scholarly journals Mediator and p300/CBP-Steroid Receptor Coactivator Complexes Have Distinct Roles, but Function Synergistically, during Estrogen Receptor α-Dependent Transcription with Chromatin Templates

2003 ◽  
Vol 23 (1) ◽  
pp. 335-348 ◽  
Author(s):  
Mari Luz Acevedo ◽  
W. Lee Kraus
Keyword(s):  
Estrogen Receptor ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Steroid Receptor ◽  
Estrogen Receptor Α ◽  
Preinitiation Complex ◽  
Transcription System ◽  
Steroid Receptor Coactivator ◽  
Transcription Reinitiation

ABSTRACT Ligand-dependent transcriptional activation by nuclear receptors involves the recruitment of various coactivators to the promoters of hormone-regulated genes assembled into chromatin. Nuclear receptor coactivators include histone acetyltransferase complexes, such as p300/CBP-steroid receptor coactivator (SRC), as well as the multisubunit mediator complexes (“Mediator”), which may help recruit RNA polymerase II to the promoter. We have used a biochemical approach, including an in vitro chromatin assembly and transcription system, to examine the functional role for Mediator in the transcriptional activity of estrogen receptor α (ERα) with chromatin templates, as well as functional interplay between Mediator and p300/CBP during ERα-dependent transcription. Using three different approaches to functionally inactivate Mediator (immunoneutralization, immunodepletion, and inhibitory polypeptides), we find that Mediator is required for maximal transcriptional activation by ligand-activated ERα. In addition, we demonstrate synergism between Mediator and p300/CBP-SRC during ERα-dependent transcription with chromatin templates, but not with naked DNA. This synergism is important for promoting the formation of a stable transcription preinitiation complex leading to the initiation of transcription. Interestingly, we find that Mediator has an additional distinct role during ERα-dependent transcription not shared by p300/CBP-SRC: namely, to promote preinitiation complex formation for subsequent rounds of transcription reinitiation. These results suggest that one functional consequence of Mediator-ERα interactions is the stimulation of multiple cycles of transcription reinitiation. Collectively, our results indicate an important role for Mediator, as well as its functional interplay with p300/CBP-SRC, in the enhancement of ERα-dependent transcription with chromatin templates.

scholarly journals Evidence for Ligand-Independent Activation of Hippocampal Estrogen Receptor-α by IGF-1 in Hippocampus of Ovariectomized Rats

Endocrinology ◽  
2016 ◽  
Vol 157 (8) ◽  
pp. 3149-3156 ◽  
Author(s):  
Elin M. Grissom ◽  
Jill M. Daniel
Keyword(s):  
Estrogen Receptor ◽  
Steroid Receptor ◽  
Estrogen Receptor Α ◽  
Female Rats ◽  
Ovariectomized Rats ◽  
Independent Manner ◽  
Steroid Receptor Coactivator ◽  
Intracerebroventricular Infusion

In the absence of ovarian estrogens, increased levels of estrogen receptor (ER)α in the hippocampus are associated with improvements in cognition. In vitro evidence indicates that under conditions of low estrogen, growth factors, including Insulin-Like Growth Factor 1 (IGF-1), can activate ERα and regulate ERα-mediated transcription through mechanisms that likely involve modification of phosphorylation sites on the receptor. The goal of the current work was to investigate a role for IGF-1 in ligand-independent activation of ERα in the hippocampus of female rats. Ovariectomized rats received a single intracerebroventricular infusion of IGF-1 and hippocampi were collected 1 or 24 hours later. After 1 h, IGF-1 increased hippocampal levels of phosphorylated ERα at serine 118 (S118) as revealed by Western blotting. Coimmunoprecipitation revealed that at 1 hour after infusion, IGF-1 increased association between ERα and steroid receptor coactivator 1, a histone acetyltransferase that increases transcriptional activity of phosphorylated ERα. IGF-1 infusion increased levels of the ERα-regulated proteins ERα, choline acetyltransferase, and brain-derived neurotrophic factor in the hippocampus 24 hours after infusion. Results indicate that IGF-1 activates ERα in ligand-independent manner in the hippocampus via phosphorylation at S118 resulting in increased association of ERα with steroid receptor coactivator 1 and elevation of ER-regulated proteins. To our knowledge, these data are the first in vivo evidence of ligand-independent actions of ERα and provide a mechanism by which ERα can impact memory in the absence of ovarian estrogens.

scholarly journals Histone H1 Represses Estrogen Receptor α Transcriptional Activity by Selectively Inhibiting Receptor-Mediated Transcription Initiation

2002 ◽  
Vol 22 (8) ◽  
pp. 2463-2471 ◽  
Author(s):  
Edwin Cheung ◽  
Alla S. Zarifyan ◽  
W. Lee Kraus
Keyword(s):  
Estrogen Receptor ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Specific Binding ◽  
Histone H1 ◽  
Estrogen Receptor Α ◽  
Transcription System ◽  
Repressive Effect

ABSTRACT Chromatin is the physiological template for many nuclear processes in eukaryotes, including transcription by RNA polymerase II. In vivo, chromatin is assembled from genomic DNA, core histones, linker histones such as histone H1, and nonhistone chromatin-associated proteins. Histone H1 is thought to act as a general repressor of transcription by promoting the compaction of chromatin into higher-order structures. We have used a biochemical approach, including an in vitro chromatin assembly and transcription system, to examine the effects of histone H1 on estrogen receptor α (ERα)-mediated transcription with chromatin templates. We show that histone H1 acts as a potent repressor of ligand- and coactivator-regulated transcription by ERα. Histone H1 exerts its repressive effect without inhibiting the sequence-specific binding of ERα to chromatin or the overall extent of targeted acetylation of nucleosomal histones by the coactivator p300. Instead, histone H1 acts by blocking a specific step in the ERα-dependent transcription process, namely, transcription initiation, without affecting transcription reinitiation. Together, our data indicate that histone H1 acts selectively to reduce the overall level of productive transcription initiation by restricting promoter accessibility and preventing the ERα-dependent formation of a stable transcription preinitiation complex.

Transcriptional activation in an improved whole-cell extract from Saccharomyces cerevisiae

1991 ◽  
Vol 11 (9) ◽  
pp. 4555-4560 ◽  
Author(s):  
M Woontner ◽  
P A Wade ◽  
J Bonner ◽  
J A Jaehning
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
In Vitro Transcription ◽  
Cell Extract ◽  
Whole Cell ◽  
Transcription System ◽  
Upstream Activation Sequence ◽  
Activation Sequence

We report an improved in vitro transcription system for Saccharomyces cerevisiae. Small changes in assay and whole-cell extraction procedures increase selective initiation by RNA polymerase II up to 60-fold over previous conditions (M. Woontner and J. A. Jaehning, J. Biol. Chem. 265:8979-8982, 1990), to levels comparable to those obtained with nuclear extracts. We have found that the simultaneous use of distinguishable templates with and without an upstream activation sequence is critical to the measurement of apparent activation. Transcription from any template was very sensitive to the concentrations of template and nontemplate DNA, extract, and activator (GAL4/VP16). Alterations in reaction conditions led to proportionately greater changes from a template lacking an upstream activation sequence; thus, the apparent ratio of activation is largely dependent on the level of basal transcription. Using optimal conditions for activation, we have also demonstrated activation by a bona fide yeast activator, heat shock transcription factor.

scholarly journals Yeast NC2 Associates with the RNA Polymerase II Preinitiation Complex and Selectively Affects Transcription In Vivo

2001 ◽  
Vol 21 (8) ◽  
pp. 2736-2742 ◽  
Author(s):  
Joseph V. Geisberg ◽  
Frank C. Holstege ◽  
Richard A. Young ◽  
Kevin Struhl
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Negative Regulator ◽  
Preinitiation Complex ◽  
Positive Role ◽  
Pol Ii ◽  
Basal Transcription Factors

ABSTRACT NC2 (Dr1-Drap1 or Bur6-Ydr1) has been characterized in vitro as a general negative regulator of RNA polymerase II (Pol II) transcription that interacts with TATA-binding protein (TBP) and inhibits its function. Here, we show that NC2 associates with promoters in vivo in a manner that correlates with transcriptional activity and with occupancy by basal transcription factors. NC2 rapidly associates with promoters in response to transcriptional activation, and it remains associated under conditions in which transcription is blocked after assembly of the Pol II preinitiation complex. NC2 positively and negatively affects approximately 17% of Saccharomyces cerevisiaegenes in a pattern that resembles the response to general environmental stress. Relative to TBP, NC2 occupancy is high at promoters where NC2 is positively required for normal levels of transcription. Thus, NC2 is associated with the Pol II preinitiation complex, and it can play a direct and positive role at certain promoters in vivo.

scholarly journals Aryl Hydrocarbon Receptor-Mediated Transcription: Ligand-Dependent Recruitment of Estrogen Receptor α to 2,3,7,8-Tetrachlorodibenzo- p-Dioxin-Responsive Promoters

2005 ◽  
Vol 25 (13) ◽  
pp. 5317-5328 ◽  
Author(s):  
Jason Matthews ◽  
Björn Wihlén ◽  
Jane Thomsen ◽  
Jan-Åke Gustafsson
Keyword(s):  
Estrogen Receptor ◽  
Aryl Hydrocarbon Receptor ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Time Course ◽  
Estrogen Receptor Α ◽  
Time Dependent ◽  
Promoter Regions ◽  
Aryl Hydrocarbon ◽  
H3 Acetylation

ABSTRACT Using chromatin immunoprecipitation assays, we studied the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated recruitment of the aryl hydrocarbon receptor (AhR) and several coregulators to the CYP1A1 promoter. AhR displayed a time-dependent recruitment, reaching a peak at 75 min and maintaining promoter occupancy for the remainder of the time course. Recruitment of AhR was followed by TIF2/SRC2, which preceded CBP, histone H3 acetylation, and RNA polymerase II (RNAPII). Simultaneous recruitment to the enhancer and the TATA box region suggests the formation of a large multiprotein complex bridging the two promoter regions. Interestingly, estrogen receptor α (ERα) displayed a TCDD- and time-dependent recruitment to the CYP1A1 promoter, which was increased by cotreatment with estradiol. Transfection in HuH7 human liver cells confirmed previously reported ERα enhancement of AhR activity. In contrast, TCDD did not induce the recruitment of ERα to the estrogen-responsive pS2 promoter, and after 120 min of cotreatment with estradiol, ERα is still present on the CYP1A1 promoter but no longer at pS2. RNA interference studies with T47D cells support a role for ERα in TCDD-dependent CYP1A1 expression. Our data suggest that ERα acts as a coregulator of AhR-mediated transcriptional activation and that the recruitment of ERα by AhR represents a novel mechanism AhR-ERα cross talk.

scholarly journals Identification of Regions within the F Domain of the Human Estrogen Receptor α that Are Important for Modulating Transactivation and Protein-Protein Interactions

2007 ◽  
Vol 21 (4) ◽  
pp. 829-842 ◽  
Author(s):  
Akiko Koide ◽  
Changqing Zhao ◽  
Misuzu Naganuma ◽  
Judith Abrams ◽  
Sarah Deighton-Collins ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Ligand Binding ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Steroid Receptor ◽  
Estrogen Receptor Α ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Steroid Receptor Coactivator ◽  
Human Estrogen Receptor

Abstract The estrogen receptor (ER)α is a biologically and clinically important ligand-modulated transcription factor. The F domain of the ERα modulates its functions in a ligand-, promoter-, and cell-specific manner. To identify the region(s) responsible for these functions, we characterized the effects of serial truncations within the F domain. We found that truncating the last 16 residues of the F domain altered the activity of the human ERα (hERα) on an estrogen response element-driven promoter in response to estradiol or 4-hydroxytamoxifen (4-OHT), its sensitivity to overexpression of the coactivator steroid receptor coactivator-1 in mammalian cells, and its interaction with a receptor-interacting domain of the coactivator steroid receptor coactivator-1 or engineered proteins (“monobodies”) that specifically bind to ERα/ligand complexes in a yeast two-hybrid system. Most importantly, the ability of the ER to induce pS2 was reduced in MDA-MB-231 cells stably expressing this truncated ER vs. the wild-type ER. The region includes a distinctive segment (residues 579–584; LQKYYIT) having a high content of bulky and/or hydrophobic amino acids that was previously predicted to adopt a β-strand-like structure. As previously reported, removal of the entire F domain was necessary to eliminate the agonist activity of 4-OHT. In addition, mutation of the vicinal glycine residues between the ligand-binding domain and F domains specifically reduced the 4-OHT-dependent interactions of the hERα ligand-binding domain and F domains with monobodies. These results show that regions within the F domain of the hERα selectively modulate its activity and its interactions with other proteins.

scholarly journals AF-2 activity and recruitment of steroid receptor coactivator 1 to the estrogen receptor depend on a lysine residue conserved in nuclear receptors.

1997 ◽  
Vol 17 (4) ◽  
pp. 1832-1839 ◽  
Author(s):  
P M Henttu ◽  
E Kalkhoven ◽  
M G Parker
Keyword(s):  
Estrogen Receptor ◽  
Nuclear Receptors ◽  
Transcriptional Activation ◽  
Alpha Helix ◽  
Steroid Receptor ◽  
Activation Function ◽  
Binding Activity ◽  
Lysine Residue ◽  
Steroid Receptor Coactivator ◽  
Helix 12

Hormone-dependent transcriptional activation by nuclear receptors depends on the presence of a conserved C-terminal amphipathic alpha-helix (helix 12) in the ligand-binding domain. Here we show that a lysine residue, which is conserved in most nuclear receptors in the predicted helix 3, is also required for estrogen-dependent transactivation. The replacement of lysine 366 with alanine appreciably reduced activation function 2 (AF-2) activity without affecting steroid- or DNA-binding activity in the mouse estrogen receptor. The mutation dramatically reduced the ability of the receptor to bind steroid receptor coactivator 1 (SRC-1) but had no effect on receptor-interacting protein 140 (RIP-140) binding, indicating that while their sites of interaction overlap, they are not entirely consistent and in keeping with the proposal that the recruitment of coactivators, such as SRC-1, is required for AF-2 activity. Although the function of RIP-140 remains to be established, RIP-140 appears to be capable of recruiting the basal transcription machinery, since overexpression of the protein markedly increased the transcriptional activity of the mutant receptor. Since the lysine residue is conserved, we propose that it is required, together with residues in helix 12, to form the surface by which members of the nuclear receptor family interact with coactivators.

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