Transcriptional transactivation functions localized to the glucocorticoid receptor N terminus are necessary for steroid induction of lymphocyte apoptosis

1992 ◽  
Vol 12 (2) ◽  
pp. 589-597
Author(s):  
E S Dieken ◽  
R L Miesfeld
Keyword(s):  
Transcriptional Regulation ◽  
Glucocorticoid Receptor ◽  
Target Genes ◽  
Dominant Negative ◽  
Receptor Expression ◽  
Transactivation Domain ◽  
Lymphocyte Apoptosis ◽  
Murine Cell Line ◽  
N Terminus ◽  
Simplex Virus

Genetic studies have suggested that transcriptional regulation of specific target genes (by either induction or repression) is the molecular basis of glucocorticoid-mediated lymphocyte apoptosis. To examine the role of transcriptional regulation more directly, we developed a complementation assay utilizing stable transfection of wild-type (wt) and mutant (nti) glucocorticoid receptor (GR) cDNA constructs into a GR-deficient S49 murine cell line (7r). Our data confirm that the level of functional GR is rate limiting for S49 apoptosis and moreover that the GR amino terminus (N terminus), which as been deleted from the nti GR, is absolutely required for complementation in this system. Surprisingly, we found that at physiological levels of receptor, expression of the nti GR in cells containing wt GR results in enhanced dexamethasone sensitivity rather than a dominant negative phenotype. One interpretation of these data is that DNA binding by wt-nti heterodimers may be functionally similar to that of wt-wt homodimers, indicating that GRE occupancy by at least one transactivation domain may be sufficient to induce the hormonal response. To determine whether acidic activating sequences such as those localized to the GR N terminus are important in the induction of lymphocyte apoptosis, we tested the activity of a chimeric receptor in which we replaced the entire GR N terminus with sequences from the herpes simplex virus VP16 protein. Our results demonstrate that 7r cells expressing VP-GR fusions are indeed steroid sensitive, strongly supporting the idea that S49 apoptosis is dependent on transcriptional regulation of specific genes which respond to acidic activating domains, implying that induction, rather than repression, may be the critical initiating event.

scholarly journals Transcriptional transactivation functions localized to the glucocorticoid receptor N terminus are necessary for steroid induction of lymphocyte apoptosis.

1992 ◽  
Vol 12 (2) ◽  
pp. 589-597 ◽  
Author(s):  
E S Dieken ◽  
R L Miesfeld
Keyword(s):  
Transcriptional Regulation ◽  
Glucocorticoid Receptor ◽  
Target Genes ◽  
Dominant Negative ◽  
Receptor Expression ◽  
Transactivation Domain ◽  
Lymphocyte Apoptosis ◽  
Murine Cell Line ◽  
N Terminus ◽  
Simplex Virus

Genetic studies have suggested that transcriptional regulation of specific target genes (by either induction or repression) is the molecular basis of glucocorticoid-mediated lymphocyte apoptosis. To examine the role of transcriptional regulation more directly, we developed a complementation assay utilizing stable transfection of wild-type (wt) and mutant (nti) glucocorticoid receptor (GR) cDNA constructs into a GR-deficient S49 murine cell line (7r). Our data confirm that the level of functional GR is rate limiting for S49 apoptosis and moreover that the GR amino terminus (N terminus), which as been deleted from the nti GR, is absolutely required for complementation in this system. Surprisingly, we found that at physiological levels of receptor, expression of the nti GR in cells containing wt GR results in enhanced dexamethasone sensitivity rather than a dominant negative phenotype. One interpretation of these data is that DNA binding by wt-nti heterodimers may be functionally similar to that of wt-wt homodimers, indicating that GRE occupancy by at least one transactivation domain may be sufficient to induce the hormonal response. To determine whether acidic activating sequences such as those localized to the GR N terminus are important in the induction of lymphocyte apoptosis, we tested the activity of a chimeric receptor in which we replaced the entire GR N terminus with sequences from the herpes simplex virus VP16 protein. Our results demonstrate that 7r cells expressing VP-GR fusions are indeed steroid sensitive, strongly supporting the idea that S49 apoptosis is dependent on transcriptional regulation of specific genes which respond to acidic activating domains, implying that induction, rather than repression, may be the critical initiating event.

scholarly journals A Model of Glucocorticoid Receptor Interaction With Coregulators Predicts Transcriptional Regulation of Target Genes

2019 ◽  
Vol 10 ◽  
Author(s):  
Federico Monczor ◽  
Antonia Chatzopoulou ◽  
Carlos Daniel Zappia ◽  
René Houtman ◽  
Onno C. Meijer ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Glucocorticoid Receptor ◽  
Target Genes ◽  
Receptor Interaction

Leukemia inhibitory factor regulates glucocorticoid receptor expression in the hypothalamic-pituitary-adrenal axis

2005 ◽  
Vol 289 (5) ◽  
pp. E857-E863 ◽  
Author(s):  
Anastasia Kariagina ◽  
Svetlana Zonis ◽  
Mahta Afkhami ◽  
Dmitry Romanenko ◽  
Vera Chesnokova
Keyword(s):  
Glucocorticoid Receptor ◽  
Hpa Axis ◽  
Leukemia Inhibitory Factor ◽  
Dominant Negative ◽  
Receptor Expression ◽  
Inhibitory Factor ◽  
Mrna Levels ◽  
Hypothalamic Pituitary Adrenal ◽  
Protein Levels

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine belonging to the gp130 family. LIF is induced peripherally and within the brain during inflammatory or chronic autoimmune diseases and is a potent stimulator of the hypothalamic-pituitary-adrenal (HPA) axis. Here we investigated the role of LIF in mediating glucocorticoid receptor (GR) expression in the HPA axis. LIF treatment (3 μg/mouse, ip) markedly decreased GR mRNA levels in murine hypothalamus (5-fold, P < 0.01) and pituitary (1.7-fold, P < 0.01) and downregulated GR protein levels. LIF decreased GR expression in murine corticotroph cell line AtT20 within 2 h, and this effect was sustained for 8 h after treatment. LIF-induced GR mRNA reduction was abrogated in AtT20 cells overexpressing dominant-negative mutants of STAT3, indicating that intact JAK-STAT signaling is required to mediate LIF effects on GR expression. Conversely, mice with LIF deficiency exhibited increased GR mRNA levels in the hypothalamus and pituitary (3.5- and 3.5-fold, respectively; P < 0.01 for both) and increased GR protein expression when compared with wild-type littermates. The suppressive effects of dexamethasone on GR were more pronounced in LIF-null animals. These data suggest that LIF maintains the HPA axis activation by decreasing GR expression and raise the possibility that LIF might contribute to the development of central glucocorticoid resistance during inflammation.

scholarly journals Characterization of the transactivation domain in the peroxisome-proliferator-activated receptor γ co-activator (PGC-1)

2007 ◽  
Vol 403 (3) ◽  
pp. 511-518 ◽  
Author(s):  
Prabodh Sadana ◽  
Edwards A. Park
Keyword(s):  
Transcriptional Activation ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Target Genes ◽  
Thyroid Hormone Receptor ◽  
Acid Oxidation ◽  
Peroxisome Proliferator ◽  
Transactivation Domain ◽  
Peroxisome Proliferator Activated Receptor ◽  
N Terminus ◽  
Cpt I

The PGC-1s (peroxisome-proliferator-activated receptor γ co-activators) are a family of transcriptional regulators that induce the expression of various metabolic genes. PGC-1 proteins stimulate genes involved in mitochondrial biogenesis, fatty acid oxidation and hepatic gluconeogenesis. Previous studies have demonstrated that the PGC-1α and β isoforms interact with nuclear receptors through the conserved LXXLL (leucine-X-X-leucine-leucine) motifs. In the present study, we have investigated the mechanisms by which these PGC-1 isoforms stimulate gene expression. We have determined that the N-terminus of PGC-1 is responsible for transcriptional activation. Two conserved peptide motifs were identified in the N-terminus of PGC-1α and β isoforms. These domains were named AD1 and AD2 (activation domain 1 and 2). Deletion of both of these motifs decreased the induction of various PGC-1-regulated genes including the PEPCK (phosphoenolpyruvate carboxykinase) and CPT-I (carnitine palmitoyltransferase-I) genes. It was determined that amino acids containing a negative charge in AD1 and the leucine residues in AD2 were important for the transcriptional induction of the PEPCK and CPT-I genes. Disruption of the AD motifs did not diminish the ability of the PGC-1α protein to associate with the PEPCK or CPT-I genes. In addition, deletion of the AD domains did not eliminate the ability of PGC-1α to interact with the thyroid hormone receptor. The data indicate that the AD1 and AD2 motifs mediate the induction of many PGC-1- responsive genes, but they do not contribute to the recruitment of PGC-1 to target genes.

scholarly journals ACTH-Dependent Regulation of MicroRNA As Endogenous Modulators of Glucocorticoid Receptor Expression in the Adrenal Gland

Endocrinology ◽  
2012 ◽  
Vol 153 (1) ◽  
pp. 212-222 ◽  
Author(s):  
Anna Riester ◽  
Orna Issler ◽  
Ariadni Spyroglou ◽  
Sharon Haramati Rodrig ◽  
Alon Chen ◽  
...  
Keyword(s):  
Adrenal Gland ◽  
Glucocorticoid Receptor ◽  
Expression Pattern ◽  
Untranslated Region ◽  
Target Genes ◽  
Receptor Expression ◽  
Acth Stimulation ◽  
Steroid Synthesis ◽  
Expression Levels ◽  
Baseline Conditions

MicroRNA (miR) are a subset of small RNA molecules, which posttranscriptionally modulate target gene expression. Although miR have been demonstrated to impact a number of processes during development and tumorigenesis, little is known about the expression and the role of miR in the adrenal gland. Because tight regulation of steroid synthesis is crucial for maintaining homeostasis upon stressful stimuli, here, we determined the miR expression pattern in mouse adrenal glands under baseline conditions, as well as 10, 30, and 60 min upon ACTH stimulation, using miR microarray. Changes in miR expression levels detected by array analysis were confirmed by real-time PCR and further analyzed by bioinformatic tools to identify miR that putatively target genes involved in adrenal function. After selecting miR, with a significant change in their expression level upon ACTH stimulation, four of the predefined miR (miR-96, miR-101a, miR-142-3p, and miR-433) were found to putatively target the glucocorticoid receptor [nuclear receptor subfamily 3, group C, member 1 (Nr3c1)]. Nr3c1 expression levels were elevated 10 min after ACTH stimulation but decreased after 60 min in comparison with baseline conditions. Modified Nr3c1-3′-untranslated region constructs were further tested by in vitro luciferase assays. Thereby, we could confirm that miR96, miR101a, miR142-3p, and miR433 target the Nr3c1-3′-untranslated region and result in a 20–40% repression of it. Taken together, ACTH stimulation could be demonstrated to acutely influence adrenal miR expression pattern in vivo; thus, potentially modulating adrenal response to acute stressors.

scholarly journals Long-range regulation of p53 DNA binding by its intrinsically disordered N-terminal transactivation domain

2018 ◽  
Vol 115 (48) ◽  
pp. E11302-E11310 ◽  
Author(s):  
Alexander S. Krois ◽  
H. Jane Dyson ◽  
Peter E. Wright
Keyword(s):  
Dna Binding ◽  
Electrostatic Interactions ◽  
Target Genes ◽  
Structural Characteristics ◽  
Full Length ◽  
Transactivation Domain ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
N Terminus ◽  
Disordered Regions

Atomic resolution characterization of the full-length p53 tetramer has been hampered by its size and the presence of extensive intrinsically disordered regions at both the N and C termini. As a consequence, the structural characteristics and dynamics of the disordered regions are poorly understood within the context of the intact p53 tetramer. Here we apply trans-intein splicing to generate segmentally 15N-labeled full-length p53 constructs in which only the resonances of the N-terminal transactivation domain (NTAD) are visible in NMR spectra, allowing us to observe this region of p53 with unprecedented detail within the tetramer. The N-terminal region is dynamically disordered in the full-length p53 tetramer, fluctuating between states in which it is free and fully exposed to solvent and states in which it makes transient contacts with the DNA-binding domain (DBD). Chemical-shift changes and paramagnetic spin-labeling experiments reveal that the amphipathic AD1 and AD2 motifs of the NTAD interact with the DNA-binding surface of the DBD through primarily electrostatic interactions. Importantly, this interaction inhibits binding of nonspecific DNA to the DBD while having no effect on binding to a specific p53 recognition element. We conclude that the NTAD:DBD interaction functions to enhance selectivity toward target genes by inhibiting binding to nonspecific sites in genomic DNA. This work provides some of the highest-resolution data on the disordered N terminus of the nearly 180-kDa full-length p53 tetramer and demonstrates a regulatory mechanism by which the N terminus of p53 transiently interacts with the DBD to enhance target site discrimination.

The Gamma Catenin/CBP Complex Maintains Survivin Transcription In β-Catenin Deficient/Depleted Cancer Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1218-1218
Author(s):  
Yong-Mi Kim ◽  
Hong Ma ◽  
Vivian Oehler ◽  
Enzi Jiang ◽  
Cu Nguyen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Combined Treatment ◽  
Specific Inhibitor ◽  
Survivin Expression ◽  
Homozygous Deletion ◽  
Dominant Negative ◽  
Transactivation Domain ◽  
Aberrant Expression

Abstract Abstract 1218 Survivin is a member of the inhibitor of apoptosis (IAP) gene family, which plays essential roles in prevention of apoptosis, cell division, and cancer development. Aberrant expression of Survivin is associated with essentially all tumors compared to their normal tissues. A number of signaling pathways are involved in the transcriptional regulation of Survivin. Previously, we demonstrated that Survivin expression is CBP/β-catenin/TCF-dependent and that ICG-001, a specific inhibitor of binding to the N-terminus of CBP, effectively attenuates Survivin expression. Now, using NCI-H28 cells, which harbor a homozygous deletion of β-catenin, we demonstrate that Survivin transcription can similarly be mediated by nuclear γ-catenin. In this study, we confirm that γ-catenin, like β-catenin, accumulates in the nucleus and that it can increase TOPFLASH reporter activity upon Wnt3a stimulation in NCI-H28 cells. In addition, overexpression of γ-catenin increases the expression of the TCF/catenin target genes Survivin and cyclinD1. This effect can be diminished by overexpression of dominant negative TCF or transactivation domain deleted γ-catenin. We demonstrate that g-catenin by binding to TCF family members and specifically recruiting the coactivator CBP drives Survivin transcription particularly in β-catenin-deficient cells indicating that γ-catenin can participate in the canonical Wnt signaling cascade. We also examined the relative expression of γ-catenin and β-catenin in 90 cases of chronic myeloid leukemia (CML) in a published gene expression microarray data base. A statistically significant negative correlation between γ-catenin and β-catenin was found in AP/BC cases (-0.389, P = 0.006). Furthermore, in subsequent independent validation studies by qPCR in 28 CP and BC patients increased g-catenin expression predominated in BC cases and was associated with concomitantly increased Survivin expression. Gene expression was 3- and 6-fold greater in BC patients as compared to CP patients, for γ-catenin and survivin, respectively. Consistent with this observation, nuclear γ-catenin accumulation was evident in this population consistent with a potential transcriptional role. Combined treatment with imatinib mesylate (IM) and ICG-001 significantly inhibited colony formation in sorted CD34+ CML progenitors (survivin+/γ-cateninhigh/β-cateninlow) isolated from one BC and one AP patient resistant to IM. Our data indicates the potential of ICG-001 to block both the CBP/γ-catenin interaction and the CBP/β-catenin interaction and this may have clinical significance in cancers in which γ-catenin plays a significant transcriptional role. Disclosures: No relevant conflicts of interest to declare.

scholarly journals An essential domain of the c-myc protein interacts with a nuclear factor that is also required for E1A-mediated transformation.

1995 ◽  
Vol 15 (3) ◽  
pp. 1536-1544 ◽  
Author(s):  
D E Brough ◽  
T J Hofmann ◽  
K B Ellwood ◽  
R A Townley ◽  
M D Cole
Keyword(s):  
Amino Acids ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Cell Transformation ◽  
Point Mutations ◽  
Dominant Negative ◽  
Nuclear Factor ◽  
Myc Oncogene ◽  
N Terminus ◽  
Essential Domain

Cell transformation by nuclear oncogenes such as c-myc presumably involves the transcriptional activation of a set of target genes that participate in the control of cell division. The function of a small evolutionarily conserved domain of the c-myc gene encompassing amino acids 129 to 145 was analyzed to explore the relationship between cell transformation and transcriptional activation. Deletion of this domain inactivated the c-myc oncogene for cell transformation while retaining the ability to activate transcription of either myc consensus binding sites or a GAL4-dependent promoter when the c-myc N-terminus was fused to the GAL4 DNA-binding domain. Point mutations that altered a conserved tryptophan (amino acid 136) within this domain had similar effects. Expression of the wt c-Myc N terminus (amino acids 1 to 262) as a GAL4 fusion was a dominant inhibitor of cell transformation by the c-myc oncogene, and this same domain also inhibited transformation by the adenovirus E1A gene. Surprisingly, deletion of amino acids 129 to 145 eliminated the dominant negative activity of GAL4-Myc on both c-myc and E1A transformation. Expression of the GAL4-Myc protein in Cos cells led to the formation of a specific complex between the Myc N terminus and a nuclear factor, and this complex was absent with the dl129-145 mutant. These results suggest that an essential domain of the c-Myc protein interacts with a specific nuclear factor that is also required for E1A transformation.

scholarly journals Caspase cleavage of Ets-1 p51 generates fragments with transcriptional dominant-negative function

2010 ◽  
Vol 426 (2) ◽  
pp. 229-241 ◽  
Author(s):  
Souhaila Choul-Li ◽  
Catherine Leroy ◽  
Gabriel Leprivier ◽  
Clélia Laitem ◽  
David Tulasne ◽  
...  
Keyword(s):  
Target Genes ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Transactivation Domain ◽  
Caspase Cleavage ◽  
Terminal Fragment ◽  
Dominant Negative Form ◽  
Target Promoters ◽  
Spliced Variant

Ets-1 is a transcription factor that plays an important role in various physiological and pathological processes, such as development, angiogenesis, apoptosis and tumour invasion. In the present study, we have demonstrated that Ets-1 p51, but not the spliced variant Ets-1 p42, is processed in a caspase-dependent manner in Jurkat T-leukaemia cells undergoing apoptosis, resulting in three C-terminal fragments Cp20, Cp17 and Cp14 and a N-terminal fragment, Np36. In vitro cleavage of Ets-1 p51 by caspase 3 produces fragments consistent with those observed in cells undergoing apoptosis. These fragments are generated by cleavage at three sites located in the exon VII-encoded region of Ets-1 p51. This region is absent from the Ets-1 p42 isoform, which therefore cannot be cleaved by caspases. In Ets-1 p51, cleavage generates C-terminal fragments containing the DNA-binding domain, but lacking the transactivation domain. The Cp17 fragment, the major cleavage product generated during apoptosis, is devoid of transcriptional activity and inhibits Ets-1 p51-mediated transactivation of target genes by competing with Ets-1 p51 for binding to Ets-binding sites present in the target promoters. In the present study, we have demonstrated that caspase cleavage of Ets-1 within the exon VII-encoded region leads to specific down-regulation of the Ets-1 p51 isoform during apoptosis. Furthermore, our results establish that caspase cleavage generates a stable C-terminal fragment that acts as a natural dominant-negative form of the full-length Ets-1 p51 protein.

Sign in / Sign up

Export Citation Format

Share Document