Light-dependent and circadian transcription dynamics in vivo recorded with a destabilized luciferase reporter in Neurospora

Abstract

We show that firefly luciferase is a stable protein when expressed at 25 °C in Neurospora, which limits its use as transcription reporter. We created a short-lived luciferase by fusing a PEST signal to its C-terminus (LUC-PEST) and applied the LUC-PEST reporter system to record in vivo transcription dynamics associated with the Neurospora circadian clock and its blue-light photosensory system over the course of several days. We show that the tool is suitable to faithfully monitor rapid, but also subtle changes in transcription in a medium to high throughput format.

Figure 1. A PEST signal reduces luciferase stability.

(A) Schematic representation of the luciferase reporter expressed under control of the promoter of the frequency gene (frq promoter). The coding region of the firefly luciferase has been codon optimized for Neurospora crassa expression . The PEST sequence fused to the 3′-end of the luciferase is indicated. The reporter gene was integrated into the genome downstream of the his-3 locus. (B) A protein stability assay in light grown cells was carried out in the presence of cycloheximide (CHX) to compare the degradation of LUC-PEST and unmodified LUC. Mycelia were grown in liquid culture at 25°C and CHX was added at t  = 0 at a final concentration of 10 µg/ml. Cells were harvested at the indicated time points, native protein extracts were prepared and luciferase activity was analyzed as described in Material and Methods. (Left) Absolute bioluminescence levels at t  = 0 are shown. (Right) Bioluminescence levels are blotted over time. Levels at t  = 0 were set to 100% (Error bars indicate to ± SEM, n  = 3). Exponential trend lines are fitted to the data (values <5% were excluded). (C) Cultures of frq::luc (blue line) and frq::luc-PEST (orange line), grown in darkness under growth-restricting conditions on solid medium in a 96-well plate, were exposed to a 1 min light pulse and then released into constant darkness. Luciferase activity was recorded in vivo for 5 hours in 20 min intervals. frq mRNA levels (black line) were determined by qPCR in WT strain grown in liquid medium. Data were normalized by setting the maxima to 100%. (Error bars indicate ± SEM, n  = 3).

Figure 2. frq-promoter driven luc-PEST exhibits high amplitude oscillations.

frq::luc (black lines) and frq::luc-PEST (blue lines) strains grown in 96-well plates were synchronized with a 1 hour light pulse and luciferase activity was measured every 15 minutes under the indicated conditions: (A) constant darkness (DD), (B) 12 h:12 h light/dark cycles (LD12∶12) and (C) 4 days in LD12∶12 followed by 3 days in DD. Representative bioluminescence records (starting at day 2) of individual cultures are shown. Data were normalized by setting the minima to 1. (D) The frq::luc-PEST reporter conveniently resolves the known differences in transcription dynamics between WT (blue) and Δvvd (red) strains. The frq::luc-PEST reporter was expressed in a Δvvd strain, which was exposed to the same light-dark regime as above. The frq::luc-PEST activity of WT grown in the same plate from part (C) is shown again for comparison (dotted line). Insert: magnification showing the ∼4 h phase delay of Δvvd after release to DD .

Figure 3. Light response and adaptation of vvd-promoter driven luciferase expression.

Luc and luc-PEST were expressed under the control of the vvd promoter. Cultures were subjected to three consecutive 15 h light steps with increasing intensity (0.35 µE, 3.5 µE and 30 µE) followed by 48 h of constant darkness. Luciferase activity was measured in 30 min intervals. (A) vvd::luc-PEST (n = 20) (B) vvd::luc (n = 4). Error bars indicate ± SEM.