Molecular Cell, Vol.

6, 723728, September, 2000, Copyright 2000 by Cell Press

Transcriptional Control: Rheostat Converted to On/Off Switch

Fabio M. V. Rossi,* Andrew M. Kringstein,* Albert Spicher,* Oivin M. Guicherit, and Helen M. Blau* * Department of Molecular Pharmacology Stanford University School of Medicine Stanford, California 94305 Ontogeny Inc. 45 Moulton Street Cambridge, Massachusetts 02138 in intact Drosophila tissues, a definitive test of this hypothesis has not been possible. The need to address the important question of whether transcriptional control suffices to induce on/off molecular switches is underscored by the discovery of a growing number of repressors and activators that bind the same sites in a mutually exclusive manner. There are ample examples that these networks are involved not only in patterning during development but also in the regulation of cellular functions in the adult (Ayer et al., 1993, 1996; Lehming et al., 1994; Mangelsdorf et al., 1995; Dubnicoff et al., 1997; Bourguignon et al., 1998; Dyson, 1998; Lemercier et al., 1998; Ohlmeyer and Kalderon, 1998; Roose et al., 1998; Xu et al., 1998; Conlan et al., 1999; Lu et al., 1999). For instance, growth and differentiation are binary decisions and their misregulation can have significant consequences such as neoplasia. To test the hypothesis that an alteration in the balance of positive and negative transcription factors that act on a given promoter is sufficient to generate an all-ornone response at the transcriptional level, we designed a system (Figure 1) in which the ratio of activators and repressors that bind to the same promoter/reporter constructs can be modulated by a single extracellular signal (inducer). Because the inducer becomes a component of the transcriptional activator or repressor, intermediate regulatory steps are circumvented. Moreover, since the regulatory elements are prokaryotic, pleiotropic effects on host regulatory elements are precluded in the eukaryotic cells examined. By analyzing the activity of individual promoter/reporter cassettes, we show that although synergistic, activators or repressors alone yield a graded (rheostat) response to a particular inducer, yet their combined effects result in a threshold (on/off) transcriptional switch. Results Essential to our studies was a means of analyzing expression levels of a single transcription unit in response to differences in dosage of an inducer (tetracycline or doxycycline). For this purpose, one copy of an inducible promoter/reporter cassette was stably integrated into individual cells. This was achieved by using low titer retroviral supernatants that infected the target cell population at a frequency of 15%, which according to the Poisson distribution results in only one copy of a randomly integrated retrovirus in 85% of infected cells. The activity of the promoter/GFP-reporter cassette was examined in the presence of either a tetR-based activator or repressor or both (Gossen and Bujard, 1992; Gossen et al., 1995; Rossi et al., 1998) (Figure 1). The transcription factors not only had opposite functions but were also engineered to differ in their allosteric response to the inducer doxycycline (dox) (Figure 1B). Thus, with increasing doses of dox, relatively less repressor and more activator bound the promoter. The inducer used here, dox, enters eukaryotic cells by passive diffusion and becomes an integral component of the transactivator or transrepressor. As a result, the concentration of inducer directly determines the level

Summary Individual cells translate concentration gradients of extracellular factors into all-or-none threshold responses leading to discrete patterns of gene expression. Signaling cascades account for some but not all such threshold responses, suggesting the existence of additional mechanisms. Here we show that all-ornone responses can be generated at a transcriptional level. A graded rheostat mechanism obtained when either transactivators or transrepressors are present is converted to an on/off switch when these factors compete for the same DNA regulatory element. Hill coefficients of doseresponse curves confirm that the synergistic responses generated by each factor alone are additive, obviating the need for feedback loops. We postulate that regulatory networks of competing transcription factors prevalent in cells and organisms are crucial for establishing true molecular on/off switches. Introduction Here we test whether an interplay of transcription factors can convert a graded to a threshold response. Interpretation of previous studies of on/off transcriptional responses in cultured cells has been complicated by intermediate steps including ligand binding to receptors and subsequent signal transduction cascades (Emilie et al., 1989; Fiering et al., 1990; Ko et al., 1990; Karttunen and Shastri, 1991). However, in Drosophila, concentration gradients of transcription factors including long-range and short-range repressors and activators have been described (Gray and Levine, 1996), and at points of intersection of these gradients, precise boundaries or stripes of gene expression are seen (Huang et al., 1997). Indeed, in some cases, genetic deletion of the repressor has led to the conversion of such sharp boundaries into gradients of gene expression (Arnosti et al., 1996; Jazwinska et al., 1999). These findings suggest that for the generation of threshold responses, the interaction of such transcriptional regulators with opposing functions is necessary, but they do not show that it is sufficient. Due to the complexity of DNA binding factors and the endogenous promoters that regulate gene expression
To whom correspondence should be addressed (e-mail: hblau@

cmgm.stanford.edu).

Molecular Cell 724

of transcription factor, eliminating the complexity often introduced by intermediate steps such as the binding of ligand to receptor and subsequent signal transduction. A distinction between graded and all-or-none transcriptional responses to the inducer (dox) was made possible by flow cytometry, which allowed an analysis of GFP distribution in large populations of individual cells (Figure 1C). Seven multimerized copies of the tetR binding site tetO were used to drive expression of the GFP reporter. First, this allowed a comparison with previous in vitro studies that had used multimerized transcription factor binding sites in the promoter (Carey et al., 1990; Lin et al., 1990). Moreover, one of the best-characterized elements involved in the establishment of sharp boundaries of gene expression in Drosophila, the eve stripe 2 enhancer, is composed of a cluster of 12 known factor binding sites, six for activators and six for repressors (Small et al., 1992). Furthermore, in this enhancer, some of the activator and repressor binding sites overlap, leading to mutually exclusive binding of the positive and negative regulators (Small et al., 1991; Stanojevic et al., 1991) as in the system presented here. Indeed, it may well be that the subset of promoters that contain a large number of binding sites for the same transcription factor are particularly well suited to on/off regulation. Three different cell populations were generated that each contained an inducible promoter/GFP-reporter cassette and expressed either a dox-regulated repressor (repressor only), a dox-regulated activator (activator only), or both (activator repressor). To assess the efficiency of gene repression, primary mouse myoblasts that harbored a single copy of an inducible promoter/ reporter cassette that expressed detectable basal levels of GFP from the tetO7-CMV promoter (HRSp-GFP) were analyzed (Rossi et al., 1998). A retrovirus encoding the repressor was then introduced and the effects on gene expression were analyzed. By contrast, to analyze gene activation by either the activator alone or in response to the combined effects of activators and repressors, a different construct was used that did not express detectable basal levels of the inducible promoter/reporter cassette (HRIgfp-hGH, Figure 1A). Critical to our experiments was that the repressor and activator bound to the tetO at the same concentration
Figure 1. Schematic of Vectors Used and Hypotheses Tested (A) Diagram of the retroviral vectors. Expression from the HRIgfphGH bicistronic reporter can be analyzed either in bulk assays using human growth hormone (hGH) or at the single-cell level by flow cytometry using green fluorescent protein (GFP). wt LTR, Moloney leukemia virus-derived LTR; IRES, internal ribosome entry site; hGH, human growth hormone; SIN LTR, self-inactivating LTR; tetO7-CMV, seven copies of the tetR binding site fused to a minimal CMV promoter. (B) Design of the system for inducing transcription. Specificity is conferred by the inducer doxycycline (dox) that acts in conjunction with prokaryotic control elements to drive transcription of a eukaryotic gene. Top panel: in the absence of dox only the repressor (tTRg) binds the tetO7-CMV promoter, resulting in maximal repression. Under these conditions, no GFP can be detected in the cells by flow cytometry. Bottom panel: in the presence of high levels of dox, only the activator (rtTAb) binds the promoter and maximal expression is observed. Dox binds to the bacterial tet repressor protein (tetR), inducing an allosteric change that alters its affinity of binding to a specific promoter element, the tet operator (tetO). The activator rtTAb (Gossen et al., 1995), a chimera of a mutant tetR and the viral activator VP16, requires dox to bind tetO, whereas dox prevents the binding of the repressor tTRg, a chimera of wild-type tetR and the KRAB effector domain of the human protein Kox-1 (Deuschle et al., 1995; Rossi et al., 1998). Since in either case tetR functions as a dimer, to allow simultaneous expression while preventing the formation of nonproductive complexes between activators and repressors, the dimerization domain of tTRg was modified as previously described (Rossi et al., 1998), which altered its sensitivity to dox (Figure 2). (C) The distinction between rheostat and on/off transcriptional responses. Rheostat mechanism, treatment of a cell population with increasing concentrations of inducer results in a homogeneous increase in expression from each transcription unit. This is evident as an increase in expression of GFP in each cell within the cell population as detected by flow cytometry. At each intermediate concentration of inducer, a graded response results in a unimodal distribution of expression (one peak). On/off switch mechanism, increasing concentrations of inducer lead to an increase in the fraction of cells within the population that expresses maximal levels of GFP. At each intermediate concentration of inducer, an all-or-none response results in a bimodal distribution of expression (two peaks) with some cells expressing no detectable GFP and some cells expressing maximal levels of GFP.

A Role for Competing Repressors and Activators 725

Figure 2. Hill Coefficients of Total Population Responses Comparison of the doseresponse curves obtained with dox in the presence of the activator only, the repressor only, or the activator and the repressor together. This analysis was possible due to modifications of the dimerization domain (Rossi et al., 1998) that increased the dox concentrations required to inhibit tTRg binding to DNA so that they are overlapping with the concentrations required to promote rtTA (Gossen et al., 1995) binding to DNA. As a result, competition for the promoter could be analyzed as described in the text. The slope of the curve obtained in the presence of both activator and repressor is steeper than the slopes of the curves obtained with either factor alone. The Hill coefficients for the three curves are also shown. (Inset) Hill equation curves for the Hill coefficient (3.2) determined empirically from the activator repressor data and for the Hill coefficient (2.9) expected in the case of a simple additive combination of the activator and repressor curves are shown to superimpose. Mean GFP fluorescence values ( / SD) are derived from triplicate cell samples treated with different concentrations of dox and analyzed by flow cytometry. For activator only and activator repressor populations, doseresponse curves comparable to those shown here were obtained when secreted hGH was assayed in lieu of GFP.

of dox, allowing the analysis of competition. Fortuitously, the genetic modification of the tetR portion of the repressor, tTRg, which we designed to prevent heterodimerization with the activator, rtTAb (Rossi et al., 1998), also led to a change in its sensitivity to dox. This was clear from the doseresponse curve observed with the modified repressor, shown in Figure 2, which differs by at least an order of magnitude from that typical of wild-type tetR (Gossen and Bujard, 1992). As a result, the doseresponse curve for repressor only cells overlapped with that of activator only cells, indicating that both regulators can bind and compete for tetO over a similar range of dox concentrations when they are coexpressed. If the repressor had retained the dose response of the wild-type tetR, essentially all repressor molecules would have been released from the DNA at dox concentrations below those which induced activator binding, precluding the competition studies carried out here. To distinguish between graded and threshold responses (Figure 1C), we analyzed the distribution of expression of GFP at the single-cell level. Because each cell harbored only one copy of the inducible promoter/

reporter cassette, the analysis of GFP levels in single cells served as an indicator of the activity of a single transcription unit. Flow cytometric analysis of the repressor only and activator only populations revealed a unimodal distribution of GFP expression at all dox concentrations tested (Figure 3A). This result indicates that either relieving tTRg-mediated repression or inducing rtTAb-mediated activation leads to a homogeneous graded increase in the amount of transcription from single transcription units in thousands of cells analyzed at the single cell level (Figure 1C). Thus, a progressive increase in the amount of inducer leads to a rheostat response, or a progressive increase in the amount of transcription per individual promoter. By contrast, the appearance of two distinct subpopulations, or an on/off switch, was observed in cells containing both activator and repressor. Treatment with dox resulted in one population of cells that lacked any detectable GFP expression and one population of cells that expressed maximal levels of GFP. With increasing concentrations of dox, a progressive decline in the GFPnegative cell population and a concomitant increase in the relative size of the GFP-positive cell population was observed (Figure 3A). These experiments show that when a combination of activators and repressors act on the same promoter, an all-or-none (on/off) response to the inducer is observed. Moreover, since either factor alone gave a graded (rheostat) response, the threshold response observed in cells that expressed both factors was not due to a dominant effect of one factor over the other but rather to their combined effects. The steeper doseresponse curve obtained with the activator and repressor together could be the result of either an additive or a cooperative interaction of the two types of factors. To distinguish between these possibilities, we calculated the Hill coefficient for each of the three doseresponse curves shown in Figure 2. In the case of a linear dose response, the Hill coefficient should have a value of 1, whereas values greater than 1 are indicative of synergism. The calculated Hill coefficients for the activator only and the repressor only curves were 1.6 and 1.8, respectively, in good agreement with previously reported synergistic effects exhibited by transcription factors (Carey et al., 1990; Lin et al., 1990). The observed steeper slope of the curve when both activator and repressor were present (Hill coefficient of approximately 3.2) can be attributed to the additive effects of the activator and repressor on the same promoter without needing to invoke cooperative interactions or feedback regulation because Hill coefficients can combine up to multiplicatively (Brown et al., 1997; Ferrell, 1997). For each concentration of the inducer tested, the distribution obtained by cell sorting revealed a range of gene expression among individual cells presumably due to the site of integration of the reporter construct. Mathematical modeling has shown previously that given such variability, a doubling of the Hill coefficient alone could shift the cell population from a unimodal to a bimodal distribution of gene expression (Ferrell and Machleder, 1998). Thus, the additive effects of the individual synergistic responses of transcriptional activators and repressors (Hill coefficent 1.6 1.8 ca. 3.2) could suffice as a mechanistic basis for the all-or-none responses observed here with increasing amounts of inducer at the single-cell level. In biology, threshold responses are common (Gurdon et al., 1998). For example, commitments to divide or to

Molecular Cell 726

Figure 3. Single-Cell Analysis by Flow Cytometry (A and B) The rheostat transcriptional response to increasing concentrations of the inducer mediated by either the activator or the repressor alone is converted to an on/off switch in single cells containing both factors. Populations of cells containing the repressor (repressor only), the activator (activator only), or both (activator repressor) were treated for 72 hr with the concentrations of dox indicated. The distribution of GFP expression in the three populations was analyzed by flow cytometry. In (A), GFP expression profiles are shown for each population. The black and the red lines mark the positions of the peak of GFP expression in uninduced and induced conditions, respectively. In the repressor only and activator only populations, increasing concentrations of dox lead to a graded increase in GFP expression in the entire cell population, as indicated by a unimodal homogeneous shift to the right of the peak. In the activator repressor population, increasing concentrations of dox lead to the expression of GFP in a subpopulation of the cells, while no GFP can be detected in the remainder of the population. This effect is indicated by the appearance of two distinct peaks in the GFP expression profiles (Figure 1C). A further increase in the dox concentration leads to an increase of the GFP-positive subpopulation at the expense of the GFPnegative subpopulation. The level of GFP expression in the positive subpopulation is equivalent at all doses of inducer, indicative of an all-or-none response. (B) shows an overlay of the GFP expression profiles shown in (A). Whereas in the repressor only and activator only populations a range of GFP expression levels can be achieved in response to changes in the concentration of the inducer, in the activator repressor population GFP can either be absent or expressed at maximal levels, but cells expressing intermediate levels of GFP are rare.

differentiate are stochastic and are characterized at any given time by a shift in the proportion of cells that activate a specific program of gene expression. The transcription of genes critical to these cell fate decisions is likely to be regulated by an all-or-none mechanism. Consistent with our hypothesis that such a mechanism can be the result of competing transcription factors is the finding that a number of regulatory networks associated with growth or differentiation include repressors and activators that bind to the same promoter in a mutually exclusive fashion. For example, Myc-Max heterodimers, which function as transcriptional activators, bind the same E box sequence as Mad-Max heterodimers, which are transcriptional repressors (Ayer et al., 1993, 1996). Similarly, the transcriptional activator E2F exists as a transcriptional repressor when it interacts with Rb family members (Dyson, 1998). In both of these cases, mitogenic and differentiation-inducing signals shift the balance of factors that either enhance or prevent the transcription of target promoters. These and other well-documented examples (Cubitus interruptus, heterodimerizing nuclear hormone receptors, MyoD/

Mist1 and MyoD/MyoR, Dorsal/DSP1 and Dorsal/ Groucho, Tcf/ -catenin and Tcf/Groucho, XBF-1, Tup1Cyc8, and Pit-1) indicate that a widely used means of regulating gene expression involves changes in the relative amounts of negative and positive transcription factors (Lehming et al., 1994; Mangelsdorf et al., 1995; Dubnicoff et al., 1997; Bourguignon et al., 1998; Lemercier et al., 1998; Ohlmeyer and Kalderon, 1998; Roose et al., 1998; Xu et al., 1998; Conlan et al., 1999; Lu et al., 1999). While in these examples binding of activators and repressors to the target promoter is thought to be mutually exclusive, it is conceivable that sharp thresholds can also be obtained with linked but nonoverlapping sites. The results presented here demonstrate that a competition of transcription factors is not only required but also sufficient to establish all-or-none switches in gene expression. Furthermore, our data predict that where a repressor and an activator compete for binding to the same promoter, a threshold response to signals that modulate that competition is likely to occur, resulting in a true molecular on/off switch.

A Role for Competing Repressors and Activators 727

Experimental Procedures Virus Production and Primary Myoblast Infection Self-inactivating (SIN) retroviruses, which delete the endogenous viral promoter sequences upon integration and are therefore transcriptionally inert, were used to deliver the reporter cassettes, allowing the regulation of the tet-inducible promoter. The HRSpGFP SIN retrovirus contains seven copies of the tetO binding site juxtaposed to a minimal CMV promoter (position 53 to 75) that drives transcription of GFP. The same vector also contains the puromycin resistance gene under the control of the SV40 promoter. Infectious particles were produced by transient transfection of the ecotropic packaging cell line NX-E (a gift from P. Achacoso and G. P. Nolan). Primary mouse myoblasts derived from C57/Bl mice were exposed to virus-containing tissue culture supernatant for 30 min at 37 C. The viral supernatant was replaced with fresh culture medium and the cells were incubated at 37 C for 48 hr prior to puromycin selection. After a week of growth in medium containing 1 g/ml puromycin, approximately 1% of the cells survived to form colonies; a low frequency suggesting single copy uptake of the retrovirus. Subsequently, these cells were infected with a virus expressing tTRg and the cell surface marker CD8 (RTRg-CD8, Figure 1A) by the same procedure and purified by flow cytometry on a Becton Dickinson Facstar based on CD8 expression. The HRIgfp-hGH SIN retrovirus contains seven tandem copies of the tetO binding site juxtaposed to a superminimal CMV promoter (position 53 to 2) that drives transcription of a bicistronic message encoding for a secreted protein, human growth hormone (hGH), and for GFP under the translational control of an internal ribosomal entry site (IRES) (Figure 1A). A GFP-positive, dox-inducible subpopulation of cells ( 15% of total, see text) was purified by flow cytometry as previously described (Kringstein et al., 1998). These cells were then either infected with a retrovirus expressing the repressor, tTRg, and the cell surface marker CD8 (RTRg-CD8, Figure 1A), or with a control virus expressing CD8 but lacking tTRg sequences. Both populations were then purified by flow cytometry based on CD8 expression, yielding a population expressing the activator repressor (RetroTet ART12) and a control population expressing the activator only.

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Determination of Hill Coefficients The raw data for triplicate samples of each of the three polyclonal myoblast populations shown in Figure 1 were fitted to the Hill coefficient equation using Sigmaplot 5.0.1 software. The equation used here to calculate the Hill coefficient is: xn ecn xn

min

(max

min)

where y is the mean GFP fluorescence value, x is the concentration of the inducer, ec is the median effective concentration, and n represents the Hill coefficient itself. The Hill coefficient expected if the actions of the activator and the repressor are additive is the product of the Hill coefficients determined for each alone (Brown et al., 1997; Ferrell, 1997).

Acknowledgments We are grateful to Charles Yanofsky, Lubert Stryer, Mark Ptashne, Dale Kaiser, and Jim Ferrell for helpful critique of the manuscript, Gerry Crabtree for stimulating discussions, Garry Nolan for the gift of the Phoenix-E cell line, and Carol Charlton for expert technical assistance. F. M. V. R. was funded by fellowship LT-623/96 from the Human Frontiers in Science Program. A. M. K. and A. S. were recipients of a Howard Hughes Medical Institute summer undergraduate research fellowship and a Swiss National Science Foundation fellowship (823A-46704), respectively. This research was supported by NIH grants HD18179, AG09521, and CA59717 to H. M. B.

Received June 1, 2000; revised July 21, 2000.

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