Graded transcriptional response to different concentrations of a single transactivator

Abstract

Threshold mechanisms of transcriptional activation are thought to be critical for translating continuous gradients of extracellular signals into discrete all-or-none cellular responses, such as mitogenesis and differentiation. Indeed, unequivocal evidence for a graded transcriptional response in which the concentration of inducer directly correlates with the level of gene expression in individual eukaryotic cells is lacking. By using a novel binary tetracycline regulatable retroviral vector system, we observed a graded rather than a threshold mechanism of transcriptional activation in two different model systems. When polyclonal populations of cells were analyzed at the single cell level, a dose-dependent, stepwise increase in expression of the reporter gene, green fluorescent protein (GFP), was observed by fluorescence-activated cell sorting. These data provide evidence that, in addition to the generally observed all-or-none switch, the basal transcription machinery also can respond proportionally to changes in concentration of extracellular inducers and trancriptional activators.

Figure 1

Binary tet-inducible reporter system. A SIN retroviral backbone was used for the reporter virus (Hermes-HRIgfp-hGH) to eliminate transcriptional interference from the viral LTR. The tet-sensitive transactivators (either tTA or rtTA) are provided constitutively from a second retrovirus [Retrotet RTAb(−) or Retrotet RTAb(+)]. Under proper dox conditions, these transactivators bind to the tet-operator sites (O7) fused upstream of the CMV core promoter, inducing transcription of a bicistronic mRNA encoding hGH and GFP from the reporter vector.

Figure 2

Purification of inducible populations. (A and B) C57 myoblasts infected with both the Hermes-HRIgfp-hGH and either the RTAb(−) or RTAb(+) were analyzed by FACS after culture for 4 days under uninduced conditions (1 μg/ml dox for RTAb(−); 0 μg/ml dox for RTAb(+); dashed curves) and induced conditions (0 μg/ml for RTAb(−); 1 μg/ml for RTAb(+); filled dark gray curves). C57 cells transduced with the reporter only were analyzed as a measure of background fluorescence (unfilled light gray curves). For both RTAb(−) and RTAb(+) systems, 15% of the cells shifted to fluorescence levels above the background provided by the reporter construct alone. These inducible subpopulations were purified by sorting and then reanalyzed (C and D). After sorting, both RTAb(−) and RTAb(+) cells demonstrated >90% inducible subpopulations when cultured under induced conditions (filled dark gray curves), yet demonstrated low background fluorescence under uninduced conditions (dashed curves). Fig. 2E shows hGH secretion from the unsorted and sorted populations from A-D, verifying that the FACS-enrichment process based on GFP expression leads to increased inducibility.

Figure 3

Dose response of binary tet-inducible system. dox dose response of hGH expression (○) and GFP expression (▵) for both RTAb(−) and RTAb(+) systems. hGH and GFP expression of RTAb(−) and RTAb(+) myoblasts cultured in eight different doses of dox were assayed after 3 days under these conditions [RTAb(−) dox doses: 0, 0.0001, 0.0002, 0.0005, 0.001, 0.005, 0.01, and 0.1 μg/ml; RTAb(+): 0, 0.001, 0.01, 0.05, 0.1, 0.5, 1, and 5 μg/ml]. Secreted hGH was measured by radioimmunoassay and normalized to total cell protein. GFP expression was determined by FACS.

Figure 4

Kinetic analysis of hGH inducibility. (A) Northern analysis of hGH transcript levels measured over time from the sorted C57 populations containing either the RTAb(−)- or RTAb(+)-based version of the reporter system. (B) Density values of the blots shown in A determined by PhosphorImaging analysis. Values are plotted as fold induction over lowest value. (C) Time course analysis of hGH protein secretion from the same cell populations as in A.

Figure 5

Kinetic analysis of GFP accumulation and single cell analysis of expression. (A) GFP expression as measured by FACS has reached a plateau after 3 days in culture at low, intermediate, and high levels of dox [RTAb(−) dox doses: 0.001 μg/ml (light gray), 0.0001 μg/ml (dark gray), and 0 μg/ml (black); RTAb(+): 0 μg/ml (light gray), 0.33 μg/ml (dark gray), and 1.0 μg/ml (black)]. (B) Overlays of histogram plots of GFP fluorescence derived from FACS analysis of the dose response in Fig. 3 at three selected doses [RTAb(-): 0.1 μg/ml (light gray), 0.001 μg/ml (dark gray), and 0 μg/ml (black); RTAb(+): 0 μg/ml (light gray), 0.1 μg/ml (dark gray), and 5 μg/ml (black)]. These overlays demonstrate the uniform shift of the population to intermediate and high expression levels as would be predicted from an individual cell-graded response. The uniform shift observed in these plots is representative of the shifts observed at each concentration of dox used in the dose response experiment in Fig. 3.

Figure 6

Idealized mechanisms of graded vs. threshold transcriptional activation. An overall graded response of a heterogeneous population observed through a bulk assay of pooled cells (A) can be the result of two distinct underlying single cell mechanisms. (B) Each individual cell might have a graded response to increasing amounts of inducer, or each of the cells might respond in an on/off threshold manner, but vary with respect to the concentration of inducer necessary to cross the threshold. (C) These two mechanisms can be distinguished by assessing the distribution of single cell expression levels in a population exposed to different levels of inducer: In a population demonstrating a true graded response, the entire population should shift homogeneously as inducer is increased. This will result in a unimodal distribution of the population at all levels of inducer. A population demonstrating an individual cell threshold response will produce a bimodal curve at intermediate levels of inducer, with one mode centered at a level corresponding to “off,” and one mode centered at “on.”