Peter J. Kushner

Estrogen receptor pathways to AP-1

Estrogen receptor (ER) binds to estrogen response elements in target genes and recruits a coactivator complex of CBP-pl60 that mediates stimulation of transcription. ER also activates transcription at AP-1 sites that bind the Jun/Fos transcription factors, but not ER. We review the evidence regarding mechanisms whereby ER increases the activity of Jun/Fos and propose two pathways of ER action depending on the ER (alpha or beta) and on the ligand. We propose that estrogen-ERalpha complexes use their activation functions (AF-1 and AF-2) to bind to the p 160 component of the coactivator complex recruited by Jun/Fos and trigger the coactivator to a higher state of activity. We propose that selective estrogen receptor modulator (SERM) complexes with ERbeta and with truncated ERalpha derivatives use their DNA binding domain to titrate histone deacetylase (HDAC)-repressor complexes away from the Jun/Fos coactivator complex, thereby allowing unfettered activity of the coactivators. Finally, we consider the possible physiological significance of ER action at AP-1 sites.

Direct acetylation of the estrogen receptor alpha hinge region by p300 regulates transactivation and hormone sensitivity.

Regulation of nuclear receptor gene expression involves dynamic and coordinated interactions with histone acetyl transferase (HAT) and deacetylase complexes. The estrogen receptor (ERα) contains two transactivation domains regulating ligand-independent and -dependent gene transcription (AF-1 and AF-2 (activation functions 1 and 2)). ERα-regulated gene expression involves interactions with cointegrators (e.g.p300/CBP, P/CAF) that have the capacity to modify core histone acetyl groups. Here we show that the ERα is acetylated in vivo.p300, but not P/CAF, selectively and directly acetylated the ERα at lysine residues within the ERα hinge/ligand binding domain. Substitution of these residues with charged or polar residues dramatically enhanced ERα hormone sensitivity without affecting induction by MAPK signaling, suggesting that direct ERα acetylation normally suppresses ligand sensitivity. These ERα lysine residues also regulated transcriptional activation by histone deacetylase inhibitors and p300. The conservation of the ERα acetylation motif in a phylogenetic subset of nuclear receptors suggests that direct acetylation of nuclear receptors may contribute to additional signaling pathways involved in metabolism and development.

Multiple signal input and output domains of the 160-kilodalton nuclear receptor coactivator proteins.

ABSTRACT Members of the 160-kDa nuclear receptor coactivator family (p160 coactivators) bind to the conserved AF-2 activation function found in the hormone binding domains of nuclear receptors (NR) and are potent transcriptional coactivators for NRs. Here we report that the C-terminal region of p160 coactivators glucocorticoid receptor interacting protein 1 (GRIP1), steroid receptor coactivator 1 (SRC-1a), and SRC-1e binds the N-terminal AF-1 activation function of the androgen receptor (AR), and p160 coactivators can thereby enhance transcriptional activation by AR. While they all interact efficiently with AR AF-1, these same coactivators have vastly different binding strengths with and coactivator effects on AR AF-2. p160 activation domain AD1, which binds secondary coactivators CREB binding protein (CBP) and p300, was previously implicated as the principal domain for transmitting the activating signal to the transcription machinery. We identified a new highly conserved motif in the AD1 region which is important for CBP/p300 binding. Deletion of AD1 only partially reduced p160 coactivator function, due to signaling through AD2, another activation domain located at the C-terminal end of p160 coactivators. C-terminal coactivator fragments lacking AD1 but containing AD2 and the AR AF-1 binding site served as efficient coactivators for full-length AR and AR AF-1. The two signal input domains (one that binds NR AF-2 domains and one that binds AF-1 domains of some but not all NRs) and the two signal output domains (AD1 and AD2) of p160 coactivators played different relative roles for two different NRs: AR and thyroid hormone receptor.

Role of direct interaction in BRCA1 inhibition of estrogen receptor activity.

The BRCA1 gene was previously found to inhibit the transcriptional activity of the estrogen receptor [ER-α] in human breast and prostate cancer cell lines. In this study, we found that breast cancer-associated mutations of BRCA1 abolish or reduce its ability to inhibit ER-α activity and that domains within the amino- and carboxyl-termini of the BRCA1 protein are required for the inhibition. BRCA1 inhibition of ER-α activity was demonstrated under conditions in which a BRCA1 transgene was transiently or stably over-expressed in cell lines with endogenous wild-type BRCA1 and in a breast cancer cell line that lacks endogenous functional BRCA1 (HCC1937). In addition, BRCA1 blocked the expression of two endogenous estrogen-regulated gene products in human breast cancer cells: pS2 and cathepsin D. The BRCA1 protein was found to associate with ER-α in vivo and to bind to ER-α in vitro, by an estrogen-independent interaction that mapped to the amino-terminal region of BRCA1 (ca. amino acid 1-300) and the conserved carboxyl-terminal activation function [AF-2] domain of ER-α. Furthermore, several truncated BRCA1 proteins containing the amino-terminal ER-α binding region blocked the ability of the full-length BRCA1 protein to inhibit ER-α activity. Our findings suggest that the amino-terminus of BRCA1 interacts with ER-α, while the carboxyl-terminus of BRCA1 may function as a transcriptional repression domain.

TRANSCRIPTIONAL ACTIVATORS DIFFER IN THEIR RESPONSES TO OVEREXPRESSION OF TATA-BOX-BINDING PROTEIN

We investigated how overexpression of human TATA-box-binding protein (TBP) affects the action of estrogen receptor (ER) and compared the response with that of other activators. When ER activates a simple promoter, consisting of a response element and either the collagenase or tk TATA box, TBP overexpression potentiates transcription. TBP potentiates only estrogen-induced and not basal transcription and does so independent of spacing between response element and TATA box. TBP overexpression also reduces autoinhibition by overexpressed ER, suggesting that one target of the autoinhibition may be TBP itself. Both AF-1 and AF-2 domains of ER are potentiated by TBP, and each domain binds TBP in vitro. Like ER, chimeric GAL4/VP16 and GAL4/Tat activators are also potentiated by TBP, as is the synergistic activation by ER and GAL4/VP16 on a complex promoter. Unlike ER, GAL4/Sp1 and GAL4/NF-I become less potent when TBP is overexpressed. Furthermore, synergy between ER and Sp1 or between ER and NF-I, whether these are supplied by transfected GAL4 fusions or by the endogenous genes, is inhibited by TBP overexpression. Thus, ER resembles VP16 in response to TBP overexpression and is different from Sp1 and NF-I, which predominate over ER in setting the response on complex promoters.

Differential SERM Effects on Corepressor Binding Dictate ERα Activity in Vivo

Selective estrogen receptor modulators (SERMs) show differential effects upon ERα activation function 1 (AF-1). Tamoxifen allows strong ERα AF-1 activity, whereas raloxifene allows less and ICI 182,780 (ICI) allows none. Here, we show that blockade of corepressor histone de-acetylase (HDAC) activity reverses the differential inhibitory effect of SERMs upon AF-1 activity in MCF-7 cells. This suggests that differential SERM repression of AF-1 involves HDAC-dependent corepressors. Consistent with this, ICI and raloxifene are more potent than tamoxifen in promoting ERα-dependent sequestration of progesterone receptor-associated corepressors. Moreover, ICI and raloxifene are more efficient than tamoxifen in promoting ERα binding to the corepressor N-CoR in vivo and in vitro. An ERα mutation (537X) that increases N-CoR binding in the presence of all SERMs blocks AF-1 activity. An ERα mutation (L379R) that decreases N-CoR binding increases AF-1 activity in the presence of ICI and raloxifene and reverses the effect of the 537X mutation. The 537X and L379R mutations also alter the ligand preference of ERα action at AP-1 sites and C3 complement, an action that also involves AF-1. Together, our results suggest that differential SERM effects on corepressor binding can explain differences in SERM effects on ERα activity. We propose a model for differential effects of SERMs on N-CoR binding.

Dynamic Stabilization of Nuclear Receptor Ligand Binding Domains by Hormone or Corepressor Binding

We have developed a novel assembly assay to examine structural changes in the ligand binding domain (LBD) of the thyroid hormone receptor (TR). Fragments including the first helix of the TR LBD interact only weakly with the remainder of the LBD in the absence of hormone, but this interaction is strongly enhanced by the addition of either hormone or the corepressor NCoR. Since neither the ligand nor the corepressor shows direct interaction with this helix, we propose that both exert their effects by stabilizing the overall structure of the LBD. Current models of activation of nuclear hormone receptors focus on a ligand-induced allosteric shift in the position of the C-terminal helix 12 that generates the coactivator binding site. Our results suggest that ligand binding also has more global effects that dynamically alter the structure of the receptor LBD.

Molecular and structural biology of thyroid hormone receptors.

1. Thyroid hormone receptors (TR) are expressed from two separate genes (α and β) and belong to the nuclear receptor superfamily, which also contains receptors for steroids, vitamins and prostaglandins.

A Splice Variant of Estrogen Receptor β Missing Exon 3 Displays Altered Subnuclear Localization and Capacity for Transcriptional Activation

There are two separate estrogen receptors (ERs), ERalpha and ERbeta. The ERbeta gene is variably spliced, and in some cases variant expression is high. Besides the full-length ERbeta (equivalent to ERbeta1), splice variants can encode proteins bearing an insert within the ligand-binding domain (beta2), a deletion of exon 3 (ERbeta1delta3) disrupting the DNA-binding domain, or both (ERbeta2delta3). Here we examine the intracellular localization and transcriptional properties of each of the ERbeta splice variants heterologously expressed in cultured cells. In accordance with ERalpha, ERbeta1 and ERbeta2 are both distributed in a reticular pattern within the nucleus after exposure to ligand. In contrast, ERbeta1delta3 and ERbeta2delta3 localize to discrete spots within the nucleus in the presence of ER agonists. In the presence of ER antagonists, the delta3 variants are distributed diffusely within the nucleus. We also show that the spots are stable nuclear structures to which the delta3 variants localize in a ligand-dependent manner. Coactivator proteins of ER colocalize with delta3 variants in the spots in the presence of agonists. The delta3 variants of ERbeta can activate luciferase reporter constructs containing an activator protein complex-1 site, but not an estrogen response element (ERE). These data suggest that without an intact DNA-binding domain, ERbeta is functionally altered, allowing localization to discrete nuclear spots and activation from activator protein-1-containing reporter genes.

Positive and negative modulation of Jun action by thyroid hormone receptor at a unique AP1 site.

We have characterized the putative AP1 site in the backbone of pUC plasmids and found unique regulatory effects. The site, which mapped to a 19-bp region around nucleotide 37, conferred transcriptional activation by Jun or Jun/Fos that was boosted up to fivefold by unliganded thyroid hormone receptor (TR). Thyroid hormone changed potentiation of the Jun response by TR into repression. Although the plasmid sequence is a near-perfect consensus AP1 site, the perfect consensus AP1 site from the human collagenase promoter did not show the same effects. Deletion of the ligand binding domain of the TR eliminated the ability of the receptor to boost Jun activity, and deletion, mutation, or changes in specificity of the DNA binding domain eliminated both its ability to potentiate Jun activity and repress with hormone. In vitro Jun/Fos complexes bound the operative plasmid fragment, and the presence of TR interfered very little with Jun/Fos binding activity. Protein interaction studies in the absence of DNA showed that TR bound Jun protein in solution either in the presence or in the absence of hormone. These observations suggest a mechanism for synergy and repression by TR through modulation of Jun activity: positive when TR is unliganded, and negative when hormone is bound. They also suggest that the presence of the plasmid element can confound studies of the regulation of linked promoters.