Molecular mechanism of light responses in Neurospora: from light-induced transcription to photoadaptation

Abstract

Blue light regulates many molecular and physiological activities in a large number of organisms. In Neurospora crassa, a eukaryotic model system for studying blue-light responses, the transcription factor and blue-light photoreceptor WHITE COLLAR-1 (WC-1) and its partner WC-2 are central to blue-light sensing. Neurospora's light responses are transient, that is, following an initial acute phase of induction, light-regulated processes are down-regulated under continuous illumination, a phenomenon called photoadaptation. The molecular mechanism(s) of photoadaptation are not well understood. Here we show that a common mechanism controls the light-induced transcription of immediate early genes (such as frq, al-3, and vvd) in Neurospora, in which light induces the binding of identical large WC-1/WC-2 complexes (L-WCC) to the light response elements (LREs) in their promoters. Using recombinant proteins, we show that the WC complexes are functional without the requirement of additional factors. In vivo, WCC has a long period photocycle, indicating that it cannot be efficiently used for repeated light activation. Contrary to previous expectations, we demonstrate that the light-induced hyperphosphorylation of WC proteins inhibits bindings of the L-WCC to the LREs. We show that, in vivo, due to its rapid hyperphosphorylation, L-WCC can only bind transiently to LREs, indicating that WCC hyperphosphorylation is a critical process for photoadaptation. Finally, phosphorylation was also shown to inhibit the LRE-binding activity of D-WCC (dark WC complex), suggesting that it plays an important role in the circadian negative feedback loop.

Figure 1.

Light induces the binding of identical L-WCCs to the LREs of frq, vvd, and al-3.(A) DNA sequence alignment of LREs in the promoters of frq, al-3, and vvd. (frq-p) frq proximal LRE; (frq-d) frq distal LRE. (B–D) EMSA assays showing the binding of the WCC to the LREs of frq (distal) (B), vvd (C), and al-3 (D). In some of EMSA assays, cold wild-type or mutated probes were added, and in some, antibodies against the WC proteins were used. (E) EMSA assay showing that the binding of the L-WCC to the frq LRE (radioactive labeled) can be efficiently competed away by the cold LREs of al-3 and vvd. For all EMSA experiments described in this figure, partially purified WCC from Neurospora (DD culture) was used. The LPs (10 min) were administered in vitro.

Figure 2.

The recombinant sf9 insect-cell-expressed WCC is sufficient to mediate the light-induced binding to the LREs. (A) SDS-PAGE gel showing the nickel column purification products of His-WC-1, His-WC-2, and the complex of WC-1 and His-WC-2 from insect cells (grown in DD) infected by respective viruses. (Control) Mock purification from uninfected cells. The asterisks indicate a nonspecific band and the possible His-WC-1 degradation products. (B) Fluorescence emission spectra analysis showing the presence of FAD in the purified WCC from insect cells. (C) EMSA assay showing the induction of the L-WCC/LRE (al-3 or vvd) complexes after an LP in vitro. (D) EMSA assay showing the side-by-side comparison of the WCC/LRE complexes (al-3 probe) using WCC purified from Neurospora or insect cells.

Figure 3.

Most of the L-WCC cannot recover back to the dark state after light activation in vitro. (A) EMSA assay showing the binding of the WCC to the frq LRE after a 10-min in vitro LP. Partially purified WCC from Neurospora (DD culture) was used. After the LP, some of the samples were incubated in the dark for the indicated time (in minutes) before they were mixed with the LRE probe. (B) Densitometric analysis of the result in A and several independent experiments.

Figure 4.

Two pulse experiments demonstrate that WC-1 has a long-period photocycle in vivo. (A) A schematic diagram depicts the time points used to harvest Neurospora cultures for the experiment shown in B. A wild-type Neurospora strain was used. (1) DD. (2) Immediately after the first LP. (3) Thirty minutes after the first LP. (4) Two hours after the first LP. (5) Four hours after the first LP. (6) Immediately after the second 15-min LP. (7) Thirty minutes after the second LP. (B) Western and Northern blot analyses showing that the second LP can result in light-induced WC-1 hyperphosphorylation and light-induced transcription. CHX was added to half of the culture to block protein synthesis. (rp-10) A ribosomal protein gene that was used as a negative control for light responses in the Northern blot analysis. (C) A schematic diagram depicts the time points used to harvest Neurospora cultures for the experiment shown in D. The second LP was administered 1, 2, or 3 h after the first LP. (1) DD. (4,7,10) One, two, and three hours in DD after the first LP, respectively. (2,5,8,11) Immediately after the first and the second LP. (3,6,9,12) Thirty minutes after the first or the second LP. (D) Western and Northern blot analyses showing that the second LP can result in light-induced WC-1 hyperphosphorylation and light-induced al-3 transcription. The wc-1^RIP, qa-WC-1 strain was used. The culture was first grown in medium containing QA for 1 d before it was removed by washing the cultures with fresh minimal medium containing 2% glucose. The first LP was administered 2 h after the removal of QA.

Figure 5.

The L-WCC binds to the LREs transiently after transfer from DD to LL. ChIP assay using WC-2 antibody showing the binding of WCC to the LREs of vvd, al-3, and frq. The densitometric analysis of three independent experiments is shown in the bottom panel.

Figure 6.

After the transfer from DD to LL, the peaks of L-WCC binding to the LREs are not correlated with the peak of WC-1 hyperphosphorylation. (A) Western and Northern blot analysis showing the kinetics of the light-induced WC-1 hyperphosphorylation and the light induction of vvd, al-3, and wc-1 mRNA after the dark-to-light transfer. Densitometric analysis of the Northern blot experiment is shown in the bottom panel. (B) ChIP assay showing the binding kinetics between WCC and LREs (al-3 and frq). (C) EMSA assay showing the binding kinetics between L-WCC and the vvd LRE. Nuclear extracts purified from cultures harvested in the indicated time points (minutes) were used in the assay. The bottom panel is the Western blot result showing the WC-1 protein for these samples.

Figure 7.

Dephosphorylation of WCC significantly enhances the binding of both L-WCC and D-WCC to the LREs. (A,B) EMSA assays showing the binding of purified Neurospora WCC to the LREs (vvd or frq) with/without the λ-phosphatase treatment. The cultures were harvested after a 15-min LP. All samples contained phosphatase buffer and were treated by the same procedure except the presence of phosphatase or phosphatase inhibitors. The top panel of A is the Western blot result showing the phosphorylation profile of WC-1 used in the EMSA assay. (C) EMSA assay showing the binding of L-WCC to the vvd LRE after an LP in vitro. The WCC was purified from cultures grown in DD.