The maize C₄-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.

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EUKARYOTIC genes respond to multiple endogenous and environmental signals, which are integrated on the promoter to control gene expression. The C₄-specific phosphoenolpyruvate carboxylase (C₄-Pepc) gene occupies a central position in primary carbon acquisition in C₄ plants such as maize (KANAI and EDWARDS 1999). C₄-Pepc is expressed in an organ- and cell-type-specific manner, is inducible by light, and is regulated by nutrient availability and the metabolic state of the cell. Thus, C₄-Pepc is an excellent gene model for studying the integrative function of promoters.

The C₄-Pepc promoter has been studied extensively, and cis-acting elements have been identified through protein-binding studies and promoter–reporter fusions in transgenic lines. YANAGISAWA and SHEEN (1998) identified binding sites for DOF (DNA binding with one finger) transcription factors that are important for leaf specificity and induction by light, 700 and 200 bp upstream from the transcription initiation site. Accordingly, a promoter fragment spanning nucleotides –570 to +81 was sufficient for restricting gene expression to the leaf mesophyll in transgenic plants (TANIGUCHI et al. 2000). In transient assays of isolated mesophyll cells, even 300 bp of promoter sequence was sufficient for strong reporter gene expression (SCHAEFFNER and SHEEN 1992).

Although cis-acting DNA elements are important for gene regulation, chromatin configuration also plays a vital role. The basic repeat structure of chromatin is the nucleosome, comprising 147 bp of DNA wound around an octamer containing two copies each of histones H2A, H2B, H3, and H4. Histone proteins have N-terminal tails that can be modified in various ways, with acetylation, methylation, and phosphorylation of histones H3 and H4 being the best characterized (KOUZARIDES 2007). The connection between histone lysine acetylation and gene expression was established 40 years ago by in vitro transcription studies (ALLFREY et al. 1964). It is now generally recognized that there is a positive correlation between the degree of histone acetylation and transcriptional activity throughout the genome. Conversely, the chromatin on transcriptionally inactive genes is mostly hypoacetylated (PFLUGER and WAGNER 2007). The N-terminal tail of histone H3 is primarily acetylated at lysines 9 (H3K9), 14 (H3K14), and 18 (H3K18), while that of H4 is acetylated at lysines 5 (H4K5), 8 (H4K8), 12 (H4K12), and 16 (H4K16). Additional acetylation sites exist on both histones, but their significance and function are mostly unknown (KURDISTANI et al. 2004; ZHANG et al. 2007). A simple model for the function of histone acetylation suggests that acetylation neutralizes the positive charge on lysine side chains and thereby reduces interaction with the negatively charged DNA backbone, allowing transcription factors better access to the DNA (IMHOF and WOLFFE 1998; DION et al. 2005). Additionally, specific triggers might store information on genes in the form of histone modification patterns that are read out by transcription factors and/or the transcription machinery. Such patterns have to be established autonomously from the final decision about gene transcription (TURNER 2007). Dependent on the crosstalk between individual modifications and the complexity of modification patterns, this signature is often referred to as a "histone code" (JENUWEIN and ALLIS 2001) or a "histone language" (BERGER 2007). Experiments in plants have documented significant regulatory complexity of histone acetylation (CHEN and TIAN 2007). Important examples are the different histone lysine residues acetylated during the potentiation and activation of the phaseolin promoter (NG et al. 2006). Moreover, acetylation patterns differ between the promoter and the transcribed region of the pea plastocyanin gene (CHUA et al. 2001), and histone acetylation contributes differentially to the two induction phases of submergence-responsive genes in rice (TSUJI et al. 2006).

We have recently shown that illumination is sufficient to trigger hyperacetylation of the N-terminal tail of histone H4 in the core promoter region of C₄-Pepc and that this occurs independently of leaf cell type and nitrogen availability (OFFERMANN et al. 2006). Furthermore, mesophyll-specific expression is associated with methylation of lysine 4 on histone H3, but not with changes in acetylation (DANKER et al. 2008). Here, we report the extension of these studies to a distal promoter region and record the modification of individual H3 and H4 lysine residues at high resolution in different organs and under diverse treatments that modulate gene activity. Our results indicate that distal promoter acetylation plays an important quantitative role in transcriptional regulation. However, acetylation in the proximal promoter and the transcribed region is strongly resistant to changes in the transcriptional state, and only selective acetylation sites are modified in response to light. The relative contribution of histone-modifying enzymes to the establishment of this pattern depends on the lysine residue and its position within the gene. The combinatorial complexity of acetylation patterns reveals specific functions for individual lysine residues that are interpreted in a position-dependent manner.

Sequences:

The C₄-Pepc gene sequence was derived from GenBank accession gi 22396 (MATSUOKA and MINAMI 1989). BAC clone gi 116268332 was used to add 5' and 3' sequence elements. The Actin-1 promoter was described by HARING et al. (2007). Subtelomeric maize sequences were derived from BURR et al. (1992).

Plant material and growth conditions:

Maize (Zea mays cv. Montello) was cultivated in growth chambers with a 16-hr photoperiod and a day/night temperature regime of 25°/20°. The plants were illuminated with Osram Superstar HQI-T 400W/DH lamps at a photon flux density of 120–180 µmol m^–2 s^–1. Seedlings were grown in soil (VM, Einheitserde, Sinntal-Jossa, Germany) for 8–10 days with varying light–dark regimes (as indicated in the figures) or in complete darkness. Re-etiolated plants were derived from illuminated plants subjected to total darkness for 72 hr.

Tissue preparation and crosslinking:

Roots were isolated from soil and washed extensively before crosslinking. Coleoptiles were manually removed from 5- to 10-day-old illuminated or etiolated seedlings. Foliar leaves were harvested and crosslinked as described previously (OFFERMANN et al. 2006) with the following modifications: 12 g of leaf material yielding sufficient chromatin for 16 precipitations under the conditions described below were vacuum infiltrated in 200 ml crosslink buffer in a 250-ml vacuum flask for 5 min. Subsequently, glycine was added to a final concentration of 125 mM, and leaves were incubated for an additional 5 min to stop the reaction. Coleoptiles (24 g) or roots (48 g) were cut into 5-mm pieces and crosslinked without vacuum for 10 min at 4° on a rotating wheel. More tissue was required to yield the same amount of chromatin that was isolated from 12 g of leaves.

Trichostatin A/-amanitin/zeatin/2-deoxyglucose treatments:

After 4 hr of illumination, 10- to 12-day-old leaves were detached under water 1 cm above the laminar joint and incubated for 3 hr (8 hr for -amanitin experiments) in solutions containing 300 µM trichostatin A (TSA) (International Clinical Services, Munich), 10 µM -amanitin, or 2-deoxyglucose (DOG) at varying concentrations as indicated in the figures, in combination with 5 µM trans-zeatin (all Sigma-Aldrich, Schnelldorf, Germany) and 16 mM KNO₃^– (SUGIHARTO et al. 1992) in tap water. Leaves for zeatin-depletion experiments were incubated in tap water only.

Chromatin immunoprecipitation:

Chromatin immunoprecipitation was performed as described by BOWLER et al. (2004) with modifications as described in OFFERMANN et al. (2006) and HARING et al. (2007). Chromatin was sheared with a Bioruptor (Diagenode, Liege, Belgium) for 10 min (setting: high, interval 30/30 sec) under constant cooling. Modified histones were detected with 5 µl anti-hyperacetylated histone H4 (Upstate 06-946, Millipore, Schwalbach, Germany), 5 µl anti-acetyl H4K5 (Upstate 07-327), 5 µl anti-acetyl H4K16 (Upstate 07-329), 5 µl anti-acetyl H3K9 (Upstate 07-352), 1 µl anti-acetyl H3K14 (Upstate 07-353), 1 µl anti-acetyl H3K18 (Upstate 07-354), and 1 µl anti-H3 C-term (ab1791, Abcam, Cambridge, UK). We also tested anti-acetyl H4K8 (Upstate 07-328 and Abcam ab 1760) and anti-acetyl H4K12 (Upstate 07-595 and Abcam ab 1761), but failed to precipitate significant amounts. The control serum for determination of background precipitation levels was derived from rabbits immunized with an unrelated protein from potato. In general, background signals never exceeded 10% of positive control levels and are therefore shown only in Figure 1.

View larger version (51K): In this window In a new window Download PPT slide   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.

 

RNA preparation and reverse transcription:

RNA isolation and reverse transcription were performed as described in OFFERMANN et al. (2006). As a direct estimate for promoter activity (DELANY 2001; REED 2003), heterogeneous nuclear RNAs (hnRNAs) were amplified from cDNA with a primer system specific for an intron. DNA synthesis in the absence of reverse transcriptase was used to exclude amplification from residual DNA contamination. A dilution series of illuminated leaf cDNA was used as a standard. Oligonucleotide sequences and conditions are given in supplemental Table 1.

Quantitative real-time PCR:

Quantitative PCR was performed on an ABI PRISM 7000 (Applied Biosystems) using SYBR Green fluorescence (Platinum SYBR Green qPCR Mix, Invitrogen) for detection. Oligonucleotide sequences and conditions are given in supplemental Table 1.

Light-induced histone acetylation on the C₄-Pepc gene:

We recently showed that light induces hyperacetylation of histone H4 (H4Hyp) on the C₄-Pepc gene in maize, independent of gene transcription (OFFERMANN et al. 2006). To further define the role of histone modification on C₄-Pepc expression, acetylation of five (H4K5, H4K16, H3K9, H3K14, H3K18) of the seven lysine residues functionally characterized to date was examined in etiolated and illuminated plants. Acetylation of H4K8 and H4K12 was not detectable even when genome hyperacetylation was artificially induced by treatment with the histone deacetylase (HDAC) inhibitor, trichostatin A (see also below). Experiments were performed at high resolution for the promoter region, at selected positions of the transcribed region, and at a position just downstream of the polyadenylation site (I). According to TANIGUCHI et al. (2000), the C₄-Pepc promoter sequence was separated into an essential proximal (–600 to +1 bp) and a distal region located farther upstream that is not essential for transcriptional activity in a transient assay system (Figure 1A). The amounts of precipitate are presented as the percentage of input material to avoid any artifacts caused by standardization (HARING et al. 2007). Results for a heterochromatic subtelomeric control region with low acetylation levels and for the constitutively active Actin-1 promoter where high acetylation was expected are shown in Figure 1, I and J.

As shown in Figure 1B, the light induction of H4Hyp could be reproduced in this assay. High acetylation of C₄-Pepc in illuminated plants was limited to the gene region and low levels were detected directly upstream from the promoter and downstream from the putative transcript polyadenylation site. Within the gene, acetylation was highest in the promoter region, and the levels dropped sharply by a factor of 5 directly behind the transcription initiation site (TIS) and recovered toward the end of the gene. When individual lysines were tested, a clear light induction of acetylation (between 3- and 10-fold depending on the position) was detectable for H4K5 (Figure 1C) and H3K9 (Figure 1E). The highest signals were again found on the promoter, with individual distribution profiles of the two modifications. H3K9 peaked in the distal part around position –2000 bp, whereas more uniform levels were detected for H4K5. The reduced acetylation described for H4Hyp in the first part of the transcribed region was also evident for these two lysines.

In contrast, acetylation of H3K18 and H4K16 was seemingly uninfluenced by light, but rather already present in etiolated leaves (Figure 1, D and G). The degree of histone acetylation was uniformly distributed over the gene,, but reduction in acetylation behind the TIS was also apparent for these modification sites. For H3K14 (Figure 1F), intermediate results were obtained with a weak light induction (less than threefold) in the most distal promoter region. Levels in the proximal promoter and the transcribed region were almost unchanged.

Precipitation with an antibody directed to an invariant domain of the C-terminal part of histone H3 (H3C) that is not subject to covalent modification was used as a measure of nucleosome occupancy on the gene (Figure 1H). In neither the promoter nor the transcribed region did nucleosome occupancy change significantly upon illumination. In both etiolated and illuminated plants, the most obvious characteristic of the H3C distribution curve was a surprising threefold increase at the start of the transcribed region, exactly where the lowest acetylation levels were found. Thus, the low acetylation in this gene region was even more pronounced when calculated relative to the number of nucleosomes that are present in the highest numbers in this region.

These results indicate the presence of both light-dependent and -independent lysine acetylation profiles throughout the C₄-Pepc gene.

Trichostatin A treatment of illuminated leaves:

We also tested the maximum inducible acetylation levels after treatment of illuminated plants with the HDAC inhibitor, trichostatin A. TSA inhibits class I and class II HDACs, but not class III HDACs (YOSHIDA et al. 1990; IMAI et al. 2000). TSA treatment induces enhanced acetylation if both a histone acetyltransferase (HAT) and a class I or class II HDAC are active at the tested gene position. For almost all tested acetylations, clear increases after TSA treatment were observed in both the promoter and the transcribed region, indicating that class I and class II HDACs control acetylation levels on the C₄-Pepc gene even in illuminated plants where the gene shows maximal activity. The amplitude of TSA induction was always lower on the proximal promoter compared to the distal promoter. H3K9 acetylation (Figure 1N) on the distal promoter constituted a notable exception, because levels were even slightly lower after TSA treatment (as compared to untreated plants), suggesting that class I or class II HDAC activity did not contribute to the regulation of this modification in illuminated plants.

In most cases, acetylation remained limited to the gene region even after TSA treatment indicating that the corresponding HATs were recruited to the promoter and the transcribed region and are absent outside of the gene. However, H3K14 and H3K18 showed significant induction of acetylation after TSA inhibition of HDAC activity even downstream from the transcribed region. These two modifications are even inducible by TSA treatment in the subtelomeric negative control region (Figure 1R), suggesting that HATs for these modifications are not specifically recruited to gene regions.

We also tested whether nucleosome occupancy is directly influenced by the acetylation status. Precipitation efficiency with the H3C antibody (Figure 1Q) was only slightly enhanced (less than twofold) in TSA-treated plants. Within the proximal promoter, this parameter remained completely uninfluenced by TSA.

These results support the observation that each specific acetylation is individually controlled with respect to absolute levels and the relative distribution over the gene. The relative contribution of HAT and HDAC activities to the adjustment of acetylation levels also depends on the lysine tested.

Re-etiolation of illuminated plants:

When maize plants are returned to the dark for extended periods after an initial illumination (re-etiolated plants), C₄-Pepc promoter activity drops to levels clearly below those observed in etiolated plants, but the light induction of transcription after a second illumination is faster (OFFERMANN et al. 2006). We wondered whether histone modifications induced by previous illumination were maintained on the gene upon transfer to the dark and therefore compared acetylation levels in illuminated, etiolated, and re-etiolated plants on seven selected gene positions. Figure 2 shows results for the core promoter representative of the other positions (shown in full in supplemental Figure S2). We found no clear differences in histone modification between the etiolated and re-etiolated states, excluding a role of the tested histone acetylation sites in light memory and preparation of the promoter for rapid reactivation in response to re-illumination.

View larger version (16K): In this window In a new window Download PPT slide   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.

 

Tissue-specific acetylation on the C₄-Pepc gene:

C₄-Pepc transcription is restricted to leaves and absent in other maize tissues. The contribution of histone acetylation to the regulation of this tissue specificity was tested by comparing acetylation levels in leaves and roots. However, roots are underground systems that are normally not exposed to light and might not express factors necessary for a light response. To further elucidate light responses and tissue-specific patterns, we also included coleoptiles in our analyses. These leaf-like organs respond to illumination by inducing chloroplast development and photosynthesis, but do not show the high bundle density typical of C₄ leaves and, consequently, do not express C₄-Pepc (LANGDALE et al. 1988; supplemental Figure S1A). Comparison of acetylation levels in the different tissues was hampered by dissimilar overall precipitation efficiencies, probably due to differences in crosslinking (data not shown). The results were therefore standardized for acetylation levels on the Actin-1 promoter because this gene showed comparable activities in the tested tissues (supplemental Figure S1A). Figure 3 shows results for one representative core promoter position, and six additional positions are shown in supplemental Figure S3. Acetylation of H3K14, H3K18, and H4K16 was reduced by <2-fold at most tested positions in both coleoptiles and roots, as compared to leaves. However, we cannot exclude the possibility that these minor changes are caused, at least in part, by the standardization method (see also DISCUSSION). More dramatic differences were observed for H4K5 (5-fold), H4Hyp (5-fold), and H3K9 (10-fold). Importantly, acetylation of these residues in coleoptiles did not increase after illumination, as has been observed before in leaves (Figure 1), suggesting that the potential for light-inducible acetylation is an important property of C₄-Pepc chromatin in leaves.

View larger version (17K): In this window In a new window Download PPT slide   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.

 

Response to inhibition of transcription:

The low levels of acetylation observed for light-regulated lysines in etiolated leaves, coleoptiles, and roots might either be directly controlled by endogenous and environmental signals or be an indirect consequence of the low transcription rates. We treated illuminated leaves with the transcription inhibitor -amanitin to discriminate between these two possibilities. C₄-Pepc promoter activity was at the detection limit 8 hr after inhibitor application (supplemental Figure S1B). The corresponding levels of H4 hyperacetylation and H3K9 acetylation at seven tested gene positions are shown in Figure 4. While acetylation remained constant on the promoter, it increased in the transcribed region after transcriptional inhibition. This indicates that low acetylation levels of these sites were not caused by low transcription rates, but were, rather, a consequence of the absence of activating stimuli.

View larger version (14K): In this window In a new window Download PPT slide   FIGURE 4.— Histone acetylation after inhibition of transcription with -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 -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.

 

Histone acetylation in response to metabolic stimuli:

In addition to its tissue specificity and light induction, the C₄-Pepc gene is also under metabolic control. Its activity is strongly regulated by nitrogen availability (transduced by the hormone zeatin) and the intracellular hexose concentration (JANG and SHEEN 1994; SUZUKI et al. 1994). We have previously shown that H4 hyperacetylation on the proximal promoter is independent of nitrogen availability and, therefore, does not change in detached leaves starved of nitrate and zeatin (OFFERMANN et al. 2006). Here, we extended the original study to cover the complete promoter and analyzed individual acetylation sites in detail. Furthermore, we included treatments with DOG, which mimics high hexose availability (JANG and SHEEN 1994). To measure transcription rates, C₄-Pepc hnRNA accumulation was monitored as described in MATERIALS AND METHODS. Transcription was strongly reduced by nitrate/zeatin depletion, while DOG-treated plants showed intermediate transcription levels (supplemental Figure S1C). On the proximal promoter and at the beginning of the transcribed region, the degree of acetylation stayed remarkably constant for all tested lysines independent of the treatment (Figure 5, B–H). However, on the distal promoter, acetylation of all tested lysines was reduced by three- to fivefold in detached plants that did not receive the nitrate/zeatin stimulus. This effect was also observed for DOG-treated plants, but the impact on acetylation levels was clearly weaker. To further test whether this weaker effect reflected incomplete promoter repression in these plants, we treated detached leaves with increasing concentrations of DOG and measured H3K9 acetylation. The high signal amplitude obtained with the corresponding antibody facilitated quantitative analyses. As shown in Figure 6A, C₄-Pepc promoter activity decreased with increasing DOG concentrations, but the control Actin-1 mRNA levels were not affected (Figure 6B). Interestingly, a gradual decrease in acetylation was evident at the two distal promoter sites that we tested (–1600 and –1300 bp), while the proximal promoter (–300 and –200 bp) or the Actin-1 promoter showed no response (Figure 6C). The data indicate that distal and proximal promoter acetylation in response to metabolic stimuli is differentially regulated. On the distal promoter, all tested acetylation sites are coregulated and the degree of acetylation is indicative of promoter activity. In contrast, acetylation on the proximal promoter is surprisingly resistant to manipulations mimicking changes in nitrogen and hexose availability.

View larger version (26K): In this window In a new window Download PPT slide   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.

 

View larger version (19K): In this window In a new window Download PPT slide   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.

 

Contribution of HDAC activity to the metabolite response:

Acetylation of H4K5 and H3K9 on the distal promoter is enhanced by illumination, but reduced by metabolic stimuli (see above). Our previous results indicated that light induction is controlled mainly by a reduction in HDAC activity, since TSA treatment of etiolated leaves in the dark is sufficient to induce acetylation levels comparable to the illuminated state (OFFERMANN et al. 2006). Furthermore, TSA-sensitive HDACs for H3K9 on the distal promoter are fully inactivated in illuminated plants (Figure 1N). We next carried out experiments to determine whether the responses to metabolic stimuli are regulated by the same mechanisms as the light response. As above, detached leaves were depleted of nitrogen/zeatin or treated with DOG. This treatment reduced distal promoter acetylation (Figures 5 and 6). After 3 hr, TSA was added to inhibit HDAC activity. If metabolic repression induces HDAC activity, which, in turn, leads to reduced acetylation, then HDAC inhibition should reverse this effect. In contrast, if reduced HAT activity is causing reduced acetylation, the levels should stay low even after HDAC inhibition.

Figure 7 shows the results for two modifications (H4Hyp and H3K9ac) on two positions on the distal promoter and a control position on the proximal promoter. H4Hyp antibodies were used instead of H4K5ac antibodies because of the very similar responses recorded with both antibodies in all previous experiments and the stronger signals obtained with the H4Hyp antibody. TSA treatment alone enhanced histone acetylation, with the exception of H3K9 acetylation on the distal promoter, as expected (see also Figure 1). In leaves that did not receive the nitrogen/zeatin treatment (–Z) or that were treated with deoxyglucose (+D) before HDAC inhibition, H4 hyperacetylation on the distal promoter was unaffected, but the level of H3K9 acetylation was reduced to less than half of the control. On the proximal promoter (–200), metabolic signals did not affect TSA-induced acetylation. This was expected because both tested modifications were not influenced by metabolic signals here (Figure 5). This result indicates that metabolic signals affect H3K9 acetylation on the distal promoter by modulating HAT activity because HDAC inhibition is not sufficient to revert the reduction in acetylation induced by the –Z and +D treatments. In contrast, the negative metabolite response of H4 hyperacetylation is apparently controlled by enhanced HDAC activity.

View larger version (23K): In this window In a new window Download PPT slide   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.

 

Three levels of stimulus-induced histone modifications on the C₄-Pepc gene:

We have previously shown that light induces hyperacetylation of histone H4 on the proximal promoter independently of other stimuli such as cell-type specificity and nutrient availability (OFFERMANN et al. 2006). Surprisingly, analyses of individual acetylation sites in this study revealed that H3K9 and H4K5 acetylation are selectively induced by light, whereas H3K14, H3K18, and H4K16 acetylation is light independent (Figure 1). The light-induced pattern is fully reversible in plants kept in the dark for prolonged periods, so no memory of previous illumination is stored at the level of histone acetylation. Since the two outermost lysine residues on the N-terminal tails of H3 and H4 are subject to light regulation, our data agree with the recent observation that H4 acetylation occurs in a progressive fashion from the body of the histone to the N terminus (EARLEY et al. 2007).

We speculated that acetylation of H3K14, H3K18, and H4K16 in etiolated leaves is involved in the formation of a poised chromatin state that allows for a fast transcriptional response to illumination. Such poised states where some, but not all, chromatin modifications associated with full gene activity are set before transcription activation have been described, e.g., for the bean phaseolin promoter (NG et al. 2006) and the pea plastocyanin gene (CHUA et al. 2001). Consequently, we expected these modifications to be absent in roots and coleoptiles, where the C₄-Pepc gene is constitutively inactive. However, significant levels were detected for all three modifications in the tested tissues. Such comparative analyses of chromatin modifications in different tissues in planta are complicated by the fact that the crosslinking efficiency and the quality of the isolated chromatin may differ between the tissues (HARING et al. 2007). Despite optimization of the experimental procedure, we always observed lower precipitation efficiencies from root chromatin and very high efficiencies from coleoptile chromatin (data not shown). We standardized our data for the level of acetylation on the Actin-1 promoter because this gene is transcribed at very similar levels in the tested tissues. Still, this does not guarantee identical acetylation levels. On the basis of this standardization method, acetylation of H3K14, H3K18, and H4K16 was reduced to approximately half in roots and coleoptiles as compared to leaves, but such slight reductions do not necessarily indicate factual differences between the tissues. Thus, acetylation of these residues does not seem to be associated with potentiating the gene for activation in etiolated leaves, but is, rather, a general property of C₄-Pepc chromatin in all tissues. Supportive of our observation, recent analyses in several organisms have suggested that H4K16 is controlled independently of other acetylation sites and constitutively acetylated in euchromatin (DION et al. 2005; BENHAMED et al. 2006; SHOGREN-KNAAK et al. 2006; ROSSI et al. 2007). Distinct from our results, H3K18 acetylation is usually associated with full gene activity on a genomewide basis in yeast, whereas H3K14 acetylation correlates only weakly with the transcriptional state (KURDISTANI et al. 2004; LIU et al. 2005).

The most obvious difference between coleoptiles and foliar leaves was the inability of coleoptiles to induce H3K9 and H4K5 acetylation upon illumination (Figure 3). Although factors necessary for light response are generally present in coleoptiles (LANGDALE et al. 1988), there was almost no change in histone acetylation in the proximal promoter region of C₄-Pepc. The presence of HATs responsible for acetylation of H4K5 and H3K9 is therefore the most evident unique property of C₄-Pepc chromatin in leaves as compared to other tissues.

Within leaves, C₄-Pepc is transcribed in mesophyll cells, but not bundle sheath cells (KAUSCH et al. 2001). However, at least in the case of H4 acetylation on the proximal promoter, light induction takes place in both cell types (OFFERMANN et al. 2006). Thus, the presence of specific HATs for light-inducible modifications might control leaf-organ specificity, but this signal is differentially interpreted in the context of other histone modifications in different cell types within the leaf. In accordance with this hypothesis, we have recently identified a shift from dimethylation to trimethylation of H3K4 on the C₄-Pepc gene, unique to mesophyll cells, that was not influenced by illumination, but instead was controlled by cell lineage (DANKER et al. 2008). Cell-type-specific histone methylation was restricted to the proximal promoter and, therefore, differed in its distribution from the light-induced acetylation that is also found in the distal promoter region (Figure 1). We conclude that histone methylation defines cell-type specificity within the leaf independently of the acetylation state.

The strong light-induced expression of C₄-Pepc in mesophyll cells of leaves is further adjusted according to the availability of nitrogen and hexose sugars (JANG and SHEEN 1994; SUZUKI et al. 1994). When manipulating these stimuli, the treatments exclusively modulated the acetylation state of the distal promoter region. Here, all tested acetylation sites were affected (Figure 5), and the degree of acetylation corresponded to the transcription rate (Figure 6). The data suggest the presence of a third independent control element for metabolite responses in the distal promoter, in addition to the factors controlling leaf and mesophyll specificity. This third element is near to a previously identified DNA methylation site that is demethylated upon gene activation (LANGDALE et al. 1991), providing independent evidence that this distant region is involved in regulation of the C₄-Pepc gene.

All the treatments that we applied influenced not only C₄-Pepc histone acetylation, but also the transcription rate. However, experiments in the presence of the RNA polymerase II inhibitor, -amanitin, showed that even complete inhibition of transcription did not affect promoter acetylation (Figure 4), resembling the highly stable histone modifications in the promoters of mammalian housekeeping genes (KOUSKOUTI and TALIANIDIS 2005). The only chromatin modifications on the C₄-Pepc gene shown thus far to be responsive to transcriptional inhibition are limited to the transcribed region and involve H3K4 trimethylation, which decreases (DANKER et al. 2008), and histone acetylation, which increases (Figure 4). The latter effect is likely related to the active deacetylation of histones during transcription elongation, which prevents spurious initiation within the transcribed region (LEE and SHILATIFARD 2007; LI et al. 2007). Therefore, the observed changes in acetylation under diverse treatments are induced directly by the respective stimulus and are not caused indirectly by low transcription rates.

Regulation of histone acetylation on the C₄-Pepc gene by HAT and HDAC enzymes:

The response of individual acetylation sites to TSA inhibition of HDAC activity allowed us to determine whether or not the same enzymes regulated acetylation of the different residues. If HDAC inhibition is sufficient to induce increased acetylation, then we assume that the corresponding HAT should be present and active in the tested genomic region, but a HDAC counteracts high acetylation. Class III HDACs are not sensitive to TSA (IMAI et al. 2000). However, in general, TSA treatment strongly induced histone acetylation throughout the C₄-Pepc gene (Figure 1), suggesting that class I and class II enzymes contribute the majority of HDAC activity on this gene. We used this concept to determine how acetylation is restricted to the gene region (Figure 1), under which conditions HAT or HDAC activities control acetylation of individual sites (Figure 7), and whether the acetylation of a specific lysine residue in a defined gene region can be controlled by different mechanisms, depending on the stimulus (Figures 1 and 7).

Our results show that acetylation of H3K14 and H3K18 increased following TSA treatment on the gene borders and even on subtelomeric DNA, where these modifications are normally rare (Figure 1), suggesting that restriction of these modifications to the promoter and the transcribed region is controlled by high HDAC activity outside the gene. In contrast, H3K9, H4K5, and H4K16 modification remained restricted to the promoter and the transcribed region in TSA-treated plants. Thus, the HATs for these sites are recruited to the gene and absent on the gene borders. Interestingly, we have recently shown that H3K9 dimethylation occurs at sites flanking the gene. This modification is also found at high levels in the acetylation gap behind the TIS (DANKER et al. 2008), supporting the conclusion that H3K9 methylation has a negative effect on histone acetylation, as described on a genomewide basis for mice (WU et al. 2007).

Despite the differences on the gene borders, HDAC inhibition increased acetylation of all tested lysines within the gene (with the exception of H3K9 acetylation on the distal promoter, which is discussed below). TSA had greater effects in the distal compared to the proximal promoter regions. This effect was most pronounced for those lysines that were not acetylated in response to light but, rather, acetylated in etiolated plants, indicating that the level of inducibility by TSA treatment correlates to the degree of regulation by diverse stimuli. It has been suggested that the chromatin of active or poised genes (prepared for transcription) is characterized by a high turnover of acetylation rather than by high absolute acetylation levels (SPENCER and DAVIE 2001; HAZZALIN and MAHADEVAN 2005). The relative increase in acetylation following exposure to TSA was used here as an indicator of the dynamics of acetylation because it reveals the contribution of HDAC and HAT activities to steady-state acetylation (CLAYTON et al. 2006). On the basis of this approach, regulated sites on the C₄-Pepc gene (H4K5 and H3K9 on the proximal promoter and all lysines on the distal promoter) tend to cycle between acetylation and deacetylation more rapidly. This rapid turnover of acetyl groups might allow prompt responses to changes in environmental and endogenous stimuli.

To define stimulus-specific characteristics of the regulation of acetylation, we further combined metabolic stimuli with TSA treatments. Our analysis of H3K9 acetylation on the distal promoter illustrated that acetylation of a specific lysine at a defined gene position can be differentially regulated according to the stimulus. On the one hand, acetylation in light-grown plants could not be enhanced by TSA, suggesting that light completely inactivated the corresponding HDAC (Figure 1N). On the other hand, metabolic stimuli controlled acetylation of the same lysine in the same gene region by reducing HAT activity, while H4 acetylation after metabolite treatments is, in turn, regulated by enhancing HDAC activity (Figure 7). Thus, H3 and H4 acetylation in response to the same stimulus are also regulated by different mechanisms.

In conclusion, a complex regulatory network involving multiple HATs and HDACs targeted to different sites on the gene controls histone acetylation on the C₄-Pepc gene. These results are unexpected because the mutation or overexpression of individual HATs and HDACs has wide-ranging effects on the acetylation of most lysines throughout the genome (TIAN et al. 2004; BENHAMED et al. 2006; ROSSI et al. 2007). In contrast, HDACs in rice are themselves expressed in a tissue-specific and environmentally regulated pattern (FU et al. 2007). A recent analysis of co-activator HATs in Arabidopsis revealed different classes with either broad or very narrow specificities for particular lysines (EARLEY et al. 2007). On the basis of the latter observation, a broad-specificity HAT might control the response of the distal promoter to metabolic stimuli, whereas illumination regulates the activity of highly specific HDACs on the complete C₄-Pepc gene.

Our results suggest that histone acetylation contributes to C₄-Pepc regulation by mechanisms that are compatible with both the "charge neutralization" and the "histone code" models (JENUWEIN and ALLIS 2001; BERGER 2007). The coregulation of all the tested sites on the distal promoter by metabolic stimuli, and the quantitative relationship between acetylation and transcription levels, argue that distal promoter acetylation has a simple role in improving transcription factor access. In contrast, the independence of proximal promoter acetylation from transcription as well as from signals other than light, and the complex regulation of acetylation by multiple HATs and HDACs, rather favor a histone acetylation code that is interpreted depending on the gene position and in the context of other modifications.

We are grateful to Fritz Kreuzaler for continuous support; Maike Stam and Max Haring for collaboration during establishment of techniques; and Richard Twyman, Klaus Grasser, and Sandy Edwards for critical reading of the manuscript. This work was supported by grants (Pe819/1) from the Deutsche Forschungsgemeinschaft to C.P.

¹ Present address: School of Biological Sciences, Washington State University, Pullman, WA 99164-4236.

ALLFREY, V. G., R. FAULKNER and A. E. MIRSKY, 1964 Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc. Natl. Acad. Sci. USA 51: 786–794.[Free Full Text]

BENHAMED, M., C. BERTRAND, C. SERVET and D.-X. ZHOU, 2006 Arabidopsis GCN5, HD1, and TAF1/HAF2 interact to regulate histone acetylation required for light-responsive gene expression. Plant Cell 18: 2893–2903.[Abstract/Free Full Text]

BERGER, S. L., 2007 The complex language of chromatin regulation during transcription. Nature 447: 407–412.[CrossRef][Medline]

BOWLER, C., G. BENVENUTO, P. LAFLAMME, D. MOLINO, A. V. PROBST et al., 2004 Chromatin techniques for plant cells. Plant J. 39: 776–789.[CrossRef][Medline]

BURR, B., F. A. BURR, E. C. MATZ and J. ROMERO-SEVERSON, 1992 Pinning down loose ends: mapping telomeres and factors affecting their length. Plant Cell 4: 953–960.[Abstract/Free Full Text]

CHEN, Z. J., and L. TIAN, 2007 Roles of dynamic and reversible histone acetylation in plant development and polyploidy. Biochim. Biophys. Acta 1769: 295–307.[Medline]

CHUA, Y. L., A. P. BROWN and J. C. GRAY, 2001 Targeted histone acetylation and altered nuclease accessibility over short regions of the pea plastocyanin gene. Plant Cell 13: 599–612.[Abstract/Free Full Text]

CLAYTON, A. L., C. A. HAZZALIN and L. C. MAHADEVAN, 2006 Enhanced histone acetylation and transcription: a dynamic perspective. Mol. Cell 23: 289–296.[CrossRef][Medline]

DANKER, T., B. DREESEN, S. OFFERMANN, I. HORST and C. PETERHANSEL, 2008 Developmental information but not promoter activity controls the methylation state of histone H3 lysine 4 on two photosynthetic genes in maize. Plant J. 53: 465–474.[CrossRef][Medline]

DELANY, A. M., 2001 Measuring transcription of metalloproteinase genes. Nuclear run-off assay vs analysis of hnRNA. Methods Mol. Biol. 151: 321–333.[Medline]

DION, M. F., S. J. ALTSCHULER, L. F. WU and O. J. RANDO, 2005 Genomic characterization reveals a simple histone H4 acetylation code. Proc. Natl. Acad. Sci. USA 102: 5501–5506.[Abstract/Free Full Text]

EARLEY, K. W., M. S. SHOOK, B. BROWER-TOLAND, L. HICKS and C. S. PIKAARD, 2007 In vitro specificities of Arabidopsis co-activator histone acetyltransferases: implications for histone hyperacetylation in gene activation. Plant J. 52: 615–626.[CrossRef][Medline]

FU, W., K. WU and J. DUAN, 2007 Sequence and expression analysis of histone deacetylases in rice. Biochem. Biophys. Res. Commun. 356: 843–850.[CrossRef][Medline]

HARING, M., S. OFFERMANN, T. DANKER, I. HORST, C. PETERHÄNSEL et al., 2007 Chromatin immunoprecipitation: quantitative analysis and data normalization. Plant Methods 2: 11.

HAZZALIN, C. A., and L. C. MAHADEVAN, 2005 Dynamic acetylation of all lysine 4-methylated histone H3 in the mouse nucleus: analysis at c-fos and c-jun. PLoS Biol. 3: e393.[CrossRef][Medline]

IMAI, S.-I., C. M. ARMSTRONG, M. KAEBERLEIN and L. GUARENTE, 2000 Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403: 795–800.[CrossRef][Medline]

IMHOF, A., and A. P. WOLFFE, 1998 Transcription: gene control by targeted histone acetylation. Curr. Biol. 8: R422–R424.[CrossRef][Medline]

JANG, J. C., and J. SHEEN, 1994 Sugar sensing in higher plants. Plant Cell 6: 1665–1679.[Abstract]

JENUWEIN, T., and C. D. ALLIS, 2001 Translating the histone code. Science 293: 1074–1080.[Abstract/Free Full Text]

KANAI, R., and G. E. EDWARDS, 1999 The biochemistry of C₄ photosynthesis, pp. 49–87 in C₄ Plant Biology, edited by R. F. SAGE and R. K. MONSON. Academic Press, San Diego.

KAUSCH, A. P., T. P. OWEN, S. J. ZACHWIEJA, A. R. FLYNN and J. SHEEN, 2001 Mesophyll-specific, light and metabolic regulation of the C₄ PPCZm1 promoter in transgenic maize. Plant Mol. Biol. 45: 1–15.[CrossRef][Medline]

KOUSKOUTI, A., and I. TALIANIDIS, 2005 Histone modifications defining active genes persist after transcriptional and mitotic inactivation. EMBO J. 24: 347–357.[CrossRef][Medline]

KOUZARIDES, T., 2007 Chromatin modifications and their function. Cell 128: 693–705.[CrossRef][Medline]

KURDISTANI, S. K., S. TAVAZOIE and M. GRUNSTEIN, 2004 Mapping global histone acetylation patterns to gene expression. Cell 117: 721–733.[CrossRef][Medline]

LANGDALE, J. A., I. ZELITCH, E. MILLER and T. NELSON, 1988 Cell position and light influence C₄ versus C₃ patterns of photosynthetic gene expression in maize. EMBO J. 7: 3643–3652.[Medline]

LANGDALE, J. A., W. C. TAYLOR and T. NELSON, 1991 Cell-specific accumulation of maize phosphoenolpyruvate carboxylase is correlated with demethylation at a specific site > 3 kb upstream of the gene. Mol. Gen. Genet. 225: 49–55.[Medline]

LEE, J. S., and A. SHILATIFARD, 2007 A site to remember: H3K36 methylation a mark for histone deacetylation. Mutat. Res. 618: 130–134.[Medline]

LI, B., M. GOGOL, M. CAREY, D. LEE, C. SEIDEL et al., 2007 Combined action of PHD and chromo domains directs the Rpd3S HDAC to transcribed chromatin. Science 316: 1050–1054.[Abstract/Free Full Text]

LIU, C. L., T. KAPLAN, M. KIM, S. BURATOWSKI, S. L. SCHREIBER et al., 2005 Single-nucleosome mapping of histone modifications in S. cerevisiae. PLoS Biol. 3: e328.[CrossRef][Medline]

MATSUOKA, M., and E. MINAMI, 1989 Complete structure of the gene for phosphoenolpyruvate carboxylase from maize. Eur. J. Biochem. 181: 593–598.[Medline]

NG, D. W. K., M. B. CHANDRASEKHARAN and T. C. HALL, 2006 Ordered histone modifications are associated with transcriptional poising and activation of the phaseolin promoter. Plant Cell 18: 119–132.[Abstract/Free Full Text]

OFFERMANN, S., T. DANKER, D. DREYMÜLLER, R. KALAMAJKA, S. TÖPSCH et al., 2006 Illumination is necessary and sufficient to induce histone acetylation independent of transcriptional activity at the C₄-specific phosphoenolpyruvate carboxylase promoter in maize. Plant Physiol. 141: 1078–1088.[Abstract/Free Full Text]

PFLUGER, J., and D. WAGNER, 2007 Histone modifications and dynamic regulation of genome accessibility in plants. Curr. Opin. Plant Biol. 10: 645–652.[CrossRef][Medline]

REED, R., 2003 Coupling transcription, splicing and mRNA export. Curr. Opin. Cell Biol. 15: 326–331.[CrossRef][Medline]

ROSSI, V., S. LOCATELLI, S. VAROTTO, G. DONN, R. PIRONA et al., 2007 Maize histone deacetylase hda101 is involved in plant development, gene transcription, and sequence-specific modulation of histone modification of genes and repeats. Plant Cell 19: 1145–1162.[Abstract/Free Full Text]

SCHAEFFNER, A. R., and J. SHEEN, 1992 Maize C₄ photosynthesis involves differential regulation of phosphoenolpyruvate carboxylase. Plant J. 2: 221–232.[Medline]

SHOGREN-KNAAK, M., H. ISHII, J. M. SUN, M. J. PAZIN, J. R. DAVIE et al., 2006 Histone H4–K16 acetylation controls chromatin structure and protein interactions. Science 311: 844–847.[Abstract/Free Full Text]

SPENCER, V. A., and J. R. DAVIE, 2001 Dynamically acetylated histone association with transcriptionally active and competent genes in the avian adult beta-globin gene domain. J. Biol. Chem. 276: 34810–34815.[Abstract/Free Full Text]

SUGIHARTO, B., J. N. BURNELL and T. SUGIYAMA, 1992 Cytokinin is required to induce the nitrogen-dependent accumulation of mRNAs for phosphoenolpyruvate carboxylase and carbonic anhydrase in detached maize leaves. Plant Physiol. 100: 153–156.[Abstract/Free Full Text]

SUZUKI, I., C. CRETIN, T. OMATA and T. SUGIYAMA, 1994 Transcriptional and posttranscriptional regulation of nitrogen-responding expression of phosphoenolpyruvate carboxylase gene in maize. Plant Physiol. 105: 1223–1229.[Abstract]

TANIGUCHI, M., K. IZAWA, M. S. B. KU, J.-H. LIN, H. SAITO et al., 2000 Binding of cell type-specific nuclear proteins to the 5'-flanking region of maize C₄ phosphoenolpyruvate carboxylase gene confers its differential transcription in mesophyll cells. Plant Mol. Biol. 44: 543–557.[CrossRef][Medline]

TIAN, L., M. P. FONG, J. J. WANG, N. E. WEI, H. JIANG et al., 2004 Reversible histone acetylation and deacetylation mediate genome-wide, promoter-dependent and locus-specific changes in gene expression during plant development. Genetics 169: 337–345.[CrossRef][Medline]

TSUJI, H., H. SAIKA, N. TSUTSUMI, A. HIRAI and M. NAKAZONO, 2006 Dynamic and reversible changes in histone H3-Lys4 methylation and H3 acetylation occurring at submergence-inducible genes in rice. Plant Cell Physiol. 47: 995–1003.[Abstract/Free Full Text]

TURNER, B. M., 2007 Defining an epigenetic code. Nat. Cell Biol. 9: 2–6.[CrossRef][Medline]

WU, J., S. H. WANG, D. POTTER, J. C. LIU, L. T. SMITH et al., 2007 Diverse histone modifications on histone 3 lysine 9 and their relation to DNA methylation in specifying gene silencing. BMC Genomics 8: 131.[CrossRef][Medline]

YANAGISAWA, S., and J. SHEEN, 1998 Involvement of maize Dof zinc finger proteins in tissue-specific and light-regulated gene expression. Plant Cell 10: 75–89.[Abstract/Free Full Text]

YOSHIDA, M., M. KIJIMA, M. AKITA and T. BEPPU, 1990 Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A. J. Biol. Chem. 265: 17174–17179.[Abstract/Free Full Text]

ZHANG, K., V. V. SRIDHAR, J. ZHU, A. KAPOOR and J. K. ZHU, 2007 Distinctive core histone post-translational modification patterns in Arabidopsis thaliana. PLoS ONE 2: e1210.[CrossRef]

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