scholarly journals Context-dependent gene regulation by transcription factor complexes

2019 ◽  
Author(s):  
Judith F. Kribelbauer ◽  
Ryan E. Loker ◽  
Siqian Feng ◽  
Chaitanya Rastogi ◽  
Namiko Abe ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Sequence Motif ◽  
Rna Seq ◽  
Binding Assays ◽  
Homeodomain Proteins ◽  
Eukaryotic Transcription ◽  
Transcription Factor Complexes ◽  
Complex Forms ◽  
Partner Proteins

ABSTRACTEukaryotic transcription factors (TFs) form complexes with various partner proteins to recognize their genomic target sites. Yet, how the DNA sequence determines which TF complex forms at any given site is poorly understood. Here we demonstrate that high-throughput in vitro binding assays coupled with unbiased computational analysis provides unprecedented insight into how complexes of homeodomain proteins adapt their stoichiometry and configuration to the bound DNA. Using inferred knowledge about minor groove width readout, we design targeted protein mutations that destabilize homeodomain binding in a complex-specific manner. By performing parallel SELEX-seq, ChIP-seq, RNA-seq and Hi-C assays, we not only reveal complex-specific functions, but also show that TF binding sites that lack a canonical sequence motif emerge as a consequence of direct interaction with functionally bound sites.

scholarly journals Reinvestigation of the dysbindin subunit of BLOC-1 (biogenesis of lysosome-related organelles complex-1) as a dystrobrevin-binding protein

2006 ◽  
Vol 395 (3) ◽  
pp. 587-598 ◽  
Author(s):  
Ramin Nazarian ◽  
Marta Starcevic ◽  
Melissa J. Spencer ◽  
Esteban C. Dell'Angelica
Keyword(s):  
Recombinant Proteins ◽  
Binding Protein ◽  
Coiled Coil ◽  
Direct Interaction ◽  
Muscle Pathology ◽  
Binding Assays ◽  
Genetic Studies ◽  
Binding Interface ◽  
Lysosome Related Organelles

Dysbindin was identified as a dystrobrevin-binding protein potentially involved in the pathogenesis of muscular dystrophy. Subsequently, genetic studies have implicated variants of the human dysbindin-encoding gene, DTNBP1, in the pathogeneses of Hermansky–Pudlak syndrome and schizophrenia. The protein is a stable component of a multisubunit complex termed BLOC-1 (biogenesis of lysosome-related organelles complex-1). In the present study, the significance of the dystrobrevin–dysbindin interaction for BLOC-1 function was examined. Yeast two-hybrid analyses, and binding assays using recombinant proteins, demonstrated direct interaction involving coiled-coil-forming regions in both dysbindin and the dystrobrevins. However, recombinant proteins bearing the coiled-coil-forming regions of the dystrobrevins failed to bind endogenous BLOC-1 from HeLa cells or mouse brain or muscle, under conditions in which they bound the Dp71 isoform of dystrophin. Immunoprecipitation of endogenous dysbindin from brain or muscle resulted in robust co-immunoprecipitation of the pallidin subunit of BLOC-1 but no specific co-immunoprecipitation of dystrobrevin isoforms. Within BLOC-1, dysbindin is engaged in interactions with three other subunits, named pallidin, snapin and muted. We herein provide evidence that the same 69-residue region of dysbindin that is sufficient for dystrobrevin binding in vitro also contains the binding sites for pallidin and snapin, and at least part of the muted-binding interface. Functional, histological and immunohistochemical analyses failed to detect any sign of muscle pathology in BLOC-1-deficient, homozygous pallid mice. Taken together, these results suggest that dysbindin assembled into BLOC-1 is not a physiological binding partner of the dystrobrevins, likely due to engagement of its dystrobrevin-binding region in interactions with other subunits.

scholarly journals Quercetin inhibits intrahepatic cholangiocarcinoma by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation via the NF-κB pathway

2021 ◽  
Author(s):  
Yinghui Song ◽  
Zhihua Zhang ◽  
Qin Chai ◽  
GuoYi Xia ◽  
Zhangtao Yu ◽  
...  
Keyword(s):  
Intrahepatic Cholangiocarcinoma ◽  
Clonogenic Assay ◽  
Direct Interaction ◽  
Rna Seq ◽  
Wound Healing Assay ◽  
Tumor Bearing ◽  
Mesenchymal Transformation ◽  
First Time

Abstract Intrahepatic cholangiocarcinoma (ICC) is a rare high-fatal hepatobiliary malignancy, the treatment option of ICC is very limited, and the prognosis is also poor. Recently, emerging evidence has shown the potential of quercetin (QE) for cancer therapy. We explored the effect and mechanism of QE on ICC in vitro and in vivo. CCK-8 assay and Clonogenic assay showed that QE could inhibit ICC cells proliferation and survival. PI staining suggested QE could induce ICC cells arrest in G1 phase. AV/PI staining suggested QE could promote ICC cells apoptosis. Wound Healing Assay and Transwell chamber experiment suggested QE could inhibit ICC cells EMT. RNA-seq, the changes in the structure of mitochondria by electron microscopy and the key markers of ferroptosis (free iron ions, MDA, SOD, GPX4) were supported QE could promote ferroptosis in ICC cells. Molecular docking showed that QE had direct interaction with NF-κB and GPX4. In vivo, treatment with QE inhibited tumor growth and prolonged survival time of tumor-bearing nude mice. Our data for the first time suggest that QE is a new ferroptosis inducer and combinative treatment of inhibiting NF-κB in ICC cells by inducing ferroptosis and inhibiting EMT, which will hopefully provide a prospective strategy for ICC patients.

Direct association of RhoA with specific domains of PKC-α

2005 ◽  
Vol 289 (4) ◽  
pp. C982-C993 ◽  
Author(s):  
Haiyan Pang ◽  
Khalil N. Bitar
Keyword(s):  
Smooth Muscle ◽  
Active Form ◽  
Direct Interaction ◽  
Full Length ◽  
Binding Assays ◽  
Glutathione S Transferase ◽  
Transfected Cells ◽  
Protein Protein Interaction ◽  
Vitro Binding

Previous studies performed at our laboratory have shown that agonist-induced contraction of smooth muscle is associated with translocation of protein kinase C (PKC)-α and RhoA to the membrane and that this interaction is due to a direct protein-protein interaction. To determine the domains of PKC-α involved in direct interaction with RhoA, His-tagged PKC-α proteins of individual domains and different combinations of PKC-α domains were used to perform in vitro binding assays with the fusion protein glutathione- S-transferase (GST)-RhoA. Coimmunoprecipitation was also performed using smooth muscle cells transfected with truncated forms of PKC-α in this study. The data indicate that RhoA directly bound to full-length PKC-α, both in vitro (82.57 ± 15.26% above control) and in transfected cells. RhoA bound in vitro to the C1 domain of PKC-α [PKC-α (C1)] (70.48 ± 20.78% above control), PKC-α (C2) (72.26 ± 29.96% above control), and PKC-α (C4) (90.58 ± 26.79% above control), but not to PKC-α (C3) (0.64 ± 5.18% above control). RhoA bound in vitro and in transfected cells to truncated forms of PKC-α, PKC-α (C2, C3, and C4), and PKC-α (C3 and C4) (94.09 ± 12.13% and 85.10 ± 16.16% above control, respectively), but not to PKC-α (C1, C2, and C3) or to PKC-α (C2 and C3) (0.47 ± 1.26% and 7.45 ± 10.76% above control, respectively). RhoA bound to PKC-α (C1 and C2) (60.78 ± 13.78% above control) only in vitro, but not in transfected cells, and PKC-α (C2, C3, and C4) and PKC-α (C3 and C4) bound well to RhoA. These data suggest that RhoA bound to fragments that may mimic the active form of PKC-α. The studies using cells transfected with truncated forms of PKC-α indicate that PKC-α (C1 and C2), PKC-α (C1, C2, and C3), and PKC-α (C2 and C3) did not associate with RhoA. Only full-length PKC-α, PKC-α (C2, C3, and C4), and PKC-α (C3 and C4) associated with RhoA. The association increased upon stimulation with acetylcholine. These results suggest that the functional association of PKC-α with RhoA may require the C4 domain.

scholarly journals An Activator Binding Module of Yeast RNA Polymerase II Holoenzyme

1999 ◽  
Vol 19 (4) ◽  
pp. 2967-2976 ◽  
Author(s):  
Young Chul Lee ◽  
Jin Mo Park ◽  
Soyoung Min ◽  
Sang Jun Han ◽  
Young-Joon Kim
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Direct Interaction ◽  
Transcriptional Activator ◽  
Polymerase Activity ◽  
Physical Interaction ◽  
Binding Assays ◽  
Activator Proteins ◽  
Rna Polymerase Ii Holoenzyme

ABSTRACT The Mediator complex of Saccharomyces cerevisiae is required for both general and regulated transcription of RNA polymerase II (PolII) and is composed of two stable subcomplexes (Srb4 and Rgr1 subcomplexes). To decipher the function of each Mediator subcomplex and to delineate the functional relationship between the subcomplexes, we characterized the compositions and biochemical activities of PolII-Mediator complexes (holoenzymes) prepared from several Mediator mutant strains of S. cerevisiae. We found that holoenzymes devoid of a functional Gal11 module were defective for activated but not basal transcription in a reconstituted in vitro system. This activation-specific defect was correlated with a crippled physical interaction to transcriptional activator proteins, which could be bypassed by artificial recruitment of a mutant holoenzyme to a promoter. Consistent with this observation, a direct interaction between Gal11 and gene-specific transcriptional activator proteins was detected by far-Western analyses and column binding assays. In contrast, the srb5 deletion mutant holoenzyme was defective for both basal and activated transcription, despite its capacity for activator binding that is comparable to that of the wild-type holoenzyme. These results demonstrate that the Gal11 module of the Rgr1 subcomplex is required for the efficient recruitment of PolII holoenzyme to a promoter via activator-specific interactions, while the Srb4 subcomplex functions in the modulation of general polymerase activity.

scholarly journals The yeast alpha2 and Mcm1 proteins interact through a region similar to a motif found in homeodomain proteins of higher eukaryotes.

1996 ◽  
Vol 16 (5) ◽  
pp. 2135-2143 ◽  
Author(s):  
J Mead ◽  
H Zhong ◽  
T B Acton ◽  
A K Vershon
Keyword(s):  
Dna Binding ◽  
Transcriptional Repression ◽  
Direct Interaction ◽  
Homeodomain Protein ◽  
Sequence Specificity ◽  
Homeodomain Proteins ◽  
Yeast Saccharomyces Cerevisiae ◽  
Transcriptional Regulatory

Homeodomain proteins are transcriptional regulatory factors that, in general, bind DNA with relatively low sequence specificity and affinity. One mechanism homeodomain proteins use to increase their biological specificity is through interactions with other DNA-binding proteins. We have examined how the yeast (Saccharomyces cerevisiae) homeodomain protein alpha2 specifically interacts with Mcm1, a MADS box protein, to bind DNA specifically and repress transcription. A patch of predominantly hydrophobic residues within a region preceding the homeodomain of alpha2 has been identified that specifies direct interaction with Mcm1 in the absence of DNA. This hydrophobic patch is required for cooperative DNA binding with Mcm1 in vitro and for transcriptional repression in vivo. We have also found that a conserved motif, termed YPWM, frequently found in homeodomain proteins of insects and mammals, partially functions in place of the patch in alpha2 to interact with Mcm1. These findings suggest that homeodomain proteins from diverse organisms may use analogous interaction motifs to associate with other proteins to achieve high levels of DNA binding affinity and specificity.

Direct binding of NuMA to tubulin is mediated by a novel sequence motif in the tail domain that bundles and stabilizes microtubules

2002 ◽  
Vol 115 (9) ◽  
pp. 1815-1824
Author(s):  
Laurence Haren ◽  
Andreas Merdes
Keyword(s):  
Cultured Cells ◽  
Direct Interaction ◽  
Sequence Motif ◽  
Tail Domain ◽  
Mitotic Spindles ◽  
Spindle Poles ◽  
Direct Binding ◽  
Multiple Poles

In mitosis, NuMA localises to spindle poles where it contributes to the formation and maintenance of focussed microtubule arrays. Previous work has shown that NuMA is transported to the poles by dynein and dynactin. So far, it is unclear how NuMA accumulates at the spindle poles following transport and how it remains associated throughout mitosis. We show here that NuMA can bind to microtubules independently of dynein/dynactin. We characterise a 100-residue domain located within the C-terminal tail of NuMA that mediates a direct interaction with tubulin in vitro and that is necessary for NuMA association with tubulin in vivo. Moreover, this domain induces bundling and stabilisation of microtubules when expressed in cultured cells and leads to formation of abnormal mitotic spindles with increased microtubule asters or multiple poles. Our results suggest that NuMA organises the poles by stable crosslinking of the microtubule fibers.

scholarly journals H-Ras Activation Promotes Cytoplasmic Accumulation and Phosphoinositide 3-Oh Kinase Association of β-Catenin in Epidermal Keratinocytes

1999 ◽  
Vol 146 (5) ◽  
pp. 967-980 ◽  
Author(s):  
Jesús Espada ◽  
Mirna Pérez-Moreno ◽  
Vania M.M. Braga ◽  
Pablo Rodriguez-Viciana ◽  
Amparo Cano
Keyword(s):  
Glycogen Synthase ◽  
Strong Association ◽  
Direct Interaction ◽  
Epidermal Keratinocytes ◽  
Binding Assays ◽  
Adenomatous Polyposis Coli Protein ◽  
Ras Activation ◽  
E Cadherin

The mechanisms underlying downregulation of the cadherin/catenin complexes and β-catenin signaling during tumor progression are not fully understood. We have analyzed the effect of oncogenic H-Ras on E-cadherin/catenin complex formation/stabilization and β-catenin distribution in epidermal keratinocytes. Microinjection or stable expression of V12Ras into keratinocytes promotes the loss of E-cadherin and α-catenin and relocalization of β-catenin to the cytoplasm and nucleus. Moreover, these effects are dependent on PI3K (phosphoinositide 3-OH kinase) activity. Interestingly, a strong association of p85α and p110α subunits of PI3K with β-catenin is induced in V12Ras-expressing keratinocytes, and in vitro binding assays show a direct interaction between β-catenin and p85α. Overexpression of either V12Ras or constitutively active p110α induces metabolic stabilization of β-catenin and promotes its accumulation in cytoplasmic and nuclear pools. In addition, the interaction of β-catenin with the adenomatous polyposis coli protein is blocked in V12Ras and p110α transformants though no changes in glycogen synthase kinase 3 β activity could be detected. Nevertheless, in V12Ras transformants the in vivo phosphorylation of β-catenin in Ser residues is strongly decreased. These results indicate that H-Ras activation induces the relocalization and cytoplasmic stabilization of β-catenin by a mechanism involving its interaction with PI3K.

scholarly journals CIF150, a Human Cofactor for Transcription Factor IID-Dependent Initiator Function

1998 ◽  
Vol 18 (1) ◽  
pp. 233-239 ◽  
Author(s):  
Jörg Kaufmann ◽  
Katharina Ahrens ◽  
Ronald Koop ◽  
Stephen T. Smale ◽  
Rolf Müller
Keyword(s):  
Transcription Factor ◽  
Core Promoter ◽  
Direct Interaction ◽  
Striking Similarity ◽  
Binding Assays ◽  
Vitro Binding ◽  
Transcription Factor Iid ◽  
Tata Box Binding Protein ◽  
Tfiid Complex

ABSTRACT The transcription factor IID (TFIID) complex is highly conserved between the Drosophila and mammalian systems. A mammalian homolog has been described for all the Drosophila TATA box-binding protein-associated factors (TAFs), with the exception of dTAFII150. We previously reported the identification of CIF, an essential cofactor for TFIID-dependent transcription from promoters containing initiator (Inr) elements. Here we describe the molecular cloning of CIF150, the human homolog of dTAFII150, and present biochemical evidence that this factor is involved in Inr activity. CIF150 is capable of mediating TFIID-dependent Inr activity in a complementation assay, and a protein fraction lacking Inr activity lacks detectable amounts of CIF150. Despite the striking similarity to dTAFII150, CIF150 does not appear to be associated with human TFIID. However, in vitro binding assays revealed a specific and direct interaction between CIF150 and hTAFII135. This interaction might be structurally important for the functional interaction between CIF150 and human TFIID, since CIF150 stabilizes TFIID binding to a core promoter.

scholarly journals Serogroup-Specific Interaction of Neisseria meningitidis Capsular Polysaccharide with Host Cell Microtubules and Effects on Tubulin Polymerization

2013 ◽  
Vol 82 (1) ◽  
pp. 265-274 ◽  
Author(s):  
Adelfia Talà ◽  
Laura Cogli ◽  
Mario De Stefano ◽  
Marcella Cammarota ◽  
Maria Rita Spinosa ◽  
...  
Keyword(s):  
Capsular Polysaccharide ◽  
Specific Interaction ◽  
Direct Interaction ◽  
Tubulin Polymerization ◽  
Binding Assays ◽  
Cell Infection ◽  
Infectious Process ◽  
Microtubule Inhibitors ◽  
Late Stages

ABSTRACTWe have previously shown that during late stages of the infectious process, serogroup B meningococci (MenB) are able to escape the phagosome ofin vitro-infected human epithelial cells. They then multiply in the cytosolic environment and spread intracellularly and to surrounding cells by exploiting the microtubule cytoskeleton, as suggested by results of infections in the presence of microtubule inhibitors and evidence of nanotubes connecting neighboring cells. In this study, by using microtubule binding assays with purified microtubule asters and bundles and microtubule bundles synthesizedin vitro, we demonstrate that the MenB capsule directly mediates the interaction between bacteria and microtubules. The direct interaction between the microtubules and the MenB capsular polysaccharide was confirmed by coimmunoprecipitation experiments. Unexpectedly, serogroup C meningococci (MenC), which have a capsular polysaccharide that differs from that of MenB only by its anomeric linkage, α(2→9) instead of α(2→8), were not able to interact with the microtubules, and the lack of interaction was not due to capsular polysaccharideO-acetylation that takes place in most MenC strains but not in MenB strains. Moreover, we demonstrate that the MenB capsular polysaccharide inhibits tubulin polymerizationin vitro. Thus, at variance with MenC, MenB may interfere with microtubule dynamics during cell infection.

Defibrotide Inhibits Platelet Activation by Cathepsin G Released From Stimulated Polymorphonuclear Leukocytes

1992 ◽  
Vol 67 (06) ◽  
pp. 660-664 ◽  
Author(s):  
Virgilio Evangelista ◽  
Paola Piccardoni ◽  
Giovanni de Gaetano ◽  
Chiara Cerletti
Keyword(s):  
Platelet Activation ◽  
Polymorphonuclear Leukocytes ◽  
Direct Interaction ◽  
Cathepsin G ◽  
In Vivo Test ◽  
Cell Functions ◽  
Test Models ◽  
Antithrombotic Effects

SummaryDefibrotide is a polydeoxyribonucleotide with antithrombotic effects in experimental animal models. Most of the actions of this drug have been observed in in vivo test models but no effects have been reported in in vitro systems. In this paper we demonstrate that defibrotide interferes with polymorphonuclear leukocyte-induced human platelet activation in vitro. This effect was not related to any direct interaction with polymorphonuclear leukocytes or platelets, but was due to the inhibition of cathepsin G, the main biochemical mediator of this cell-cell cooperation. Since cathepsin G not only induces platelet activation but also affects some endothelial cell functions, the anticathepsin G activity of defibrotide could help to explain the antithrombotic effect of this drug.

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