scholarly journals Glucocorticoid Receptor Phosphorylation Differentially Affects Target Gene Expression

2008 ◽  
Vol 22 (8) ◽  
pp. 1754-1766 ◽  
Author(s):  
Weiwei Chen ◽  
Thoa Dang ◽  
Raymond D. Blind ◽  
Zhen Wang ◽  
Claudio N. Cavasotto ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Target Genes ◽  
Divalent Cations ◽  
Phosphorylation Site ◽  
Transcriptional Response ◽  
Receptor Occupancy ◽  
Wild Type ◽  
Interacting Protein

Abstract The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.

scholarly journals A Novel Isoform of Human LZIP Negatively Regulates the Transactivation of the Glucocorticoid Receptor

2009 ◽  
Vol 23 (11) ◽  
pp. 1746-1757 ◽  
Author(s):  
Hyereen Kang ◽  
Yoon Suk Kim ◽  
Jesang Ko
Keyword(s):  
Amino Acids ◽  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Target Genes ◽  
Transmembrane Domain ◽  
Negative Regulator ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Basic Leucine Zipper

Abstract The human leucine zipper protein (LZIP) is a basic leucine zipper transcription factor that is involved in leukocyte migration, tumor suppression, and endoplasmic reticulum stress-associated protein degradation. Although evidence suggests a diversity of roles for LZIP, its function is not fully understood, and the subcellular localization of LZIP is still controversial. We identified a novel isoform of LZIP and characterized its function in ligand-induced transactivation of the glucocorticoid receptor (GR) in COS-7 and HeLa cells. A novel isoform of human LZIP designated as “sLZIP” contains a deleted putative transmembrane domain (amino acids 229–245) of LZIP and consists of 345 amino acids. LZIP and sLZIP were ubiquitously expressed in a variety of cell lines and tissues, with LZIP being much more common. sLZIP was mainly localized in the nucleus, whereas LZIP was located in the cytoplasm. Unlike LZIP, sLZIP was not involved in the chemokine-mediated signal pathway. sLZIP recruited histone deacetylases (HDACs) to the promoter region of the mouse mammary tumor virus luciferase reporter gene and enhanced the activities of HDACs, resulting in suppression of expression of the GR target genes. Our findings suggest that sLZIP functions as a negative regulator in glucocorticoid-induced transcriptional activation of GR by recruitment and activation of HDACs.

scholarly journals Localization of glucocorticoid receptor interacting protein 1 in murine tissues using two novel polyclonal antibodies

2001 ◽  
pp. 323-333 ◽  
Author(s):  
R Puustinen ◽  
N Sarvilinna ◽  
T Manninen ◽  
P Tuohimaa ◽  
T Ylikomi
Keyword(s):  
Mammary Gland ◽  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Hormone Receptors ◽  
Target Genes ◽  
Polyclonal Antibodies ◽  
Nuclear Staining ◽  
Dependent Manner ◽  
Interacting Protein ◽  
Cytoplasmic Expression

OBJECTIVE: Glucocorticoid receptor interacting protein 1 (GRIP1) is a coactivator that binds to the nuclear hormone receptors in a ligand-dependent manner and mediates transcriptional activation of the target genes. The aim of this study was to investigate GRIP1 expression in various murine tissues and whether the protein is nuclear, cytoplasmic, or both. DESIGN: Two novel polyclonal antibodies against amino acids 34-47 and 468-481 of GRIP1 were raised and characterized in order to study the GRIP1 expression with immunohistochemistry. RESULTS: Transient transfection studies with COS cells showed a clearly nuclear staining pattern and also immunohistochemical localization of GRIP1 was mainly nuclear, but cytoplasmic expression was seen as well. GRIP1 was expressed in epithelial cells of the submandibular gland, gastrointestinal tract, pancreas, kidney, uterus, mammary gland, testis, prostate, trachea, lungs and adrenal gland. GRIP1 was also detected in stromal cells of colon, rectum, urinary bladder, vagina, uterus, mammary gland and trachea, and to a lesser extent in esophagus, ureter, urethra, thymus and spleen. Smooth muscle cells of the gastrointestinal and urinary tract, uterus, epididymis, prostrate and bronchioles expressed GRIP1. Blood vessels of many organs, capsule of the kidney and prostate, mesovarium, adipocytes of the mammary gland, pericardium and cartilage of the trachea were also GRIP1-positive. Liver, thyroid gland and striated muscle did not express GRIP1. CONCLUSIONS: GRIP1 was expressed in a wide variety of murine organs, and expression varied between cell types and organs. In addition to mainly nuclear localization of endogenous GRIP1, cytoplasmic expression was seen as well.

scholarly journals Evaluating the Influence of a G-Quadruplex Prone Sequence on the Transactivation Potential by Wild-Type and/or Mutant P53 Family Proteins through a Yeast-Based Functional Assay

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 277
Author(s):  
Paola Monti ◽  
Vaclav Brazda ◽  
Natália Bohálová ◽  
Otília Porubiaková ◽  
Paola Menichini ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Relative Activity ◽  
Gene Organization ◽  
Functional Assay ◽  
Wild Type ◽  
P53 Family ◽  
Transactivation Potential ◽  
G Quadruplex

P53, P63, and P73 proteins belong to the P53 family of transcription factors, sharing a common gene organization that, from the P1 and P2 promoters, produces two groups of mRNAs encoding proteins with different N-terminal regions; moreover, alternative splicing events at C-terminus further contribute to the generation of multiple isoforms. P53 family proteins can influence a plethora of cellular pathways mainly through the direct binding to specific DNA sequences known as response elements (REs), and the transactivation of the corresponding target genes. However, the transcriptional activation by P53 family members can be regulated at multiple levels, including the DNA topology at responsive promoters. Here, by using a yeast-based functional assay, we evaluated the influence that a G-quadruplex (G4) prone sequence adjacent to the p53 RE derived from the apoptotic PUMA target gene can exert on the transactivation potential of full-length and N-terminal truncated P53 family α isoforms (wild-type and mutant). Our results show that the presence of a G4 prone sequence upstream or downstream of the P53 RE leads to significant changes in the relative activity of P53 family proteins, emphasizing the potential role of structural DNA features as modifiers of P53 family functions at target promoter sites.

scholarly journals Crosstalk between p53 and TGF-β Signalling

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Rebecca Elston ◽  
Gareth J. Inman
Keyword(s):  
Transcriptional Activation ◽  
Cancer Biology ◽  
Target Genes ◽  
Tumour Progression ◽  
Signal Transduction Pathway ◽  
Mirna Biogenesis ◽  
Mutant P53 ◽  
Wild Type ◽  
P53 Family ◽  
Wild Type P53

Wild-type p53 and TGF-β are key tumour suppressors which regulate an array of cellular responses. TGF-β signals in part via the Smad signal transduction pathway. Wild-type p53 and Smads physically interact and coordinately induce transcription of a number of key tumour suppressive genes. Conversely mutant p53 generally subverts tumour suppressive TGF-β responses, diminishing transcriptional activation of key TGF-β target genes. Mutant p53 can also interact with Smads and this enables complex formation with the p53 family member p63 and blocks p63-mediated activation of metastasis suppressing genes to promote tumour progression. p53 and Smad function may also overlap during miRNA biogenesis as they can interact with the same components of the Drosha miRNA processing complex to promote maturation of specific subsets of miRNAs. This paper investigates the crosstalk between p53 and TGF-β signalling and the potential roles this plays in cancer biology.

scholarly journals A DNA Region Recognized by the Nitric Oxide-Responsive Transcriptional Activator NorR Is Conserved in β- and γ-Proteobacteria

2004 ◽  
Vol 186 (23) ◽  
pp. 7980-7987 ◽  
Author(s):  
Andrea Büsch ◽  
Anne Pohlmann ◽  
Bärbel Friedrich ◽  
Rainer Cramm
Keyword(s):  
Nitric Oxide ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Ralstonia Eutropha ◽  
Upstream Region ◽  
Nitric Oxide Reductase ◽  
Wild Type ◽  
Genome Database ◽  
Nitric Oxide Metabolism ◽  
Signaling Domain

ABSTRACT The σ54-dependent regulator NorR activates transcription of target genes in response to nitric oxide (NO) or NO-generating agents. In Ralstonia eutropha H16, NorR activates transcription of the dicistronic norAB operon that encodes NorA, a protein of unknown function, and NorB, a nitric oxide reductase. A constitutively activating NorR derivative (NorR′), in which the N-terminal signaling domain was replaced by MalE, specifically bound to the norAB upstream region as revealed by gel retardation analysis. Within a 73-bp DNA segment protected by MalE-NorR′ in a DNase I footprint assay, three conserved inverted repeats, GGT-(N7)-ACC (where N is any base), that we consider to be NorR-binding boxes were identified. Mutations altering the spacing or the base sequence of these repeats resulted in an 80 to 90% decrease of transcriptional activation by wild-type NorR. Genome database analyses demonstrate that the GT-(N7)-AC core of the inverted repeat is found in several proteobacteria upstream of gene loci encoding proteins of nitric oxide metabolism, including nitric oxide reductase (NorB), flavorubredoxin (NorV), NO dioxygenase (Hmp), and hybrid cluster protein (Hcp).

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 Interdependent Recruitment of SAGA and Srb Mediator by Transcriptional Activator Gcn4p

2005 ◽  
Vol 25 (9) ◽  
pp. 3461-3474 ◽  
Author(s):  
Hongfang Qiu ◽  
Cuihua Hu ◽  
Fan Zhang ◽  
Gwo Jiunn Hwang ◽  
Mark J. Swanson ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Wild Type ◽  
General Transcription Factors ◽  
Tata Element ◽  
High Level ◽  
Target Promoters ◽  
Activation Sequence

ABSTRACT Transcriptional activation by Gcn4p is enhanced by the coactivators SWI/SNF, SAGA, and Srb mediator, which stimulate recruitment of TATA binding protein (TBP) and polymerase II to target promoters. We show that wild-type recruitment of SAGA by Gcn4p is dependent on mediator but independent of SWI/SNF function at three different promoters. Recruitment of mediator is also independent of SWI/SNF but is enhanced by SAGA at a subset of Gcn4p target genes. Recruitment of all three coactivators to ARG1 is independent of the TATA element and preinitiation complex formation, whereas efficient recruitment of the general transcription factors requires the TATA box. We propose an activation pathway involving interdependent recruitment of SAGA and Srb mediator to the upstream activation sequence, enabling SWI/SNF recruitment and the binding of TBP and other general factors to the promoter. We also found that high-level recruitment of Tra1p and other SAGA subunits is independent of the Ada2p/Ada3p/Gcn5p histone acetyltransferase module but requires Spt3p in addition to subunits required for SAGA integrity. Thus, while Tra1p can bind directly to Gcn4p in vitro, it requires other SAGA subunits for efficient recruitment in vivo.

scholarly journals The methylated-DNA binding protein MBD2 enhances NGFI-A (egr-1)-mediated transcriptional activation of the glucocorticoid receptor

2014 ◽  
Vol 369 (1652) ◽  
pp. 20130513 ◽  
Author(s):  
Ian C. G. Weaver ◽  
Ian C. Hellstrom ◽  
Shelley E. Brown ◽  
Stephen D. Andrews ◽  
Sergiy Dymov ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Protein A ◽  
Site Directed Mutagenesis ◽  
Signalling Pathways ◽  
Hek 293 ◽  
293 Cells ◽  
Exon 1 ◽  
Inducible Protein

Variations in maternal care in the rat influence the epigenetic state and transcriptional activity of glucocorticoid receptor (GR) gene in the hippocampus. The mechanisms underlying this maternal effect remained to be defined, including the nature of the relevant maternally regulated intracellular signalling pathways. We show here that increased maternal licking/grooming (LG), which stably enhances hippocampal GR expression, paradoxically increases hippocampal expression of the methyl-CpG binding domain protein-2 (MBD2) and MBD2 binding to the exon 1 7 GR promoter. Knockdown experiments of MBD2 in hippocampal primary cell culture show that MBD2 is required for activation of exon 1 7 GR promoter. Ectopic co-expression of nerve growth factor-inducible protein A (NGFI-A) with MBD2 in HEK 293 cells with site-directed mutagenesis of the NGFI-A response element within the methylated exon 1 7 GR promoter supports the hypothesis that MBD2 collaborates with NGFI-A in binding and activation of this promoter. These data suggest a possible mechanism linking signalling pathways, which are activated by behavioural stimuli and activation of target genes.

A Novel 2 Amino Acids Deletion in the Leucine Zipper Domain of C/EBPε Leads to Neutrophil-Specific Granule Deficiency (SGD)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4101-4101
Author(s):  
Tadayuki Akagi ◽  
Taizo Wada ◽  
Masahiro Muraoka ◽  
Tomoko Toma ◽  
Kenzo Kaji ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Nih3t3 Cells ◽  
Endogenous Expression ◽  
Leucine Zipper Domain ◽  
Type C ◽  
Specific Granule

Abstract Neutrophil-specific granule deficiency (SGD) is a rare congenital disorder. Neutrophils of SGD patients show abnormal bilobed nuclei and lack secondary and tertiary granule gene expression. Since neutrophils of SGD patients are deficient for bactericidal activity, the patients display immunodeficiency and suffer from frequent and severe infections. The transcription factor CCAAT/enhancer binding protein epsilon (C/EBPε) is one of the major regulators of granulopoiesis and is known to be the responsible gene for the disease. At least two homozygous germline mutations (5bp deletion and A-nucleotide insertion) of C/EBPε, which are functionally defective, have been reported. Here, we report a novel in-frame deletion of the C/EBPεgene in a 55 years-old female patient with a life-long history of recalcitrant skin infections with ulcer and scar formation. Peripheral blood smear showed characteristic changes, including reduction of cytoplasmic granules and increased bilobed nuclei of the neutrophils, monocytosis, absence of eosinophils and increased basophils. Flow cytometric examination revealed significant reduction of CD16 and abnormal expression of CD14 on the neutrophil surface. Sequencing of the C/EBPε gene revealed a homozygous deletion of 6 base-pairs (arginine and serine residues deletion; ΔRS) within the leucine zipper domain. Activation of luciferase driven by G-CSF receptor promoter was not induced by ΔRS; and endogenous expression of granule genes, including B9, NGAL and lactoferrin, was induced by wild-type C/EBPε but not by ΔRS in NIH3T3 cells, suggesting that transcriptional activity of ΔRS was diminished. GFP-tagged ΔRS, as well as wild-type C/EBPε, were localized to the nucleus in NIH3T3 cells, and DNA binding activity of ΔRS, which was determined by biotin-labeled DNA pulldown assay, was intact. Meanwhile, ΔRS together with Gata1 and PU.1 were not able to induce expression of eosinophil major basic protein (MBP) in NIH3T3 cells. Of note, wild-type C/EBPε associated with Gata1; however, ΔRS did not interact with Gata1, indicating that the ΔRS likely impairs protein-protein interaction of other transcription factors resulting in loss of transcriptional activation. These results further support the importance of the leucine zipper domain of C/EBPε for its essential function and indicate multiple molecular mechanisms leading to SGD. Disclosures No relevant conflicts of interest to declare.

scholarly journals MED14 and MED1 Differentially Regulate Target-Specific Gene Activation by the Glucocorticoid Receptor

2006 ◽  
Vol 20 (3) ◽  
pp. 560-572 ◽  
Author(s):  
Weiwei Chen ◽  
Inez Rogatsky ◽  
Michael J. Garabedian
Keyword(s):  
Glucocorticoid Receptor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Leucine Zipper ◽  
Target Genes ◽  
Gene Activation ◽  
Specific Gene ◽  
Small Interfering Rnas ◽  
Mrna Induction ◽  
Specific Regulation

Abstract The Mediator subunits MED14 and MED1 have been implicated in transcriptional regulation by the glucocorticoid receptor (GR) by acting through its activation functions 1 and 2. To understand the contribution of these Mediator subunits to GR gene-specific regulation, we reduced the levels of MED14 and MED1 using small interfering RNAs in U2OS-hGR osteosarcoma cells and examined the mRNA induction by dexamethasone of four primary GR target genes, interferon regulatory factor 8 (IRF8), ladinin 1, IGF-binding protein 1 (IGFBP1), and glucocorticoid-inducible leucine zipper (GILZ). We found that the GR target genes differed in their requirements for MED1 and MED14. GR-dependent mRNA expression of ladinin 1 and IRF8 required both MED1 and MED14, whereas induction of IGFBP1 mRNA by the receptor was dependent upon MED14, but not MED1. In contrast, GILZ induction by GR was largely independent of MED1 and MED14, but required the p160 cofactor transcriptional intermediary factor 2. Interestingly, we observed higher GR occupancy at GILZ than at the IGFBP1 or IRF8 glucocorticoid response element (GREs). In contrast, recruitment of MED14 compared with GR at IGFBP1 and IRF8 was higher than that observed at GILZ. At GILZ, GR and RNA polymerase II were recruited to both the GRE and the promoter, whereas at IGFBP1, RNA polymerase II occupied the promoter, but not the GRE. Thus, MED14 and MED1 are used by GR in a gene-specific manner, and the requirement for the Mediator at GILZ may be bypassed by increased GR and RNA polymerase II occupancy at the GREs. Our findings suggest that modulation of the Mediator subunit activities would provide a mechanism for promoter selectivity by GR.

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