The use of inducible engrailed fusion proteins to study the cellular functions of eukaryotic transcription factors

Methods 26 (2002) 270–280 www.academicpress.com

The use of inducible engrailed fusion proteins to study the cellular functions of eukaryotic transcription factors Elaine R. Vickers and Andrew D. Sharrocks* School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT, UK Accepted 2 February 2002

Abstract Transcription factors determine cell lineages, control cell fate, and regulate cellular responses to stimuli. Many methods are currently used to study the function of transcription factors in a cellular context and several of these involve overexpressing a constitutively active form of the protein and studying its effects. Here we outline an alternative approach involving the inducible expression of dominant-negative transcription factors in human cell lines. Dominant-negative transcription factors can be used to investigate the effect of signaling pathways on complex cellular processes that are regulated by a particular transcription factor. Potent dominant-negative transcription factors can be created by using fusions to the engrailed repressor domain. These fusion proteins can be coupled to inducible expression systems such as the ecdysone-inducible system. The ability to control protein expression has several benefits including the ability to create stable cell lines that express potentially cytotoxic proteins. Therefore when used in tandem, these two methods constitute a new and improved approach for dissecting the cellular role and transcriptional targets of many transcription factors. Here we illustrate this integrated approach by using a conditional dominant-negative Elk-1 protein to identify candidate Elk-1-regulated target genes. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Ecdysone; Engrailed; Muristerone; Ponasterone A; Transcription factor

1. Introduction The analysis of transcription factor function is a burgeoning field, and yet one fraught with complications. In the postgenomic era it is essential to understand which complement of genes is regulated by specific transcription factors. Many transcription factors have multiple roles depending on the cell type, timing and duration of activation, cell cycle status, and also the developmental state of the organism. Transcription factors that are thought to be involved in cell cycle entry such as the ETS-domain transcription factor Elk-1 are maintained under strict control to avoid aberrant cell division (reviewed in [1,2]). Overexpression studies are often used to study transcription factors but these approaches are unlikely to mirror normal physiological functions. Despite this, many studies have been carried

*

Corresponding author. Fax: +44-161-275-5082. E-mail address: [email protected] (A.D. Sharrocks).

out in which stable cell lines have been created that express a transcription factor in vast excess compared with the normal endogenous levels. Such studies are particularly valuable in studying the roles of transcription factors that are overexpressed and/or hyperactivated in cancers (e.g., [3,4]). However, in many cases this approach might not be optimal, as cells may evolve to cope with the continuous high-level production of transcription factors that are usually present only at low levels and/or are active only for short periods. Furthermore, clonal selection and the characteristics of the protein expressed might alter the physiology of the cell line such that it responds to stimuli in a fashion dramatically different from that of the parental line. An alternative strategy is to use an inducible expression system. Several inducible expression systems now exist for use in mammalian cells ([5–7]; see below). A major advantage of these systems is that experiments are carried out in a single cell line, thereby circumventing potential problems arising from clonal selection and the resulting interclonal heterogeneity.

1046-2023/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 6 - 2 0 2 3 ( 0 2 ) 0 0 0 3 1 - 2

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A second problem facing researchers studying transcription factor function is that transcription factors are not always in their active state in the absence of appropriate cofactors or posttranslational modifications. Thus, it is often desirable to couple an inducible system with a ‘‘dominant-negative’’ or a ‘‘constitutively active’’ version of a transcription factor. These can be produced by fusion to potent repressor domains (e.g., engrailed [8]; see below) or activation domains (e.g., VP16; see [9]). Here we review the use of inducible systems and transcription factor fusions to study transcription factor function. In particular we focus on the ecdysone-inducible system and engrailed fusion proteins. We demonstrate the applicability of this coupled system to the study of the mammalian transcription factor Elk-1.

2. Ecdysone-inducible expression Although various inducible expression systems exist, not all exhibit the tight regulation or rapid kinetics required to study transcription factor function (reviewed in [7]). Two of the systems that are able to achieve this are those regulated by tetracycline and ecdysone. Tetracycline-inducible systems are useful in a variety of situations (e.g., [7,10–14]) and improvements have been made to rectify the problems of leaky expression and inducibility (reviewed in [6]). However, the ecdysoneinducible expression system consistently exhibits lower levels of leaky expression and, therefore, for potentially toxic proteins, represents the method of choice for setting up an inducible cell line. The ecdysone system represents a precise and effective way of controlling the expression of a gene of interest [5]. This system makes use of a response pathway used by Drosophila melanogaster to trigger metamorphosis. A heterodimer of the ecdysone receptor and the product of the ultraspiricle gene (USP) mediates this morphological change. When ecdysone is present, this heterodimer binds to ecdysone response elements and drives transcription. As neither ecdysone receptors nor response elements are present in mammalian cells, the introduction of these components into a mammalian cell line confers ecdysone responsiveness to the cells and leads to the transcription of any genes under the control of a suitable ecdysone-responsive promoter. The advantages of this system include the fact that there is little or no background activation of the gene of interest, that a high level of inducible expression is possible, and that ecdysone and ecdysone analogs are inert in mammalian cells. To increase the level of gene induction attainable with this system, several modifications have been made both to the receptor heterodimer and to the promoter response element (Fig. 1) [5]. The use of RXR rather than USP conferred added inducibility, as did the modification of EcR by N-terminally fusing it to the VP16 acti-

Fig. 1. Schematic representation of the ecdysone-inducible mammalian expression system. The integrated expression cassette for the VgEcR and RXR nuclear hormone receptors is shown at the top. These are constitutively expressed from the CMV (PCMV) and RSV (PRSV) promoters, respectively. The integrated pIND(SP1) cassette is shown at the bottom. Target genes encoding the protein under study are expressed from a promoter containing multimerized ecdysone response elements (E/GRE) that is activated by the VgEcR/RXR heterodimer on ponasterone A treatment. The integrated expression cassettes are linked to constitutively expressed genes conferring zeocin (VgEcR/ RXR) and neomycin [pIND(SP1)] resistance (not shown).

vation domain. Due to the slight possibility of there being endogenous mammalian proteins that can bind to ecdysone elements a novel ecdysone-specific response element (E/GRE) is used in this system. This response element can be bound by EcR/RXR heterodimers in which the EcR receptor has three single-residue mutations in its coding sequence leading to an altered DNA binding specificity (the resulting heterodimer is termed VgEcR/RXR and the plasmid encoding it, pVgRXR). A suitable cell line is transfected with pVgRXR (which contains a gene conferring zeocin resistance) and the production of VgEcR/RXR is verified. The gene of interest is then cloned into the pIND(SP1) vector using the multiple cloning site which is located downstream of five E/GREs. The resulting vector is transfected into the pVgRXR cell line and selection of stable transfectants is possible using the neomycin resistance gene within the pIND(SP1) vector. Surviving clones are treated with an ecdysone analog, ponasterone A, and assayed for the inducible expression of the gene of interest. A modification of the system uses coexpression of green fluorescent protein (GFP) as an efficient and simple way of identifying inducible clones. Coexpression is achieved either by using a polycistronic vector [15,16] or by using a single vector containing two separate sets of ecdysone response elements that drive the expression of GFP and the gene of interest [17].

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the dissection of the specific components of the apoptotic machinery activated by the protein of interest before cell death ensues [18–21]. The proapoptotic transcription factor c-Myc represents an important transcription factor that has been studied using the ecdysone system by inducibly expressing myc antisense RNA [22]. A similar study in which antisense cyclin D1 was inducibly expressed allowed the characterization of the function of cyclin D1 at various stages of the cell cycle [23]. In a variation of the ecdysone system, cells transiently transfected with pVgRXR and pIND vectors were used [24]. In the latter experiments the generation of a stable cell line was unnecessary as a BID–GFP fusion protein was expressed from the pIND vector. BID is a proapoptotic member of the Bcl2 family and, by following the phenotypic changes of the green fluorescing cells, it was possible to follow the sequence of apoptotic events triggered by the presence of BID. A further useful property of the ecdysone-inducible system is that it has proved possible to modulate the level of expression from the pIND vector by varying the

Despite the relative youth of the ecdysone-inducible expression system, it has already been used to study a variety of biological processes (Table 1). In the majority of cases inducible expression represents a simple and effective way of studying the effects of the absence or presence of a gene of interest in a specific cellular environment. Proteins such as transcription factors, membrane proteins, kinases, receptors, and cell cycle regulators have all been placed under the control of ecdysone response elements in a wide variety of cell lines (see Table 1 for examples). Inducible expression appears to be especially useful in the controlled expression of proteins that either are required for cell survival or actively lead to cell death. It is impossible to produce useful stable cell lines overexpressing proapoptotic proteins. Any surviving cells will have modified the integrated vector to ensure that the proapoptotic gene is not expressed, or the cells themselves will have mutated to survive the apoptotic signals and will therefore not represent a good physiological model. The ecdysone-inducible system therefore allows

Table 1 Applications of the ecdysone-inducible mammalian expression systema Biological Process

Protein/gene

Cell line

Application

Reference

Apoptosis

53BP2

EcR-293

[25]

*c-myc

M14

BID

MCF7/Fas

Bax FACL4 and COX-2

Jurkat EcR-293

Role of p53 interacting protein 53BP2 in determining cell survival Expression of antisense c-myc leads to apoptosis in melanomas Effect of transiently transfected BID-GFP fusions on apoptosis Mechanism of Bax-induced apoptosis Role of fatty acid-CoA ligase 4 (FACL4), cyclooxygenase 2 (COX-2) in protecting against apoptosis

Signal transduction

Calnuc LMP1

EcR-CHO EcR-293

Role of Calnuc in establishing the Golgi Ca2þ pool Epstein–Barr virus latent membrane protein 1 (LMP1) activation of the p38 mitogen-activated protein kinase pathway

[68,69] [70]

Cell cycle regulation

Cyclin D1

RKO1

[23]

*p53 *KLF4

H1299 RKO

p21Waf1 and p27Kip1

RKO

Used antisense cyclin D1 to analyze its role in phosphorylating cell cycle proteins Role of p53 in G2 arrest Regulation of proliferation rate and cell cycle progression by Kruppel-like factor 4 (KLF4) Effects of p21Waf1 and p27Kip1 on cell cycle distribution and sensitivity to paclitaxel-mediated cytotoxicity

*CREB

3T3-L1

p204

C2C12

*SREBP

M19

Differentiation

a

Constitutively active and dominant-negative CREB constructs used to probe their role in induction of adipocyte differentiation Role of p204 in myoblast differentiation and interferon signaling Titrated muristerone levels to demonstrate specific effects of different sterol regulatory element-binding proteins (SREBPs)

[22] [24] [21] [67]

[71] [16] [72]

[73]

[74] [75]

M14, human melanoma cell line; MCF7/Fas, MCF breast carcinoma cells stably expressing Fas; RKO1, human colon carcinoma cell line; H1299, non-small-cell lung cancer cell line, null for p53 expression; M19, mutant CHO cells with a deletion in the sterol regulatory element-binding proteins (SREBPs). Transcription factors are indicated by asterisks.

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concentration of the inducing agent and the duration of induction. This is particularly important for studying proteins that act in a dose-dependent manner. An example of this is in a study of the p53 interacting protein 53BP2 [25], in which the level of induction of this protein could be varied between 6- and 140-fold by varying the inducer over a 10-fold range (0.5–5 lM). In this circumstance a 6-fold induction of protein is physiologically relevant as it is similar to that seen when the cells are exposed to UV irradiation. As a result of previous experiments using transient transfections it had been presumed that 53BP2 caused apoptotic cell death. However, when using the inducible cell line it became apparent that at physiological levels 53BP2 is unable to trigger apoptosis, but rather is able to sensitize cells to apoptotic stimuli.

3. The engrailed repressor domain A variety of different methods can be used to specifically block gene expression. Antisense RNA or DNA and ribozymes can be used to disrupt mRNA expression [26–29]. Alternatively, dominant-negative effects can be achieved by employing techniques such as the expression of mutant heterodimerization partners of receptor proteins [30] and the use of dominant-negative repressors of transcription. One of the best characterized mechanisms for producing dominant-negative transcription factors is to delete the activation domain so that the protein is transcriptionally inert, but the DNA binding ability of the protein still enables it to compete with endogenous protein for DNA binding sites. However, this approach has problems when applied to the study of members of large transcription factor families due to their overlapping DNA binding specificities. DNA binding is often regulated by inter- and intramolecular protein–protein interactions which may be lost in truncated proteins [31]. A better approach is therefore to use fusions between the intact transcription factor and a powerful repression domain such as that found in the Drosophila engrailed protein. Engrailed is a Drosophila homeodomain protein required for proper segmentation and for maintenance of posterior compartment identity. It is known to efficiently repress the activity of a variety of transcriptional activators and the repressor activity of Engrailed (En) lies in amino acids 2–298 which are distant from the DNA binding homeodomain [8]. More detailed analysis of the functional domains of En has defined two regions within this N-terminal portion of the protein that are involved in active repression. The first is an alanine-rich region spanning residues 228–282 which appears to be responsible for the majority of the activity of En in vitro [32,33]. A second domain, eh1 (amino acids 172–186), appears to be of greater importance in vivo [34].

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The eh1 domain acts via recruitment of Groucho (Gro) [33,35]. Gro lacks DNA binding activity and acts as a corepressor for several transcription factors including Dorsal, Engrailed, and Runt domain proteins (reviewed in [36]). Gro, in turn, recruits the histone deacetylase Rpd3 [37] and a vertebrate homolog of Gro interacts with the human homologue of Rpd3 (HDAC1) [38], thus leading to transcriptional repression through histone deacetylation and modification of chromatin structure. The N-terminal region of En (residues 2–298) was initially shown to function as a portable repressor domain by fusing it to the DNA binding domain of the glucocorticoid receptor. This fusion protein mediated active repression of transcription [8]. The En repressor domain has since been fused to several other transcription factors to produce dominant-negative regulators of transcription. For example, En fusions have been used extensively to analyze c-Myb, an important regulator of cell growth and differentiation in hematopoiesis [39]. c-Myb null mice are known to die at Day 15 in utero due to the failure of hepatic erythropoiesis [40]. Therefore a dominant-negative version of c-Myb represented a useful alternative. The creation of a cell line expressing a conditionally active version of Myb– En facilitated the identification and analysis of Myb activity responsible for the effects seen in Myb-deficient mice. The fusion protein efficiently and specifically inhibited transcription from a reporter encoding a strong Myb binding site and was then used to demonstrate that Myb–En leads to rapid downregulation of bcl-2 and subsequent triggering of apoptosis [41]. A similar conditionally active c-Myb–En fusion protein has been used in a cytotoxic T-cell line [42]. In both studies the c-Myb–En protein was rendered conditionally active by fusing it to a modified estrogen receptor hormone binding domain [43]. The resulting protein represses a c-Myb-responsive reporter only in the presence of a synthetic steroid, 4-hydroxytamoxifen (4-OHT) [41,42]. The c-Myb–En fusion is toxic to cells and therefore the ability to conditionally activate the fusion protein was necessary to study the transcriptional and cell cycle effects of c-Myb–En induction. The Myb–En fusion protein has been used by other workers to further study the role of Myb in fibroblasts [44]. It has also been used in conjunction with a c-Myc– En fusion to investigate the function of both Myb and cMyc in vascular smooth muscle cell proliferation [45]. These studies demonstrate the effectiveness and versatility of En fusion proteins.

4. Methods The inducible expression of a dominant-negative form of a transcription factor can be used both to

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determine the complement of genes regulated by that transcription factor in response to a specific stimulus and to assess the global effects of blocking transcription factor action. Here we wished to study the targets and cellular processes regulated by the transcription factor Elk-1. Elk-1 plays a pivotal role in the translocation of mitogenic and stress signals from the external and internal cellular environment into transcriptional changes in the nucleus [1,2]. Many signaling molecules, such as various growth factors, relay their signals via the mitogen-activated protein kinase (MAPK) cascades. These cascades lead to the phosphorylation of Elk-1 (and other members of the ternary complex factor (TCF) subfamily of ETS-domain transcription factors), which then activates the transcription of target genes. Although much work has been previously carried out on the activation of the c-fos protooncogene by Elk-1, the global effects of Elk-1 activation have not previously been studied. One reason for this is that disruption or overactivation of such an important signaling molecule might have diverse repercussions that could be near impossible to dissect. As an activator of immediate-early genes, Elk-1 executes its transcriptional effects with rapid and transient kinetics. Continual overexpression of Elk-1 would therefore not be effective in elucidating its role in the response to external signals. Furthermore, the long-term effects of Elk-1 overexpression in the absence of signaling would be very different from those caused by transient activation of endogenous levels of this protein. This article outlines the approach taken in our laboratory to study the role of Elk-1 in the cellular response

to mitogenic stimuli. The use of a constitutively active transcription factor would clarify the effects of Elk-1 activation, but would not be of use in determining the role of Elk-1 in response to diverse cellular signals. We therefore produced a stable cell line that can inducibly express a dominant-negative version of Elk-1. This system is particularly useful for studying the cellular responses to Elk-1 activation following mitogenic and stress stimuli, which might lead to very different endpoints. The expression of dominant-negative Elk-1 will lead to the inhibition of Elk-1-responsive genes and hence downstream cellular responses (Fig. 2A). Therefore if uninduced cells and those induced to express the dominant-negative transcription factor are both stimulated with a mitogen, comparison of the gene expression profiles of the two cell populations should reveal which genes are normally regulated by endogenous Elk-1. The inducible expression of a dominant-negative transcription therefore represents a sensitive tool for the analysis of a range of cellular responses involving the activation of Elk-1. We used the ecdysone-inducible system in which Elk1 fusions were made with the engrailed repression domain (Elk–En) and were used to block signaling through Elk-1 to subsequent downstream events (Fig. 2A). The methods used to set up the inducible expression system and assess alterations in the response of cells to stimuli at the level of gene expression are outlined below. The pIND/ecdysone-inducible expression system is illustrated in Fig. 1. EcR293 cells (HEK 293 cells that have been stably transfected with the vector pVgRXR) were supplied by Invitrogen. The VgRXR vector results

Fig. 2. Experimental approach to identify novel targets of the transcription factor Elk-1. (A) Epidermal growth factor (EGF) is known to stimulate Elk-1-mediated transcription through activation of the ERK mitogen-activated protein kinase (MAPK) cascade. The induction of Elk–En will result in blocking of this pathway in the nucleus at the level of Elk-1. (B) Comparison of gene expression profiles by micro-macroarray analysis in EcR293(Elk–En) cells that have been incubated with or without ponasterone A and then treated with EGF. mRNAs that exhibit different expression profiles in these populations represent potential target genes.

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in the constitutive expression of the modified EcR/RXR heterodimer. DNA encoding Elk-1 fused to En residues 2–298 with a C-terminal FLAG epitope tag (Elk–En) was cloned into the pIND(SP1) vector using the Hin dIII/XbaI sites (pAS1406). There are now a variety of alternative vectors supplied by Invitrogen that are compatible with the EcR cell lines. This study used the pIND(SP1) vector, which has three Sp1 binding sites for enhanced expression of the gene of interest and the neomycin resistance gene to aid selection of stable integrants. Plasmids are also available that impart hygromycin rather than neomycin resistance, or contain DNA encoding the V5 epitope and polyhistidine tag which are fused to the C-terminal end of the protein to be transcribed and allow easy detection of gene expression. Further modifications include the addition of GFP to pIND to facilitate testing the inducible expression capabilities of cell lines that have been previously transfected with VgRXR plasmid (see above).

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Fig. 3. Dose-dependent Elk–En induction by ponasterone A. EcR293(Elk–En) cells were grown in DMEM, 10% FBS, 600 lg/ml G418, and 400 lg/ml zeocin for 24 h. Ponasterone A was added to the media for a further 24 h at the concentrations shown. Elk–En expression was analyzed by Western blotting using a mouse anti-FLAG M2 antibody (Sigma) followed by detection by ECL (Pierce).

By varying the concentration of ponasterone A different levels of Elk–En expression were induced (Fig. 3). All clones exhibiting inducible expression of Elk–En were further analyzed by reporter gene analysis as detailed below. 4.2. Reporter gene analysis

4.1. Construction of the inducible cell line 1. EcR293 cells were plated out onto 10-cm dishes at a density of 2  106 cells/dish. Cells were cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Life Technologies)/400 lg/ml zeocin at 37 °C in a humidified 5% CO2 atmosphere. 2. Twenty-four hours later cells were washed with 3 ml DMEM supplemented with 10% FBS and transfected with 10 lg linearized pIND(SP1) vector encoding the Elk–En fusion protein (pAS1406). Forty microliters of Superfect transfection reagent (Qiagen) was used in a mixture with 300 ll Optimem (Life Technologies). Cells were incubated at 37 °C for 3 h, washed with DMEM/10% FBS, and grown in 10 ml DMEM/10% FBS containing 400 lg/ml zeocin. Vector DNA was linearized before transfection using SspI restriction enzyme to promote integration into the chromosomal DNA. 3. Seventy-two hours later the cells were trypsinised and plated out again at a density of 5  105 cells/ dish in DMEM containing 10% FBS, 600 lg/ml G418, and 400 lg/ml zeocin. Approximately 10 days later 30 individual cell foci were isolated using cloning cylinders (Sigma) and transferred to six-well plates. 4. All selected clones were expanded and tested for inducible expression using Western blotting to detect FLAG-tagged protein. Five of the thirty clones expanded were found to exhibit inducible expression of Elk–En [EcR293(Elk–En)]. Positive clones were defined as those in which Elk–En expression was below detection levels in untreated cells but in which FLAG-tagged protein could be seen after the cells were incubated in 5 lM ponasterone A for 24 h.

Reporter gene analysis represents a sensitive tool to study the activity of transcription factors. This approach can be used to verify that the En-fusion proteins function as transcriptional repressors in the predicted manner. Cells are transfected with a vector encoding a protein that is easily detectable or has an enzymatic activity that can be easily monitored. Commonly used reporter genes include those encoding b-galactosidase [46], firefly luciferase [47], alkaline phosphatase [48,49], chloramphenicol acetyltransferase [50], and green fluorescent protein [51]. The gene is placed under the control of transcription factor response elements such that when the transcription factor of interest is expressed and is active, the reporter gene is transcribed. The reporter gene used in this system is the firefly luciferase gene, which emits light when a suitable substrate is added. The light output can then be quantified using a luminometer and the intensity of light emitted is proportional to the level of luciferase expression. Here we use a serum response element (SRE)-driven luciferase gene that is regulated by an Elk-1–SRF complex in response to MAPK activation via mitogenic and stress stimuli (Fig. 4A) (reviewed in 1, 2). 1. EcR293(Elk–En) cells were plated out onto tissue culture six-well plates (Costar) where each well has a diameter of 3 cm. The cells were plated at a density of 4  105 cells per well in 3 ml DMEM, 10% FBS, 600 lg/ml G418, and 400 lg/ml zeocin and incubated at 37 °C in a humidified atmosphere with 5% CO2 . 2. The cells were then transfected using Superfect (Qiagen) with two reporter plasmids: the first contains the firefly luciferase gene under the control of two serum response elements (pSRE-luc, 250 ng); the second drives constitutive expression of b-galactosidase for

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Fig. 4. Reporter gene analysis of transcriptional repression by Elk–En. (A) Schematic representation of SRE-driven reporter gene regulated by Elk-1. The firefly luciferase gene is under the control of two tandem serum response elements (SREs). Elk-1-VP16 acts as a constitutively active form of Elk-1. (B) EcR293(Elk–En) cells were transfected with 250 ng of a SRE-driven luciferase reporter (SRE-Luc) (lanes 1–3) plus 500 ng of a plasmid encoding Elk–VP16 (lanes 2 and 3). In lane 3 the cells were also treated with 5 lM ponasterone A to induce Elk–En expression. Cells were lysed 24 h later and the cell lysates assayed for luciferase activity. Transfection efficiency was monitored by cotransfecting all cells with a b-galactosidase expression vector. The luciferase activities relative to SRE-Luc vector alone (means  SD, n ¼ 3) are presented. Elk–VP16 causes activation of the SRE-Luc reporter which is reduced back to near-basal levels on induction of Elk–En expression with ponasterone A.

normalization of transfection efficiency (pCH110, 500 ng). Some cells were also transfected with 500 ng of a vector encoding a constitutively active form of Elk-1 (full-length Elk-1 fused to the VP16 activation domain [52]) to activate the luciferase reporter construct. 3. After a 2-h incubation with plasmid DNAs and transfection reagent, the cells were washed and left in DMEM containing 0.5% FBS. At this point ponasterone A was added to the medium to a final concentration of 5 lM, which leads to maximal expression of Elk–En in this system (Fig. 3). Twenty-four hours later the cells were lysed and assayed for luciferase and b-galactosidase activity using the dual light reporter gene assay system (PE Applied Biosystems). Light emissions were measured for 10 s with a Turner TD-20/20 luminometer.

All five stable cell lines were tested in reporter assays. The cell line that was chosen for further studies exhibited a high activation potential of the reporter by Elk–VP16 (25-fold), (Fig. 4B), which coincided with a very low basal level of Elk–En expression. In addition it was also possible to efficiently inhibit Elk–VP16-induced reporter activity by treating the cells with ponasterone A to induce Elk–En (Fig. 4B). As exogenous Elk–VP16 is highly efficient at activating SREs it is presumed that the level of Elk–En activity exhibited by the cell line would also be sufficient to repress any activation of SREs by endogenous ternary complex factors. The results from reporter assays demonstrate not only that Elk–En expression is induced by ponasterone A treatment, but also that this system can be used to mediate the repression of target genes. Reporter assays using alternative response elements not bound by TCFs demonstrate that Elk–En specifically represses SREs and does not cause global repression of transcription (data not shown). To assess the ability of Elk–En to repress endogenous immediate-early (IE) gene expression and to verify that the stable cell line exhibits normal kinetics of IE gene induction it is possible to use reverse transcriptionpolymerase chain reaction (RT-PCR), Northern blotting, or RNase protection assays. c-fos is an IE gene known to be activated by Elk-1 as well as by many other transcription factors (reviewed in [53]), and the kinetics of c-fos induction in response to mitogenic stimuli have been characterized in many systems (for an example see [54]). RT-PCR analysis of c-fos expression would therefore provide an indication of the ability of Elk–En to repress endogenous gene expression. However, controls should also be run in parallel such as for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), whose transcription should not be altered by the activities of Elk–En. 4.3. cDNA arrays Many techniques have been developed to identify genes differentially expressed between cell populations. These include differential display analysis, subtractive RT-PCR, serial analysis of gene expression (SAGE), representational difference analysis (RDA), and chromatin immunoselection. However, the method of choice is now the use of macro- or microarrays. A variety of arrays are available that are useful in a wide range of situations where monitoring the expression levels of hundreds or even thousands of genes is desirable (for reviews see [55–62]). For the purposes of this study Clontech nylon membrane-based arrays represent an ideal tool. These arrays represent a compromise between the desire to screen the expression of large numbers of genes and

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the desire not to resort to unknown sequences. All the cDNA fragments spotted onto these arrays represent known genes, and are available as general human, mouse, and rat arrays, as well as those containing genes associated with specific areas of biological research such as cancer, apoptosis, cell cycle, neurobiology, and toxicology. Here we selected a cancerbased array due to the central position of Elk-1 in mitogenic signaling, a process that, when aberrant, is associated with the development of the majority of cancers. The Clontech Cancer 1.2 array contains 1176 cDNA fragments from genes thought to be associated with cancer. The basis of a protocol to identify differentially regulated genes as a result of Elk–En expression in the presence of EGF is represented in Fig. 2B and outlined below. 1. Two T75 flasks of EcR293(Elk–En) cells are seeded with 2  106 cells/flask and grown for 48 h in DMEM with 10% FBS, 600 lg/ml G418, and 400 lg/ml zeocin. The cells are then washed twice with serum-containing medium and serum starved for a further 48 h in DMEM with 0.5% FBS. 2. Six hours prior to the end of this period, ponasterone A is added to one flask of cells to a final concentration of 5 lM. This allows Elk–En expression in these cells but helps avoid any cell cycle or cell proliferation effects of the protein that may occur with prolonged expression. 3. After 6 h both flasks of cells are stimulated with 50 nM epidermal growth factor (EGF, Sigma) for 20 min. Both flasks are then harvested by washing the cells with PBS, adding trypsin–EDTA (Life Technologies), and centrifuging to collect the cell pellet. If not being used immediately the cells are snap-frozen in liquid nitrogen and stored at )80 °C. 4. Total RNA is isolated from the cell pellets (approximately 1  107 cells) and enriched for mRNA using the Clontech Atlas Pure total RNA labeling system according to the manufacturer’s instructions. This is followed by cDNA synthesis with gene-specific primers and incorporation of [a-32 P]dCTP (3000 Ci/ mmol). These primers restrict cDNA synthesis to those genes represented on the array, lead to a stronger hybridization signal from the final experiment, and also limit nonspecific hybridization which would cause a high background. 5. The radiolabeled cDNA probes are then hybridized to separate arrays followed by phosphorimaging and statistical normalization of the results to determine the expression levels of the genes spotted on the array. The levels of expression on each array are then compared in silico to identify differentially expressed genes in each cell population. There are several approaches to normalizing data from array experiments. The two simplest methods are to normalize the signal of each spot to the sum of the

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signals from the entire array and to normalize each spot to the signal from the housekeeping genes represented on the array. Both approaches should, if the hybridization has proceeded correctly, give the same result: the majority of genes should give a 1:1 ratio of expression between the two arrays. All data are presented on a logarithmic scale ðlog2 xÞ as the variability in gene expression levels within the individual cDNA pools is of several orders of magnitude. It is generally accepted that a ratio greater than 2:1 between the two arrays signifies differential regulation of a gene. Plotting the normalized data from the two arrays on a graph permits visual identification of differentially regulated genes (Fig. 5). Most genes are clustered around the line representing a 1:1 ratio of expression. However, genes that are highly upregulated in cDNA pool 1 (C02d, C03k) and two downregulated genes (B13g, C10l) can be observed as outliers from this cluster. The genes differentially expressed in the two cell populations are either those that are normally upregulated by Elk-1 in response to EGF stimulation or those whose expression has been altered directly or indirectly by Elk–En, independently of EGF. As Elk–En is a transcriptional repressor it cannot directly activate genes; however, there may be some genes that, due to their downregulation by Elk–En, release other genes from their normal state of transcriptional repression. This would lead to the apparent upregulation of some genes by Elk–En. As an extension of this approach, the treatment of cells with activators of different pathways (e.g., the JNK and p38 MAPK cascades) instead of with EGF would permit the evaluation of the role of Elk-1 in response to alternative stimuli. It is possible that target genes identified by this approach might not be specific to Elk-1 but might be

Fig. 5. Graphic representation of cDNA array data. Pools 1 and 2 are from arrays probed with cDNAs from EcR293(Elk–En) cells that were left untreated or treated with ponasterone A, respectively. The normalized data from each array are plotted on separate axes to compare the expression data from the two arrays. The line representing a 1:1 expression ratio between the two samples is shown on the graph. Two upregulated (C03k, C02d) and two downregulated (B13g, C10l) genes in the ponasterone-treated cell line are circled.

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upregulated by virtue of containing ETS-binding sites and being normally regulated by different ETS-domain proteins. An elegant control for these experiments would therefore be to contruct a second stable cell line that inducibly expresses a mutant form of dominant negative Elk-1. In the case of Elk-1, protein–protein interactions with SRF play a major role in its recruitment to DNA (reviewed in [1,2]). Single-amino-acid mutations in the region of Elk-1 involved in these protein–protein interactions are sufficient to disrupt this interaction. These mutants exhibit a diminished ability to bind to the c-fos SRE, while behaving like the wildtype protein in their capacity to bind to high-affinity ETS binding sites [63]. A cell line that inducibly expresses such a mutant form of Elk–En would therefore be useful to rule out nonspecific repression of genes through binding via the ETS-domain alone. The mutant could also be used to differentiate between genes regulated by Elk-1 as part of a complex and those potentially regulated via autonomous DNA binding by the transcription factor. An alternative control could involve the creation of mutants defective in DNA binding to rule out indirect effects caused by the titration of essential coregulatory partner proteins. The inclusion of cell lines expressing appropriate mutant proteins would greatly increase the confidence in the validity of any target genes identified using this approach. A second approach for validating the authenticity of target genes as direct targets is to use chromatin immunoprecipitation assays with transcription factor-specific antibodies to demonstrate promoter occupancy. Alternatively, microarray studies can be carried out in the presence of protein synthesis inhibitors to differentiate between immediate and secondary effects of the particular transcription factor. However, such inhibitors could be used only after the inducible transcription factor has been produced, limiting the use of this approach to a subset of applications.

5. Limitations of the system The coupling of the ecdysone-inducible system to the expression of transcription factor-engrailed fusion proteins represents a powerful tool to study the regulatory profiles for specific transcription factors. However, the system has several limitations. One major limitation of the EcR system is the restricted number of cell types that can be obtained commercially pretransfected with VgRXR: only EcRCV1, EcR-293, and EcR-CHO are presently available. Therefore to study specific cell types a stable cell line expressing the VgEcR/RXR heterodimers must be produced before transfecting the cells with the pIND vector containing the gene of interest. This process is both laborious and time-consuming. The development of a

single vector containing both halves of the expression system would be a significant advancement, although technical difficulties such as the size of the resulting vector might preclude such improvements. The ecdysone system can lead to vast overexpression of the gene of interest unless the concentration of inducer is carefully titrated to give expression levels within the physiological range of that protein. This degree of control over the level of protein expression is often not necessary when dealing with a transcriptional repressor rather than a transcriptional activator; the basal level of expression of many genes, especially IE genes, is essentially zero. However, it is plausible that massive overexpression of any protein may have cytotoxic consequences. It is therefore recommended that a control cell line is used that expresses a mutant form of the engrailed fusion protein to rule out nonspecific cytotoxicity. The expression of the gene of interest can be detected in some cases after 30 min but is often detectable after 6 h posttreatment with muristerone or ponasterone A. Although this is sufficiently rapid induction for many systems, in the analysis of IE gene induction or any enzymatic or transcriptional process with rapid and transient kinetics, this is inadequate. Unless the timing of expression can be narrowed down to minutes rather than hours, many of the direct effects of expression of the gene will already have declined by the time the cells are analyzed. In the case of an IE gene such as c-fos, transcription is initiated within 15 min of stimulation and upregulation of gene expression lasts less than 1 h. A system where protein gradually accumulates to detectable levels in a matter of hours is not flexible enough either to determine the specific effects of a protein that could regulate a gene such as c-fos or to distinguish between direct and indirect effects of transcription factor upregulation. In the case of cytotoxic proteins where inducible expression is a necessity, the fusion of the protein of interest with the hormone binding domain of a steroid receptor would add an additional level of control. Leaky expression with the ecdysone-inducible system does appear to be minimal, but any expression of a potentially cytotoxic gene may be deleterious to the cells. Many authors have reported the use of nuclear receptor hormone binding domain fusions to control the activity of proteins such as transcription factors and protein kinases (reviewed in [64]). The addition of a hormone or synthetic steroid to the cell medium leads to activation of the fusion protein. The kinetics of activation appear to be in the region of minutes rather than hours [65]. By combining the ecdysone-inducible system with hormone binding domain fusions, it should be possible to create a more stringent dual- controlled system. There are, however, drawbacks to this system, as the presence of the additional polypeptide fused to the protein may

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cause steric hindrance of interactions with cofactors, causing altered function, and the addition of a hormone to the cell medium may cause pleiotropic effects. The use of the En repressor domain is highly advantageous when active repression is required rather than inhibition through competition for binding sites and/or cofactors. However, the overexpression of these fusion proteins might cause problems with specificity in the case of large transcription factor families where DNA binding specificities are similar. For example, Elk1 is highly homologous to the two other members of the TCF subfamily of ETS-domain transcription factors, SAP-1a and SAP-2/Net/ERP. Although these proteins do differ in terms of their DNA binding specificities, they can all be recruited to the c-fos SRE by SRF. Hence it is probable that Elk–En is able to repress Elk-1-, SAP1a-, and SAP-2-mediated transcription at SREs. Nevertheless, the realization that Pax-5 can recruit Elk-1 and SAP-2, but not SAP-1, to the mb-1 promoter [66] does suggest that these three proteins may have distinct roles at different promoters which would be revealed by the comparison of Elk–En and similar SAP-1-/SAP-2fusion proteins.

6. Conclusions Despite the problems highlighted above, the inducible expression of a dominant-negative transcription factor represents a useful tool in the analysis of transcription factor function. We demonstrate that Elk–En fusion proteins can be inducibly expressed by the ecdysone system. These fusion proteins act as specific transcriptional repressors and their presence results in differential expression of endogenous genes. Indeed, the En repressor domain is a powerful and specific repressor of transcription, which could theoretically be fused to a wide range of transcription factors whose functions have not yet been fully elucidated. As many transcription factors are tightly regulated in mammalian cells and regulated via a range of parallel signal transduction pathways it is possible that the use of dominant-negative transcription factors will be a more versatile tool than constitutively active proteins. The additional level of control afforded by inducible expression systems also aids in the analysis of complex processes such as apoptosis, proliferation, senescence, and differentiation. Many of the consequences of transcription factor regulation are transient and therefore the production of cells that exhibit constitutive expression of these proteins are of little use in elucidating the temporal role of transcription factor activation. Overall, the inducible regulation of a dominant-negative transcription factor represents a powerful and versatile tool, which, when coupled to new techniques such as cDNA microand macroarrays, should allow the unraveling of the

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complex signaling pathways that exist in eukaryotic cells.

Acknowledgments We thank Paul Shore for comments on the manuscript. E.R.V. is supported by a William Ross Cancer Research Campaign (CRC) studentship. A.D.S. is a Research Fellow of the Lister Institute of Preventive Medicine.

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