Molecular Genetic Analysis of Glucocorticoid Signaling Using the Cre/loxP System

2000 ◽  
Vol 381 (9-10) ◽  
pp. 961-964 ◽  
Author(s):  
Holger M. Reichardt ◽  
François Tronche ◽  
Anton Bauer ◽  
Günther Schütz
Keyword(s):  
Stress Response ◽  
Dna Binding ◽  
Gene Targeting ◽  
Protein Interaction ◽  
Molecular Genetic ◽  
Molecular Genetic Analysis ◽  
Emotional Behavior ◽  
Protein Protein Interaction ◽  
Stress Erythropoiesis ◽  
Physiological Processes

Abstract Glucocorticoids (GC) are involved in a plethora of physiological processes that range from the regulation of the stress response and the control of the immune system to modulation of behavior. Most GC effects are mediated by the glucocorticoid receptor (GR) via activation and repression of gene expression. Whereas in most cases activation requires DNA binding of the receptor, repression is usually mediated by protein-protein interaction with other transcription factors. To decipher the molecular mode of action of GR, mice were generated by gene targeting carrying a point mutation in one of the dimerization domains, thus abrogating DNA binding by GR. Analysis of these mice demonstrated that thymocyte apoptosis and stress erythropoiesis require the DNA binding-dependent function of GR, whereas lung development and the anti-inflammatory activity of GR are mediated by protein-protein interaction. Furthermore, to study the role of GC in the brain, mice were generated specifically lacking GR function in the nervous system. Using these mice we demonstrated that GR is essential for the regulation of the HPA-axis and the stress response, as well as for the control of emotional behavior. Taken together, gene targeting using the Cre/loxP system proved to be highly valuable for the analysis of both molecular mechanism and tissue-specific functions of the GR.

Molecular genetic analysis of glucocorticoid and mineralocorticoid signaling in development and physiological processes

Steroids ◽  
1996 ◽  
Vol 61 (4) ◽  
pp. 236-239 ◽  
Author(s):  
Stefan A. Berger ◽  
Timothy J. Cole ◽  
Wolfgang Schmid ◽  
Günther Schütz
Keyword(s):  
Genetic Analysis ◽  
Molecular Genetic ◽  
Molecular Genetic Analysis ◽  
Physiological Processes

scholarly journals Methods for addressing the protein-protein interaction between histone deacetylase 6 and ubiquitin

2017 ◽  
Author(s):  
Carolina dos S. Passos ◽  
Nathalie Deschamps ◽  
Yun Choi ◽  
Robert E. Cohen ◽  
Remo Perozzo ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Stress Response ◽  
Small Molecules ◽  
Histone Deacetylase ◽  
Protein Interaction ◽  
Potential Role ◽  
Histone Deacetylase 6 ◽  
Protein Protein Interaction ◽  
Binding Function

AbstractHistone deacetylase 6 (HDAC6) is a cytoplasmic HDAC isoform able to remove acetyl groups from cellular substrates such as α-tubulin. In addition to the two deacetylase domains, HDAC6 has a C-terminal zinc-finger ubiquitin (Ub)-binding domain (ZnF-UBP) able to recognize free Ub. HDAC6-Ub interaction is thought to function in regulating the elimination of misfolded proteins during stress response through the aggresome pathway. Small molecules inhibiting deacetylation by HDAC6 were shown to reduce aggresomes, but the interplay between HDAC6 catalytic activity and Ub-binding function is not fully understood. Here we describe two methods to measure the HDAC6-Ub interaction in vitro using full-length HDAC6. Both methods were effective for screening inhibitors of the HDAC6-Ub protein-protein interaction independently of the catalytic activity. Our results suggest a potential role for the HDAC6 deacetylase domains in modulating HDAC6-Ub interaction. This new aspect of HDAC6 regulation can be targeted to address the roles of HDAC6-Ub interaction in normal and disease conditions.

Functional interference between the ubiquitous and constitutive octamer transcription factor 1 (OTF-1) and the glucocorticoid receptor by direct protein-protein interaction involving the homeo subdomain of OTF-1

1992 ◽  
Vol 12 (11) ◽  
pp. 4960-4969
Author(s):  
E Kutoh ◽  
P E Strömstedt ◽  
L Poellinger
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Protein Interaction ◽  
Binding Activity ◽  
Dependent Manner ◽  
Protein Protein Interaction ◽  
Dna Binding Activity ◽  
Functional Interference

The ubiquitous and constitutive octamer transcription factor OTF-1 (Oct 1) is the target of positive regulation by the potent herpes simplex virus trans-activator VP16, which forms a complex with the homeodomain of OTF-1. Here we present evidence that the glucocorticoid receptor can negatively regulate OTF-1 function by a mechanism that is independent of DNA binding. In vivo-expressed glucocorticoid receptor inhibited in a hormone-dependent manner activation of a minimal promoter construct carrying a functional octamer site. Moreover, expression of the receptor in vivo resulted in hormone-dependent repression of OTF-1-dependent DNA-binding activity in nuclear extract. In vitro, the DNA-binding activity of partially purified OTF-1 was repressed following incubation with purified glucocorticoid receptor. Cross-linking and immunoprecipitation experiments indicated that the functional interference may be due to a strong association between these two proteins in solution. Finally, preliminary evidence indicates that the homeo subdomain of OTF-1 that directs formation of a complex with VP16 may also be critical for interaction with the glucocorticoid receptor. Thus, OTF-1 is a target for both positive and negative regulation by protein-protein interaction. Moreover, the functional interference between OTF-1 and the glucocorticoid receptor represents a novel regulatory mechanism in the cross-coupling of signal transduction pathways of nuclear receptors and constitutive transcription factors.

Molecular Characterization of a PU.1 Transcription Complex Formed on the IL-1β Proximal Promoter.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3547-3547
Author(s):  
Trang Hoang ◽  
Benoit Grondin ◽  
Martin Lefrancois ◽  
Marianne St Denis ◽  
Daniel G. Tenen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stress Response ◽  
Dna Binding ◽  
Leucine Zipper ◽  
Protein Complexes ◽  
Proximal Promoter ◽  
Stress Exposure ◽  
Protein Protein Interaction ◽  
Nuclear Extracts

Abstract The gene coding for the pro-inflammatory cytokine IL-1β is induced at the transcription level in differentiating macrophages and in stress response. Interestingly, PU.1 and C/EBPβ, two transcription factors implicated in IL-1β gene expression are not induced by stress exposure, while c-Jun is strongly induced. Strikingly, this upregulation of c-Jun is required for IL-1β induction, as cells expressing a c-Jun antisense construct fail to respond to stress exposure. We have mapped the induction of IL-1β gene expression to its proximal promoter and show that it is mediated by the transcriptional synergy between C/EBPβ, c-Jun and PU.1 via specific DNA binding sites for C/EBPβ and PU.1 only. To elucidate how PU.1 and C/EBPβ cooperate with c-Jun at the molecular level, we have optimized a DNA binding assay based on IL-1β promoter fragments immobilized on beads to isolate protein complexes from nuclear extracts, which were subsequently eluted and identified by Western blotting. We show that PU.1 or C/EBPβ alone directly bind this promoter fragment via specific sequences while purified recombinant c-Jun fails to do so. However, the presence of either PU.1 or C/EBPβ on the promoter allows for a recruitment of c-Jun to the DNA template, mediated by direct protein-protein interaction. Interestingly, the leucine zipper domain of c-Jun is essential for its interaction with C/EBPβ while dispensable for PU.1 interaction in vitro whereas its basic domain is required for both interactions. Furthermore, we show that PU.1 and C/EBPβ cooperatively bind the IL-1β promoter, resulting in a synergistic recruitment of c-Jun. Finally, we show that the strength of interaction of c-Jun mutants with PU.1 or C/EBPβ determine the strength of transcription output and c-Jun mutants that fail to associate with either PU.1 or C/EBPβ are transcriptionally inactive. In contrast, c-Jun mutants exhibiting increased homodimerization are more active that the wild type protein. Taken together, our data suggest that c-Jun homodimers can be targeted to the IL-1β promoter in the absence of a specific DNA binding element, and conclude that PU.1 and C/EBPβ are specifically tethered to the IL-1β promoter while c-Jun cooperatively binds these proteins and acts as a transcriptional co-activator. We propose a mechanism based on an initial binding of PU.1 and C/EBPβ to the IL-1β promoter followed by a cooperative recruitment of c-Jun, resulting in transcriptional synergy and IL-1β gene expression in stress response.

scholarly journals Point Mutations in Two EVI1 Zn Fingers Abolish EVI1-GATA1 Interaction and Allow Erythroid Differentiation of Murine Bone Marrow Cells

2006 ◽  
Vol 26 (20) ◽  
pp. 7658-7666 ◽  
Author(s):  
Leopoldo Laricchia-Robbio ◽  
Raffaella Fazzina ◽  
Donglan Li ◽  
Ciro R. Rinaldi ◽  
Kisaly K. Sinha ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Binding ◽  
Protein Interaction ◽  
Chromosomal Abnormalities ◽  
Point Mutations ◽  
Constitutive Expression ◽  
Erythroid Differentiation ◽  
Murine Bone Marrow ◽  
Protein Protein Interaction

ABSTRACT EVI1 is an aggressive nuclear oncoprotein deregulated by recurring chromosomal abnormalities in myelodysplastic syndrome (MDS). The expression of the corresponding gene is a very poor prognostic marker for MDS patients and is associated with severe defects of the erythroid lineage. We have recently shown that the constitutive expression of EVI1 in murine bone marrow results in a fatal disease with features characteristic of MDS, including anemia, dyserythropoiesis, and dysmegakaryopoiesis. These lineages are regulated by the DNA-binding transcription factor GATA1. EVI1 has two zinc finger domains containing seven motifs at the N terminus and three motifs at the C terminus. Supported by results of assays utilizing synthetic DNA promoters, it was earlier proposed that erythroid-lineage repression by EVI1 is based on the ability of this protein to compete with GATA1 for DNA-binding sites, resulting in repression of gene activation by GATA1. Here, however, we show that EVI1 is unable to bind to classic GATA1 sites. To understand the mechanism utilized by EVI1 to repress erythropoiesis, we used a combination of biochemical assays, mutation analyses, and in vitro bone marrow differentiation. The results indicate that EVI1 interacts directly with the GATA1 protein rather than the DNA sequence. We further show that this protein-protein interaction blocks efficient recognition or binding to DNA by GATA1. Point mutations that disrupt the geometry of two zinc fingers of EVI1 abolish the protein-protein interaction, leading to normal erythroid differentiation of normal murine bone marrow in vitro.

scholarly journals Oligomerization and Binding of the Dnmt3a DNA Methyltransferase to Parallel DNA Molecules

2011 ◽  
Vol 286 (27) ◽  
pp. 24200-24207 ◽  
Author(s):  
Renata Z. Jurkowska ◽  
Arumugam Rajavelu ◽  
Nils Anspach ◽  
Claus Urbanke ◽  
Gytis Jankevicius ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Protein Interaction ◽  
Dna Methyltransferase ◽  
Structural Studies ◽  
Dna Molecules ◽  
Protein Protein Interaction ◽  
Dna Molecule ◽  
Dna Binding Site ◽  
In Cells

Structural studies showed that Dnmt3a has two interfaces for protein-protein interaction in the heterotetrameric Dnmt3a/3L C-terminal domain complex: the RD interface (mediating the Dnmt3a-3a contact) and the FF interface (mediating the Dnmt3a-3L contact). Here, we demonstrate that Dnmt3a-C forms dimers via the FF interface as well, which further oligomerize via their RD interfaces. Each RD interface of the Dnmt3a-C oligomer creates an independent DNA binding site, which allows for binding of separate DNA molecules oriented in parallel. Because Dnmt3L does not have an RD interface, it prevents Dnmt3a oligomerization and binding of more than one DNA molecule. Both interfaces of Dnmt3a are necessary for the heterochromatic localization of the enzyme in cells. Overexpression of Dnmt3L in cells leads to the release of Dnmt3a from heterochromatic regions, which may increase its activity for methylation of euchromatic targets like the differentially methylated regions involved in imprinting.

scholarly journals Single-molecule dynamics and genome-wide transcriptomics reveal that NF-κB (p65)-DNA binding times can be decoupled from transcriptional activation

2018 ◽  
Author(s):  
Andrea Callegari ◽  
Christian Sieben ◽  
Alexander Benke ◽  
David M. Suter ◽  
Beat Fierz ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Single Molecule ◽  
Protein Interaction ◽  
Transcriptional Activation ◽  
Dna Binding Domain ◽  
Protein Protein Interaction ◽  
Genome Wide ◽  
Binding Time ◽  
Transcriptional Output

AbstractTranscription factors (TFs) regulate gene expression in both prokaryotes and eukaryotes by recognizing and binding to specific DNA promoter sequences. In higher eukaryotes, it remains unclear how the duration of TF binding to DNA relates to downstream transcriptional output. Here, we address this question for the transcriptional activator NF-κB (p65), by live-cell single molecule imaging of TF-DNA binding kinetics and genome-wide quantification of p65-mediated transcription. We used mutants of p65, perturbing either the DNA binding domain (DBD) or the protein-protein transactivation domain (TAD). We found that p65-DNA binding time was predominantly determined by its DBD and directly correlated with its transcriptional output as long as the TAD is intact. Surprisingly, mutation or deletion of the TAD did not modify p65-DNA binding stability, suggesting that the p65 TAD generally contributes neither to the assembly of an “enhanceosome,” nor to the active removal of p65 from putative specific binding sites. However, TAD removal did reduce p65-mediated transcriptional activation, indicating that protein-protein interactions act to translate the long-lived p65-DNA binding into productive transcription.Author SummaryTo control transcription of a certain gene or a group of genes, both eukaryotes and prokaryotes express specialized proteins, transcription factors (TFs). During gene activation, TFs bind gene promotor sequences to recruit the transcriptional machinery including DNA polymerase II. TFs are often multi-subunit proteins containing a DNA-binding domain (DBD) as well as a protein-protein interaction interface. It was suggested that the duration of a TF-DNA binding event 1) depends on these two subunits and 2) dictates the outcome, i.e. the amount of mRNA produced from an activated gene. We set out to address these hypotheses using the transcriptional activator NF-κB (p65) as well as a number of mutants affecting different functional subunits. Using a combination of live-cell microscopy and RNA sequencing, we show that p65 DNA-binding time indeed correlates with the transcriptional output, but that this relationship depends on, and hence can be uncoupled by altering, the protein-protein interaction capacity. Our results suggest that, while p65 DNA binding times are dominated by the DBD, a transcriptional output can only be achieved with a functional protein-protein interaction subunit.

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