scholarly journals Differentiation-Dependent Interactions between RUNX-1 and FLI-1 during Megakaryocyte Development

2009 ◽  
Vol 29 (15) ◽  
pp. 4103-4115 ◽  
Author(s):  
Hui Huang ◽  
Ming Yu ◽  
Thomas E. Akie ◽  
Tyler B. Moran ◽  
Andrew J. Woo ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Fetal Liver ◽  
Gel Filtration ◽  
Cell Types ◽  
Protein Protein Interactions ◽  
Megakaryocyte Differentiation

ABSTRACT The transcription factor RUNX-1 plays a key role in megakaryocyte differentiation and is mutated in cases of myelodysplastic syndrome and leukemia. In this study, we purified RUNX-1-containing multiprotein complexes from phorbol ester-induced L8057 murine megakaryoblastic cells and identified the ets transcription factor FLI-1 as a novel in vivo-associated factor. The interaction occurs via direct protein-protein interactions and results in synergistic transcriptional activation of the c-mpl promoter. Interestingly, the interaction fails to occur in uninduced cells. Gel filtration chromatography confirms the differentiation-dependent binding and shows that it correlates with the assembly of a complex also containing the key megakaryocyte transcription factors GATA-1 and Friend of GATA-1 (FOG-1). Phosphorylation analysis of FLI-1 with uninduced versus induced L8057 cells suggests the loss of phosphorylation at serine 10 in the induced state. Substitution of Ser10 with the phosphorylation mimic aspartic acid selectively impairs RUNX-1 binding, abrogates transcriptional synergy with RUNX-1, and dominantly inhibits primary fetal liver megakaryocyte differentiation in vitro. Conversely, substitution with alanine, which blocks phosphorylation, augments differentiation of primary megakaryocytes. We propose that dephosphorylation of FLI-1 is a key event in the transcriptional regulation of megakaryocyte maturation. These findings have implications for other cell types where interactions between runx and ets family proteins occur.

The role of transcription factors, chromatin structure and DNA replication in 5 S RNA gene regulation

1994 ◽  
Vol 107 (8) ◽  
pp. 2055-2063 ◽  
Author(s):  
A.P. Wolffe
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Protein Interactions ◽  
Multigene Family ◽  
Cell Types ◽  
Protein Protein Interactions ◽  
Eukaryotic Genes ◽  
Rna Genes

Differential expression of the oocyte and somatic 5 S RNA genes during Xenopus development can be explained by changes in transcription factor and histone interactions with the two types of gene. Both factors and histones bind 5 S RNA genes with specificity. Protein-protein interactions determine the stability of potentially transcriptionally active or repressed nucleoprotein complexes. A decline in transcription factor abundance, differential binding of transcription factors to oocyte and somatic 5 S genes, and increased competition with the histones for association with DNA during early embryogenesis, can account for the developmental decision to selectively repress the oocyte genes, while retaining the somatic genes in the transcriptionally active state. The 5 S ribosomal genes of Xenopus are perhaps the simplest eukaryotic genes to show regulated expression during development. A large multigene family (oocyte 5 S DNA) is transcriptionally active in oocytes but is repressed in somatic cells, whereas a small multigene family (somatic 5 S DNA) is active in both cell types. A potential molecular mechanism to explain the developmental switch that turns off oocyte 5 S DNA transcription has been experimentally reconstructed in vitro and more recently tested in vivo. Central to this mechanism is the specific association of both transcription factors and histones with 5 S RNA genes. How the interplay of histones and transcription factors is thought to affect transcription, and how their respective contributions might change during development from an oocyte, to an embryo and eventually to a somatic cell is the focus of this review.

c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein

1994 ◽  
Vol 14 (9) ◽  
pp. 6021-6029
Author(s):  
R Metz ◽  
A J Bannister ◽  
J A Sutherland ◽  
C Hagemeier ◽  
E C O'Rourke ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
High Concentrations ◽  
Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

scholarly journals In-vivo crystals reveal protein interactions

2019 ◽  
Author(s):  
Eleanor R. Martin ◽  
Alessandro Barbieri ◽  
Robert C. Ford ◽  
Robert C. Robinson
Keyword(s):  
Protein Interactions ◽  
Protein Function ◽  
Pdz Domain ◽  
Cell Types ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
Reporter System ◽  
X Ray Crystallography

AbstractCrystallisation of recombinant proteins has been fundamental to our understanding of protein function, dysfunction, and molecular recognition. However, this information has often been gleaned under non-physiological extremes of protein, salt, and H+ concentrations. Here, we describe the development of the robust iBox-PAK4cat system that spontaneously crystallises in several mammalian cell types. The developments described here allow the quantitation of in-vivo protein-protein interactions using a novel GFP-linked reporter system. Here, we have combined this assay with in-vitro X-ray crystallography and molecular dynamics studies characterise the molecular determinants of the interaction between NHERF1 PDZ2 and CFTR, a protein complex pertinent to the genetic disease cystic fibrosis. These studies have revealed the crystal structure of the extended PDZ domain of NHERF1, and indicated, contrary to previous reports, that residue selection at −1 and −3 PDZ-binding motif positions influence the affinity and specificity of the interaction. The results presented here demonstrate that the iBox-PAK4cat assay could easily be utilised to screen other protein-protein interactions.

scholarly journals c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein.

1994 ◽  
Vol 14 (9) ◽  
pp. 6021-6029 ◽  
Author(s):  
R Metz ◽  
A J Bannister ◽  
J A Sutherland ◽  
C Hagemeier ◽  
E C O'Rourke ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
High Concentrations ◽  
Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

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

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.

Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

2005 ◽  
Vol 83 (4) ◽  
pp. 535-547 ◽  
Author(s):  
Gareth N Corry ◽  
D Alan Underhill
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Current Knowledge ◽  
Nuclear Architecture ◽  
Subnuclear Localization ◽  
Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

scholarly journals Ways into Understanding HIF Inhibition

Cancers ◽  
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.

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

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.

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.

scholarly journals Bromodomain protein inhibition: a novel therapeutic strategy in rheumatic diseases

RMD Open ◽  
2018 ◽  
Vol 4 (2) ◽  
pp. e000744 ◽  
Author(s):  
Kerstin Klein
Keyword(s):  
Animal Models ◽  
Rheumatic Diseases ◽  
Transcriptional Activation ◽  
Therapeutic Strategy ◽  
Cell Types ◽  
Protein Inhibition ◽  
Different Cell Types ◽  
Novel Therapeutic

The reading of acetylation marks on histones by bromodomain (BRD) proteins is a key event in transcriptional activation. Small molecule inhibitors targeting bromodomain and extra-terminal (BET) proteins compete for binding to acetylated histones. They have strong anti-inflammatory properties and exhibit encouraging effects in different cell types in vitro and in animal models resembling rheumatic diseases in vivo. Furthermore, recent studies that focus on BRD proteins beyond BET family members are discussed.

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