Genes and transcription factors, including nuclear receptors: Methods of studying their interactions

REVIEW ARTICLE Genes and transcription factors, including nuclear receptors: Methods of studying their interactions STEN Z. CEKAN STOCKHOLM, SWEDEN Abbreviations: bp ⫽ base pair; CAT ⫽chloramphenicol transferase; cDNA ⫽ complementary DNA; mRNA⫽ messenger RNA; EMSA ⫽ electrophoretic motility shift assay; ER ⫽ estrogen receptor; ERE ⫽ estrogen-responsive element; ERR ⫽ estrogenrelated (orphan) receptor; GRIP-1 ⫽ glucocorticoid receptor–interacting protein; GST ⫽ glutathione S-transferase; GR ⫽ glucocorticoid receptor; GTH ⫽ glutathione; HSP ⫽ heat-shock protein; IPTG ⫽ isopropylthio-␤-D-galactopyranoside; LBD ⫽ ligand-binding domain; Luc ⫽ luciferase; NR ⫽ nuclear receptor; PAGE ⫽ polyacrylamide gel electrophoresis; PR ⫽ progesterone receptor; RAR ⫽ retinoic acid receptor; RE ⫽ response element; SDS ⫽ sodium dodecyl sulfate; SFRE ⫽ SF1-responsive element; SPR ⫽ surface plasmon resonance; SRC ⫽ steroid receptor coactivator; TATA box ⫽ thymineadenine-thymine-adenine sequence; TF ⫽ transcription factor; TIC ⫽ transcription-initiation complex; TIF ⫽ transcription intermediary factor; TBBP ⫽ TATA box– binding protein; TR ⫽ thyroid hormone receptor; TRE ⫽ thyroid hormone response element; TRAP ⫽ TR-associated protein

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n previous articles, the authors reviewed quantitative measurements of nuclear receptors and their mRNAs.1,2 These measurements have dealt with the posttranscriptional situation when mRNAs are formed and corresponding receptor molecules are created. In this article, pretranscription events are addressed. Interactions of NRs with various TFs are discussed. Such interactions lead to active complexes that form a basis for the transcription of a gene. Emphasis is placed on the methods that have been used for the interaction studies. Even if the list of methods is incomplete and the descriptions sketchy, it is hoped that this overview will be useful to all who study or wish to study receptor-TF interactions. MECHANISM OF GENE TRANSCRIPTION

It is well known that the expression of an activated gene consists of the transcription into a corresponding From the Department of Woman and Child Health, Division of Reproductive Endocrinology, Karolinska Institute. Submitted for publication March 8, 2002; revision submitted April 23, 2002; accepted May 8, 2002. Reprint requests: Sten Z. Cekan, PhD, Reproductive Endocrinology, Karolinska Hospital L5, 171 76 Stockholm, Sweden; e-mail: [email protected]. J Lab Clin Med 2002;140:215-27. Copyright © 2002 by Mosby, Inc. All rights reserved. 0022-2143/2002 $35.00 ⫹ 0 5/1/127370 doi:10.1067/mlc.2002.127370

mRNA, followed by a translation process that gives rise to a protein preprogrammed by the gene (see Reference 3 for review). In the activation of a gene, three types of entities are involved. One consists of the gene itself and of regulatory nucleotide sequences present in the DNA, mostly upstream from the gene proper, before the site of transcription initiation. These sequences are called “cisacting”. Among them, the core promoter is located close to the transcription-initiation site. This promoter directs the transcription of the gene, which may take place at very low levels, even constitutively, in the absence of other regulatory elements. A part of the core promoter is the TATA box, located 25 to 35 bp upstream from the transcription-initiation site. Another sequence sometimes taken as part of a larger promoter is a RE that modulates the transcription in response to specific external stimuli (eg, steroids). REs are usually located in a short distance upstream from the promoter. This, however, is no general rule. Multiple REs have even been found 2000 bases from the promoter.4 However, as a result of the folding of the DNA chain in the TIC (also called the preinitiation complex), these distant elements come into close proximity to the promoter and the transcription-initiation site. The same is true of the enhancers, which may be located very distantly from the gene, upstream and downstream; their function is to increase the level of transcription. The second entity is the complex of “trans-acting”, TFs that must be attached to the regulatory sequences 215

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described above to make possible the transcription of a gene. In this review, NRs will be dealt with as a major group of TFs. Transcription processes and TFs associated with cell-surface receptors will not be included. The third entity is the enzyme RNA polymerase II, which executes the transcription of protein-encoding genes.3,5 The RNA polymerase II can only act when the complex of transcription factors attached to the gene and its cis-acting sequences — the TIC — is complete and assumes an appropriate conformation in a nucleosome (DNA-histone unit).6 NRs as transcription factors. Among the trans-acting factors needed for the initiation of transcription, NRs (eg, steroid hormone receptors) are of special importance. They occur and act attached to the regulatory elements of a gene, and they form a link with other TFs of the TIC. NRs are distinctly different from cellsurface receptors (eg, those for gonadotropins) that act on the gene by way of signal transduction through the cytoplasm.3 NRs may be considered basic transcription factors. Their activity is mostly ligand-dependent.3,7 The ligands are small molecules that diffuse through the cell membrane and bind to their specific receptors. The binding results in receptor activation by dimerization. The receptor changes its shape (tertiary structure) and binds subsequently to the corresponding RE on the one hand and other TFs on the other. The NRs form a superfamily characterized by great similarities in protein structure. The NRs include receptors with known ligands, such as those for estradiol (estrogen), progesterone, testosterone (androgen), thyroid hormone, glucocorticoids, mineralocorticoids, retinoic acid, vitamin D, and ecdysone,8,9 as well as a series of “orphan” receptors for which ligands have not yet been found. Similarities exist in the structures of all NRs. All have distinct domains — for instance, for ligand binding (important in receptor activation) and for DNA binding (where binding to an RE takes place).10,11 TFs other than NRs. Hundreds of TFs have been described so far. The basic ones are the proteins associated with the TATA box, TBBPs: TFIIA, TFIIB, TFIID, TFIIE, TFIIG, and TFIIH. They are general coactivators that form the basis of a TIC with RNA polymerase II.8,12,13 Other TFs contributing to the formation of a TIC are mostly modular proteins with distinct functional domains — activation domain, DNA-binding domain,12 and NR interaction domain.14,15 There are basically two groups of TFs.16 One, called coactivators (cofactors, or adapters), actively support and accelerate transcription. Another group impedes transcription. They are called corepressors (repressors,

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inhibitors or silencers).8 The ratio of coactivators and corepressors, their down-regulation and up-regulation, directs the acceleration or retardation of transcription.16 Some coactivators and corepressors will be mentioned below. NRs have an ambivalent position. They act mostly as coactivators when activated by a ligand. However, in the unliganded state they may function as corepressors for other NRs. Interactions of some coactivators with NRs depend mostly on the preceding activation of a NR by a ligand. Such is the case with ER. When this receptor is activated by estradiol, the coactivators SRC, cAMP response element-binding protein, and others14,15,17,18 may be attached. Many coactivators are “promiscuous” — that is, they mediate transcription of genes for several NRs.15 HSPs are repressors at the receptor level. Their binding to receptors (by default) prevents formation of an active receptor-RE complex. The HSPs are dissociated from the receptor as soon as a ligand is present. The ligand is bound to the receptor, and the receptor is dimerized.15 Important representatives of the HSP family are HSP 90, HSP 70, and HSP 56.8,12 Other examples of corepressors are the silencing mediator for retinoid and thyroid hormone receptors (SMRT) and the nuclear receptor corepressor (N-CoR), proteins that interact, respectively, with unliganded members of the TR and RAR families. The interaction, however, is disturbed by a ligand.16 Exogenous compounds may influence the transcription process. For example, RU-486 (mifepristone), although bound to PR, blocks the activation of the transcription process by recruiting corepressors. Thus RU486 acts as antagonist of gene expression. In certain cases and in certain tissues, the expression of corepressors is low, and RU-486 becomes an agonist promoting gene activation.15,16 p55 Histone acetyl transferase (HAT) functions as a coactivator in that it acetylates conserved lysine residues in the H-terminal domain of histones (the DNAskeleton proteins). As a result of this action, the nucleosome structure is loosened (opened up), making the DNA more accessible to transcription factors.15 On the other hand, histone deacetylases are corepressors in the sense that in deacetylating histones, they pack nucleosomes tight (chromatin becomes more condensed) and shut down gene expression.3,15,19 Histone deacetylation seems to be mediated by DNA methylation. Therefore the DNA methyl transferase has the property of a corepressor.3 Transcription complexes. Before the transcription of a gene is initiated, a cascade of events results in the formation of a TIC, a complex of an activated receptor

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Table I. Methods of studying DNA-protein interactions Method

Sequence-specific DNA-affinity chromatography EMSA* Southwestern method One-hybrid system

Transient transfection* Three-plasmid method Template commitment assay Transcription (in vitro cell-free) assay

Principle

In a final purification step, a protein is captured on a Sepharose-bound DNA sequence. Slowing down of a DNA-protein complex in electrophoresis is used to identify the protein. A cDNA library is expressed in E. coli on agar. After transfer onto nitrocellulose, a TF is detected with a probe of labeled DNA. Transformed yeast contains an RE and a reporter gene. Plasmid containing cDNA library fused with a sequence coding for the activation domain of GAL4 is incorporated into the yeast. After expression, positive clones are sequenced. To test functionality of a receptor, one plasmid containing a receptor cDNA and another carrying an RE and a reporter gene are both incorporated into a cell for expression. As above, but a third plasmid is involved containing a sequence coding for a TF. The formation of RE-receptor-TF complex is investigated. Assay in two steps. In the first step, a DNA sequence is bound to a TF. In the second step, a competition of a changed DNA, or of another TF, is explored. A DNA sequence is transcribed. The variables are either mutations of DNA or various TFs.

*Method frequently used.

and other transcription factors bound to the promoter, response element, and other gene-regulatory elements. The elements of the complex are bound together by Van der Waals forces and hydrogen bonds.12,20 The assembly of the TIC and the resulting transcription are driven by a phosphorylation system.12 On the whole, the formation of a functional TIC is a result of competition between coactivators and corepressors.16 The gene and DNA-regulatory sequences are kept in a silent state under the influence of repressors. When activated, they shed the corepressor(s) and undergo a profound steric change in forming an active TIC. One type of change is the remodeling of the chromatin, loosening the nucleosome structure by acetylation (see above). The second type is the bending and twisting of the chromatin and DNA molecules to yield a conformation that is suitable for the attachment of the RNA polymerase II and for the transcription. It appears that not just the region proximal to the initiation site is involved. Even distant regulatory sequences (upstream and downstream) — the enhancers — can form complexes (called enhancesomes) that, as a result of the bending of chromatin, end up in the vicinity of the initiation site and participate in the transcription.12 Hence they execute long-range control of gene expression.3 Various models of transcription complexes may be seen in a number of publications.8,15,17,21-27 METHODS OF STUDYING DNA-TF AND TF-TF INTERACTIONS

In the investigation of interactions of TFs (including NRs) with DNA, or binding between TFs, most analytical methods deal with the interactions of pairs of molecules. Sometimes three or more entities are stud-

ied. Studies of the conformation of entire complexes or their parts are less frequent. In the investigations of new TFs, several methods, complementing and supporting each other, are frequently used. Many of the methods described below involve recombinant DNA techniques that are used in molecular biology. Readers unfamiliar with these techniques and with the nomenclature are advised to consult appropriate textbooks. Wherever indicated, the methods will be described with examples. To show methodological possibilities in the studies of NR-associated TFs, even those methods that have not yet been related to NRs will be described. All methods and examples will be described without going into laboratory details, which may be found in the publications quoted. Interaction of DNA and protein (receptor). The methods employed in this section are listed in Table I and described in more detail below. Sequence-specific DNA-affinity chromatography is used as a final step in the purification of TFs.12 After prepurification, a cell extract is run through a column of, for example, polystyrene beads onto which relevant DNA radiolabeled sequences of a gene are covalently bound. When the cell extract is passed through the column, the protein/receptor forms a complex with the DNA and is retained in the column. The retained DNAprotein complex is identified on salt-gradient elution and electrophoresis of the fractions. The binding to a DNA sequence makes this method rather specific. EMSA (aka gel-shift assay, gel retardation assay) is a method used to identify TFs.3,12 As schematically indicated in Fig 1, a mixture of TF-containing proteins is separated into fractions on a ion-exchange column. Each fraction is incubated with a radioactive DNA

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Fig 1. A flow chart depicting the series of events in EMSA.

fragment of interest as a probe and then subjected to gel electrophoresis. In one or more fractions, the DNA sequence picks the targeted protein by forming a TFDNA complex. The complex has reduced mobility compared with the DNA fragment, as detected on autoradiography. Among other uses, the method has been employed in the study of binding between ERs ␣ and ␤ and the ERE,28-31 or ERR and SFRE.32-35 EMSA has also been used to study ER ␣ and ␤ heterodimers; the monomers and the dimer were located with specific antibodies.36 The Southwestern method is used for the detection and isolation of a TF. As shown in Fig 2, a cDNA library or a gene coding for a transcription factor is cloned into a bacteriophage as expression vector. The

construct is transfected into Escherichia coli and expressed. The expressed library is plated out onto agar in culture dishes. The incubation results in well-separated phage plaques. After transfer to nitrocellulose filters, the TF is detected with a radiolabeled DNA probe containing a binding site (eg, part of an enhancer).3,37,38 Localization of colonies on the agar plate may be facilitated by the formation of color on the filter, if the lacZ (␤-galactosidase) gene has been incorporated into the bacteriophage and the filters have been impregnated with the substrate IPTG. In that case, positive clones would be detected with the ␤-galactosidase color reaction.38 A one-hybrid system can be used to detection and identify a TF in the following fashion (Fig 3): A DNA

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Fig 2. Schematic representation of the Southwestern method.

Fig 3. Model of the one-hybrid system.

construct consisting of tandem copies of a known binding sequence (eg, an RE), a promoter, and reporter sequences (eg his3, histidine biosynthesis gene; and lacZ, galactosidase gene) is integrated into the yeast genome by way of a plasmid. Thereafter, the yeast cells are transformed with plasmids containing a cDNA library of TFs fused with a cDNA sequence encoding an activation domain of the GAL4 gene (see description of

the two-hybrid method, below) needed for the expression of the reporter gene. The yeast cells are plated out on selective media. The colonies are located on the plate on the basis of the expression of reporter genes. The positive clones are isolated, and the cDNA insert encoding for a TF is identified by means of sequencing.3,39,40 Transient transfection (aka two-plasmid assay, func-

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Fig 4. A flow chart depicting the series of events in the transient transfection assay.

tionality assay,41 in vivo assay for TF activity,12 cotransfection,42 ␤-Galactosidase assay43) is used to study the interaction of a receptor with a binding sequence (eg, RE) of a gene. With this method, the functionality of the receptor preparation can also be examined, usually in a hormone-dependent manner (in the absence or presence of ligand).42 Two expression plasmids are cotransfected into a receptor-deficient cell line (Fig 4). The first plasmid contains the gene expressing a receptor protein; the other (the reporter plasmid) holds a receptor-binding gene and a reporter gene. If the expressed receptor binds to the binding sequence in the reporter plasmid, the reporter gene is activated and transcribed into the corresponding mRNA, which is translated into a reporter compound in the cytoplasm of the host cell. The cells are cultured and harvested, after which the reporter compound is analyzed quantitatively.42 If lacZ is the reporter gene, ␤-galactosidase is the reporter compound. The method was instrumental in the process of isolating and indentifying ER-␤. The reporter plasmid contained a vitellogenin promoter ERE and alkaline phosphatase gene, and the expression plasmid was constructed by means of insertion of a clone supposedly coding for ER-␤. After incubation of transfected CHO-K1 cells, the medium was assayed for alkaline phosphatase activity on the basis of chemiluminiscence. This experiment was carried out in both the

presence and absence of estradiol to test the susceptibility of the receptor preparation to be transactivated by a ligand.44 As another example, a plasmid encoding the nuclear orphan receptor ERR-1 was produced. A sequence of SFRE was cloned into the reporter plasmid containing a CAT reporter gene. Both plasmids were transfected into HeLa cells, after which the activity (acetylation of chloramphenicol) of the reporter gene was measured.32 Other examples of the interactions studied as follows: ER with CAT-ERE,34,45 ER, ERR1, ERR2 or ERR3 with ERE-Luc or TRE-Luc46,47 (Luc⫺measured on the basis of luminescence); GR with a promoter/enhancer sequence-CAT,10 TR with TRE-CAT.24,42 In another version of this method, yeast was used as a host. In these cases, lacZ gene coding for ␤-galactosidase41 was inserted as reporter. Examples are as follows: ER with lacZ-ERE;43 GR with lacZ-GRE41 (triamcinolone was used as ligand). Three-plasmid, or transcriptional interference (squelching) method is an extension of the transienttransfection technique described above. It was designed to study how the binding of a receptor to the cognate RE is influenced by another protein (TF), a protein that could either enhance or squelch binding.15,16,48 Three vectors are used. Two are expression vectors, each containing cDNA sequences coding for the two protein

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components. The third plasmid (the reporter) contains the response element, together with a strong promoter and a reporter gene. All three plasmids are transfected into suitable cells. If the two proteins interact and form a complex that binds to the response element, transcription takes place. The end point is measurement of RNA or of expressed protein. For example, squelching or synergism of various TFs (or their parts) with the binding of ER to the ERE was investigated in HeLa cells. The runoff RNAs were isolated by means of gel eletrophoresis and quantified on S1 endonuclease analysis.48 Another example: three plasmids were cotransfected into yeast. The first contained estrogen RE and reporter CAT gene, the second plasmid held an ER DNA sequence, and the third contained a sequence coding for SPT6, a protein that modulates the transcriptional activity of ER by interacting with the hormone-binding domain of ER.20 In another experiment, COS7 cells were transfected with expression vectors for glucocorticoid receptor and Stat5 (a signal transducer and activator of transcription). As reporter gene, a luciferase construct (ERELuc) was used. The expression was measured on luminiscence assays.49 Template commitment assays50 and competition prebinding assays,51 both in vitro assays, are extensions of the transcription assays. They show which sequences are essential to transcription by highlighting changes (mutations) of a template on the formation of a TIC, indicating the importance of individual TFs. The characteristic feature of these assays is an incubation in two steps. In a study of template sequences (all containing a gene and a strong promoter),51 the first, modified template was preincubated with a cell extract containing RNA polymerase and a limited number of TIFs. The second, wild-type template was then added, and the mixture was allowed to further incubate. Synthesis of RNA was initiated by the addition of nucleoside triphosphates. It could be seen whether the second template was able to challenge (compete with) the first one — that is, whether the first (modified) template was capable of stably binding the TFs to prevent transcription of the second (wild-type) template. The results were established by autoradiography of gels. Alternatively, it may be determined which TF is preferentially bound (committed) to a DNA template. Two templates, closely related but producing RNAs distinctly separated by gel electrophoresis, are incubated with RNA polymerase II and several TFs. The incubation is carried out in two runs, in which various combinations of TFs are used. The TF that firmly occupies (commits) the template in the first run and

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excludes from transcription the other TFs in the second run is the one considered essential to the formation of a TIC.50 Using the template commitment assay, it was demonstrated that steroid receptors (as a type of TF) can stabilize the formation of TIC and thus activate targetgene expression.8 The basis of transcription assays is the cell-free in vitro transcription of DNA-TF complexes (see References 8 and 52 for lists of papers). In these flexible systems, the effects of mutations (by deletion or substitution) of DNA sequences can be explored, or the importance to transcription of individual TFs can be studied. In the first case, cell extracts containing all necessary TFs are incubated with a mutated template (a modified DNA sequence containing a TATA box and a promoter) in parallel with a wild (unmodified) template. The incubation is done in the presence of RNA polymerase. The transcription is initiated by the addition of nucleoside triphosphates (one of them may be radiolabeled). The runoff RNAs are identified on gel electrophoresis, and their quantity may be assessed by radioactivity counting51,53 or with a blot analyzer.13 In the other case, the template is known, and the aim is to identify those basic TFs that are essential for in vitro transcription by forming a stable TIC. For instance, one in vitro transcription system utilized a template, RNA polymerase II, and TFII factors A, B, D, E, and F. It appeared that a newly isolated TFIIG was required for a full transcription.50 The results were quantified by autoradiography of gel-fractionated RNA. Another example is the study of TFIIIC in viral infection.54 The stability of the TIC may be tested with the so-called heparin-challenge assay. If the TIC is resistant, heparin has no effect when added after the TIC has been formed. However, when added to the incubation mixture in the process of TIC formation, no transcription takes place.50 Protein–protein interactions (interactions of NRs with transcription factors). Table II provides a list of methods

described in this section. Immunoprecipitation seems to be the simplest method for the study of receptor-TF interactions. An example: Sulfur 35–labeled ER prepared by in vitro translation and activated by estradiol was incubated with a TF called SP1. The complex was treated with the reversible cross-linker dithio-bis (succinimidyl propionate) and, after further incubation, immunoprecipitated with anti-ER or anti-Sp1 antibodies. The complex was bound to agarose A and washed, after which the cross-links were reversed with SDS buffer and the eluted proteins resolved on electrophoresis.34

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Table II. Methods of studying protein-protein interactions Method

Immunoprecipitation Pull-down assay* Far-Western assay DNA-dependent assay

Ternary EMSA Two-hybrid system*

Three-hybrid system Surface plasmon resonance

Principle

Complex of a labeled NR with a TF is precipitated with monoclonal antibodies, bound to protein A agarose, eluted, and isolated on electrophoresis. A GST-protein 1 is bound to Sepharose-GTH. Protein 1 binds protein 2. The complex is eluted and separated on electrophoresis. A protein mixture is separated on electrophoresis and probed with a radiolabeled sequence of another protein. Radiolabeled receptor is bound to biotinylated RE. The complex is formed with streptavidinsepharose. After washing, a TF is bound to the receptor. The resulting complex is subjected to electrophoresis. A binary complex of labeled DNA segment with a receptor (see EMSA) is bound to a TF. Plasmid 1 contains a gene encoding protein X and the binding domain of GAL4; plasmid 2 carries a gene of protein Y and the activation domain of GAL4. The plasmids are cotransfected into yeast. If X interacts with Y, a yeast reporter gene is expressed. As above; an additional plasmid contains a sequence of a coactivator or corepresor. One protein is immobilized on a biosensor chip and the second protein is allowed to flow over the surface.

*Method frequently used.

The pull-down assay (aka the interaction assay14 or the GST-dependent protein-protein–interaction assay,55 is designed to test interaction between two proteins (eg, a receptor and a TF) in a more sophisticated way than immunoprecipitation is capable of doing. In the pull-down assay, the in vitro avid binding of GTH to GST is used in such a way that one protein is fused with GST (carried out using recombinant techniques56,57), and the fused protein is bound, by way of GST, to GTH previously attached to Sepharose (or agarose14) beads (Fig 5). The second protein is labeled (often with sulfur 35). If the two proteins interact with each other, a complex forms on the Sepharose beads. The complex of two proteins with GST can be eluted by competition with an excess of free GTH56 or by boiling in the presence of SDS.57 It is then subjected to SDS electrophoresis. If, in the course of the preparation of the fused protein, a sequence is incorporated for a site-specific protease (eg, thrombin), the GST-protein bond can be cleaved and the two-protein complex isolated.56 The interaction between a receptor and a TF can be studied in two ways. In the first variant, the receptor is fused with GST and the TF is radiolabeled. In the second model, it is the other way around. When radioactive labeling is not used, the two proteins–GST complex can be detected with fluorography.21 The pull-down assay has been used in a number of studies. Examples of interactions investigated are as follows: ER (with and without activation by estradiol) with SRC1,7,18,58 ER with Sp1,34 hormone-binding domain of ER with a potential coactivator SPT6;20 ER-␣ with TIF proteins59 or with GR interactin protein 1,25

Fig 5. A flow chart depicting the series of events in the pull-down assay.

ER-␣ and ER-␤ with TRAP220 (NR-binding subunit of the mammalian mediator complex) or TIF2 (p160 coactivator);60 PR (activated by progesterone) with SRC1;61 the orphan member of the steroid hormone receptor superfamily chicken ovalbumin upstream pro-

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moter-transcription factor (COUP-TFC) with TFIIB21 or ER;62 TR-␤ with TFIIB;63 TR-␤ with TRAP220 or TIF2;64 TR-␤ or TR-␤ LBD with GRIP1 boxes in NID (nuclear receptor interaction domain of p160 family coactivator;14) TR or GR with sequences of GRIP1;55 androgen receptor AR with ARA70;65 hormone binding domain of GR with transcription activator GRIP 1;66 ER-␣ or ER-␤ with SHP (orphan nuclear receptor).67 A competition of a coactivator with an RE for a receptor was studied in a system consisting of a sequence of coactivator SRC-1 (fused with GST), TRE, and labeled TR.68 The far-Western assay is another alternative for the detection of interaction of two proteins. In principle, a protein mixture (cell extract or a mixture of cloned proteins) is subjected to gel electrophoresis. The separated proteins are transferred onto nitrocellulose. A protein probe is then used to identify the protein under investigation. The probe is very often radioactive (phosphorus 32 or sulfur 35) and fused with GST.69,70 (The GST part of the molecule serves to purify the probe on GTH-Sepharose and should not inhibit the binding to the protein to be detected.) The protocol may be reversed by the separation of a mixture of GSTfusion proteins on electrophoresis and probing with a sulfur 35–labeled protein.71 Instead of radioactivity, probe-enzyme chimeras (eg, alkaline phosphatase–labeled probe72 or monoclonal antibodies against the probe) may be used.73 One way to visualize the antibody complex is to use iodine 125–protein A21 that binds to the Fc region of immunoglobulin molecules. An example: A nuclear extract was resolved on SDSPAGE and transferred onto a nitrocellulose filter; this was probed with phosphorus 32–labeled AF2 (a sequence of the ER hormone-binding domain), and one of the p160 proteins was found interacting with ER.69 The DNA-dependent assay for protein-protein interactions was developed to investigate putative coactivators or corepressors.74 Its principle is as follows: The biotinylated sequence of an RE is incubated with a phosphorus 32–labeled receptor (liganded or nonliganded). The DNA-receptor complex is captured on streptavidin-agarose and washed. A cell extract containing a TF is added, after which the slurry is incubated and washed. The complex DNA-receptor-TF is isolated on PAGE, transferred to a nitrocellulose membrane, and subjected to autoradiography. This system was developed for the RAR74 and applied, for example, in the study of binding of TRAP, TIF-2, or both to NRs (sulfur 35–labeled heterodimer of thyroid receptor and retinoid X receptor) by way of receptor binding to a biotinylated sequence of the corresponding response element.64 Ternary EMSA is an expansion of the EMSA de-

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scribed above in the section on DNA-TF interations. It is also a variant of the DNA-dependent assay described in the preceding paragraphs. An NR is bound to a radiolabeled DNA segment. This binary complex is exposed to another TF, and a ternary complex, RE-NRTF, is formed. The complexes are separated on electrophoresis and detected with autoradiography. For example, a labeled double-stranded ERE sequence was incubated with ER proteins (obtained in vitro translation), and further incubated with TRAP or TIF-2 (both fused with GST, the GST having no part in the binding).60 In another study, a labeled RE segment was associated with TR/retinoic X receptor heterodimers, followed by TRAP or TIF-2 proteins.64 The two-hybrid system (aka the interaction trap system3) is a powerful in vivo method for the testing of protein-protein interactions. Its principle3,12,75,76 follows from this example (Fig 6): A TF called GAL4 is needed for the expression of the lacZ gene encoding ␤-galactosidase. GAL4 contains a DNA-binding domain and an activation domain. These domains are separately placed into two expression plasmids. Plasmid 1 contains a sequence coding for protein X (eg, a receptor) fused with a sequence coding for the DNAbinding domain of GAL4. Plasmid 2 contains a sequence coding for protein Y (eg, a TF) fused with a sequence for the activation domain of GAL4. Protein Y may be encoded by a known gene or one originating from a cDNA library. The plasmids are cotransformed into a yeast strain containing a reporter gene, a promoter, and a GAL4 binding site. If the expressed proteins X and Y lock into (interact with) each other, the binding and activation domains of GAL4 are brought together, the complex is bound to the genome, and the promoter of the reporter gene in the host cell is activated. After plating on a medium, colonies containing XY can be visualized with the ␤-galactosidase color reaction. Interactions of steroid receptors are usually studied in the presence of ligand. Examples of the interactions studied are as follows: ER with coactivator RIP-140;77 ER with coactivators RIP140, SRC1, TIF1, TIF218; ER with SRC1 (luciferase gene was used as reporter)7 PR with SRC-1;61 hormone-binding domain of GR with GRIP1;66 ER␣ or ER␤ with SHP (orphan NR).67 The three-hybrid system is an expansion of the twohybrid system that includes an additional plasmid coding for a third “partner” in the interactions. It can be designed to test the influence of the third partner on the binding of two proteins, the third partner being another protein that, in stepping between the first and second partners, functions as a coactivator or corepressor. The third partner can also be an enzyme (eg, a kinase) that

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Fig 6. A model of the two-hybrid system.

by phosphorylation of one of the partners makes possible their interaction.39,78 In SPR, one of the reactants in a protein-protein interaction study is immobilized on the surface of a biosensor chip in a BIOCORE instrument (www.biocore.se). The second reactant is injected with a continuous flow over the surface. The SPR biosensor has the ability to monitor the formation and breakdown of the formed complexes in real time, and it is possible to determine kinetic rate constants and affinity constants of the interactions.11,79,80 As examples, the studies may be mentioned on the TRAP (bound to biosensor) and the competing AF-2 domain of thyroid receptor and TIF-2,64 or a similar study concerning the ER-␣ and ER-␤ binding preferences to the TRAP, TIF2,60 or TBP.81 Conformational studies of TFs. In conformation studies, the shape of portions of TFs in their association with a ligand, DNA, or another TF are studied to visualize and explain the character of binding. A few examples: X-ray crystallographic studies of LBD revealed ligand-induced conformational changes in the AF-2 region of nuclear receptors.8 A modeling described the docking of estradiol into the pocket of the ligandbinding domain of ER.82 A model of interactions of DNA with several TFs and RNA polymerase II was proposed on the basis of crystallographic and electron microscopic findings.12 The complex of LBD of TR-␤ with a peptide sequence of coactivator GRIP1 (p160 family) was ob-

tained in a crystalline form and subjected to crystallographic analysis (Cu K ␣-radiation). It appeared that ligand (T3) binding leads to the formation of a hydrophobic groove within the LBD of the TR, a groove interacting with the sequence of GRIP-1.14 A similar sudy was carried out with the LBD of ER.83 On the basis of computer modeling, a three-dimensional structure of the LBD of GR was proposed.84 Crystallographic studies showed how the binding of various ligands could induce different NR conformations, thereby modulating their transcriptional activity.83 Circular dichroism spectroscopy was used for the study of a conformational change caused by the interaction of N-terminal regions of ER-␣ and ER-␤ with TBBP.81 CONCLUSIONS

This article is an overview of the methods most commonly used in the study of interactions of receptors with TFs and among TFs themselves. Many of the methods take advantage of molecular biology techniques, such as recombinant DNA techniques, hybridization, and others. To understand the background of some of the methods described above, it is essential to be familiar with the molecular biology techniques and the corresponding nomenclature. The aim of the interaction studies is to give us possibilities to better grasp the processes that activate or silence a gene in its function. None of the methods

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alone can provide an answer to all questions. The methods usually complement and verify each other, as may be seen in the vast majority of studies cited. No comparisons have been made concerning the usefulness, practicability, or precision of the methods described. Each method has its own justification in a given study. All laboratories that currently conduct interaction research use their own sets of methods. For laboratories newly entering the field, the frequency of use of individual methods in earlier studies by others may be a criterion in method selection. I am grateful to Professor H. Eriksson and Associate Professor L. Sahlin of the Department of Woman and Child Health, Karolinska Institute, for their valuable comments on the manuscript.

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