Evidence for two modes of cooperative DNA binding in vivo that do not involve direct protein–protein interactions

SWI4 and SWI6 play a crucial role in START-specific transcription in Saccharomyces cerevisiae. SWI4 and SWI6 form a specific complex on the SCB (SWI4/6-dependent cell cycle box) sequences which have been found in the promoters of HO and G1 cyclin genes. Overproduction of SWI4 eliminates the SWI6 dependency of HO transcription in vivo and results in a new SWI6-independent, SCB-specific complex in vitro, which is heterogeneous and reacts with SWI4 antibodies. The C terminus of SWI4 is not required for SWI6-independent binding of SWI4 to SCB sequences, but it is necessary and sufficient for association with SWI6. Both SWI4 and SWI6 contain two copies of a 33-amino-acid TPLH repeat, which has been implicated in protein-protein interactions in other proteins. These repeats are not required for the SWI4-SWI6 association. Alanine substitutions in both TPLH repeats of SWI6 reduce its activity but do not affect the stability of the protein or its association with SWI4. However, these mutations reduce the ability of the SWI4/6 complex to bind DNA. Deletion of the lucine zipper motif in SWI6 also allows SWI4/6 complex formation, but it eliminates the DNA-binding ability of the SWI4/6 complex. This indicates that the integrity of two different regions of SWI6 is required for DNA binding by the SWI4/6 complex. From these data, we propose that the sequence-specific DNA-binding domain resides in SWI4 but that SWI6 controls the accessibility of this domain in the SWI4/6 complex.

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Myogenic Basic Helix-Loop-Helix Proteins and Sp1 Interact as Components of a Multiprotein Transcriptional Complex Required for Activity of the Human Cardiac α-Actin Promoter

Molecular and Cellular Biology ◽

10.1128/mcb.19.4.2577 ◽

1999 ◽

Vol 19 (4) ◽

pp. 2577-2584 ◽

Cited By ~ 61

Author(s):

Elzbieta Biesiada ◽

Yasuo Hamamori ◽

Larry Kedes ◽

Vittorio Sartorelli

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

Serum Response Factor ◽

Multiprotein Complex ◽

Protein Protein Interactions ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Transcriptional Complex

ABSTRACT Activation of the human cardiac α-actin (HCA) promoter in skeletal muscle cells requires the integrity of DNA binding sites for the serum response factor (SRF), Sp1, and the myogenic basic helix-loop-helix (bHLH) family. In this study we report that activation of the HCA correlates with formation of a muscle-specific multiprotein complex on the promoter. We provide evidence that proteins eluted from the multiprotein complex specifically react with antibodies directed against myogenin, Sp1, and SRF and that the complex can be assembled in vitro by using the HCA promoter and purified MyoD, E12, SRF, and Sp1. In vitro and in vivo assays revealed a direct association of Sp1 and myogenin-MyoD mediated by the DNA-binding domain of Sp1 and the HLH motif of myogenin. The results obtained in this study indicate that protein-protein interactions and the cooperative DNA binding of transcriptional activators are critical steps in the formation of a transcriptionally productive multiprotein complex on the HCA promoter and suggest that the same mechanisms might be utilized to regulate the transcription of muscle-specific and other genes.

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A Simple Method to Detect the Inhibition of Transcription Factor-DNA Binding Due to Protein–Protein Interactions In Vivo

Genes ◽

10.3390/genes10090684 ◽

2019 ◽

Vol 10 (9) ◽

pp. 684 ◽

Cited By ~ 1

Author(s):

Guangzhe Yang ◽

Dong Chao ◽

Zhenhua Ming ◽

Jixing Xia

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

Regulatory Elements ◽

Inhibitory Effect ◽

Protein Protein Interactions ◽

Mobility Shift ◽

Simple Method ◽

Mobility Shift Assay ◽

Aureobasidin A

Binding of transcription factors (TFs) to cis-regulatory elements (DNA) could modulate the expression of downstream genes, while interactions between TFs and other proteins might inhibit them binding to DNA. Nowadays, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) approaches are usually employed to detect the inhibitory effect. However, EMSA might not reflect the inhibitory effect in vivo. ChIP requires preparation of specific antibody or stable genetic transformation and complicated experimental steps, making it laborious and time-consuming. Here, based on the yeast one-hybrid (Y1H) system, we present a simple method to detect the inhibition of TF–DNA binding due to protein–protein interactions in vivo. When interactions between TFs and other proteins inhibit TFs binding to DNA, the reporter (Aureobasidin A resistance) gene is not activated, thereby inhibiting yeast growth on media containing the AbA antibiotic. Two examples were tested with the newly developed method to demonstrate its feasibility. In conclusion, this method provides an alternative strategy for detecting the inhibition of DNA-binding of TFs due to their interactions with other proteins in vivo.

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Heterodimerization of Msx and Dlx homeoproteins results in functional antagonism.

Molecular and Cellular Biology ◽

10.1128/mcb.17.5.2920 ◽

1997 ◽

Vol 17 (5) ◽

pp. 2920-2932 ◽

Cited By ~ 189

Author(s):

H Zhang ◽

G Hu ◽

H Wang ◽

P Sciavolino ◽

N Iler ◽

...

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

Expression Patterns ◽

Reciprocal Inhibition ◽

Limb Bud ◽

Protein Protein Interactions ◽

Dna Binding Sites ◽

Dlx Genes ◽

Functional Antagonism

Protein-protein interactions are known to be essential for specifying the transcriptional activities of homeoproteins. Here we show that representative members of the Msx and Dlx homeoprotein families form homo- and heterodimeric complexes. We demonstrate that dimerization by Msx and Dlx proteins is mediated through their homeodomains and that the residues required for this interaction correspond to those necessary for DNA binding. Unlike most other known examples of homeoprotein interactions, association of Msx and Dlx proteins does not promote cooperative DNA binding; instead, dimerization and DNA binding are mutually exclusive activities. In particular, we show that Msx and Dlx proteins interact independently and noncooperatively with homeodomain DNA binding sites and that dimerization is specifically blocked by the presence of such DNA sites. We further demonstrate that the transcriptional properties of Msx and Dlx proteins display reciprocal inhibition. Specifically, Msx proteins act as transcriptional repressors and Dlx proteins act as activators, while in combination, Msx and Dlx proteins counteract each other's transcriptional activities. Finally, we show that the expression patterns of representative Msx and Dlx genes (Msx1, Msx2, Dlx2, and Dlx5) overlap in mouse embryogenesis during limb bud and craniofacial development, consistent with the potential for their protein products to interact in vivo. Based on these observations, we propose that functional antagonism through heterodimer formation provides a mechanism for regulating the transcriptional actions of Msx and Dlx homeoproteins in vivo.

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Analysis of the SWI4/SWI6 protein complex, which directs G1/S-specific transcription in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.1069-1077.1993 ◽

1993 ◽

Vol 13 (2) ◽

pp. 1069-1077

Author(s):

J Sidorova ◽

L Breeden

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Binding ◽

Protein Interactions ◽

Protein Protein Interactions ◽

C Terminus ◽

Specific Complex ◽

Dependent Cell ◽

The Stability

SWI4 and SWI6 play a crucial role in START-specific transcription in Saccharomyces cerevisiae. SWI4 and SWI6 form a specific complex on the SCB (SWI4/6-dependent cell cycle box) sequences which have been found in the promoters of HO and G1 cyclin genes. Overproduction of SWI4 eliminates the SWI6 dependency of HO transcription in vivo and results in a new SWI6-independent, SCB-specific complex in vitro, which is heterogeneous and reacts with SWI4 antibodies. The C terminus of SWI4 is not required for SWI6-independent binding of SWI4 to SCB sequences, but it is necessary and sufficient for association with SWI6. Both SWI4 and SWI6 contain two copies of a 33-amino-acid TPLH repeat, which has been implicated in protein-protein interactions in other proteins. These repeats are not required for the SWI4-SWI6 association. Alanine substitutions in both TPLH repeats of SWI6 reduce its activity but do not affect the stability of the protein or its association with SWI4. However, these mutations reduce the ability of the SWI4/6 complex to bind DNA. Deletion of the lucine zipper motif in SWI6 also allows SWI4/6 complex formation, but it eliminates the DNA-binding ability of the SWI4/6 complex. This indicates that the integrity of two different regions of SWI6 is required for DNA binding by the SWI4/6 complex. From these data, we propose that the sequence-specific DNA-binding domain resides in SWI4 but that SWI6 controls the accessibility of this domain in the SWI4/6 complex.

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Mutations on the DNA-binding surface of TATA-binding protein can specifically impair the response to acidic activators in vivo.

Molecular and Cellular Biology ◽

10.1128/mcb.15.10.5461 ◽

1995 ◽

Vol 15 (10) ◽

pp. 5461-5469 ◽

Cited By ~ 38

Author(s):

M Lee ◽

K Struhl

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

Convex Surface ◽

Binding Protein ◽

Tata Binding Protein ◽

Protein Protein Interactions ◽

Activator Proteins ◽

Tata Element ◽

Binding Surface

The TATA-binding protein (TBP) contains a concave surface that interacts specifically with TATA promoter elements and a convex surface that mediates protein-protein interactions with general and gene-specific transcription factors. Biochemical experiments suggest that interactions between activator proteins and TBP are important in stimulating transcription by the RNA polymerase II machinery. To gain insight into the role of TBP in mediating transcriptional activation in vivo, we implemented a genetic strategy in Saccharomyces cerevisiae that involved the use of a TBP derivative with altered specificity for TATA elements. By genetically screening a set of TBP mutant libraries that were biased to the convex surface that mediates protein-protein interactions, we identified TBP derivatives that are impaired in the response to three acidic activators (Gcn4, Gal4, and Ace1) but appear normal for constitutive polymerase II transcription. A genetic complementation assay indicates that the activation-defective phenotypes reflect specific functional properties of the TBP derivatives rather than an indirect effect on transcription. Surprisingly, three of the four activation-defective mutants affect residues that directly contact DNA. Moreover, all four mutants are defective for TATA element binding, but they interact normally with an acidic activation domain and TFIIB. In addition, we show that a subset of TBP derivatives with mutations on the DNA-binding surface of TBP are also compromised in their responses to acidic activators in vivo. These observations suggest that interactions at the TBP-TATA element interface can specifically affect the response to acidic activator proteins in vivo.

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Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

10.26434/chemrxiv.12052869.v1 ◽

2020 ◽

Author(s):

James Frederich ◽

Ananya Sengupta ◽

Josue Liriano ◽

Ewa A. Bienkiewicz ◽

Brian G. Miller

Keyword(s):

Protein Interactions ◽

Neurological Diseases ◽

Adapter Proteins ◽

Protein Protein Interactions ◽

Specific Expression ◽

Individual Client ◽

Isoform Specificity ◽

Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

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Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism

Nature Communications ◽

10.1038/s41467-021-22253-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Christopher R. Horne ◽

Hariprasad Venugopal ◽

Santosh Panjikar ◽

David M. Wood ◽

Amy Henrickson ◽

...

Keyword(s):

Sialic Acid ◽

Dna Binding ◽

Protein Interactions ◽

Acid Metabolism ◽

Environmental Changes ◽

Dna Interaction ◽

Protein Protein Interactions ◽

Nutrient Source ◽

Cryo Electron Microscopy ◽

Close Proximity

AbstractBacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)3-repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA)3-repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.

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Ways into Understanding HIF Inhibition

Cancers ◽

10.3390/cancers13010159 ◽

2021 ◽

Vol 13 (1) ◽

pp. 159

Author(s):

Tina Schönberger ◽

Joachim Fandrey ◽

Katrin Prost-Fingerle

Keyword(s):

Protein Interactions ◽

Target Genes ◽

Protein Protein Interactions ◽

Low Oxygen ◽

Drug Candidates ◽

Open Questions ◽

Wide Range ◽

Hif Pathway

Hypoxia is a key characteristic of tumor tissue. Cancer cells adapt to low oxygen by activating hypoxia-inducible factors (HIFs), ensuring their survival and continued growth despite this hostile environment. Therefore, the inhibition of HIFs and their target genes is a promising and emerging field of cancer research. Several drug candidates target protein–protein interactions or transcription mechanisms of the HIF pathway in order to interfere with activation of this pathway, which is deregulated in a wide range of solid and liquid cancers. Although some inhibitors are already in clinical trials, open questions remain with respect to their modes of action. New imaging technologies using luminescent and fluorescent methods or nanobodies to complement widely used approaches such as chromatin immunoprecipitation may help to answer some of these questions. In this review, we aim to summarize current inhibitor classes targeting the HIF pathway and to provide an overview of in vitro and in vivo techniques that could improve the understanding of inhibitor mechanisms. Unravelling the distinct principles regarding how inhibitors work is an indispensable step for efficient clinical applications and safety of anticancer compounds.

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EEF1A2 interacts with HSP90AB1 to promote lung adenocarcinoma metastasis via enhancing TGF-β/SMAD signalling

British Journal of Cancer ◽

10.1038/s41416-020-01250-4 ◽

2021 ◽

Author(s):

Liqing Jia ◽

Xiaolu Ge ◽

Chao Du ◽

Linna Chen ◽

Yanhong Zhou ◽

...

Keyword(s):

Lung Adenocarcinoma ◽

Protein Interactions ◽

Elongation Factor ◽

Protein Translation ◽

Protein Protein Interactions ◽

Promising Target ◽

Tgfβ Receptor ◽

Mesenchymal Transformation

Abstract Background Eukaryotic protein translation elongation factor 1α2 (EEF1A2) is an oncogene that promotes the progression of breast and pancreatic cancer. In this study, we aimed to elucidate the oncogenic function of EEF1A2 in the metastasis of lung adenocarcinoma (LUAD). Methods Immunohistochemistry and western blot were used to study EEF1A2 expression levels in LUAD tissues and cells, respectively. The role of EEF1A2 in LUAD progression were investigated in vitro and in vivo. We identified potential EEF1A2-binding proteins by liquid chromatography-electrospray mass spectrometry (LC-MS)/MS. Protein–protein interactions were determined by immunofluorescence and co-immunoprecipitation (Co-IP). Results In this study, we report that EEF1A2 mediates the epithelial–mesenchymal transformation (EMT), to promote the metastasis of LUAD cells in vitro and in vivo. Moreover, EEF1A2 interacts with HSP90AB1 to increase TGFβ Receptor (TβR)-I, and TβRII expression, followed by enhanced SMAD3 and pSMAD3 expression and nuclear localisation, which promotes the EMT of LUAD cells. Overexpression of EEF1A2 in cancer tissues is associated with poor prognosis and short survival of patients with LUAD. Conclusions These findings underscore the molecular functions of EEF1A2 in LUAD metastasis and indicate that EEF1A2 represents a promising target in the treatment of aggressive LUAD.

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