Transcriptional infidelity promotes heritable phenotypic change in a bistable gene network

Abstract

Bistable epigenetic switches are fundamental for cell fate determination in unicellular and multicellular organisms. Regulatory proteins associated with bistable switches are often present in low numbers and subject to molecular noise. It is becoming clear that noise in gene expression can influence cell fate. Although the origins and consequences of noise have been studied, the stochastic and transient nature of RNA errors during transcription has not been considered in the origin or modeling of noise nor has the capacity for such transient errors in information transfer to generate heritable phenotypic change been discussed. We used a classic bistable memory module to monitor and capture transient RNA errors: the lac operon of Escherichia coli comprises an autocatalytic positive feedback loop producing a heritable all-or-none epigenetic switch that is sensitive to molecular noise. Using single-cell analysis, we show that the frequency of epigenetic switching from one expression state to the other is increased when the fidelity of RNA transcription is decreased due to error-prone RNA polymerases or to the absence of auxiliary RNA fidelity factors GreA and GreB (functional analogues of eukaryotic TFIIS). Therefore, transcription infidelity contributes to molecular noise and can effect heritable phenotypic change in genetically identical cells in the same environment. Whereas DNA errors allow genetic space to be explored, RNA errors may allow epigenetic or expression space to be sampled. Thus, RNA infidelity should also be considered in the heritable origin of altered or aberrant cell behaviour.

Figure 1. Stochastic Switching in the lac Bistable Gene Network

(A) Under maintenance conditions, the lac operon is OFF when the lac repressor is bound to the lac operator (indicated by the solid red line) and the inducer TMG remains extracellular; stochastic events that lead to a transient derepression of the lac operon will result in a burst of lac operon functions and the appearance of permease will initiate an autocatalytic positive-feedback response (indicated by solid blue lines), which will heritably maintain the ON state (TMG induces an allosteric transition in lac repressor, indicated by the dashed red line, so that it no longer binds to the lac operator), and the cell will exhibit green fluorescence. (B) Cells that were originally ON (filled symbols) or OFF (open symbols) were sub-cultured and grown in media containing various concentrations of TMG. Circles denote the wild-type strain; squares denote the ΔgreA ΔgreB strain. The shaded region shows the maintenance concentration of TMG. Each value is the average ± SD from four to six independent cultures. (C) Uninduced (OFF) wild-type (top panel) and ΔgreA ΔgreB cells (bottom panel) were diluted and grown in media containing 3 μM (light blue), 6 μM (red), and 30 μM TMG (dark blue). After 42 h growth, fluorescence microscopy was performed to determine the frequency of epigenetically ON cells. Shown are average fluorescence values per cell, with background fluorescence subtracted (Figure S1), for a representative set of 350 cells per treatment; each circle represents an individual cell.