Developmental and environmental signals induce distinct histone acetylation profiles on distal and proximal promoter elements of the C4-Pepc gene in maize

Abstract

The maize C(4)-Pepc gene is expressed in an organ- and cell-type-specific manner, inducible by light and modulated by nutrient availability and the metabolic state of the cell. We studied the contribution of histone acetylation at five lysine residues to the integration of these signals into a graduated promoter response. In roots and coleoptiles, where the gene is constitutively inactive, three of the five lysines were acetylated and the modifications showed unique patterns with respect to their distribution on the gene. A similar pattern was observed in etiolated leaves, where the gene is poised for activation by light. Here, illumination selectively induced the acetylation of histone H4 lysine 5 and histone H3 lysine 9 in both the promoter and the transcribed region, again with unique distribution patterns. Induction was independent of transcription and fully reversible in the dark. Nitrate and hexose availability modulated acetylation of all five lysines restricted to a distal promoter region, whereas proximal promoter acetylation was highly resistant to these stimuli. Our data suggest that light induction of acetylation is controlled by regulating HDAC activity, whereas metabolic signals regulate HAT activity. Acetylation turnover rates were high in the distal promoter and the transcribed regions, but low on the proximal promoter. On the basis of these results, we propose a model with three levels of stimulus-induced histone modifications that collectively adjust promoter activity. The results support a charge neutralization model for the distal promoter and a stimulus-mediated, but transcription-independent, histone acetylation pattern on the core promoter, which might be part of a more complex histone code.

Figure 1.—

Histone acetylation profile of the C₄-Pepc gene. (A) Gene structure survey (intron line and exon block diagram). Shaded area, proximal promoter (−600 to +1 bp); solid vertical line, TIS; 1–10, exons 1–10; P, polyadenylation site; I, intergenic region. Numbers on x-axis represent the position in base pairs relative to the transcription initiation site. (B–H) Amounts of chromatin precipitated with antibodies specific for tri- and tetra-acetylated isoforms of histone H4 (H4Hyp), histone H4 lysine 5 acetylation (H4K5ac), histone H4 lysine 16 acetylation (H4K16ac), histone H3 lysine 9 acetylation (H3K9ac), histone H3 lysine 14 acetylation (H3K14ac), histone H3 lysine 18 acetylation (H3K18ac), or an invariant C-terminal epitope on histone H3 (H3C) in etiolated (dashed line) or illuminated (solid line) leaves. Dotted lines represent background precipitation with an unrelated serum. (I and J) Amounts of chromatin precipitated with antibodies as specified above on the telomere and the Actin-1 loci in illuminated (solid columns) and etiolated (shaded columns) leaves. (K–Q) Amounts of chromatin precipitated with antibodies as specified above in TSA-treated (solid line) and control (dashed line) leaves. (R and S) Amounts of chromatin precipitated with antibodies as specified above on the telomere and the Actin-1 loci in TSA-treated (shaded columns) and control (solid columns) leaves. Data points shown in B–J are based on four independent experiments; data points shown in K–S are based on two independent experiments. Vertical lines indicate standard errors.

Figure 2.—

Light-mediated histone acetylation. (A) Comparison of histone acetylation in illuminated (solid columns), etiolated (shaded columns), and re-etiolated (open columns) leaves at a representative position (−200 bp) in the proximal promoter of C₄-Pepc. For all other tested positions, see supplemental Figure S2. Amount of chromatin precipitated with antibodies is as specified in Figure 1.

Figure 3.—

Comparison of histone acetylation in illuminated leaves (solid columns), etiolated coleoptiles (darkly shaded columns), illuminated coleoptiles (lightly shaded columns), and roots (open columns) at a representative position (−200 bp) in the proximal promoter of C₄-Pepc. For all other tested positions, see supplemental Figure S3. Due to the generally enhanced precipitation efficiency in coleoptiles compared to leaves, the amount of chromatin precipitated with the antibodies was standardized for the amount of Actin-1 promoter chromatin precipitated with the same antibody. Data points are based on four independent experiments. Vertical lines indicate standard errors.

Figure 4.—

Histone acetylation after inhibition of transcription with α-amanitin. Amount of chromatin precipitated with an antibody specific for (A) tri- and tetra-acetylated isoforms of histone H4 (H4Hyp) or (B) histone H3 lysine 9 acetylation (H3K9ac) in α-amanitin-treated (solid columns) or control leaves (open columns) on representative positions in the distal (−1600 and −1300 bp) or proximal (−200 bp) promoter at the beginning (450 bp), middle (1900 bp), and end (4300 bp) of the transcribed region or at an intergenic position (5900 bp). Data points are based on at least three independent experiments. Vertical lines indicate standard errors.

Figure 5.—

Histone acetylation on the distal and proximal C₄-Pepc promoter after zeatin depletion and DOG repression. (A) Gene structure survey (intron line and exon block diagram). Shaded area, proximal promoter (−600 to +1 bp), solid vertical line, transcription initiation site; 1 and 2, exons 1 to 2. Numbers on x-axis represent the position in base pairs relative to the transcription initiation site. (B–H) Amounts of chromatin precipitated with antibodies specific for tri- and tetra-acetylated isoforms of histone H4 (H4Hyp), histone H4 lysine 5 acetylation (H4K5ac), histone H4 lysine 16 acetylation (H4K16ac), histone H3 lysine 9 acetylation (H3K9ac), histone H3 lysine 14 acetylation (H3K14ac), histone H3 lysine 18 acetylation (H3K18ac), or an invariant C-terminal epitope on histone H3 (H3C) in zeatin-depleted (dotted line), DOG-repressed (dashed line), or control leaves (solid line). Data points are based on four independent experiments. Vertical lines indicate standard errors.

Figure 6.—

Metabolic repression of promoter activity and histone acetylation. Quantification of (A) C₄-Pepc hnRNA and (B) Actin-1 mRNA in illuminated leaves after treatment with increasing DOG concentrations. Numbers are relative units of the maximum hnRNA or mRNA abundance in control leaves (0 mm DOG, open columns). (C) Histone H3 lysine 9 acetylation on each of two representative positions on the distal (−1600 and −1300 bp) and proximal (−300 and −200 bp) promoter of C₄-Pepc and on the Actin-1 promoter. Open columns, control without DOG; lightly shaded columns, 0.625 mm DOG; darkly shaded columns, 12.5 mm DOG; solid columns, 25 mm DOG. Data points are based on four independent experiments. Vertical lines indicate standard errors.

Figure 7.—

Changes in histone acetylation after metabolic repression and subsequent HDAC inhibition. Illuminated leaves were treated with 25 mm deoxyglucose (+D) or starved nitrate/zeatin (−Z) for 3 hr. Afterward, TSA was added to the solution and plants were incubated for another 3 hr. As a control, illuminated leaves were incubated in the presence (+) or absence of TSA (−) without prior DOG administration or zeatin depletion. Columns represent amounts of chromatin precipitated with antibodies specific for tri- and tetra-acetylated isoforms of histone H4 (H4Hyp) or histone H3 lysine 9 acetylation (H3K9ac) on three representative chromatin positions in the distal (−1600 and −1300 bp) and proximal (−200 bp) promoter region. Data points are based on three independent experiments. Vertical lines indicate standard errors.