Self-assembled monolayer-assisted silicon nanowire biosensor for detection of protein-DNA interactions in nuclear extracts from breast cancer cell

The large number of estrogen receptor (ER) binding sites of various sequence patterns requires a sensitive detection to differentiate between subtle differences in ER-DNA binding affinities. A self-assembled monolayer (SAM)-assisted silicon nanowire (SiNW) biosensor for specific and highly sensitive detection of protein-DNA interactions, remarkably in nuclear extracts prepared from breast cancer cells, is presented. As a typical model, estrogen receptor element (ERE, dsDNA) and estrogen receptor alpha (ERα, protein) binding was adopted in the work. The SiNW surface was coated with a vinyl-terminated SAM, and the termination of the surface was changed to carboxylic acid via oxidation. DNA modified with amine group was subsequently immobilized on the SiNW surface. Protein-DNA binding was finally investigated by the functionalized SiNW biosensor. X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) were employed to characterize the stepwise functionalization of the SAM and DNA on bare silicon surface, and to visualize protein-DNA binding on the SiNW surface, respectively. We observed that ERα had high sequence specificity to the SiNW biosensor which was functionalized with three different EREs including wild-type, mutant and scrambled DNA sequences. We also demonstrate that the specific DNA-functionalized SiNW biosensor was capable of detecting ERα as low as 10 fM. Impressively, the developed SiNW biosensor was able to detect ERα-DNA interactions in nuclear extracts from breast cancer cells. The SAM-assisted SiNW biosensor, as a label-free and highly sensitive tool, shows a potential in studying protein-DNA interactions.     

Stability of the ligand-estrogen receptor interaction depends on estrogen response element flanking sequences and cellular factors

To determine whether accessory proteins mediate the ligand- and DNA sequence-dependent specificity of estrogen receptor (ER) interaction with DNA, the binding of partly purified vs highly purified bovine ER to various estrogen response elements (EREs) was measured in the presence of different ER ligands. Partly purified estradiol-liganded ER (E₂-ER) binds cooperatively to stereoaligned tandem EREs flanked by naturally occurring AT-rich sequences, with a stoichiometry of one E₂-ER dimer per ERE. In contrast, highly purified E₂-ER binds with a 10-fold lower affinity and non-cooperatively to EREs flanked by the AT-rich region. Moreover, the binding stoichiometry of highly purified E₂-ER was 0.5 E₂-ER dimer, or one monomer per ERE, independent of the ERE flanking sequence. Interestingly, the binding of ER liganded with the antiestrogen 4-hydroxytamoxifen (4-OHT-ER) was non-cooperative with an apparent stoichiometry of 0.5 4-OHT-ER dimer per ERE, regardless of ER purity or ERE flanking sequence. We recently showed that when 4-OHT-ER binds DNA, one molecule of 4-OHT dissociates from the dimeric 4-OHT-ER-ERE complex, accounting for the reduced apparent binding stoichiometry. In contrast, ER covalently bound by tamoxifen aziridine (TAz) gave an ERE binding stoichiometry of one TAz-ER dimer per ERE, and TAz-ER binds cooperatively to multiple AT-rich EREs, regardless of the purity of the receptor. We have obtained evidence that purification of ER removes an accessory protein(s) that interacts with ER in a sequence- and/or DNA conformational-dependent manner, resulting in stabilization of E₂, but not 4-OHT, in the ligand binding domain when the receptor binds to DNA. We postulate that retention of ligand by ER maintains the receptor in a conformation necessary to achieve high-affinity, cooperative ERE binding.     

Uterine estrogen receptor-DNA complexes: effects of different ERE sequences, ligands, and receptor forms

Previous studies used the gel retardation assay to examine the binding of the mouse estrogen receptor (ER) to the estrogen-responsive element (ERE) from the vitellogenin A2 gene (VitA2ERE). Multiple specific complexes were formed when the ER was bound to various estrogen agonists or antagonists, or in the absence of bound hormone. The ERE from the human PS2 gene, which varies from the consensus ERE by one base change in the right arm, was used in this study to determine the effect of DNA sequence on ER-ERE interaction with various ligand-receptor complexes. Partially purified ligand-free soluble ER showed a 3-fold lower affinity for the PS2ERE than for the VitA2ERE, suggesting a possible influence of the imperfect DNA sequence on certain binding interactions. However, multiple complexes of similar affinity were formed with the PS2 sequence by nuclear ER regardless of the agonist or antagonist bound. In gel retardation experiments, antagonist (LY117018) nuclear ER complexes bound to either PS2 or VitA2ERE migrated more slowly than agonist complexes, indicating that the slower migrating form of the complex was not due to the DNA sequence. Interestingly, soluble ER bound by LY 117018 did not produce this decreased mobility complex, suggesting that it was specific to the nuclear form of the ER antagonist complex. Receptor activation has been linked with exposure to increased temperature, resulting in an ER form that has an increased affinity for DNA. The binding of molybdate-stabilized nonactivated 8S ER to VitA2ERE was studied to determine the effect of temperature on ER binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

An explanation for observed estrogen receptor binding to single-stranded estrogen-responsive element DNA.

Estrogen-inducible genes contain an enhancer called the estrogen response element (ERE), a double-stranded inverted repeat. The estrogen receptor (ER) is generally thought to bind to the double-stranded ERE. However, some reports provide evidence that an ER homodimer can bind a single strand of the ERE and suggest that single-stranded ERE binding is the preferred binding mode for ER. Since these two models describe quite different mechanisms of receptor action, we have attempted to reconcile the observations. Analyzing DNA structure by nuclease sensitivity, we found that two identical molecules of a single strand of DNA containing the ERE sequence can partially anneal in an antiparallel manner. Bimolecular annealing produces double-stranded inverted repeats, with adjacent unannealed tails. The amount of annealing correlates exactly with the ability of ER to bind bimolecular EREs. Either strand of an ERE could anneal to itself in a way that would bind ER. We conclude that ER binds only the annealed double-stranded ERE both in vitro and in vivo.     

A single d(GpG) cisplatin adduct on the estrogen response element decreases the binding of the estrogen receptor

Both cisplatin and the estrogen receptor (ER) are known to bend DNA. The influence of the bending of sequences by the d(GpG)cisPt adduct binding of ER to estrogen response element (ERE)-like sequences was examined. Three ERE-like oligonucleotides with different affinities for ER and which include a GG in the linker sequence were designed in order to form a single central d(GpG)cisPt adduct. Using electrophoretic mobility shift assay and Scatchard analysis, it was shown that the presence of a single d(GpG)cisPt adduct in the linker sequence decreases the ER affinity for DNA. These results do not support a critical role of a DNA bend in the initial recognition of ERE by ER. Then, the platination of DNA outside of the ERE half-sites decreases the interaction of ER with ERE.     

A fractal analysis of protein to DNA binding kinetics using biosensors

A fractal analysis of a confirmative nature only is presented for the binding of estrogen receptor (ER) in solution to its corresponding DNA (estrogen response element, ERE) immobilized on a sensor chip surface [J. Biol. Chem. 272 (1997) 11384], and for the cooperative binding of human 1,25-dihydroxyvitamin D(3) receptor (VDR) to DNA with the 9-cis-retinoic acid receptor (RXR) [Biochemistry 35 (1996) 3309]. Ligands were also used to modulate the first reaction. Data taken from the literature may be modeled by using a single- or a dual-fractal analysis. Relationships are presented for the binding rate coefficient as a function of either the analyte concentration in solution or the fractal dimension that exists on the biosensor surface. The binding rate expressions developed exhibit a wide range of dependence on the degree of heterogeneity that exists on the surface, ranging from sensitive (order of dependence equal to 1.202) to very sensitive (order of dependence equal to 12.239). In general, the binding rate coefficient increases as the degree of heterogeneity or the fractal dimension of the surface increases. The predictive relationships presented provide further physical insights into the reactions occurring on the biosensor surface. Even though these reactions are occurring on the biosensor surface, the relationships presented should assist in understanding and in possibly manipulating the reactions occurring on cellular surfaces.     

Quantitative characterization of the interaction between purified human estrogen receptor alpha and DNA using fluorescence anisotropy

In an effort to better define the molecular mechanisms of the functional specificity of human estrogen receptor α, we have carried out equilibrium binding assays to study the interaction of the receptor with a palindromic estrogen response element derived from the vitellogenin ERE. These assays are based on the observation of the fluorescence anisotropy of a fluorescein moiety covalently bound to the target oligonucleotide. The low anisotropy value due to the fast tumbling of the free oligonucleotide in solution increases substantially upon binding the receptor to the labeled ERE. The quality of our data are sufficient to ascertain that the binding is clearly cooperative in nature, ruling out a simple monomer interaction and implicating a dimerization energetically coupled to DNA binding in the nanomolar range. The salt concentration dependence of the affinity reveals formation of high stoichiometry, low specificity complexes at low salt concentration. Increasing the KCl concentration above 200 mM leads to specific binding of ER dimer. We interpret the lack of temperature dependence of the apparent affinity as indicative of an entropy driven interaction. Finally, binding assays using fluorescent target EREs bearing mutations of each of the base pairs in the palindromic ERE half-site indicate that the energy of interaction between ER and its target is relatively evenly distributed throughout the site.     

The DNA binding domain of estrogen receptor alpha is required for high-affinity nuclear interaction induced by estradiol

Estrogen receptor alpha (ER) is a member of the nuclear hormone receptor family, which upon binding estrogen shows increased apparent affinity for nuclear components (tight nuclear binding). The nuclear components that mediate this tight nuclear binding have been proposed to include both ER-DNA interactions and ER-protein interactions. In this paper, we demonstrate that tight nuclear binding of ER upon estrogen occupation requires ER-DNA interactions. Hormone-bound ER can be extracted from the nucleus in low-salt buffer using various polyanions, which mimic the phosphate backbone of DNA. The importance of specific ER-DNA interactions in mediating tight nuclear binding is also supported by the 380-fold lower concentration of the ERE oligonucleotide necessary to extract estrogen-occupied ER from the nucleus compared to the polyanions. We also demonstrate that estrogen-induced tight nuclear binding requires both the nuclear localization domain and the DNA binding domain of ER. Finally, enzymatic degradation of nuclear DNA allows us to recover 45% of tight nuclear-bound ER. We further demonstrate that ER-AIB1 interaction is not required for estrogen-induced tight nuclear binding. Taken together, we propose a model in which tight nuclear binding of the estrogen-occupied ER is predominantly mediated by ER-DNA interactions. The effects of estrogen binding on altering DNA binding in whole cells are proposed to occur through estrogen-induced changes in ER-chaperone protein interactions, which alter the DNA accessibility of ER but do not directly change the affinity of the ER for DNA, which is similar for both unoccupied and occupied ER.     

The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer

Extracts containing wild-type or mutant human estrogen receptor (ER) have been used to study the binding of ER to its responsive element (ERE). Estradiol (E2) or the antiestrogen hydroxytamoxifen is required for ER binding as assayed by gel retardation. The DNA binding domain (DBD) encompasses the highly conserved region C. Both intact ER-E2 complexes and ER mutants truncated for the hormone binding domain (HBD) bind as dimers to an ERE. However, an HBD-truncated ER binds less tightly to an ERE than an intact ER-E2 complex. The DBD and HBD contain a constitutive and a stronger ER-induced dimerization function, respectively. Thus, in addition to inducing the activation function associated with the HBD, estrogen plays a crucial role in the formation of stable ER dimers that bind tightly to ERE.     

DNA binding analysis of glucocorticoid receptor specificity mutants.

The glucocorticoid receptor (GR) DNA binding domain consists of several conserved amino acids and folds into two zinc finger-like structures. Previous transactivation experiments indicated that three amino acids residing in this region, Gly, Ser and Val, appear to be critical for target-site discrimination. Based on the solved crystal structure, these residues are at the beginning of an amphipathic alpha-helix that interacts with the DNA's major groove; of these, only valine, however, contacts DNA. In order to examine their functional role directly, we have substituted these residues for the corresponding amino acids from the estrogen receptor (ER), overexpressed and purified the mutant proteins, and assayed their binding specificity and affinity by gel mobility shifts using glucocorticoid or estrogen response elements (GRE or ERE, respectively) as DNA probes. We find that all three residues are indeed required to fully switch GR's specificity to an ERE. The contacting valine in GR is of primary importance. The corresponding residue in ER, alanine, is less important for specificity, while glutamic acid, four amino acids towards the N-terminus, is most critical for ER discrimination. Finally, we show that the GR DNA binding domain carrying all three ER-specific mutations has a significantly higher affinity for an ERE than the ER DNA binding domain itself. We interpret these results in the context of both the data presented here and the crystal structure of the GR DNA binding domain complexed to a GRE.