Arabidopsis GCN5, HD1, and TAF1/HAF2 interact to regulate histone acetylation required for light-responsive gene expression

Abstract

We previously showed that Arabidopsis thaliana histone acetyltransferase TAF1/HAF2 is required for the light regulation of growth and gene expression, and we show here that histone acetyltransferase GCN5 and histone deacetylase HD1/HDA19 are also involved in such regulation. Mutation of GCN5 resulted in a long-hypocotyl phenotype and reduced light-inducible gene expression, whereas mutation of HD1 induced opposite effects. The double mutant gcn5 hd1 restored a normal photomorphogenic phenotype. By contrast, the double mutant gcn5 taf1 resulted in further loss of light-regulated gene expression. gcn5 reduced acetylation of histones H3 and H4, mostly on the core promoter regions, whereas hd1 increased acetylation on both core and more upstream promoter regions. GCN5 and TAF1 were both required for H3K9, H3K27, and H4K12 acetylation on the target promoters, but H3K14 acetylation was dependent only on GCN5. Interestingly, gcn5 taf1 had a cumulative effect mainly on H3K9 acetylation. On the other hand, hd1 induced increased acetylation on H3K9, H3K27, H4K5, and H4K8. GCN5 was also shown to be directly associated with the light-responsive promoters. These results suggest that acetylation of specific histone Lys residues, regulated by GCN5, TAF1, and HD1, is required for light-regulated gene expression.

Figure 1.

gcn5 and hd1 Mutations Affect the Light Repression of Hypocotyl Elongation. (A) Seven-day-old seedling hypocotyl lengths of wild-type Ws, gcn5, and hd1plantlets grown in darkness or under white light (16 h/d at 120 μmol·m⁻²·s⁻¹) or continuous red (10 μmol·m⁻²·s⁻¹) far-red (5 μmol·m⁻²·s⁻¹), or blue (18 μmol·m⁻²·s⁻¹) light. (B) Seven-day-old far-red light–grown seedling phenotypes. Left, comparison of the gcn5-1 allele with the wild type (Ws); right, comparison of Ws, gcn5, gcn5 complemented with GCN5 cDNA [gcn5 (C)], hd1, and the double mutant gcn5 hd1-1. Means of 20 plantlets are given. Error bars represent sd values. The measures were analyzed by Student's t test at α = 0.05.

Figure 2.

The gcn5 and hd1 Mutations Have Opposite Effects on Light-Inducible Gene Expression. (A) RNA gel analysis of CAB2 and RBCS-1A gene expression in both light- and dark-grown gcn5 and Ws seedlings. Actin mRNA levels were analyzed to normalize gel loading. (B) RNA gel analysis of CAB2 and RBCS-1A gene expression in both light- and dark-grown hd1 and Ws seedlings. rRNA transferred to the membranes was used to normalize gel loading. (C) RNA gel analysis of CAB2 and RBCS-1A gene expression in light-grown Ws, gcn5, gcn5 complemented (C), the gcn5-2 hd1 double mutant, and hd1 seedlings. Signal quantifications relative to the wild type (arbitrarily given as 100) are presented at right of the gels. Black bars, RBCS-1A; white bars, CAB2.

Figure 3.

Genetic Relationship between gcn5 and taf1 Mutations. (A) Phenotypes of taf1 gcn5 double mutants with rosette leaves (3 weeks) grown in the greenhouse. Bars = 0.5 cm. (B) Top, expression of the light-inducible genes CAB2, RBCS-1A, and CHS in the double mutant compared with that in Ws and the single mutants. Bottom, comparison of expression levels of IAA3 and CHS in Ws, gcn5, taf1, and hd1. (C) Comparison of 7-d-old seedling hypocotyl lengths of taf1 hy5 (top) and gcn5 hy5 (bottom) double mutants grown under continuous far-red light (5 μmol·m⁻²·s⁻¹). Average lengths (in millimeters) are indicated at bottom with sd values from at least 20 plantlets (in parentheses).

Figure 4.

Acetylation State of Histones H3 and H4 of CAB2, RBCS-1A, IAA3, and CHS in Ws, gcn5, and hd1 Seedlings. Nuclei were extracted from cross-linked 12-d-old light-grown seedlings, sonicated, and immunoprecipitated with antibodies specific to total histone H3, acetylated histone H3, and acetylated histone H4. The immunoprecipitates were analyzed by real-time PCR. The primer sets (arrowheads) are numbered for each gene (open boxes represent the translated regions), and the positions of the primers relative to the initiation ATG codon are indicated. The relative amounts of the PCR products compared with input chromatin from wild-type extracts (arbitrarily given as 100) are shown below the genes. Black bars, acetylated histone H3; white bars, acetylated histone H4. Error bars represent sd values from at least three repetitions.

Figure 5.

Acetylation Profiles of the N-Terminal Tails of Histones H3 and H4 on Light-Responsive Promoters in taf1, gcn5, hd1, and taf1 gcn5 Compared with Wild Type Ws. As indicated, ChIP with specific antibodies against histones H3K9, H3K14, and H3K27 and H4K5, H4K8, H4K12, and H4K16 were used. The immunoprecipitated promoter fragments of RBCS-1A (black boxes), IAA3 (white boxes), and CHS (gray boxes) were quantified by real-time PCR. The relative amounts of the promoter fragments compared with input chromatin from wild-type extracts were arbitrarily given as 100. Error bars represent sd values from at least three repetitions.

Figure 6.

GCN5 Was Associated with the Target Light-Responsive Promoters. (A) Protein gel blot analysis of nuclear extracts from wild-type (Ws) and gcn5 rosette leaves and protein extracts of E. coli transformed by a plasmid expressing GCN5 (I, induced by isopropylthio-β-galactoside; NI, noninduced). (B) Real-time PCR quantification of RBCS-1A, CAB2, and CHS core promoter regions (as in Figure 4) immunoprecipitated by the GCN5 antibodies or by the preimmune serum. The signals are given as percentage of the input chromatin. Error bars represent sd values from at least three repetitions.