scholarly journals RIP-140 interacts with multiple nuclear receptors by means of two distinct sites.

1996 ◽  
Vol 16 (11) ◽  
pp. 6029-6036 ◽  
Author(s):  
F L'Horset ◽  
S Dauvois ◽  
D M Heery ◽  
V Cavaillès ◽  
M G Parker
Keyword(s):  
Nuclear Receptors ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Target Genes ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Vitro Binding ◽  
Mutant Receptors

We have characterized two distinct binding sites, called site 1 and site 2, in the nuclear protein RIP-140 which interact with the ligand binding domain of the estrogen receptor both in solution and when the receptor is bound to DNA. Both sites are capable of independently interacting with other nuclear receptors, including the thyroid hormone and retinoic acid receptors, but they are not identical since the interaction with retinoid X receptor is mediated primarily by site 1. The interaction is enhanced by agonists but not by antagonists, and the in vitro binding activities to a number of mutant receptors correlate with their abilities to stimulate transcription in vivo. When RIP-140 is fused to heterologous DNA binding domains, it is able to stimulate the transcription of reporter genes in both yeast and mammalian cells. Thus, RIP-140 is likely to function as a bridging protein between receptors and the basal transcription machinery and thereby stimulate the transcription of target genes.

Cooperative interactions between paired domain and homeodomain

Development ◽  
1996 ◽  
Vol 122 (9) ◽  
pp. 2639-2650 ◽  
Author(s):  
S. Jun ◽  
C. Desplan
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Target Genes ◽  
Cooperative Interaction ◽  
Paired Domain ◽  
Cooperative Interactions ◽  
Dna Binding Domains ◽  
Binding Domains

The Pax proteins are a family of transcriptional regulators involved in many developmental processes in all higher eukaryotes. They are characterized by the presence of a paired domain (PD), a bipartite DNA binding domain composed of two helix-turn-helix (HTH) motifs, the PAI and RED domains. The PD is also often associated with a homeodomain (HD) which is itself able to form homo- and hetero-dimers on DNA. Many of these proteins therefore contain three HTH motifs each able to recognize DNA. However, all PDs recognize highly related DNA sequences, and most HDs also recognize almost identical sites. We show here that different Pax proteins use multiple combinations of their HTHs to recognize several types of target sites. For instance, the Drosophila Paired protein can bind, in vitro, exclusively through its PAI domain, or through a dimer of its HD, or through cooperative interaction between PAI domain and HD. However, prd function in vivo requires the synergistic action of both the PAI domain and the HD. Pax proteins with only a PD appear to require both PAI and RED domains, while a Pax-6 isoform and a new Pax protein, Lune, may rely on the RED domain and HD. We propose a model by which Pax proteins recognize different target genes in vivo through various combinations of their DNA binding domains, thus expanding their recognition repertoire.

Gene regulation by Sp1 and Sp3

2004 ◽  
Vol 82 (4) ◽  
pp. 460-471 ◽  
Author(s):  
Lin Li ◽  
Shihua He ◽  
Jian-Min Sun ◽  
James R Davie
Keyword(s):  
Gene Regulation ◽  
Transcription Factors ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Cellular Localization ◽  
Target Genes ◽  
In Vivo Studies ◽  
Binding Domains

The Sp family of transcription factors is united by a particular combination of three conserved Cys2His2 zinc fingers that form the sequence-specific DNA-binding domain. Within the Sp family of transcription factors, Sp1 and Sp3 are ubiquitously expressed in mammalian cells. They can bind and act through GC boxes to regulate gene expression of multiple target genes. Although Sp1 and Sp3 have similar structures and high homology in their DNA binding domains, in vitro and in vivo studies reveal that these transcription factors have strikingly different functions. Sp1 and Sp3 are able to enhance or repress promoter activity. Regulation of the transcriptional activity of Sp1 and Sp3 occurs largely at the post-translational level. In this review, we focus on the roles of Sp1 and Sp3 in the regulation of gene expression.Key words: Sp1, Sp3, gene regulation, sub-cellular localization.

scholarly journals Role of Papillomavirus E1 Initiator Dimerization in DNA Replication

2005 ◽  
Vol 79 (13) ◽  
pp. 8661-8664 ◽  
Author(s):  
Stephen Schuck ◽  
Arne Stenlund
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Severe Effect ◽  
Origin Of Dna Replication ◽  
Initiation Of Dna Replication

ABSTRACT Viral initiator proteins are polypeptides that form oligomeric complexes on the origin of DNA replication (ori). These complexes carry out a multitude of functions related to initiation of DNA replication, and although many of these functions have been characterized biochemically, little is understood about how the complexes are assembled. Here we demonstrate that loss of one particular interaction, the dimerization between E1 DNA binding domains, has a severe effect on DNA replication in vivo but has surprisingly modest effects on most individual biochemical activities in vitro. We conclude that the dimer interaction is primarily required for initial recognition of ori.

scholarly journals Fused protein domains inhibit DNA binding by LexA.

1992 ◽  
Vol 12 (7) ◽  
pp. 3006-3014 ◽  
Author(s):  
E A Golemis ◽  
R Brent
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
In Vitro Assays ◽  
Dimerization Domain ◽  
Activation Domains ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Operator Binding

Many studies of transcription activation employ fusions of activation domains to DNA binding domains derived from the bacterial repressor LexA and the yeast activator GAL4. Such studies often implicitly assume that DNA binding by the chimeric proteins is equivalent to that of the protein donating the DNA binding moiety. To directly investigate this issue, we compared operator binding by a series of LexA-derivative proteins to operator binding by native LexA, by using both in vivo and in vitro assays. We show that operator binding by many proteins such as LexA-Myc, LexA-Fos, and LexA-Bicoid is severely impaired, while binding of other LexA-derivative proteins, such as those that carry bacterially encoded acidic sequences ("acid blobs"), is not. Our results also show that DNA binding by LexA derivatives that contain the LexA carboxy-terminal dimerization domain (amino acids 88 to 202) is considerably stronger than binding by fusions that lack it and that heterologous dimerization motifs cannot substitute for the LexA88-202 function. These results suggest the need to reevaluate some previous studies of activation that employed LexA derivatives and modifications to recent experimental approaches that use LexA and GAL4 derivatives to detect and study protein-protein interactions.

THE MODE OF ACTION OF THE ANTIBIOTIC PAROMOMYCIN ON STAPHYLOCOCCUS AUREUS

1966 ◽  
Vol 12 (6) ◽  
pp. 1157-1165 ◽  
Author(s):  
A. von Seefried ◽  
D. C. Jordan
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Binding Sites ◽  
Bactericidal Action ◽  
Plate Method ◽  
Vitro Binding ◽  
Resistant Mutants ◽  
Intracellular Binding

Paromomycin (Humatin, Parke Davis & Co.), a broad-spectrum aminoglycosidic antibiotic, inhibits the incorporation of amino acids into the trypsinsoluble protein fraction of Staphylococcus aureus 257. Protein synthesis is inhibited immediately, but the synthesis of cell-wall mucopeptide and alcohol-soluble proteins and lipids is not affected for approximately 35 min after antibiotic addition to actively growing cells. Paromomycin, at the ribosomal level, prevents the attachment of amino acyl-s-RNA and causes accumulation of m-RNA.Divalent cations (Ca++ and Mg++) antagonize the bactericidal action of paromomycin and interfere with the in vivo binding of the antibiotic on both the cell surface and the intracellular binding sites. In vitro binding to free ribosomes can be prevented and reversed by both monovalent and divalent cations.Using a "cylinder-plate" method, involving the displacement of antibiotic from cellular fractions by 0.2 M MgCl2, the antibiotic can be recovered from the ribosomes, cytoplasm, and the cell wall of paromomycin-sensitive S. aureus cells, but is not found in any of these fractions isolated from paromomycin-resistant cells developed from the sensitive parent strain. The resistant mutants apparently have lost the ability to adsorb and transport the antibiotic into the cell.

scholarly journals A Multiplicity of Coactivators Is Required by Gcn4p at Individual Promoters In Vivo

2003 ◽  
Vol 23 (8) ◽  
pp. 2800-2820 ◽  
Author(s):  
Mark J. Swanson ◽  
Hongfang Qiu ◽  
Laarni Sumibcay ◽  
Anna Krueger ◽  
Soon-ja Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chromatin Immunoprecipitation ◽  
Target Gene ◽  
Target Genes ◽  
Yeast Cells ◽  
Histone Acetyltransferases ◽  
Vitro Binding ◽  
Paf1 Complex

ABSTRACT Transcriptional activators interact with multisubunit coactivators that modify chromatin structure or recruit the general transcriptional machinery to their target genes. Budding yeast cells respond to amino acid starvation by inducing an activator of amino acid biosynthetic genes, Gcn4p. We conducted a comprehensive analysis of viable mutants affecting known coactivator subunits from the Saccharomyces Genome Deletion Project for defects in activation by Gcn4p in vivo. The results confirm previous findings that Gcn4p requires SAGA, SWI/SNF, and SRB mediator (SRB/MED) and identify key nonessential subunits of these complexes required for activation. Among the numerous histone acetyltransferases examined, only that present in SAGA, Gcn5p, was required by Gcn4p. We also uncovered a dependence on CCR4-NOT, RSC, and the Paf1 complex. In vitro binding experiments suggest that the Gcn4p activation domain interacts specifically with CCR4-NOT and RSC in addition to SAGA, SWI/SNF, and SRB/MED. Chromatin immunoprecipitation experiments show that Mbf1p, SAGA, SWI/SNF, SRB/MED, RSC, CCR4-NOT, and the Paf1 complex all are recruited by Gcn4p to one of its target genes (ARG1) in vivo. We observed considerable differences in coactivator requirements among several Gcn4p-dependent promoters; thus, only a subset of the array of coactivators that can be recruited by Gcn4p is required at a given target gene in vivo.

scholarly journals The LIM-only protein FHL2 interacts with β-catenin and promotes differentiation of mouse myoblasts

2002 ◽  
Vol 159 (1) ◽  
pp. 113-122 ◽  
Author(s):  
Bernd Martin ◽  
Richard Schneider ◽  
Stefanie Janetzky ◽  
Zoe Waibler ◽  
Petra Pandur ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Muscle Development ◽  
Target Genes ◽  
Myogenic Differentiation ◽  
Myoblast Differentiation ◽  
Lim Domains ◽  
Vitro Binding ◽  
Specific Proteins

FHL2 is a LIM-domain protein expressed in myoblasts but down-regulated in malignant rhabdomyosarcoma cells, suggesting an important role of FHL2 in muscle development. To investigate the importance of FHL2 during myoblast differentiation, we performed a yeast two-hybrid screen using a cDNA library derived from myoblasts induced for differentiation. We identified β-catenin as a novel interaction partner of FHL2 and confirmed the specificity of association by direct in vitro binding tests and coimmunoprecipitation assays from cell lysates. Deletion analysis of both proteins revealed that the NH2-terminal part of β-catenin is sufficient for binding in yeast, but addition of the first armadillo repeat is necessary for binding FHL2 in mammalian cells, whereas the presence of all four LIM domains of FHL2 is needed for the interaction. Expression of FHL2 counteracts β-catenin–mediated activation of a TCF/LEF-dependent reporter gene in a dose-dependent and muscle cell–specific manner. After injection into Xenopus embryos, FHL2 inhibited the β-catenin–induced axis duplication. C2C12 mouse myoblasts stably expressing FHL2 show increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. These data imply that FHL2 is a muscle-specific repressor of LEF/TCF target genes and promotes myogenic differentiation by interacting with β-catenin.

scholarly journals Physical and Functional Interactions between Cellular Retinoic Acid Binding Protein II and the Retinoic Acid-Dependent Nuclear Complex

1999 ◽  
Vol 19 (10) ◽  
pp. 7158-7167 ◽  
Author(s):  
Laurent Delva ◽  
Jean-Noël Bastie ◽  
Cécile Rochette-Egly ◽  
Radhia Kraïba ◽  
Nicole Balitrand ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Nuclear Receptors ◽  
Mammalian Cells ◽  
Specific Response ◽  
Independent Manner ◽  
Control Of Gene Expression ◽  
Steady State Levels ◽  
And Control

ABSTRACT Two sorts of proteins bind to, and mediate the developmental and homeostatic effects of, retinoic acid (RA): the RAR and RXR nuclear receptors, which act as ligand-dependent transcriptional regulators, and the cellular RA binding proteins (CRABPI and CRABPII). CRABPs are generally known to be implicated in the synthesis, degradation, and control of steady-state levels of RA, yet previous and recent data have indicated that they could play a role in the control of gene expression. Here we show for the first time that, both in vitro and in vivo, CRABPII is associated with RARα and RXRα in a ligand-independent manner in mammalian cells (HL-60, NB-4, and MCF-7). In the nucleus, this protein complex binds the RXR-RAR-specific response element of an RA target gene (RARE-DR5). Moreover, in the presence of retinoids that bind both the nuclear receptors and CRABPII, enhancement of transactivation by RXRα-RARα heterodimers is observed in the presence of CRABPII. Thus, CRABPII appears to be a novel transcriptional regulator involved in RA signaling.

scholarly journals Mapping transcription factor occupancy using minimal numbers of cells in vitro and in vivo

2017 ◽  
Author(s):  
Luca Tosti ◽  
James Ashmore ◽  
Boon Siang Nicholas Tan ◽  
Benedetta Carbone ◽  
Tapan K Mistri ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Regulatory Networks ◽  
Embryonic Stem ◽  
Specific Cell ◽  
Dna Interactions

AbstractThe identification of transcription factor (TF) binding sites in the genome is critical to understanding gene regulatory networks (GRNs). While ChIP-seq is commonly used to identify TF targets, it requires specific ChIP-grade antibodies and high cell numbers, often limiting its applicability. DNA adenine methyltransferase identification (DamID), developed and widely used in Drosophila, is a distinct technology to investigate protein-DNA interactions. Unlike ChIP-seq, it does not require antibodies, precipitation steps or chemical protein-DNA crosslinking, but to date it has been seldom used in mammalian cells due to technical impediments. Here we describe an optimised DamID method coupled with next generation sequencing (DamID-seq) in mouse cells, and demonstrate the identification of the binding sites of two TFs, OCT4 and SOX2, in as few as 1,000 embryonic stem cells (ESCs) and neural stem cells (NSCs), respectively. Furthermore, we have applied this technique in vivo for the first time in mammals. Oct4 DamID-seq in the gastrulating mouse embryo at 7.5 days post coitum (dpc) successfully identified multiple Oct4 binding sites proximal to genes involved in embryo development, neural tube formation, mesoderm-cardiac tissue development, consistent with the pivotal role of this TF in post-implantation embryo. This technology paves the way to unprecedented investigations of TF-DNA interactions and GRNs in specific cell types with limited availability in mammals including in vivo samples.

beta3-tubulin is directly repressed by the engrailed protein in Drosophila

Development ◽  
1997 ◽  
Vol 124 (13) ◽  
pp. 2527-2536 ◽  
Author(s):  
N. Serrano ◽  
H.W. Brock ◽  
F. Maschat
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Transgenic Line ◽  
Target Genes ◽  
Regulatory Protein ◽  
Polytene Chromosomes ◽  
First Intron ◽  
Direct Target

In Drosophila, Engrailed is a nuclear regulatory protein with essential roles during embryonic development. Although Engrailed is a transcription factor, little progress has been achieved in identifying its target genes. We report here the identification of an effector gene, the beta3-tubulin gene, as a direct target of Engrailed. The cytological location of beta3-tubulin, 60C, is a strong site of Engrailed binding on polytene chromosomes. Immunostaining analysis of a transgenic line containing a P[beta3-tubulin-lacZ] construct shows an additional site of Engrailed binding at the location of the transgene. Molecular analysis allowed identification of several Engrailed binding sites, both in vitro and in vivo, within the first intron of the beta3-tubulin locus. Engrailed binding sites identified in vitro are active in larvae. Furthermore, expression of beta3-tubulin is derepressed in the ectoderm of engrailed mutant embryos. Repression of beta3-tubulin by Engrailed is also obtained when Engrailed is ectopically expressed in embryonic mesoderm. Finally, two different sets of Engrailed binding sites are shown to be involved in the early and late regulation of beta3-tubulin by Engrailed during embryogenesis.

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