Light-mediated control of DNA transcription in yeast

Abstract

A variety of methods exist for inducible control of DNA transcription in yeast. These include the use of native yeast promoters or regulatory elements that are responsive to small molecules such as galactose, methionine, and copper, or engineered systems that allow regulation by orthogonal small molecules such as estrogen. While chemically regulated systems are easy to use and can yield high levels of protein expression, they often provide imprecise control over protein levels. Moreover, chemically regulated systems can affect many other proteins and pathways in yeast, activating signaling pathways or physiological responses. Here, we describe several methods for light mediated control of DNA transcription in vivo in yeast. We describe methodology for using a red light and phytochrome dependent system to induce transcription of genes under GAL1 promoter control, as well as blue light/cryptochrome dependent systems to control transcription of genes under GAL1 promoter or LexA operator control. Light is dose dependent, inexpensive to apply, easily delivered, and does not interfere with cellular pathways, and thus has significant advantages over chemical systems.

Figure 1

Schematic showing split transcription factor reconstitution using optical dimerizers. (A) Red light switches PhyB into the Pfr form, enabling binding to PIF3. The PhyB/PIF3 interaction allows reconstitution of a split Gal4 transcription factor and switches on DNA transcription. Far-red light reverts PhyB to the Pr form, which is unable to bind to PIF3 and halts transcription. (B) Blue light enables binding of CRY2 to CIB1, bringing together a split Gal4 transcription factor controlling DNA transcription. Dark reversion of CRY2 dissociates the interaction with CIB1 and halts Gal4-dependent transcription.

Figure 2

Establishment of parameters for use of PhyB/PIF3 split Gal4 system. PJ69-4a yeast cultures expressing PhyBNT-GalBD and GalAD-PIF3 were diluted to OD₆₀₀ = 0.2 and grown for indicated times in the dark with indicated amounts of PCB before red light exposure (2.8 mW/cm², 660 nm). (A) PCB titration. Yeast were grown 4 hours in the dark with indicated amounts of PCB, then illuminated with red light (5 s pulse). Cultures were incubated in the dark for an additional hour, then analyzed for β-galactosidase reporter activity. (B) Light pulse titration. Yeast cultures were grown for 4 hours in the dark with 2 µM PCB, then exposed to red light for indicated amounts of time. Samples were returned to the dark for 3 hours, then analyzed for β-galactosidase reporter activity. (C) Yeast were grown for indicated times in the dark before exposing to red light (5 sec pulse) then returned to the dark for 1 hour before analysis of β-galactosidase reporter activity.

Figure 3

Induction of protein expression using PhyB/PIF3 split Gal4 system. (A) PJ69-4a yeast cultures expressing PhyBNT-Gal4BD, Gal4AD-PIF3, and the GalUAS-YFP reporter were diluted to OD₆₀₀ = 0.2 and grown for 4 hrs in the dark with 2 µM PCB then exposed to a pulse of red light (5 s, 2.8 mW/cm², 660 nm). Cultures were returned to the dark, then flashed with far-red light (5 s, 6.9 mW/cm²) at indicated times to halt transcription. Samples were then returned to the dark, and all samples were harvested simultaneously 140 minutes after the initial red light exposure. The graph below show integrated intensities of immunoblot bands (arbitrary units). (B) Yeast cultures as in (A) were incubated with indicated amounts of PCB at OD₆₀₀ = 0.2 and grown for 6 hours, treated with a pulse of red light (5 s, 2.8 mW/cm²) each hour. All samples were harvested simultaneously at the end of the 6-hour incubation period.

Figure 4

Control of protein expression using CRY2/CIB1 split Gal4 system. (A) PJ69-4a cultures expressing Gal4BD-CRY2, Gal4AD-CIB1, and the GalUAS-YFP reporter were inoculated at OD₆₀₀ = 0.2 and grown for 3 hours in the dark. Blue light flashes were then applied (2 s, every 4 minutes, 2.7 mW/cm²), and samples were taken at indicated times for immunoblot analysis. (B) Samples as in (A) were grown for 5 hours with blue light flashes (2 s, every 4 minutes, 2.7 mW/cm²). After 5 hours, the samples were placed in the dark for indicated amounts of time, then harvested for immunoblot analysis (top). The graph below shows the integrated intensity of blot bands (arbitrary units). (C) PJ69-4a containing pRH120 (which expresses Gal4BD-CRY2PHR and Gal4ADCIB1 from a single plasmid) and a GalUAS-YFP reporter were treated as in (A).

Figure 5

Orthogonal light-regulated LexA-VP16 transcription system. (A) PJ69-4a yeast expressing GalBD-CRY2 or GalBD-CRY2PHR and VP16-CIB1 or VP16-CIBN were tested for activation of a β-galactosidase reporter after a 5 hr incubation in the dark (‘D’) or exposure to blue light pulses (‘L’) (1 s, every 4 min, 1.9 mW/cm²). (B) PJ69-4a yeast expressing GalBD-CRY2PHR, VP16-CIB1, and a GalUAS-YFP reporter were exposed for indicated times to blue light pulses (1 s, every 4 min, 1.7 mW/cm²). (C) W303-1A yeast expressing LexA-CRY2PHR, VP16-CIBN, and a pSH18-34 reporter (containing 8 LexA operators driving expression of LacZ) were incubated in the dark or exposed to blue light pulses (2 s, every 4 min, 1.9 mW/cm²) for 3 hours, then extracts were assayed for β-galactosidase activity. (D) W303-1A yeast expressing LexA-CRY2PHR, VP16-CIBN, and a LexA-8xop- SIC1^0p construct, containing a hyperstable Sic1 protein, were kept in the dark (left) or exposed to blue light pulses (2 s, every 4 min, 2.7 mW/cm²) for 4 hours (right).