Core promoter acetylation is not required for high transcription from the phosphoenolpyruvate carboxylase promoter in maize

Abstract

Background: Acetylation of promoter nucleosomes is tightly correlated and mechanistically linked to gene activity. However, transcription is not necessary for promoter acetylation. It seems, therefore, that external and endogenous stimuli control histone acetylation and by this contribute to gene regulation. Photosynthetic genes in plants are excellent models with which to study the connection between stimuli and chromatin modifications because these genes are strongly expressed and regulated by multiple stimuli that are easily manipulated. We have previously shown that acetylation of specific histone lysine residues on the photosynthetic phosphoenolpyruvate carboxylase (Pepc) promoter in maize is controlled by light and is independent of other stimuli or gene activity. Acetylation of upstream promoter regions responds to a set of other stimuli which include the nutrient availability of the plant. Here, we have extended these studies by analysing histone acetylation during the diurnal and circadian rhythm of the plant.

Results: We show that histone acetylation of individual lysine residues is removed from the core promoter before the end of the illumination period which is an indication that light is not the only factor influencing core promoter acetylation. Deacetylation is accompanied by a decrease in gene activity. Pharmacological inhibition of histone deacetylation is not sufficient to prevent transcriptional repression, indicating that deacetylation is not controlling diurnal gene regulation. Variation of the Pepc promoter activity during the day is controlled by the circadian oscillator as it is maintained under constant illumination for at least 3 days. During this period, light-induced changes in histone acetylation are completely removed from the core promoter, although the light stimulus is continuously applied. However, acetylation of most sites on upstream promoter elements follows the circadian rhythm.

Conclusion: Our results suggest a central role of upstream promoter acetylation in the quantitative regulation of gene expression in this model gene. Induced core promoter acetylation is dispensable for the highest gene expression in the diurnal and circadian rhythm.

Figure 1

Diurnal pattern of phosphoenolpyruvate carboxylase (Pepc). Quantification of (A) Pepc hnRNA and (B) Pepc mRNA through one day/night cycle. Numbers are units relative to levels in leaves harvested 4 h after illumination. The orange line shows the transcription level in re-etiolated leaves (lowest Pepc promoter activity) for comparison. (C) Comparison of hnRNA levels and RNA Polymerase II abundance in the Pepc coding region. Data points are based on at least three independent experiments. Vertical lines indicate standard errors.

Figure 2

Diurnal histone acetylation pattern on the upstream and core phosphoenolpyruvate carboxylase (Pepc) promoter. (A) Transcription levels, (B) nucleosome density and (C) histone acetylation in leaves 4 h after illumination (dark green columns), 16 h after illumination (light green columns), or re-etiolated leaves (orange columns), respectively. Values are H3K9, H3K14, H3K18, H3K23, H3K27, H4K5 and H4K16 histone acetylation levels on an upstream (-1300 bp) and a core (-200 bp) promoter position of Pepc. Data are standardized for acetylation levels on the Actin-1 promoter. For better orientation, the 1.0 level is emphasized by a black line. H3C = chromatin precipitated with an antibody to an invariant epitope on histone H3 as an indicator for nucleosome density. Zein = Intergenic region within the Zein gene cluster. Data points are based on four independent experiments. Vertical lines indicate standard errors.

Figure 3

Diurnal transcription after HDAC inhibition. (A) Quantification of phosphoenolpyruvate carboxylase (Pepc) heterogeneous nuclear RNA after treatment with the histone deacetylase inhibitor Trichostatin A. Numbers are values relative to leaves from plants 4 h after illumination. (B) H4K5 acetylation at an upstream (-1300 bp) and a core (-200 bp) promoter position of Pepc. Black columns = leaves from plants 4 h after illumination; light grey columns = leaves from plants 16 h after illumination. Data points are based on four independent experiments. Vertical lines indicate standard errors.

Figure 4

Circadian transcription profile of phosphoenolpyruvate carboxylase (Pepc). Quantification of (A) Pepc hnRNA and (B) Pepc mRNA during 56 hours of constant illumination. After a normal 16 h light period, illumination was extended for an additional 40 h without any dark period or temperature shift. Dark periods during previous growth are symbolized by the light grey bar under each chart. Numbers are values relative to transcription levels in leaves harvested 4 hours after illumination. Data points are based on four independent experiments. Vertical lines indicate standard errors.

Figure 5

Circadian histone acetylation profile of the upstream and core phosphoenolpyruvate carboxylase (Pepc) promoter. (A) Transcription levels and (B) histone acetylation under constant illumination (free-running conditions). After a normal 16 h light period, illumination was extended for an additional 40 h without any dark period or temperature shift. Dark periods during previous growth are symbolized by the light grey bar under each chart. The amount of Pepc heterogeneous nuclear RNA was standardized for the amount of Actin-1 mRNA (A). The amount of promoter chromatin precipitated with the antibody indicated in the figure was standardized for the amount of Actin-1 promoter chromatin precipitated with the same antibody. For a better orientation, the 1.0 level is emphasized by a black line. Data for an upstream (-1300 bp) and a core promoter (-200 bp) position are shown. Data points are based on four independent experiments. Vertical lines indicate standard errors.