Estrogen Receptor DNA-binding Domain Research Articles

Importance of the Sequence-Directed DNA Shape for Specific Binding Site Recognition by the Estrogen-Related Receptor.

Most nuclear receptors (NRs) bind DNA as dimers, either as hetero- or as homodimers on DNA sequences organized as two half-sites with specific orientation and spacing. The dimerization of NRs on their cognate response elements (REs) involves specific protein–DNA and protein–protein interactions. The estrogen-related receptor (ERR) belongs to the steroid hormone nuclear receptor (SHR) family and shares strong similarity in its DNA-binding domain (DBD) with that of the estrogen receptor (ER). In vitro, ERR binds with high affinity inverted repeat REs with a 3-bps spacing (IR3), but in vivo, it preferentially binds to single half-site REs extended at the 5′-end by 3 bp [estrogen-related response element (ERREs)], thus explaining why ERR was often inferred as a purely monomeric receptor. Since its C-terminal ligand-binding domain is known to homodimerize with a strong dimer interface, we investigated the binding behavior of the isolated DBDs to different REs using electrophoretic migration, multi-angle static laser light scattering (MALLS), non-denaturing mass spectrometry, and nuclear magnetic resonance. In contrast to ER DBD, ERR DBD binds as a monomer to EREs (IR3), such as the tff1 ERE-IR3, but we identified a DNA sequence composed of an extended half-site embedded within an IR3 element (embedded ERRE/IR3), where stable dimer binding is observed. Using a series of chimera and mutant DNA sequences of ERREs and IR3 REs, we have found the key determinants for the binding of ERR DBD as a dimer. Our results suggest that the sequence-directed DNA shape is more important than the exact nucleotide sequence for the binding of ERR DBD to DNA as a dimer. Our work underlines the importance of the shape-driven DNA readout mechanisms based on minor groove recognition and electrostatic potential. These conclusions may apply not only to ERR but also to other members of the SHR family, such as androgen or glucocorticoid, for which a strong well-conserved half-site is followed by a weaker one with degenerated sequence.

Cloning, Characterization, and Expression Profile of Estrogen Receptor in Common Chinese Cuttlefish, Sepiella japonica.

Sex steroid hormones are widely detected in molluscs and play important roles in sex determination, gonadal tissue maturation, and gametogenesis. Nevertheless, the signaling pathways of sex steroids in cephalopod have not yet been clearly elucidated. In the present study, a full-length sequence encoding the estrogen receptor (ER) was isolated from common Chinese cuttlefish, Sepiella japonica. The sjER cDNA clone was found to contain 1,788 nucleotides including a 1,470 bp open reading frame encoding 489 amino acid (aa) residues. The deduced ER protein consisted of six nuclear receptor characteristic domains. Based on a phylogenetic analysis, the ER DNA-binding domain and ligand-binding domain are highly conserved compared to other mollusc ERs. Highest aa identities were found for sjER with common octopus (Octopus vulgaris) ER (89%) and pacific oyster (Crassostrea gigas) ER (61%). Tissue expression analysis confirmed that sjER was widely distributed among tissues and predominantly expressed in the brain, liver, gonad (testis and ovary), and other accessory sexual gland (nidamental gland). The ER expression was temporally upregulated in the brain, liver, and ovary during the early sexual maturation period in S. japonica, which is coincident with the fluctuation of ovary estradiol content. These suggest that sjER may be involved in regulating the reproductive cycle of S. japonica. A fusion protein transient transfections assay showed that sjER was mainly located in the nucleus, suggesting a possible orthodox working mechanism of S. japonica ER in the nucleus through a ligand-dependent activation of specific gene transcription.

Mapping protein binding stability on nucleosomal DNA by single-molecule approach

The conformation of nucleosomal DNA is significantly different from that of a canonical B-form double stranded DNA (dsDNA), and is generally regarded to be less flexible and less accessible than free dsDNA due to the tight association of histone cores. Previous studies have demonstrated that the key mechanism involved in nucleosomal DNA-protein interaction is the protein accessibility to the DNA binding site. In this work, we used single molecule assays to measure the stability of two transcriptional factors (glucocorticoid receptor DNA binding domain (GRDBD) and estrogen receptor DNA-binding domain (ERDBD)) bound to their binding sites on different positions of the nucleosomal DNA. Interestingly, the results demonstrated that the nucleosomal DNA-GRDBD binding is not always consistent with the histone shielding effect, but adjusted by additional structural changes. Furthermore, the changes of these DNA-GRDBD interaction profiles were confirmed using molecular modeling and docking approaches based on their crystal structures. Very differently, ERDBD essentially is unable to bind to the nucleosomal DNA anywhere including the unblocked positions. We thus have concluded that the nucleosomal DNA-protein interaction is regulated not only by the histone shielding of the DNA binding sites, but also by the conformational changes of the nucleosomal DNA.

Systematic Mapping of Posttranslational Modifications in Human Estrogen Receptor-α with Emphasis on Novel Phosphorylation Sites

A systematic study of posttranslational modifications of the estrogen receptor isolated from the MCF-7 human breast cancer cell line is reported. Proteolysis with multiple enzymes, mass spectrometry, and tandem mass spectrometry achieved very high sequence coverage for the full-length 66-kDa endogenous protein from estradiol-treated cell cultures. Nine phosphorylated serine residues were identified, three of which were previously unreported and none of which were previously observed by mass spectrometry by any other laboratory. Two additional modified serine residues were identified in recombinant protein, one previously reported but not observed here in endogenous protein and the other previously unknown. Although major emphasis was placed on identifying new phosphorylation sites, N-terminal loss of methionine accompanied by amino acetylation and a lysine side chain acetylation (or possibly trimethylation) were also detected. The use of both HPLC-ESI and MALDI interfaced to different mass analyzers gave higher sequence coverage and identified more sites than could be achieved by either method alone. The estrogen receptor is critical in the development and progression of breast cancer. One previously unreported phosphorylation site identified here was shown to be strongly dependent on estradiol, confirming its potential significance to breast cancer. Greater knowledge of this array of posttranslational modifications of estrogen receptor, particularly phosphorylation, will increase our understanding of the processes that lead to estradiol-induced activation of this protein and may aid the development of therapeutic strategies for management of hormone-dependent breast cancer.

The plasticity of estrogen receptor–DNA complexes: Binding affinity and specificity of estrogen receptors to estrogen response element half-sites separated by variant spacers

The consensus estrogen response element (cERE) contains a palindromic sequence of two 6-base pair (bp) half-sites separated by a spacer size of 3 bp. This study investigates the extent to which estrogen receptors, ERα and ERβ can bind target sequences not considered as conventional EREs. We determined the effect of spacer size ( n = 0–4) on the binding affinity and conformation of ERα and ERβ in these complexes and the effect of HMGB1 on the complexation. We find (1) both receptors bind similarly and with progressively reduced affinity to cERE n, as n differs from 3; (2) however, both receptors bind as strongly to the cERE with no spacer (cERE0) as to cERE3; (3) HMGB1 enhances ER binding affinity in all complexes, resulting in strong and comparable binding affinities in all complexes examined; (4) the full-length ER binding differs strikingly from similar binding studies for the ER DNA binding domain (ERDBD), with the full-length ER dimer exhibiting strong binding affinity, enormous plasticity and retaining binding cooperativity as the spacer size varies; (5) both protease digestion profiles and monoclonal antibody binding assays indicate the conformation of the receptor in the ER/ERE complex is sensitive to the spacer size; (6) the ER/cERE0 complex appears to be singularly different than the other ER/cERE n complexes in binding and conformation. This multifaceted approach reinforces the notion of the plasticity in ER binding and leads to the hypothesis that in most cases, the minimum requirement for estrogen receptor binding is the ERE half-site, in which one or more cofactors, such as HMGB1, can cooperate to decrease ER binding specificity, while increasing its binding affinity.

Quantification of Cysteine Oxidation in Human Estrogen Receptor by Mass Spectrometry

Redox-dependent modifications of sulfhydryl groups within the two Cys4 zinc fingers of the estrogen receptor DNA-binding domain (ER-DBD) result in structural damage and loss of ER DNA-binding function, which parallels the situation observed in many ER-positive breast cancers. Quantitation of the redox status of cysteinyl thiols within ER-DBD employed cysteine-specific oxidants to induce varying degrees of oxidation in recombinant ER, followed by differential alkylation with the stable isotopic labeling reagents [12C2]-iodoacetic acid and [13C2]-bromoacetic acid. Subsequent proteolysis with LysC/Asp-N generated diagnostic peptides of which the C-terminal peptide of the second zinc finger is most strongly detected by mass spectrometry (MS) and serves as a suitable marker of ER-DBD redox status. Data were collected from two different MALDI-MS instruments: a time-of-flight and a linear ion trap (vMALDI-LIT). An analogous but larger synthetic peptide treated with three isotopic variants of the alkylating reagent modeled isotopic overlaps that might complicate the relative quantitation of cysteine oxidation. Despite the isotopic overlaps, excellent relative quantitation was achieved from MS data obtained from both instruments. This was also true of tandem MS/MS data from the vMALDI-LIT, which should facilitate selected reaction monitoring. Relative quantitation by MS also closely matched data from immunochemical methods.

Disruption of estrogen receptor DNA-binding domain and related intramolecular communication restores tamoxifen sensitivity in resistant breast cancer

A serious obstacle to successful treatment of estrogen receptor (ER)-positive human breast cancer is cell resistance to tamoxifen (TAM) therapy. Here we show that the electrophile disulfide benzamide (DIBA), an ER zinc finger inhibitor, blocks ligand-dependent and -independent cell growth of TAM-resistant breast cancer in vitro and in vivo. Such inhibition depends on targeting disruption of the ER DNA-binding domain and its communication with neighboring functional domains, facilitating ERalpha dissociation from its coactivator AIB1 and concomitant association with its corepressor NCoR bound to chromatin. DIBA does not affect phosphorylation of HER2, MAPK, AKT, and AIB1, suggesting that DIBA-modified ERalpha may induce a switch from agonistic to antagonistic effects of TAM on resistant breast cancer cells.

A conserved lysine in the estrogen receptor DNA binding domain regulates ligand activation profiles at AP-1 sites, possibly by controlling interactions with a modulating repressor.

BackgroundEstrogen receptors alpha and beta (ERα and ERβ) differentially activate genes with AP-1 elements. ERα activates AP-1 targets via activation functions with estrogens (the AF-dependent pathway), whereas ERβ, and a short version of ERα (ERα DBD-LBD) activate only with anti-estrogens (AF-independent pathway). The DNA binding domain (DBD) plays an important role in both pathways, even though neither pathway requires ERE recognition.ResultsMutations of a highly conserved DBD lysine (ERα.K206A/G), lead to super-activation of AP-1 through activation function dependent pathways, up to 200 fold. This super-activity can be elicited either through ER AFs 1 or 2, or that of a heterologous activation function (VP16). The homologous substitution in ERβ, K170A, or in ERα DBD-LBD leads to estrogen-dependent AP-1 activation and loss of the usually potent anti-estrogen effects. Each of numerous K206 substitutions in ERα, except K206R, eliminates anti-estrogen activation and this loss correlates perfectly with a loss of ability to titrate a repressive function from the RU486 bound progesterone receptor.ConclusionWe conclude that ER DBDs contain a complex regulatory function that influences ligand activation profiles at AP-1. This function, which requires the integrity of the conserved lysine, both allows for activation at AP-1 with anti-estrogens (with ERβ and ERα DBD-LBD), and prevents ERα from becoming superactive at AP-1 with estrogens. We discuss the possibility that a repressor interaction with the DBD both mediates the AF-independent pathway and dampens the AF dependent pathway. Mutations in the conserved lysine might, by this model, disrupt the binding or function of the repressor.

The Role of the C-terminal Extension (CTE) of the Estrogen Receptor α and β DNA Binding Domain in DNA Binding and Interaction with HMGB

HMGB-1/-2 are coregulatory proteins that facilitate the DNA binding and transcriptional activity of steroid receptor members of the nuclear receptor family of transcription factors. We investigated the influence and mechanism of action of HMGB-1/-2 (formerly known as HMG-1/-2) on estrogen receptor alpha (ERalpha) and ERbeta. Both ER subtypes were responsive to HMGB-1/-2 with respect to enhancement of receptor DNA binding affinity and transcriptional activity in cells. Responsiveness to HMGB-1/-2 was dependent on the C-terminal extension (CTE) region of the ER DNA binding domain (DBD) and correlated with a direct protein interaction between HMGB-1/-2 and the CTE. Thus the previously reported higher DNA binding affinity and transcription activity of ERalpha as compared with ERbeta is not due to a lack of ERbeta interaction with HMGB-1/-2. Using chimeric receptor DBDs, the higher intrinsic DNA binding affinity of ERalpha than ERbeta was shown to be due to a unique property of the ERalpha CTE, independent of HMGB-1/-2. The CTE of both ER subtypes was also shown to be required for interaction with ERE half-sites. These studies reveal the importance of the CTE and HMGB-1/-2 for ERalpha and ERbeta interaction with their cognate target DNAs.

Suppression of breast cancer by chemical modulation of vulnerable zinc fingers in estrogen receptor.

Current antiestrogen therapy for breast cancer is limited by the mixed estrogenic and antiestrogenic activity of selective estrogen receptor modulators. Here we show that the function of zinc fingers in the estrogen receptor DNA-binding domain (DBD) is susceptible to chemical inhibition by electrophilic disulfide benzamide and benzisothiazolone derivatives, which selectively block binding of the estrogen receptor to its responsive element and subsequent transcription. These compounds also significantly inhibit estrogen-stimulated cell proliferation, markedly reduce tumor mass in nude mice bearing human MCF-7 breast cancer xenografts, and interfere with cell-cycle and apoptosis regulatory gene expression. Functional assays and computational analysis support a molecular mechanism whereby electrophilic agents preferentially disrupt the vulnerable C-terminal zinc finger, thus suppressing estrogen receptor-mediated breast carcinoma progression. Our results provide the proof of principle for a new strategy to inhibit breast cancer at the level of DNA binding, rather than the classical antagonism of estrogen binding.

Essential cysteine-alkylation strategies to monitor structurally altered estrogen receptor as found in oxidant-stressed breast cancers

Oxidant-induced structural modifications within the cysteine-rich DNA-binding domain (DBD) of the overexpressed estrogen receptor (ER) likely contribute to its loss of DNA-binding function and altered transcriptional activity during human breast cancer development. Using recombinant ER protein as a model, procedures to detect such endogenously produced structural changes in the two Cys 4-type zinc fingers within the DBD of ER extracted from breast cancer cells are being developed. Unfortunately, ex vivo oxidation of these ER-DBD cysteine residues can occur during routine ER purification and preparation procedures. Also, cysteine residues readily undergo thiol–disulfide exchange reactions that can result in artificial oxidation and incorrect disulfide bond assignments. These problems can be circumvented by an initial irreversible alkylation of all free thiols followed by reduction of any disulfides and treatment with a second alkylating agent, prior to proteolysis and high-performance liquid chromatography mass spectrometry analysis of peptides in the doubly alkylated ER digest, to differentiate between the originally free and the disulfide-bonded cysteine residues. Although the use of chemically identical but isotopically different alkylating agents was more effective than the use of chemically different alkylating agents, subsequent problems were encountered with incomplete alkylation of particular Cys residues in the native ER protein. To overcome this limitation, the initial alkylation was accompanied by denaturation and the second alkylation was carried out during the proteolytic digestion. These improved analytical strategies should facilitate the monitoring of structurally altered endogenous ER produced within oxidant-stressed human breast cancer cells.

Cloning, sequencing and phylogenetic/phenetic classification of an estrogen receptor alpha (α) subtype of sheepshead minnow ( Cyprinodon variegatus)

The estrogen receptor (ER) is a key component of the reproductive system of both teleosts and tetrapods. In this study, the sequence and evolutionary relationship of sheepshead minnow ( Cyprinodon variegatus) ER were examined. Total RNA from livers of adult laboratory-reared gravid female C. variegatus was reverse-transcribed to prepare cDNA. Nested pairs of gene-specific degenerative primers derived from conserved amino acid sequences of the ER DNA binding domain were used to amplify an internal fragment of the ER cDNA of C. variegatus using polymerase chain reaction (PCR) followed by rapid amplification of cDNA ends (RACE). The amplified cDNA products were inserted into pGEM T ®-Easy Vector for cloning and sequencing. The cloned ER cDNA segments gave a 524-amino-acid ER sequence, which represents ∼80% of the sequence. The use of phylip for phylogenetic analysis with the maximum parsimony method and for phenetic analysis with the neighbor-joining method, along with bootstrap resampling, using 24 known sequences of α and β ER subtypes (both teleosts and tetrapods) indicated that the ER cDNA sequence of C. variegatus has strong homology to the alpha-subtype (ERα) of other teleostean fish, especially the closely related killifish species, Japanese medaka ( Oryzias latipes). Because ERα was the only subtype found, it appears that the α-subtype is predominant in C. variegatus liver.

Mutations in the Estrogen Receptor DNA-binding Domain Discriminate between the Classical Mechanism of Action and Cross-talk with Stat5b and Activating Protein 1 (AP-1)

Estrogen receptors (ERs) efficiently potentiate the transcriptional activity of prolactin-activated Stat5b through a mechanism that involves the ER DNA-binding domain (DBD) and the hinge domain. We have identified residues within the DBD of ER that are critical for the functional interaction of ER with Stat5b. We show that disruption of the second zinc finger structure abrogated cross-talk between ER and Stat5b, while the structure of the first zinc finger was not important. Furthermore, we confirm that intact DNA binding activity was not required for potentiation of Stat5b activity and that the dimerization of ER did not seem to be involved. Ligand-bound ERs also modulated activating protein 1-dependent transcription, and our data demonstrate that both zinc finger structures of the ER DBD are important for an intact response. We show that introduction of various point mutations within the DBD altered the response of the receptor to 17beta-estradiol and to the estrogen antagonists 4-hydroxytamoxifen and ICI 182,870 on the collagenase promoter. These findings provide new insights into the mechanisms by which ERs act in cross-talk with non-related transcription factors.

Estrogen regulates a tissue-specific calpain in the anterior pituitary

A PCR-based cDNA subtraction hybridization was performed to identify the genes stimulated by estrogen in the pituitary. A novel tissue-specific calpain (nCL-2 ′), previously shown to be expressed mainly in the stomach, was markedly induced in the pituitary after estrogen treatment. The 5 ′-flanking region of the calpain nCL-2 ′ gene was analyzed to assess the molecular mechanism of estrogen regulation. Sequence analysis of the nCL-2 ′ promoter (1.9 kb) revealed a perfectly palindromic putative estrogen-response element (ERE), GGTCATGCTGACC. In transient transfection studies, the nCL-2 ′ promoter was highly responsive to estrogen in the presence of estrogen receptor (ER). Transcriptional activation by estrogen was prevented by an ERE mutation as well as by mutations in the ER DNA-binding domain. An ER antagonist, ICI 182780, blocked estrogen inducibility of the nCL-2 ′ promoter. We conclude that the nCL-2 ′ form of calpain is expressed in the pituitary and upregulated by estrogen at the transcription level.

Cross-talk between ERs and signal transducer and activator of transcription 5 is E2 dependent and involves two functionally separate mechanisms.

Steroid hormone receptors and signal transducers and activators of transcription (STAT) factors constitute two distinct families of transcription factors activated by different signaling pathways. In previous reports, cross-talk between STAT5 and several steroid receptors has been demonstrated. We investigated putative cross-talk between ERalpha and ERbeta and STAT5. ERalpha and ERbeta were found to potently repress PRL-induced STAT5 transcriptional activity on a beta-casein promoter construct in a ligand-dependent manner. This down-regulation was found to rely on direct physical interaction between the ERs and STAT5, mediated via the ER DNA-binding domain (DBD). The contact between the ER DBD and STAT5 is highly specific; the interaction is abolished if the ERalpha DBD is replaced with the DBD of a closely related steroid receptor. The physical interaction, however, is insufficient to confer the repression of STAT5 activity, which in addition requires the ligand-activated C-terminal part of the ERs, although these domains are not in direct contact with STAT5. Negative cross-talk between ERs and STAT5 is thus mediated via several functionally separated domains of the ERs. Our findings may enhance the understanding of mechanisms of regulation of the different hormonal signaling pathways occurring during different functional events in tissues coexpressing ERs and STAT5.

External Surface Display of Proteins Linked to DNA-Binding Domains

A novel system (DBDX) was developed which allows the external surface display on filamentous bacteriophage of proteins fused to either the N- or the C-terminus of a DNA-binding protein. In conjunction with helper phage infection, expression of proteins fused to the estrogen receptor DNA-binding domain (DBD) in a phagemid vector containing the DNA sequence recognized by the DBD resulted in the production of phage particles which display the fusion protein through the phage pVIII coat on the external surface of the particle. The viability of the technique was established with several model systems: particles displaying the C-terminal domain of N-cadherin or the biotinylation domain of propionyl coenzyme A carboxylase fused to the C-terminus of the DBD were found to be bound specifically by antibody or streptavidin, respectively. Human κ constant region cDNA was selected from a N-terminal DBD fusion lymphocyte cDNA library after two rounds of selection with anti-κ antibody. This display system may complement currently available bacterial selection techniques.

High level expression of full-length estrogen receptor in Escherichia coli is facilitated by the uncoupler of oxidative phosphorylation, CCCP

The expression of high levels of full-length human estrogen receptor α (hERα) in Escherichia coli has proven difficult. We found that expression of the ER DNA binding domain is highly toxic to E. coli, resulting in rapid loss of the expression plasmid. Using a tightly regulated arabinose expression system and the antibiotic Timentin, we were able to overcome ER toxicity and express substantial levels of ER. The expressed ER exhibited protease cleavage at a single site near the N-terminus of the hinge region. Of the many measures we tested to eliminate ER cleavage, only addition of carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), an uncoupler of oxidative phosphorylation, completely blocked intracellular proteolysis of the ER. Using CCCP and our expression methods, full-length FLAG epitope-tagged hERα (fER) was expressed in E. coli at ∼1 mg/l. The fER was purified to homogeneity in a single step by immunoaffinity chromatography with anti-FLAG monoclonal antibody. Purified full-length bacterial fER binds 17β-estradiol with the same affinity as hER expressed in human cells ( K D∼0.5 nM). At high concentrations of fER (20 nM), a bell-shaped estrogen binding curve with a Hill coefficient of 1.7 was seen. Bacterially-expressed fER exhibits a reduced affinity for the estrogen response element (ERE). Anti-FLAG antibody restores high affinity binding of the fER to the ERE, suggesting that impaired dimerization may be responsible for the reduced affinity of bacterially-expressed fER for the ERE. The use of Timentin and CCCP may provide a general method for high level bacterial expression of steroid/nuclear receptors and other proteins important in hormone action.

Preferential oxidation of zinc finger 2 in estrogen receptor DNA-binding domain prevents dimerization and, hence, DNA binding.

For approximately one-third of estrogen receptor (ER)-positive breast cancer patients, extracted tumor ER is unable to bind to its cognate DNA estrogen response element (ERE), an effect that is partly reversible by the thiol-reducing agent dithiothreitol (DTT). Full-length (67 kDa) ER or its 11 kDa recombinant DNA-binding domain (ER-DBD) is also susceptible to loss of structure and function by the action of oxidants such as diamide and hydrogen peroxide; however, prior DNA binding by ER or ER-DBD protects against this oxidant induced loss of function. The ER-DBD contains two (Cys)(4)-liganded zinc finger motifs that cooperate to stabilize a rigid DNA-binding recognition helix and a flexible helix-supported dimerization loop, respectively. Comparisons between synthetic peptide analogues of each zinc finger and recombinant ER-DBD in the presence of zinc by electrophoretic mobility shift assay, circular dichroism, and mass spectrometry confirm that cooperativity between these zinc fingers is required for both ER-DBD structure (alpha-helicity) and function (dimeric DNA binding). Rapid proteolytic digestion of monomeric, non-DNA-bound ER-DBD followed by HPLC-MS analysis of the resulting peptides demonstrates that zinc inhibits thiol oxidation of the DNA-binding finger, but not the finger supporting the flexible dimerization loop, which remains sensitive to internal disulfide formation. These findings indicate that the loss of ER DNA-binding function in extracts from some primary breast tumors and in ER or ER-DBD exposed to thiol-reacting oxidants results from this asymmetric zinc finger susceptibility to disulfide formation that prevents dimerization. Although ER-DBD contains several strategically located methionine residues, they are less susceptible to oxidation than the thiol groups and, thus, afford no protection against cysteine oxidation and consequent loss of ER DNA-binding function.

Tamoxifen-Bound Estrogen Receptor (ER) Strongly Interacts with the Nuclear Matrix Protein HET/SAF-B, a Novel Inhibitor of ER-Mediated Transactivation

The estrogen receptor (ER) is a ligand-dependent transcription factor that acts in a cell- and promoter-specific manner. Evidence suggests that the activity of the ER can be regulated by a number of other stimuli (e.g. growth factors) and that the effects of the ER are modulated by nuclear factors termed coregulators. While the interplay among these factors may in part explain the pleiotropic effects elicited by the ER, there are several other less well described mechanisms of control, such as interactions with the nuclear matrix. Here we report that the nuclear matrix protein/scaffold attachment factor HET/SAF-B is an ER-interacting protein. ER and HET/SAF-B interact in in vitro binding assays, with HET binding to both the ER DNA-binding domain and the hinge region. Coimmunoprecipitation experiments reveal that HET/SAF-B and ER associate in cell lines in the presence or absence of estradiol, but binding is increased by the antiestrogen tamoxifen. HET/SAF-B enhances tamoxifen antagonism of estrogen-induced ER-mediated transactivation, but at high concentrations can inhibit both estrogen and tamoxifen-induced ER activity. HET/SAF-B-mediated repression of ER activity is dependent upon interaction with the ER-DBD. While the existence of high-affinity binding sites for the ER in the nuclear matrix has been known for some time, we now provide evidence of a specific nuclear matrix protein binding to the ER. Furthermore, our data showing that HET/SAF-B binds to ER particularly strongly in the presence of tamoxifen suggests that it may be important for the antagonist effect of tamoxifen.

Tamoxifen-bound estrogen receptor (ER) strongly interacts with the nuclear matrix protein HET/SAF-B, a novel inhibitor of ER-mediated transactivation.

The estrogen receptor (ER) is a ligand-dependent transcription factor that acts in a cell- and promoter-specific manner. Evidence suggests that the activity of the ER can be regulated by a number of other stimuli (e.g. growth factors) and that the effects of the ER are modulated by nuclear factors termed coregulators. While the interplay among these factors may in part explain the pleiotropic effects elicited by the ER, there are several other less well described mechanisms of control, such as interactions with the nuclear matrix. Here we report that the nuclear matrix protein/scaffold attachment factor HET/SAF-B is an ER-interacting protein. ER and HET/SAF-B interact in in vitro binding assays, with HET binding to both the ER DNA-binding domain and the hinge region. Coimmunoprecipitation experiments reveal that HET/SAF-B and ER associate in cell lines in the presence or absence of estradiol, but binding is increased by the antiestrogen tamoxifen. HET/SAF-B enhances tamoxifen antagonism of estrogen-induced ER-mediated transactivation, but at high concentrations can inhibit both estrogen and tamoxifen-induced ER activity. HET/SAF-B-mediated repression of ER activity is dependent upon interaction with the ER-DBD. While the existence of high-affinity binding sites for the ER in the nuclear matrix has been known for some time, we now provide evidence of a specific nuclear matrix protein binding to the ER. Furthermore, our data showing that HET/SAF-B binds to ER particularly strongly in the presence of tamoxifen suggests that it may be important for the antagonist effect of tamoxifen.