Transcriptional Control Fabio M.V. Rossi, Andrew M. Kringstein, Albert Spicher, Oivin M. Guicherit, Helen M. Blau  Molecular Cell  Volume 6, Issue 3, Pages 723-728 (September 2000) DOI: 10.1016/S1097-2765(00)00070-8

Figure 1 Schematic of Vectors Used and Hypotheses Tested (A) Diagram of the retroviral vectors. Expression from the HRIgfp-hGH 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. Molecular Cell 2000 6, 723-728DOI: (10.1016/S1097-2765(00)00070-8)

Figure 2 Hill Coefficients of Total Population Responses Comparison of the dose–response 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, dose–response curves comparable to those shown here were obtained when secreted hGH was assayed in lieu of GFP. Molecular Cell 2000 6, 723-728DOI: (10.1016/S1097-2765(00)00070-8)

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 GFP-negative 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. Molecular Cell 2000 6, 723-728DOI: (10.1016/S1097-2765(00)00070-8)