scholarly journals Glucocorticoid Receptor Activation of the IκBα Promoter within Chromatin

2001 ◽  
Vol 12 (11) ◽  
pp. 3365-3374 ◽  
Author(s):  
Bonnie J. Deroo ◽  
Trevor K. Archer
Keyword(s):  
Transcription Factor ◽  
Glucocorticoid Receptor ◽  
Restriction Enzyme ◽  
Chromatin Structure ◽  
Receptor Activation ◽  
Model Systems ◽  
Micrococcal Nuclease ◽  
Open Chromatin ◽  
Glucocorticoid Treatment

The glucocorticoid receptor (GR) is a ligand-activated transcription factor that induces expression of many genes. The GR has been useful for understanding how chromatin structure regulates steroid-induced transcription in model systems. However, the effect of glucocorticoids on chromatin structure has been examined on few endogenous mammalian promoters. We investigated the effect of glucocorticoids on the in vivo chromatin structure of the glucocorticoid-responsive IκBα gene promoter, the inhibitor of the ubiquitous transcription factor, nuclear factor kappa B (NFκB). Glucocorticoids inhibit NFκB activity in some tissues by elevating the levels of IκBα. We found that glucocorticoids activated the IκBα promoter in human T47D/A1-2 cells containing the GR. We then investigated the chromatin structure of the IκBα promoter in the absence and presence of glucocorticoids with the use of micrococcal nuclease, restriction enzyme, and deoxyribonuclease (DNaseI) analyses. In untreated cells, the promoter assembles into regularly positioned nucleosomes, and glucocorticoid treatment did not alter nucleosomal position. Restriction enzyme accessiblity studies indicated that the IκBα promoter is assembled as phased nucleosomes that adopt an “open” chromatin architecture in the absence of hormone. However, glucocorticoids may be required for transcription factor binding, because DNaseI footprinting studies suggested that regulatory factors bind to the promoter upon glucocorticoid treatment.

scholarly journals FoxA1 Binding Directs Chromatin Structure and the Functional Response of a Glucocorticoid Receptor-Regulated Promoter

2009 ◽  
Vol 29 (20) ◽  
pp. 5413-5425 ◽  
Author(s):  
Sergey Belikov ◽  
Carolina Åstrand ◽  
Örjan Wrange
Keyword(s):  
Transcription Factor ◽  
Glucocorticoid Receptor ◽  
Chromatin Structure ◽  
Partial Agonist ◽  
Dimethyl Sulfate ◽  
Specific Response ◽  
Hormone Regulation ◽  
Context Specific ◽  
In Vivo Footprinting

ABSTRACT Reconstitution of the glucocorticoid receptor (GR)-regulated mouse mammary tumor virus (MMTV) promoter in Xenopus oocytes was used to monitor the effects of different transcription factor contexts. Three constitutively binding factors, nuclear factor 1 (NF1), octamer transcription factor 1 (Oct1), and the Forkhead box A1 (FoxA1), were shown to act in concert, to direct the chromatin structure, and to enhance the GR response. FoxA1 has a dominant effect in the absence of hormone and induces a cluster of DNase I-hypersensitive sites in the segment comprising bp −400 to +25. This FoxA1-mediated chromatin remodeling does not induce MMTV transcription, as opposed to that of the GR. However, the robust FoxA1-dependent chromatin opening has the following drastic functional consequences on the hormone regulation: (i) GR-DNA binding is facilitated, as revealed by dimethyl sulfate in vivo footprinting, leading to increased hormone-induced transcription, and (ii) the GR antagonist RU486 is converted into a partial agonist in the presence of FoxA1 via ligand-independent GR activation. We conclude that FoxA1 mediates a preset chromatin structure and directs a context-specific response of a nuclear receptor. Furthermore, the alternative nucleosome arrangement induced by GR and FoxA1 implies this to be determined by constitutive binding of transcription factors rather than by the DNA sequence itself.

scholarly journals Inhibitors of Histone Deacetylase and DNA Methyltransferase Synergistically Activate the Methylated Metallothionein I Promoter by Activating the Transcription Factor MTF-1 and Forming an Open Chromatin Structure

2002 ◽  
Vol 22 (23) ◽  
pp. 8302-8319 ◽  
Author(s):  
Kalpana Ghoshal ◽  
Jharna Datta ◽  
Sarmila Majumder ◽  
Shoumei Bai ◽  
Xiaocheng Dong ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Histone Deacetylase ◽  
Chromatin Structure ◽  
Dna Methyltransferase ◽  
Combined Treatment ◽  
Hdac Inhibitors ◽  
Prolonged Treatment ◽  
Open Chromatin ◽  
Chip Assay

ABSTRACT Inhibitors of DNA methyltransferase (Dnmt) and histone deacetylases (HDAC) synergistically activate the methylated metallothionein I gene (MT-I) promoter in mouse lymphosarcoma cells. The cooperative effect of these two classes of inhibitors on MT-I promoter activity was robust following demethylation of only a few CpG dinucleotides by brief exposure to 5-azacytidine (5-AzaC) but persisted even after prolonged treatment with the nucleoside analog. HDAC inhibitors (trichostatin A [TSA] and depsipeptide) either alone or in combination with 5-AzaC did not facilitate demethylation of the MT-I promoter. Treatment of cells with HDAC inhibitors increased accumulation of multiply acetylated forms of H3 and H4 histones that remained unaffected after treatment with 5-AzaC. Chromatin immunoprecipitation (ChIP) assay showed increased association of acetylated histone H4 and lysine 9 (K9)-acetyl H3 with the MT-I promoter after treatment with TSA, which was not affected following treatment with 5-AzaC. In contrast, the association of K9-methyl histone H3 with the MT-I promoter decreased significantly after treatment with 5-AzaC and TSA. ChIP assay with antibodies specific for methyl-CpG binding proteins (MBDs) demonstrated that only methyl-CpG binding protein 2 (MeCP2) was associated with the MT-I promoter, which was significantly enhanced after TSA treatment. Association of histone deacetylase 1 (HDAC1) with the promoter decreased after treatment with TSA or 5-AzaC and was abolished after treatment with both inhibitors. Among the DNA methyltransferases, both Dnmt1 and Dnmt3a were associated with the MT-I promoter in the lymphosarcoma cells, and association of Dnmt1 decreased with time after treatment with 5-AzaC. Treatment of these cells with HDAC inhibitors also increased expression of the MTF-1 (metal transcription factor-1) gene as well as its DNA binding activity. In vivo genomic footprinting studies demonstrated increased occupancy of MTF-1 to metal response elements of the MT-I promoter after treatment with both inhibitors. Analysis of the promoter by mapping with restriction enzymes in vivo showed that the MT-I promoter attained a more open chromatin structure after combined treatment with 5-AzaC and TSA as opposed to treatment with either agent alone. These results implicate involvement of multifarious factors including modified histones, MBDs, and Dnmts in silencing the methylated MT-I promoter in lymphosarcoma cells. The synergistic activation of this promoter by these two types of inhibitors is due to demethylation of the promoter and altered association of different factors that leads to reorganization of the chromatin and the resultant increase in accessibility of the promoter to the activated transcription factor MTF-1.

scholarly journals Cisplatin-mediated activation of glucocorticoid receptor induces platinum resistance via MAST1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chaoyun Pan ◽  
JiHoon Kang ◽  
Jung Seok Hwang ◽  
Jie Li ◽  
Austin C. Boese ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Glucocorticoid Receptor ◽  
Tumor Growth ◽  
Nuclear Translocation ◽  
Mapk Pathway ◽  
Underlying Mechanism ◽  
Patient Treatment ◽  
Platinum Resistance ◽  
Platinum Based Chemotherapy

AbstractAgonists of glucocorticoid receptor (GR) are frequently given to cancer patients with platinum-containing chemotherapy to reduce inflammation, but how GR influences tumor growth in response to platinum-based chemotherapy such as cisplatin through inflammation-independent signaling remains largely unclear. Combined genomics and transcription factor profiling reveal that MAST1, a critical platinum resistance factor that reprograms the MAPK pathway, is upregulated upon cisplatin exposure through activated transcription factor GR. Mechanistically, cisplatin binds to C622 in GR and recruits GR to the nucleus for its activation, which induces MAST1 expression and consequently reactivates MEK signaling. GR nuclear translocation and MAST1 upregulation coordinately occur in patient tumors collected after platinum treatment, and align with patient treatment resistance. Co-treatment with dexamethasone and cisplatin restores cisplatin-resistant tumor growth, whereas addition of the MAST1 inhibitor lestaurtinib abrogates tumor growth while preserving the inhibitory effect of dexamethasone on inflammation in vivo. These findings not only provide insights into the underlying mechanism of GR in cisplatin resistance but also offer an effective alternative therapeutic strategy to improve the clinical outcome of patients receiving platinum-based chemotherapy with GR agonists.

scholarly journals Acute Glucocorticoid Administration Rapidly Suppresses Basal and Stress-Induced Hypothalamo-Pituitary-Adrenal Axis Activity

Endocrinology ◽  
2012 ◽  
Vol 153 (1) ◽  
pp. 200-211 ◽  
Author(s):  
Marcus H. Andrews ◽  
Susan A. Wood ◽  
Richard J. Windle ◽  
Stafford L. Lightman ◽  
Colin D. Ingram
Keyword(s):  
Glucocorticoid Receptor ◽  
Receptor Activation ◽  
Inhibitory Effect ◽  
Corticosterone Secretion ◽  
Glucocorticoid Administration ◽  
Corticosterone Response ◽  
Activity Marker ◽  
Corticosterone Release ◽  
Dose Dependent

Hypothalamo-pituitary-adrenal (HPA) axis activity is subject to negative feedback control by glucocorticoids. Although the rapid component of this feedback is widely considered to contribute to regulation of dynamic HPA activity, few in vivo data exist on the temporal and pharmacological characteristics of this phenomenon. Thus, frequent automated blood sampling was undertaken in rats to determine the effects of acute glucocorticoid administration on basal and stress-induced corticosterone secretion. The glucocorticoid agonist methylprednisolone (5–2000 μg) or dexamethasone (5–500 μg) injected iv at the peak of the diurnal rhythm caused dose-dependent suppression of basal corticosterone secretion, which was attenuated by the glucocorticoid receptor antagonist RU38486. With 50 μg methylprednisolone, the onset of this suppression occurred at 40 min and remained significant for 120 min. However, although higher doses led to a greater and more sustained suppression of endogenous corticosterone, the response was delayed by the emergence of an initial stimulatory response that imposed a finite minimum delay. A corticosterone response to injection of CRH (1 μg, iv) during the period of maximal suppression indicated a suprapituitary site for the inhibitory effect glucocorticoid activation. This mechanism was supported by glucocorticoid injection immediately before a psychological stress (30 min, white noise); methylprednisolone caused dose-dependent attenuation of stress-induced corticosterone release and expression of the activity marker c-fos mRNA in the paraventricular nucleus but did not block the pituitary response to CRH. Thus, in rats, glucocorticoid receptor activation rapidly suppresses basal and stress-induced HPA activity that operates, at least in part, through a central mechanism of action.

scholarly journals Rhythmic SAF-A Binding Underlies Circadian Transcription of the Bmal1 Gene

2008 ◽  
Vol 28 (10) ◽  
pp. 3477-3488 ◽  
Author(s):  
Yoshiaki Onishi ◽  
Syuji Hanai ◽  
Tomoya Ohno ◽  
Yasuhiro Hara ◽  
Norio Ishida
Keyword(s):  
Chromatin Structure ◽  
Matrix Protein ◽  
Open Chromatin ◽  
Nuclear Matrix Protein ◽  
Actual Mechanism ◽  
Gene Assay ◽  
Flanking Region ◽  
Circadian Transcription

ABSTRACT Although Bmal1 is a key component of the mammalian clock system, little is understood about the actual mechanism of circadian Bmal1 gene transcription, particularly at the chromatin level. Here we discovered a unique chromatin structure within the Bmal1 promoter. The RORE region, which is a critical cis element for the circadian regulation of the Bmal1 gene, is comprised of GC-rich open chromatin. The 3′-flanking region of the promoter inhibited rhythmic transcription in the reporter gene assay in vitro even in the presence of RORα and REV-ERBα. We also found that the nuclear matrix protein SAF-A binds to the 3′-flanking region with circadian timing, which was correlated with Bmal1 expression by footprinting in vivo. These results suggest that the unique chromatin structure containing SAF-A is required for the circadian transcriptional regulation of the Bmal1 gene in cells.

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.

scholarly journals Uteroplacental insufficiency alters hepatic expression, phosphorylation, and activity of the glucocorticoid receptor in fetal IUGR rats

2005 ◽  
Vol 289 (5) ◽  
pp. R1348-R1353 ◽  
Author(s):  
Mariana Baserga ◽  
Merica A. Hale ◽  
Robert A. McKnight ◽  
Xing Yu ◽  
Christopher W. Callaway ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Glucocorticoid Receptor ◽  
Chromatin Structure ◽  
P53 Protein ◽  
Mrna Levels ◽  
Artery Ligation ◽  
Pregnant Rat ◽  
Hepatic Expression ◽  
Protein Levels

Uteroplacental insufficiency (UPI) induces persistent changes in hepatic gene expression secondary to altered chromatin dynamics in the intrauterine growth- restricted (IUGR) rat liver. The glucocorticoid receptor (GR) is a transcription factor that when activated can induce changes in chromatin structure. To begin the process of identifying pathways by which IUGR affects chromatin structure, we hypothesized that UPI in the rat induces a significant increase in endogenous glucocorticoids (corticosterone) and increases GR expression and activation. To prove our hypothesis, we induced IUGR through bilateral uterine artery ligation of the pregnant rat. At day 1, UPI significantly increased corticosterone levels and was associated with increased total GR mRNA and protein levels in the liver, as well as increased hepatic phosphorylation of GR serine 211. Moreover, cyclin-dependent kinase 2 (CDK2) cyclinA/CDK2 protein levels, which selectively phosphorylate GR serine 211, were also significantly increased. To assess activity of the GR, we measured protein levels of the transcription factor p53 whose levels are downregulated, at least in part, by active GR. In this study, UPI decreased p53 protein and its downstream target Bax mRNA levels. We conclude that UPI in rats affects GR expression and activity in the liver. We speculate that these alterations early in life may contribute to the changes in chromatin structure and gene expression previously described in the IUGR liver.

scholarly journals LGD-5552, an Antiinflammatory Glucocorticoid Receptor Ligand with Reduced Side Effects, in Vivo

Endocrinology ◽  
2008 ◽  
Vol 149 (5) ◽  
pp. 2080-2089 ◽  
Author(s):  
Francisco J. López ◽  
Robert J. Ardecky ◽  
Bruce Bebo ◽  
Khalid Benbatoul ◽  
Louise De Grandpre ◽  
...  
Keyword(s):  
Side Effects ◽  
Glucocorticoid Receptor ◽  
Therapeutic Window ◽  
Glucocorticoid Treatment ◽  
Receptor Ligand ◽  
Monocyte Chemoattractant Protein 1 ◽  
Inflammatory Conditions ◽  
Differential Gene Regulation ◽  
Antiinflammatory Effects

Treatment of inflammation is often accomplished through the use of glucocorticoids. However, their use is limited by side effects. We have examined the activity of a novel glucocorticoid receptor ligand that binds the receptor efficiently and strongly represses inflammatory gene expression. This compound has potent antiinflammatory activity in vivo and represses the transcription of the inflammatory cytokine monocyte chemoattractant protein-1 and induces the antiinflammatory cytokine IL-10. The compound demonstrates differential gene regulation, compared with commonly prescribed glucocorticoids, effectively inducing some genes and repressing others in a manner different from the glucocorticoid prednisolone. The separation between the antiinflammatory effects of LGD-5552 and the side effects commonly associated with glucocorticoid treatment suggest that this molecule differs significantly from prednisolone and other steroids and may provide a safer therapeutic window for inflammatory conditions now commonly treated with steroidal glucocorticoids.

scholarly journals ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Gabriel E. Zentner ◽  
Sivakanthan Kasinathan ◽  
Beibei Xin ◽  
Remo Rohs ◽  
Steven Henikoff
Keyword(s):  
Transcription Factor ◽  
High Throughput Sequencing ◽  
Temporal Analysis ◽  
Micrococcal Nuclease ◽  
Time Resolved ◽  
Calcium Dependent ◽  
Consensus Motifs ◽  
Dna Shape ◽  
Kinetics Of

Abstract Chromatin endogenous cleavage (ChEC) uses fusion of a protein of interest to micrococcal nuclease (MNase) to target calcium-dependent cleavage to specific genomic loci in vivo. Here we report the combination of ChEC with high-throughput sequencing (ChEC-seq) to map budding yeast transcription factor (TF) binding. Temporal analysis of ChEC-seq data reveals two classes of sites for TFs, one displaying rapid cleavage at sites with robust consensus motifs and the second showing slow cleavage at largely unique sites with low-scoring motifs. Sites with high-scoring motifs also display asymmetric cleavage, indicating that ChEC-seq provides information on the directionality of TF-DNA interactions. Strikingly, similar DNA shape patterns are observed regardless of motif strength, indicating that the kinetics of ChEC-seq discriminates DNA recognition through sequence and/or shape. We propose that time-resolved ChEC-seq detects both high-affinity interactions of TFs with consensus motifs and sites preferentially sampled by TFs during diffusion and sliding.

scholarly journals Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of theHPRT Promoter

2001 ◽  
Vol 21 (22) ◽  
pp. 7682-7695 ◽  
Author(s):  
Chien Chen ◽  
Thomas P. Yang
Keyword(s):  
Chromatin Structure ◽  
Transcription Initiation ◽  
Dimethyl Sulfate ◽  
Dnase I ◽  
Minimal Promoter ◽  
Micrococcal Nuclease ◽  
Promoter Regions ◽  
Permeabilized Cells ◽  
Active Allele

ABSTRACT Differential chromatin structure is one of the hallmarks distinguishing active and inactive genes. For the X-linked human hypoxanthine phosphoribosyltransferase gene (HPRT), this difference in chromatin structure is evident in the differential general DNase I sensitivity and hypersensitivity of the promoter regions on active versus inactive X chromosomes. Here we characterize the nucleosomal organization responsible for the differential chromatin structure of the active and inactive HPRT promoters. The micrococcal nuclease digestion pattern of chromatin from the active allele in permeabilized cells reveals an ordered array of translationally positioned nucleosomes in the promoter region except over a 350-bp region that is either nucleosome free or contains structurally altered nucleosomes. This 350-bp region includes the entire minimal promoter and all of the multiple transcription initiation sites of the HPRT gene. It also encompasses all of the transcription factor binding sites identified by either dimethyl sulfate or DNase I in vivo footprinting of the active allele. In contrast, analysis of the inactive HPRT promoter reveals no hypersensitivity to either DNase I or a micrococcal nuclease and no translational positioning of nucleosomes. Although nucleosomes on the inactive promoter are not translationally positioned, high-resolution DNase I cleavage analysis of permeabilized cells indicates that nucleosomes are rotationally positioned over a region of at least 210 bp on the inactive promoter, which coincides with the 350-bp nuclease-hypersensitive region on the active allele, including the entire minimal promoter. This rotational positioning of nucleosomes is not observed on the active promoter. These results suggest a model in which the silencing of the HPRT promoter during X chromosome inactivation involves remodeling a transcriptionally competent, translationally positioned nucleosomal array into a transcriptionally repressed architecture consisting of rotationally but not translationally positioned nucleosomal arrays.

Sign in / Sign up

Export Citation Format

Share Document