Erik M. Jorgensen ◽

Michael Krause

Keyword(s):

Dna Binding ◽

Binding Activity ◽

Binding Domain ◽

Dominant Negative ◽

Mutant Protein ◽

Dna Binding Domain ◽

Mesodermal Cell ◽

C Elegans ◽

Dna Binding Activity ◽

A Charge

Twist is a transcription factor that is required for mesodermal cell fates in all animals studied to date. Mutations of this locus in humans have been identified as the cause of the craniofacial disorder Saethre-Chotzen syndrome. The Caenorhabditis elegans Twist homolog is required for the development of a subset of the mesoderm. A semidominant allele of the gene that codes for CeTwist, hlh-8, has defects that occur earlier in the mesodermal lineage than a previously studied null allele of the gene. The semidominant allele has a charge change (E29K) in the basic DNA-binding domain of CeTwist. Surprisingly, the mutant protein retains DNA-binding activity as both a homodimer and a heterodimer with its partner E/Daughterless (CeE/DA). However, the mutant protein blocks the activation of the promoter of a target gene. Therefore, the mutant CeTwist may cause cellular defects as a dominant negative protein by binding to target promoters as a homo- or heterodimer and then blocking transcription. Similar phenotypes as those caused by the E29K mutation were observed when amino acid substitutions in the DNA-binding domain that are associated with the human Saethre-Chotzen syndrome were engineered into the C. elegans protein. These data suggest that Saethre-Chotzen syndrome may be caused, in some cases, by dominant negative proteins, rather than by haploinsufficiency of the locus.

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A single amino acid change in CUP2 alters its mode of DNA binding.

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4778 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4778-4787 ◽

Cited By ~ 28

Author(s):

C Buchman ◽

P Skroch ◽

W Dixon ◽

T D Tullius ◽

M Karin

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Binding Activity ◽

Binding Domain ◽

Single Amino Acid ◽

Dna Binding Domain ◽

Sequence Motifs ◽

Wild Type ◽

Mobility Shift ◽

Single Amino Acid Change

CUP2 is a copper-dependent transcriptional activator of the yeast CUP1 metallothionein gene. In the presence of Cu+ and Ag+) ions its DNA-binding domain is thought to fold as a cysteine-coordinated Cu cluster which recognizes the palindromic CUP1 upstream activation sequence (UASc). Using mobility shift, methylation interference, and DNase I and hydroxyl radical footprinting assays, we examined the interaction of wild-type and variant CUP2 proteins produced in Escherichia coli with the UASc. Our results suggest that CUP2 has a complex Cu-coordinated DNA-binding domain containing different parts that function as DNA-binding elements recognizing distinct sequence motifs embedded within the UASc. A single-amino-acid substitution of cysteine 11 with a tyrosine results in decreased Cu binding, apparent inactivation of one of the DNA-binding elements and a dramatic change in the recognition properties of CUP2. This variant protein interacts with only one part of the wild-type site and prefers to bind to a different half-site from the wild-type protein. Although the variant has about 10% of wild-type DNA-binding activity, it appears to be completely incapable of activating transcription.

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