The Neurospora crassa White Collar-1 dependent blue light response requires acetylation of histone H3 lysine 14 by NGF-1

Abstract

Blue light-induced transcription in Neurospora crassa is regulated by the White Collar-1 (WC-1) photoreceptor. We report that residue K14 of histone H3 associated with the light-inducible albino-3 (al-3) promoter becomes transiently acetylated after photoinduction. This acetylation depends on WC-1. The relevance of this chromatin modification was directly evaluated in vivo by construction of a Neurospora strain with a mutated histone H3 gene (hH3(K14Q)). This strain phenocopies a wc-1 blind mutant and shows a strong reduction of light-induced transcriptional activation of both al-3 and vivid (vvd), another light-inducible gene. We mutated Neurospora GCN Five (ngf-1), which encodes a homologue of the yeast HAT Gcn5p, to generate a strain impaired in H3 K14 acetylation and found that it was defective in photoinduction. Together, our findings reveal a direct link between histone modification and light signaling in Neurospora and contribute to the developing understanding of the molecular mechanisms operating in light-inducible gene activation.

Figure 1.

The al-3 LRR is sufficient to confer light inducibility on the hph reporter gene. Top, schematic representation of the reporter region of pBG3 plasmid, containing the LRR of the al-3 promoter fused to hph, a gene that confers resistance to hygromycin B. The sequence coordinates are relative to the transcription start site. Bottom, host wild-type strain (WT; FGSC462) and a representative transformant (BG3) obtained by transformation with plasmid pBG3 were grown on solid medium plus increasing amounts of Hyg in the dark or light (see Materials and Methods). Only the BG3 strain is able to grow in the presence of Hyg in the light.

Figure 2.

Light induces a transient increase in acetylation of histone H3. (A) Acetylated histones accumulate 20 min after a light pulse. Left, representative PCR coamplifications of the al-3 LRR and the actin promoter (internal control) from chromatin immunoprecipitations of Neurospora WT (FGSC987) with antibodies directed against acH3 IP or H3 IP. INPUT represents the sample before immunoprecipitation. Right, histograms derived from three independent amplifications with two independent immunoprecipitations. (B) H3 K14 is the target for transient acetylation after a light pulse. Left, representative PCR coamplifications of the al-3 and actin promoter regions 20 min after a light pulse (see A) after immunoprecipitation with antibodies directed against acH3 IP or H3 acetylated specifically on K14 (acK14H3 IP) Right, histograms derived from three independent amplifications with two independent immunoprecipitations.

Figure 3.

Light-inducible acetylation of H3 K14 requires the presence of WC-1, the blue light photoreceptor. Left, representative PCR coamplifications of al-3 and actin promoter regions (see Figure 2A) after chromatin immunoprecipitation from a wc-1 mutant strain (FGSC3081) with antibodies directed against histone H3 K14 (acK14H3 IP). Right, histograms derived from three independent amplifications with two independent immunoprecipitations.

Figure 4.

A histone H3 K14Q mutation reduces the light response. (A) Phenotypes of Neurospora strains with wild-type histone H3 (WT; N644) and a dominant histone H3 K14Q substitution (hH3^K14Q; N3095). The hH3^K14Q mutant phenocopied wc-1 (FGSC3081) and ngf-1^RIP1 (N2842). Strains were grown on minimal medium for 1 wk in the dark, induced under saturating light, and the production of carotenoids was observed after 6 h at 4°C. (B) Northern analysis of al-3, vvd, and actin (control) revealed light-induced expression in WT. This regulation was abolished in wc-1 and reduced in hH3^K14Q mutants. Densitometric analysis for al-3 (left) and vvd (right) revealed a five- or sevenfold reduction, respectively, in the hH3^K14Q strain compared with wild type (below).

Figure 5.

NGF-1 is the Neurospora homologue of the GCN5 histone acetyltransferase. Alignment of Neurospora NGF-1 (NcNGF1) with homologues from S. cerevisiae (ScGcn5), M. grisea (MgGCN5), and G. zeae (GzGCN5). Bold letters indicate identity among proteins. Mutations resulting in substitutions between wild-type NGF-1 and the predicted amino acid sequence of the mutant allele, ngf-1^RIP1, are indicated (+). Predicted nonsense codons are also shown (‡). Solid and dashed underlines indicate the catalytic HAT domain and bromodomain, respectively.

Figure 6.

The ngf-1^RIP1 mutant is defective in light inducibility. (A) Loss of light-inducible H3 K14 acetylation in the ngf-1^RIP1 mutant. Left, representative PCR coamplifications of al-3 and actin promoter regions after immunoprecipitation of chromatin from the ngf-1^RIP1 mutant with antibodies directed against histone H3 K14 (acK14H3 IP). Right, histograms derived from three independent amplifications with two independent immunoprecipitations. (B) Loss of mRNA light inducibility in the ngf-1^RIP1 mutant. Northern analyses (blots shown on top; densitometric results on bottom) revealed loss of response to light in the ngf-1^RIP1 mutant for al-3 and vvd; vvd expression was absent, whereas al-3 mRNA was constitutively expressed in both dark and light conditions.