scholarly journals The Transcriptional Coactivator Cbp Interacts with β-Catenin to Activate Gene Expression

2000 ◽  
Vol 149 (2) ◽  
pp. 249-254 ◽  
Author(s):  
Ken-Ichi Takemaru ◽  
Randall T. Moon
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Terminal Region ◽  
Creb Binding Protein ◽  
Transcriptional Coactivator ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid System ◽  
Recruitment System

β-Catenin plays a pivotal role in the transcriptional activation of Wnt-responsive genes by binding to TCF/LEF transcription factors. Although it has been suggested that the COOH-terminal region of β-catenin functions as an activation domain, the mechanisms of activation remain unclear. To screen for potential transcriptional coactivators that bind to the COOH-terminal region of β-catenin, we used a novel yeast two-hybrid system, the Ras recruitment system (RRS) that detects protein–protein interactions at the inner surface of the plasma membrane. Using this system, we isolated the CREB-binding protein (CBP). Armadillo (Arm) repeat 10 to the COOH terminus of β-catenin is involved in binding to CBP, whereas β-catenin interacts directly with the CREB-binding domain of CBP. β-Catenin synergizes with CBP to stimulate the activity of a synthetic reporter in vivo. Conversely, β-catenin–dependent transcriptional activation is repressed by E1A, an antagonist of CBP function, but not by an E1A mutant that does not bind to CBP. The activation of Wnt target genes such as siamois and Xnr3 in Xenopus embryos is also sensitive to E1A. These findings suggest that CBP provides a link between β-catenin and the transcriptional machinery, and possibly mediates the oncogenic function of β-catenin.

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.

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 TULP1 and TUB Are Required for Specific Localization of PRCD to Photoreceptor Outer Segments

2020 ◽  
Vol 21 (22) ◽  
pp. 8677
Author(s):  
Lital Remez ◽  
Ben Cohen ◽  
Mariela J. Nevet ◽  
Leah Rizel ◽  
Tamar Ben-Yosef
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Outer Segment ◽  
Protein Protein Interactions ◽  
Photoreceptor Outer Segment ◽  
Photoreceptor Outer Segments ◽  
Outer Segments ◽  
Yeast Two Hybrid System ◽  
Specific Localization ◽  
Recruitment System

Photoreceptor disc component (PRCD) is a small protein which is exclusively localized to photoreceptor outer segments, and is involved in the formation of photoreceptor outer segment discs. Mutations in PRCD are associated with retinal degeneration in humans, mice, and dogs. The purpose of this work was to identify PRCD-binding proteins in the retina. PRCD protein-protein interactions were identified when implementing the Ras recruitment system (RRS), a cytoplasmic-based yeast two-hybrid system, on a bovine retina cDNA library. An interaction between PRCD and tubby-like protein 1 (TULP1) was identified. Co-immunoprecipitation in transfected mammalian cells confirmed that PRCD interacts with TULP1, as well as with its homolog, TUB. These interactions were mediated by TULP1 and TUB highly conserved C-terminal tubby domain. PRCD localization was altered in the retinas of TULP1- and TUB-deficient mice. These results show that TULP1 and TUB, which are involved in the vesicular trafficking of several photoreceptor proteins from the inner segment to the outer segment, are also required for PRCD exclusive localization to photoreceptor outer segment discs.

scholarly journals Cubitus interruptus Requires DrosophilaCREB-Binding Protein To Activate wingless Expression in theDrosophila Embryo

2000 ◽  
Vol 20 (5) ◽  
pp. 1616-1625 ◽  
Author(s):  
Yang Chen ◽  
R. H. Goodman ◽  
Sarah M. Smolik
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Binding Protein ◽  
Point Mutations ◽  
Creb Binding Protein ◽  
Wild Type ◽  
Interaction Domain ◽  
Cubitus Interruptus

ABSTRACT CREB-binding protein (CBP) serves as a transcriptional coactivator in multiple signal transduction pathways. The Drosophilahomologue of CBP, dCBP, interacts with the transcription factors Cubitus interruptus (CI), MAD, and Dorsal (DL) and functions as a coactivator in several signaling pathways during Drosophiladevelopment, including the hedgehog (hh),decapentaplegic (dpp), and Tollpathways. Although dCBP is required for the expression of thehh target genes, wingless (wg) andpatched (ptc) in vivo, and potentiatesci-mediated transcriptional activation in vitro, it is not known that ci absolutely requires dCBP for its activity. We used a yeast genetic screen to identify several ci point mutations that disrupt CI-dCBP interactions. These mutant proteins are unable to transactivate a reporter gene regulated by cibinding sites and have a lower dCBP-stimulated activity than wild-type CI. When expressed exogenously in embryos, the CI point mutants cannot activate endogenous wg expression. Furthermore, a CI mutant protein that lacks the entire dCBP interaction domain functions as a negative competitor for wild-type CI activity, and the expression of dCBP antisense RNAs can suppress CI transactivation in Kc cells. Taken together, our data suggest that dCBP function is necessary forci-mediated transactivation of wg duringDrosophila embryogenesis.

scholarly journals Direct Protein Interactions Are Responsible for Ikaros-GATA and Ikaros-Cdk9 Cooperativeness in Hematopoietic Cells

2013 ◽  
Vol 33 (16) ◽  
pp. 3064-3076 ◽  
Author(s):  
Stefania Bottardi ◽  
Lionel Mavoungou ◽  
Vincent Bourgoin ◽  
Nazar Mashtalir ◽  
El Bachir Affar ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Transcription Elongation ◽  
Hematopoietic Cells ◽  
Elongation Factor ◽  
Gene Promoters ◽  
Zinc Finger Domain

Ikaros (Ik) is a critical regulator of hematopoietic gene expression. Here, we established that the Ik interactions with GATA transcription factors and cyclin-dependent kinase 9 (Cdk9), a component of the positive transcription elongation factor b (P-TEFb), are required for transcriptional activation of Ik target genes. A detailed dissection of Ik-GATA and Ik-Cdk9 protein interactions indicated that the C-terminal zinc finger domain of Ik interacts directly with the C-terminal zinc fingers of GATA1, GATA2, and GATA3, whereas the N-terminal zinc finger domain of Ik is required for interaction with the kinase and T-loop domains of Cdk9. The relevance of these interactions was demonstratedin vivoin COS-7 and primary hematopoietic cells, in which Ik facilitated Cdk9 and GATA protein recruitment to gene promoters and transcriptional activation. Moreover, the oncogenic isoform Ik6 did not efficiently interact with Cdk9 or GATA proteinsin vivoand perturbed Cdk9/P-TEFb recruitment to Ik target genes, thereby affecting transcription elongation. Finally, characterization of a novel nuclear Ik isoform revealed that Ik exon 6 is dispensable for interactions with Mi2 and GATA proteins but is essential for the Cdk9 interaction. Thus, Ik is central to the Ik-GATA-Cdk9 regulatory network, which is broadly utilized for gene regulation in hematopoietic cells.

scholarly journals GAC63, a GRIP1-Dependent Nuclear Receptor Coactivator

2005 ◽  
Vol 25 (14) ◽  
pp. 5965-5972 ◽  
Author(s):  
Yong-Heng Chen ◽  
Jeong Hoon Kim ◽  
Michael R. Stallcup
Keyword(s):  
Estrogen Receptor ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Gene Promoter ◽  
Dependent Manner ◽  
Protein Protein Interactions ◽  
Promoter Regions ◽  
Nuclear Receptor Coactivator ◽  
Binding Domains

ABSTRACT Nuclear receptors (NRs) regulate target gene transcription through the recruitment of multiple coactivator complexes to the promoter regions of target genes. One important coactivator complex includes a p160 coactivator (GRIP1, SRC-1, or ACTR) and its downstream coactivators (e.g., p300, CARM1, CoCoA, and Fli-I), which contribute to transcriptional activation by protein acetylation, protein methylation, and protein-protein interactions. In this study, we identified a novel NR coactivator, GAC63, which binds to the N-terminal region of p160 coactivators as well as the ligand binding domains of some NRs. GAC63 enhanced transcriptional activation by NRs in a hormone-dependent and GRIP1-dependent manner in transient transfection assays and cooperated synergistically and selectively with other NR coactivators, including GRIP1 and CARM1, to enhance estrogen receptor function. Endogenous GAC63 was recruited to the estrogen-responsive pS2 gene promoter of MCF-7 cells in response to the hormone. Reduction of the endogenous GAC63 level by small interfering RNA inhibited transcriptional activation by the hormone-activated estrogen receptor. Thus, GAC63 is a physiologically relevant part of the p160 coactivator signaling pathway that mediates transcriptional activation by NRs.

scholarly journals Subunit Oligomerization and Substrate Recognition of the Escherichia coli ClpYQ (HslUV) Protease Implicated by In Vivo Protein-Protein Interactions in the Yeast Two-Hybrid System

2003 ◽  
Vol 185 (8) ◽  
pp. 2393-2401 ◽  
Author(s):  
Yi-Ying Lee ◽  
Chiung-Fang Chang ◽  
Chueh-Ling Kuo ◽  
Meng-Ching Chen ◽  
Chien Hung Yu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hybrid System ◽  
Protein Interactions ◽  
Large Subunit ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

ABSTRACT The Escherichia coli ClpYQ (HslUV) is an ATP-dependent protease that consists of an ATPase large subunit with homology to other Clp family ATPases and a peptidase small subunit related to the proteasomal β-subunits of eukaryotes. Six identical subunits of both ClpY and ClpQ self-assemble into an oligomeric ring, and two rings of each subunit, two ClpQ rings surrounded by single ClpY rings, form a dumbbell shape complex. The ClpYQ protease degrades the cell division inhibitor, SulA, and a positive regulator of capsule transcription, RcsA, as well as RpoH, a heat shock sigma transcription factor. Using the yeast-two hybrid system, we explored the in vivo protein-protein interactions of the individual subunits of the ClpYQ protease involved in self-oligomerization, as well as in recognition of specific substrates. Interactions were detected with ClpQ/ClpQ, ClpQ/ClpY, and ClpY/SulA. No interactions were observed in experiments with ClpY/ClpY, ClpQ/RcsA, and ClpQ/SulA. However, ClpY, lacking domain I (ClpYΔI) was able to interact with itself and with intact ClpY. The C-terminal region of ClpY is important for interaction with other ClpY subunits. The previously defined PDZ-like domains at the C terminus of ClpY, including both D1 and D2, were determined to be indispensable for substrate binding. Various deletion and random point mutants of SulA were also made to verify significant interactions with ClpY. Thus, we demonstrated in vivo hetero- and homointeractions of ClpQ and ClpY molecules, as well as a direct association between ClpY and substrate SulA, thereby supporting previous in vitro biochemical findings.

scholarly journals CIITA is a transcriptional coactivator that is recruited to MHC class II promoters by multiple synergistic interactions with an enhanceosome complex

2000 ◽  
Vol 14 (9) ◽  
pp. 1156-1166
Author(s):  
Krzysztof Masternak ◽  
Annick Muhlethaler-Mottet ◽  
Jean Villard ◽  
Madeleine Zufferey ◽  
Viktor Steimle ◽  
...  
Keyword(s):  
Mhc Class Ii ◽  
Protein Interactions ◽  
Class Ii ◽  
Hereditary Disease ◽  
Transcriptional Coactivator ◽  
Protein Protein Interactions ◽  
Mhc I ◽  
Mhc Ii ◽  
Class Ii Mhc

By virtue of its control over major histocompatibility complex class II (MHC-II) gene expression, CIITA represents a key molecule in the regulation of adaptive immune responses. It was first identified as a factor that is defective in MHC-II deficiency, a hereditary disease characterized by the absence of MHC-II expression. CIITA is a highly regulated transactivator that governs all spatial, temporal, and quantitative aspects of MHC-II expression. It has been proposed to act as a non-DNA-binding transcriptional coactivator, but evidence that it actually functions at the level of MHC-II promoters was lacking. By means of chromatin immunoprecipitation assays, we show here for the first time that CIITA is physically associated with MHC-II, as well asHLA–DM, Ii, MHC-I, and β2mpromoters in vivo. To dissect the mechanism by which CIITA is recruited to the promoter, we have developed a DNA-dependent coimmunoprecipitation assay and a pull-down assay using immobilized promoter templates. We demonstrate that CIITA recruitment depends on multiple, synergistic protein–protein interactions with DNA-bound factors constituting the MHC-II enhanceosome. CIITA therefore represents a paradigm for a novel type of regulatory and gene-specific transcriptional cofactor.

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.

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