Developmental and Environmental Signals Induce Distinct Histone Acetylation Proﬁles on Distal and Proximal Promoter Elements of the C4-Pepc Gene in Maize

Sascha Offermann,1 Bjo¨rn Dreesen, Ina Horst, Tanja Danker, Michal Jaskiewicz and Christoph Peterhansel2 RWTH Aachen, Biology I, 52056 Aachen, Germany Manuscript received January 25, 2008 Accepted for publication May 29, 2008

ABSTRACT

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.

UKARYOTIC genes respond to multiple endoge- fragment spanning nucleotides À570 to 181 was suffi- E nous and environmental signals, which are in- cient for restricting gene expression to the leaf mesophyll tegrated on the promoter to control gene expression. in transgenic plants (Taniguchi et al. 2000). In transient The C4-specific phosphoenolpyruvate carboxylase (C4- assays of isolated mesophyll cells, even 300 bp of Pepc) gene occupies a central position in primary car- promoter sequence was sufficient for strong reporter bon acquisition in C4 plants such as maize (Kanai and gene expression (Schaeffner and Sheen 1992). Edwards 1999). C4-Pepc is expressed in an organ- and Although cis-acting DNA elements are important for cell-type-specific manner, is inducible by light, and is gene regulation, chromatin configuration also plays a regulated by nutrient availability and the metabolic vital role. The basic repeat structure of chromatin is the state of the cell. Thus, C4-Pepc is an excellent gene model nucleosome, comprising 147 bp of DNA wound around for studying the integrative function of promoters. an octamer containing two copies each of histones H2A, The C4-Pepc promoter has been studied extensively, and H2B, H3, and H4. Histone proteins have N-terminal cis-acting elements have been identified through protein- tails that can be modified in various ways, with acetyla- binding studies and promoter–reporter fusions in trans- tion, methylation, and phosphorylation of histones H3 genic lines. Yanagisawa and Sheen (1998) identified and H4 being the best characterized (Kouzarides binding sites for DOF (DNA binding with one finger) 2007). The connection between histone lysine acetyla- transcription factors that are important for leaf specificity tion and gene expression was established 40 years ago by and induction by light, 700 and 200 bp upstream from in vitro transcription studies (Allfrey et al. 1964). It is the transcription initiation site. Accordingly, a promoter now generally recognized that there is a positive cor- relation between the degree of histone acetylation and transcriptional activity throughout the genome. Con- 1 Present address: School of Biological Sciences, Washington State versely, the chromatin on transcriptionally inactive University, Pullman, WA 99164-4236. fluger agner 2Corresponding author: RWTH Aachen, Biology I, Worringer Weg 1, genes is mostly hypoacetylated (P and W 52056 Aachen, Germany. E-mail [email protected] 2007). The N-terminal tail of histone H3 is primarily

Genetics 179: 1891–1901 (August 2008) 1892 S. Offermann et al. acetylated at lysines 9 (H3K9), 14 (H3K14), and 18 MATERIALS AND METHODS (H3K18), while that of H4 is acetylated at lysines 5 Sequences: The C4-Pepc gene sequence was derived from (H4K5), 8 (H4K8), 12 (H4K12), and 16 (H4K16). GenBank accession gi 22396 (Matsuoka and Minami 1989). Additional acetylation sites exist on both histones, but BAC clone gi 116268332 was used to add 59 and 39 sequence their significance and function are mostly unknown elements. The Actin-1 promoter was described by Haring et al. (Kurdistani et al. 2004; Zhang et al. 2007). A simple (2007). Subtelomeric maize sequences were derived from Burr et al. (1992). model for the function of histone acetylation suggests Plant material and growth conditions: Maize (Zea mays cv. that acetylation neutralizes the positive charge on lysine Montello) was cultivated in growth chambers with a 16-hr pho- side chains and thereby reduces interaction with the toperiod and a day/night temperature regime of 25°/20°. The negatively charged DNA backbone, allowing transcrip- plants were illuminated with Osram Superstar HQI-T 400W/ À2 À1 tion factors better access to the DNA (Imhof and DH lamps at a photon flux density of 120–180 mmol m s . olffe ion Seedlings were grown in soil (VM, Einheitserde, Sinntal-Jossa, W 1998; D et al. 2005). Additionally, specific Germany) for 8–10 days with varying light–dark regimes (as triggers might store information on genes in the form indicated in the figures) or in complete darkness. Re-etiolated of histone modification patterns that are read out by plants were derived from illuminated plants subjected to total transcription factors and/or the transcription machin- darkness for 72 hr. ery. Such patterns have to be established autonomously Tissue preparation and crosslinking: Roots were isolated urner from soil and washed extensively before crosslinking. Coleop- from the final decision about gene transcription (T tiles were manually removed from 5- to 10-day-old illuminated 2007). Dependent on the crosstalk between individual or etiolated seedlings. Foliar leaves were harvested and cross- modifications and the complexity of modification pat- linked as described previously (Offermann et al. 2006) with terns, this signature is often referred to as a ‘‘histone the following modifications: 12 g of leaf material yielding sufficient chromatin for 16 precipitations under the condi- code’’ ( Jenuwein and Allis 2001) or a ‘‘histone lan- erger tions described below were vacuum infiltrated in 200 ml guage’’ (B 2007). Experiments in plants have doc- crosslink buffer in a 250-ml vacuum flask for 5 min. Subse- umented significant regulatory complexity of histone quently, glycine was added to a final concentration of 125 mm, acetylation (Chen and Tian 2007). Important examples and leaves were incubated for an additional 5 min to stop the are the different histone lysine residues acetylated 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 during the potentiation and activation of the phaseolin rotating wheel. More tissue was required to yield the same promoter (Ng et al. 2006). Moreover, acetylation pat- amount of chromatin that was isolated from 12 g of leaves. terns differ between the promoter and the transcribed Trichostatin A//a-amanitin//zeatin//2-deoxyglucose treat- region of the pea plastocyanin gene (Chua et al. 2001), ments: After 4 hr of illumination, 10- to 12-day-old leaves were and histone acetylation contributes differentially to the detached under water 1 cm above the laminar joint and incubated for 3 hr (8 hr for a-amanitin experiments) in two induction phases of submergence-responsive genes solutions containing 300 mm trichostatin A (TSA) (Interna- in rice (Tsuji et al. 2006). tional Clinical Services, Munich), 10 mm a-amanitin, or 2- We have recently shown that illumination is sufficient deoxyglucose (DOG) at varying concentrations as indicated in m to trigger hyperacetylation of the N-terminal tail of the figures, in combination with 5 m trans-zeatin (all Sigma- m À ugiharto histone H4 in the core promoter region of C -Pepc and Aldrich, Schnelldorf, Germany) and 16 m KNO3 (S 4 et al. 1992) in tap water. Leaves for zeatin-depletion experi- that this occurs independently of leaf cell type and ments were incubated in tap water only. nitrogen availability (Offermann et al. 2006). Further- Chromatin immunoprecipitation: Chromatin immunopre- more, mesophyll-specific expression is associated with cipitation was performed as described by Bowler et al. (2004) with modifications as described in Offermann et al. (2006) methylation of lysine 4 on histone H3, but not with aring anker and H et al. (2007). Chromatin was sheared with a changes in acetylation (D et al. 2008). Here, we Bioruptor (Diagenode, Liege, Belgium) for 10 min (setting: report the extension of these studies to a distal promoter high, interval 30/30 sec) under constant cooling. Modified region and record the modification of individual H3 histones were detected with 5 ml anti-hyperacetylated histone and H4 lysine residues at high resolution in different H4 (Upstate 06-946, Millipore, Schwalbach, Germany), 5 ml organs and under diverse treatments that modulate anti-acetyl H4K5 (Upstate 07-327), 5 ml anti-acetyl H4K16 (Upstate 07-329), 5 ml anti-acetyl H3K9 (Upstate 07-352), 1 ml gene activity. Our results indicate that distal promoter anti-acetyl H3K14 (Upstate 07-353), 1 ml anti-acetyl H3K18 acetylation plays an important quantitative role in tran- (Upstate 07-354), and 1 ml anti-H3 C-term (ab1791, Abcam, scriptional regulation. However, acetylation in the prox- Cambridge, UK). We also tested anti-acetyl H4K8 (Upstate 07- imal promoter and the transcribed region is strongly 328 and Abcam ab 1760) and anti-acetyl H4K12 (Upstate 07- resistant to changes in the transcriptional state, and only 595 and Abcam ab 1761), but failed to precipitate significant amounts. The control serum for determination of background selective acetylation sites are modified in response to precipitation levels was derived from rabbits immunized with light. The relative contribution of histone-modifying an unrelated protein from potato. In general, background enzymes to the establishment of this pattern depends on signals never exceeded 10% of positive control levels and are the lysine residue and its position within the gene. The therefore shown only in Figure 1. RNA preparation and reverse transcription: RNA isolation combinatorial complexity of acetylation patterns reveals and reverse transcription were performed as described in specific functions for individual lysine residues that are Offermann et al. (2006). As a direct estimate for promoter interpreted in a position-dependent manner. activity (Delany 2001; Reed 2003), heterogeneous nuclear Regulation of Histone Acetylation on the Maize C4-Pepc Gene 1893 RNAs (hnRNAs) were amplified from cDNA with a primer bp, whereas more uniform levels were detected for system specific for an intron. DNA synthesis in the absence of H4K5. The reduced acetylation described for H4Hyp in reverse transcriptase was used to exclude amplification from the first part of the transcribed region was also evident residual DNA contamination. A dilution series of illuminated leaf cDNA was used as a standard. Oligonucleotide sequences for these two lysines. and conditions are given in supplemental Table 1. In contrast, acetylation of H3K18 and H4K16 was Quantitative real-time PCR: Quantitative PCR was per- seemingly uninfluenced by light, but rather already formed on an ABI PRISM 7000 (Applied Biosystems) using present in etiolated leaves (Figure 1, D and G). The de- SYBR Green fluorescence (Platinum SYBR Green qPCR Mix, gree of histone acetylation was uniformly distributed Invitrogen) for detection. Oligonucleotide sequences and conditions are given in supplemental Table 1. 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 RESULTS most distal promoter region. Levels in the proximal pro- Light-induced histone acetylation on the C4-Pepc moter and the transcribed region were almost unchanged. gene: We recently showed that light induces hyper- Precipitation with an antibody directed to an in- acetylation of histone H4 (H4Hyp) on the C4-Pepc gene variant domain of the C-terminal part of histone H3 in maize, independent of gene transcription (Offer- (H3C) that is not subject to covalent modification was mann et al. 2006). To further define the role of histone used as a measure of nucleosome occupancy on the modification on C4-Pepc expression, acetylation of five gene (Figure 1H). In neither the promoter nor the (H4K5, H4K16, H3K9, H3K14, H3K18) of the seven transcribed region did nucleosome occupancy change lysine residues functionally characterized to date was significantly upon illumination. In both etiolated and examined in etiolated and illuminated plants. Acetyla- illuminated plants, the most obvious characteristic of tion of H4K8 and H4K12 was not detectable even when the H3C distribution curve was a surprising threefold genome hyperacetylation was artificially induced by increase at the start of the transcribed region, exactly treatment with the histone deacetylase (HDAC) inhib- where the lowest acetylation levels were found. Thus, itor, trichostatin A (see also below). Experiments were the low acetylation in this gene region was even more performed at high resolution for the promoter region, pronounced when calculated relative to the number of at selected positions of the transcribed region, and at a nucleosomes that are present in the highest numbers in position just downstream of the polyadenylation site (I). this region. According to Taniguchi et al. (2000), the C4-Pepc pro- These results indicate the presence of both light- moter sequence was separated into an essential proxi- dependent and -independent lysine acetylation profiles mal (À600 to 11 bp) and a distal region located farther throughout the C4-Pepc gene. upstream that is not essential for transcriptional activity Trichostatin A treatment of illuminated leaves: We in a transient assay system (Figure 1A). The amounts of also tested the maximum inducible acetylation levels precipitate are presented as the percentage of input after treatment of illuminated plants with the HDAC material to avoid any artifacts caused by standardization inhibitor, trichostatin A. TSA inhibits class I and class II (Haring et al. 2007). Results for a heterochromatic HDACs, but not class III HDACs (Yoshida et al. 1990; subtelomeric control region with low acetylation levels Imai et al. 2000). TSA treatment induces enhanced and for the constitutively active Actin-1 promoter where acetylation if both a histone acetyltransferase (HAT) high acetylation was expected are shown in Figure 1, I and a class I or class II HDAC are active at the tested and J. gene position. For almost all tested acetylations, clear As shown in Figure 1B, the light induction of H4Hyp increases after TSA treatment were observed in both the could be reproduced in this assay. High acetylation of promoter and the transcribed region, indicating that C4-Pepc in illuminated plants was limited to the gene class I and class II HDACs control acetylation levels on region and low levels were detected directly upstream the C4-Pepc gene even in illuminated plants where the from the promoter and downstream from the putative gene shows maximal activity. The amplitude of TSA transcript polyadenylation site. Within the gene, acety- induction was always lower on the proximal promoter lation was highest in the promoter region, and the levels compared to the distal promoter. H3K9 acetylation dropped sharply by a factor of 5 directly behind the (Figure 1N) on the distal promoter constituted a nota- transcription initiation site (TIS) and recovered toward ble exception, because levels were even slightly lower the end of the gene. When individual lysines were after TSA treatment (as compared to untreated plants), tested, a clear light induction of acetylation (between suggesting that class I or class II HDAC activity did not 3- and 10-fold depending on the position) was detect- contribute to the regulation of this modification in able for H4K5 (Figure 1C) and H3K9 (Figure 1E). The illuminated plants. highest signals were again found on the promoter, with In most cases, acetylation remained limited to the individual distribution profiles of the two modifications. gene region even after TSA treatment indicating that H3K9 peaked in the distal part around position À2000 the corresponding HATs were recruited to the promoter 1894 S. Offermann et al. and the transcribed region and are absent outside of the mally not exposed to light and might not express factors gene. However, H3K14 and H3K18 showed significant necessary for a light response. To further elucidate light induction of acetylation after TSA inhibition of HDAC responses and tissue-specific patterns, we also included activity even downstream from the transcribed region. coleoptiles in our analyses. These leaf-like organs re- These two modifications are even inducible by TSA spond to illumination by inducing chloroplast develop- treatment in the subtelomeric negative control region ment and photosynthesis, but do not show the high (Figure 1R), suggesting that HATs for these modifica- bundle density typical of C4 leaves and, consequently, do tions are not specifically recruited to gene regions. not express C4-Pepc (Langdale et al. 1988; supplemental We also tested whether nucleosome occupancy is Figure S1A). Comparison of acetylation levels in the dif- directly influenced by the acetylation status. Precipita- ferent tissues was hampered by dissimilar overall pre- tion efficiency with the H3C antibody (Figure 1Q) was cipitation efficiencies, probably due to differences in only slightly enhanced (less than twofold) in TSA- crosslinking (data not shown). The results were therefore treated plants. Within the proximal promoter, this param- standardized for acetylation levels on the Actin-1 promoter eter remained completely uninfluenced by TSA. because this gene showed comparable activities in the These results support the observation that each spe- tested tissues (supplemental Figure S1A). Figure 3 shows cific acetylation is individually controlled with respect to results for one representative core promoter position, absolute levels and the relative distribution over the and six additional positions are shown in supplemental gene. The relative contribution of HATand HDAC activ- Figure S3. Acetylation of H3K14, H3K18, and H4K16 was ities to the adjustment of acetylation levels also depends reduced by ,2-fold at most tested positions in both on the lysine tested. coleoptiles and roots, as compared to leaves. However, Re-etiolation of illuminated plants: When maize we cannot exclude the possibility that these minor changes plants are returned to the dark for extended periods are caused, at least in part, by the standardization method after an initial illumination (re-etiolated plants), C4-Pepc (see also discussion). More dramatic differences were promoter activity drops to levels clearly below those observed for H4K5 (5-fold), H4Hyp (5-fold), and H3K9 observed in etiolated plants, but the light induction of (10-fold). Importantly, acetylation of these residues in transcription after a second illumination is faster coleoptiles did not increase after illumination, as has (Offermann et al. 2006). We wondered whether histone been observed before in leaves (Figure 1), suggesting modifications induced by previous illumination were that the potential for light-inducible acetylation is an maintained on the gene upon transfer to the dark and important property of C4-Pepc chromatin in leaves. therefore compared acetylation levels in illuminated, Response to inhibition of transcription: The low etiolated, and re-etiolated plants on seven selected gene levels of acetylation observed for light-regulated lysines positions. Figure 2 shows results for the core promoter in etiolated leaves, coleoptiles, and roots might either representative of the other positions (shown in full in be directly controlled by endogenous and environmen- supplemental Figure S2). We found no clear differences tal signals or be an indirect consequence of the low in histone modification between the etiolated and re- transcription rates. We treated illuminated leaves with etiolated states, excluding a role of the tested histone the transcription inhibitor a-amanitin to discriminate be- acetylation sites in light memory and preparation of tween these two possibilities. C4-Pepc promoter activity the promoter for rapid reactivation in response to re- was at the detection limit 8 hr after inhibitor application illumination. (supplemental Figure S1B). The corresponding levels Tissue-specific acetylation on the C4-Pepc gene: C4- of H4 hyperacetylation and H3K9 acetylation at seven Pepc transcription is restricted to leaves and absent in tested gene positions are shown in Figure 4. While other maize tissues. The contribution of histone acety- acetylation remained constant on the promoter, it in- lation to the regulation of this tissue specificity was tested creased in the transcribed region after transcriptional by comparing acetylation levels in leaves and roots. inhibition. This indicates that low acetylation levels of However, roots are underground systems that are nor- these sites were not caused by low transcription rates, < igure F 1.—Histone acetylation profile of the C4-Pepc gene. (A) Gene structure survey (intron line and exon block diagram). Shaded area, proximal promoter (À600 to 11 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. Regulation of Histone Acetylation on the Maize C4-Pepc Gene 1895 but were, rather, a consequence of the absence of acti- the C4-Pepc gene is also under metabolic control. Its vating stimuli. activity is strongly regulated by nitrogen availability Histone acetylation in response to metabolic stimuli: (transduced by the hormone zeatin) and the intracel- In addition to its tissue specificity and light induction, lular hexose concentration ( Jang and Sheen 1994; 1896 S. Offermann et al.

Figure 2.—Light-mediated histone acetylation. (A) Com- parison of histone acetylation in illuminated (solid columns), etiolated (shaded columns), and re-etiolated (open columns) leaves at a representative position (À200 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.

Suzuki et al. 1994). We have previously shown that H4 hyperacetylation on the proximal promoter is indepen- Figure 4.—Histone acetylation after inhibition of tran- dent of nitrogen availability and, therefore, does not scription with a-amanitin. Amount of chromatin precipitated change in detached leaves starved of nitrate and zeatin with an antibody specific for (A) tri- and tetra-acetylated iso- (Offermann et al. 2006). Here, we extended the orig- forms of histone H4 (H4Hyp) or (B) histone H3 lysine 9 acetylation (H3K9ac) in a-amanitin-treated (solid columns) or inal study to cover the complete promoter and analyzed control leaves (open columns) on representative positions individual acetylation sites in detail. Furthermore, we in the distal (À1600 and À1300 bp) or proximal (À200 bp) included treatments with DOG, which mimics high promoter at the beginning (450 bp), middle (1900 bp), hexose availability ( Jang and Sheen 1994). To measure and end (4300 bp) of the transcribed region or at an intergenic position (5900 bp). Data points are based on at least transcription rates, C4-Pepc hnRNA accumulation was materials and methods three independent experiments. Vertical lines indicate stan- monitored as described in . dard errors. 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 remarkably constant for all tested lysines independent transcribed region, the degree of acetylation stayed 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 (Fig- Figure 3.—Comparison of histone acetylation in illumi- ure 6B). Interestingly, a gradual decrease in acetylation nated leaves (solid columns), etiolated coleoptiles (darkly was evident at the two distal promoter sites that we tested shaded columns), illuminated coleoptiles (lightly shaded columns), and roots (open columns) at a representative position (À1600 and À1300 bp), while the proximal promoter (À200 bp) in the proximal promoter of C4-Pepc. For all other (À300 and À200 bp) or the Actin-1 promoter showed no tested positions, see supplemental Figure S3. Due to the gen- response (Figure 6C). The data indicate that distal and erally enhanced precipitation efficiency in coleoptiles com- proximal promoter acetylation in response to metabolic pared to leaves, the amount of chromatin precipitated with stimuli is differentially regulated. On the distal pro- the antibodies was standardized for the amount of Actin-1 promoter chromatin precipitated with the same antibody. Data moter, all tested acetylation sites are coregulated and points are based on four independent experiments. Vertical the degree of acetylation is indicative of promoter activ- lines indicate standard errors. ity. In contrast, acetylation on the proximal promoter Regulation of Histone Acetylation on the Maize C4-Pepc Gene 1897 is surprisingly resistant to manipulations mimicking changes in nitrogen and hexose availability. 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 re- corded 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 (ÀZ) or that were treated with deoxyglucose (1D) 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 (À200), 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 igure F 5.—Histone acetylation on the distal and proximal result indicates that metabolic signals affect H3K9 C4-Pepc promoter after zeatin depletion and DOG repression. (A) Gene structure survey (intron line and exon block dia- acetylation on the distal promoter by modulating HAT gram). Shaded area, proximal promoter (À600 to 11 bp), activity because HDAC inhibition is not sufficient to solid vertical line, transcription initiation site; 1 and 2, exons revert the reduction in acetylation induced by the ÀZ 1 to 2. Numbers on x-axis represent the position in base pairs and 1D treatments. In contrast, the negative metabolite relative to the transcription initiation site. (B–H) Amounts of response of H4 hyperacetylation is apparently con- chromatin precipitated with antibodies specific for tri- and tetra-acetylated isoforms of histone H4 (H4Hyp), histone trolled by enhanced HDAC activity. 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 DISCUSSION 18 acetylation (H3K18ac), or an invariant C-terminal epitope Three levels of stimulus-induced histone modifica- on histone H3 (H3C) in zeatin-depleted (dotted line), DOG- repressed (dashed line), or control leaves (solid line). Data tions on the C4-Pepc gene: We have previously shown that points are based on four independent experiments. Vertical light induces hyperacetylation of histone H4 on the prox- lines indicate standard errors. imal promoter independently of other stimuli such as cell- 1898 S. Offermann et al.

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 (À1600 and À1300 bp) and proximal (À300 and À200 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. type specificity and nutrient availability (Offermann We speculated that acetylation of H3K14, H3K18, and et al. 2006). Surprisingly, analyses of individual acetyla- H4K16 in etiolated leaves is involved in the formation of tion sites in this study revealed that H3K9 and H4K5 a poised chromatin state that allows for a fast transcrip- acetylation are selectively induced by light, whereas tional response to illumination. Such poised states where H3K14, H3K18, and H4K16 acetylation is light inde- some, but not all, chromatin modifications associated pendent (Figure 1). The light-induced pattern is fully with full gene activity are set before transcription ac- reversible in plants kept in the dark for prolonged tivation have been described, e.g., for the bean phaseo- periods, so no memory of previous illumination is lin promoter (Ng et al. 2006) and the pea plastocyanin stored at the level of histone acetylation. Since the two gene (Chua et al. 2001). Consequently, we expected outermost lysine residues on the N-terminal tails of H3 these modifications to be absent in roots and coleop- and H4 are subject to light regulation, our data agree tiles, where the C4-Pepc gene is constitutively inactive. with the recent observation that H4 acetylation occurs However, significant levels were detected for all three in a progressive fashion from the body of the histone to modifications in the tested tissues. Such comparative the N terminus (Earley et al. 2007). analyses of chromatin modifications in different tissues

Figure 7.—Changes in histone acetylation after metabolic repression and subsequent HDAC inhibition. Illuminated leaves were treated with 25 mm deoxyglucose (1D) or starved nitrate/zeatin (ÀZ) for 3 hr. Afterward, TSA was added to the solution and plants were incubated for an- other 3 hr. As a control, illuminated leaves were incubated in the presence (1) or absence of TSA (À) 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 (À1600 and À1300 bp) and proximal (À200 bp) promoter region. Data points are based on three independent experiments. Vertical lines indicate standard errors. Regulation of Histone Acetylation on the Maize C4-Pepc Gene 1899 in planta are complicated by the fact that the cross- clude that histone methylation defines cell-type specific- linking efficiency and the quality of the isolated chro- ity within the leaf independently of the acetylation state. matin may differ between the tissues (Haring et al. 2007). The strong light-induced expression of C4-Pepc in Despite optimization of the experimental procedure, mesophyll cells of leaves is further adjusted according to we always observed lower precipitation efficiencies from the availability of nitrogen and hexose sugars ( Jang and root chromatin and very high efficiencies from cole- Sheen 1994; Suzuki et al. 1994). When manipulating optile chromatin (data not shown). We standardized these stimuli, the treatments exclusively modulated the our data for the level of acetylation on the Actin-1 acetylation state of the distal promoter region. Here, all promoter because this gene is transcribed at very similar tested acetylation sites were affected (Figure 5), and the levels in the tested tissues. Still, this does not guarantee degree of acetylation corresponded to the transcription identical acetylation levels. On the basis of this stan- rate (Figure 6). The data suggest the presence of a third dardization method, acetylation of H3K14, H3K18, and independent control element for metabolite responses H4K16 was reduced to approximately half in roots and in the distal promoter, in addition to the factors con- coleoptiles as compared to leaves, but such slight trolling leaf and mesophyll specificity. This third ele- reductions do not necessarily indicate factual differ- ment is near to a previously identified DNA methylation ences between the tissues. Thus, acetylation of these site that is demethylated upon gene activation (Langdale residues does not seem to be associated with po- et al. 1991), providing independent evidence that this tentiating the gene for activation in etiolated leaves, distant region is involved in regulation of the C4-Pepc but is, rather, a general property of C4-Pepc chromatin in gene. all tissues. Supportive of our observation, recent analy- All the treatments that we applied influenced not only ses in several organisms have suggested that H4K16 is C4-Pepc histone acetylation, but also the transcription controlled independently of other acetylation sites and rate. However, experiments in the presence of the RNA constitutively acetylated in euchromatin (Dion et al. polymerase II inhibitor, a-amanitin, showed that even 2005; Benhamed et al. 2006; Shogren-Knaak et al. 2006; complete inhibition of transcription did not affect pro- Rossi et al. 2007). Distinct from our results, H3K18 moter acetylation (Figure 4), resembling the highly acetylation is usually associated with full gene activity on stable histone modifications in the promoters of mam- a genomewide basis in yeast, whereas H3K14 acetylation malian housekeeping genes (Kouskouti and Talianidis correlates only weakly with the transcriptional state 2005). The only chromatin modifications on the C4-Pepc (Kurdistani et al. 2004; Liu et al. 2005). gene shown thus far to be responsive to transcriptional The most obvious difference between coleoptiles and inhibition are limited to the transcribed region and foliar leaves was the inability of coleoptiles to induce involve H3K4 trimethylation, which decreases (Danker H3K9 and H4K5 acetylation upon illumination (Figure et al. 2008), and histone acetylation, which increases 3). Although factors necessary for light response are (Figure 4). The latter effect is likely related to the active generally present in coleoptiles (Langdale et al. 1988), deacetylation of histones during transcription elonga- there was almost no change in histone acetylation in the tion, which prevents spurious initiation within the tran- proximal promoter region of C4-Pepc. The presence of scribed region (Lee and Shilatifard 2007; Li et al. HATs responsible for acetylation of H4K5 and H3K9 is 2007). Therefore, the observed changes in acetylation therefore the most evident unique property of C4-Pepc under diverse treatments are induced directly by the chromatin in leaves as compared to other tissues. respective stimulus and are not caused indirectly by low Within leaves, C4-Pepc is transcribed in mesophyll transcription rates. cells, but not bundle sheath cells (Kausch et al. 2001). Regulation of histone acetylation on the C4-Pepc gene However, at least in the case of H4 acetylation on the by HAT and HDAC enzymes: The response of in- proximal promoter, light induction takes place in both dividual acetylation sites to TSA inhibition of HDAC cell types (Offermann et al. 2006). Thus, the presence activity allowed us to determine whether or not the same of specific HATs for light-inducible modifications might enzymes regulated acetylation of the different residues. control leaf-organ specificity, but this signal is differen- If HDAC inhibition is sufficient to induce increased tially interpreted in the context of other histone modi- acetylation, then we assume that the corresponding fications in different cell types within the leaf. In HATshould be present and active in the tested genomic accordance with this hypothesis, we have recently iden- region, but a HDAC counteracts high acetylation. Class tified a shift from dimethylation to trimethylation of III HDACs are not sensitive to TSA (Imai et al. 2000). H3K4 on the C4-Pepc gene, unique to mesophyll cells, However, in general, TSA treatment strongly induced that was not influenced by illumination, but instead was histone acetylation throughout the C4-Pepc gene (Figure controlled by cell lineage (Danker et al. 2008). Cell- 1), suggesting that class I and class II enzymes contrib- type-specific histone methylation was restricted to the ute the majority of HDAC activity on this gene. We used proximal promoter and, therefore, differed in its distri- this concept to determine how acetylation is restricted bution from the light-induced acetylation that is also to the gene region (Figure 1), under which conditions found in the distal promoter region (Figure 1). We con- HATor HDAC activities control acetylation of individual 1900 S. Offermann et al. sites (Figure 7), and whether the acetylation of a specific ure 1N). On the other hand, metabolic stimuli con- lysine residue in a defined gene region can be controlled acetylation of the same lysine in the same gene trolled by different mechanisms, depending on the region by reducing HAT activity, while H4 acetylation stimulus (Figures 1 and 7). after metabolite treatments is, in turn, regulated by Our results show that acetylation of H3K14 and enhancing HDAC activity (Figure 7). Thus, H3 and H4 H3K18 increased following TSA treatment on the gene acetylation in response to the same stimulus are also borders and even on subtelomeric DNA, where these regulated by different mechanisms. modifications are normally rare (Figure 1), suggesting In conclusion, a complex regulatory network involv- that restriction of these modifications to the promoter ing multiple HATs and HDACs targeted to different sites and the transcribed region is controlled by high HDAC on the gene controls histone acetylation on the C4-Pepc activity outside the gene. In contrast, H3K9, H4K5, and gene. These results are unexpected because the muta- H4K16 modification remained restricted to the pro- tion or overexpression of individual HATs and HDACs moter and the transcribed region in TSA-treated plants. has wide-ranging effects on the acetylation of most Thus, the HATs for these sites are recruited to the gene lysines throughout the genome (Tian et al. 2004; and absent on the gene borders. Interestingly, we have Benhamed et al. 2006; Rossi et al. 2007). In contrast, recently shown that H3K9 dimethylation occurs at sites HDACs in rice are themselves expressed in a tissue- flanking the gene. This modification is also found at specific and environmentally regulated pattern (Fu et al. high levels in the acetylation gap behind the TIS 2007). A recent analysis of co-activator HATs in Arabi- (Danker et al. 2008), supporting the conclusion that dopsis revealed different classes with either broad or H3K9 methylation has a negative effect on histone very narrow specificities for particular lysines (Earley acetylation, as described on a genomewide basis for et al. 2007). On the basis of the latter observation, a mice (Wu et al. 2007). broad-specificity HAT might control the response of the Despite the differences on the gene borders, HDAC distal promoter to metabolic stimuli, whereas illumina- inhibition increased acetylation of all tested lysines tion regulates the activity of highly specific HDACs on within the gene (with the exception of H3K9 acetylation the complete C4-Pepc gene. on the distal promoter, which is discussed below). TSA Our results suggest that histone acetylation contrib- had greater effects in the distal compared to the proxi- utes to C4-Pepc regulation by mechanisms that are com- mal promoter regions. This effect was most pronounced patible with both the ‘‘charge neutralization’’ and the for those lysines that were not acetylated in response to ‘‘histone code’’ models ( Jenuwein and Allis 2001; light but, rather, acetylated in etiolated plants, indicat- Berger 2007). The coregulation of all the tested sites on ing that the level of inducibility by TSA treatment the distal promoter by metabolic stimuli, and the correlates to the degree of regulation by diverse stimuli. quantitative relationship between acetylation and tran- It has been suggested that the chromatin of active or scription levels, argue that distal promoter acetylation poised genes (prepared for transcription) is character- has a simple role in improving transcription factor ized by a high turnover of acetylation rather than by access. In contrast, the independence of proximal pro- high absolute acetylation levels (Spencer and Davie moter acetylation from transcription as well as from 2001; Hazzalin and Mahadevan 2005). The relative signals other than light, and the complex regulation of increase in acetylation following exposure to TSA was acetylation by multiple HATs and HDACs, rather favor used here as an indicator of the dynamics of acetylation a histone acetylation code that is interpreted depend- because it reveals the contribution of HDAC and HAT ing on the gene position and in the context of other activities to steady-state acetylation (Clayton et al. modifications. 2006). On the basis of this approach, regulated sites We are grateful to Fritz Kreuzaler for continuous support; Maike on the C4-Pepc gene (H4K5 and H3K9 on the proximal Stam and Max Haring for collaboration during establishment of promoter and all lysines on the distal promoter) tend to techniques; and Richard Twyman, Klaus Grasser, and Sandy Edwards cycle between acetylation and deacetylation more rap- for critical reading of the manuscript. This work was supported by idly. This rapid turnover of acetyl groups might allow grants (Pe819/1) from the Deutsche Forschungsgemeinschaft to C.P. prompt responses to changes in environmental and LITERATURE CITED endogenous stimuli. To define stimulus-specific characteristics of the Allfrey, V. G., R. Faulkner and A. E. Mirsky, 1964 Acetylation and methylation of histones and their possible role in the regu- regulation of acetylation, we further combined meta- lation of RNA synthesis. Proc. Natl. Acad. Sci. USA 51: 786–794. bolic stimuli with TSA treatments. Our analysis of H3K9 Benhamed, M., C. Bertrand,C.Servet and D.-X. Zhou, acetylation on the distal promoter illustrated that acet- 2006 Arabidopsis GCN5, HD1, and TAF1/HAF2 interact to regulate histone acetylation required for light-responsive gene ex- ylation of a specific lysine at a defined gene position can pression. Plant Cell 18: 2893–2903. be differentially regulated according to the stimulus. Berger, S. L., 2007 The complex language of chromatin regulation On the one hand, acetylation in light-grown plants during transcription. Nature 447: 407–412. Bowler, C., G. Benvenuto,P.Laflamme,D.Molino,A.V.Probst could not be enhanced by TSA, suggesting that light et al., 2004 Chromatin techniques for plant cells. Plant J. 39: completely inactivated the corresponding HDAC (Fig- 776–789. Regulation of Histone Acetylation on the Maize C4-Pepc Gene 1901

Burr,B.,F.A.Burr,E.C.Matz and J. Romero-Severson, 1992 Pin- Matsuoka, M., and E. Minami, 1989 Complete structure of the ning down loose ends: mapping telomeres and factors affecting gene for phosphoenolpyruvate carboxylase from maize. Eur. J. Bi- their length. Plant Cell 4: 953–960. ochem. 181: 593–598. Chen, Z. J., and L. Tian, 2007 Roles of dynamic and reversible his- Ng, D. W. K., M. B. Chandrasekharan and T. C. Hall, 2006 Or- tone acetylation in plant development and polyploidy. Biochim. dered histone modifications are associated with transcriptional Biophys. Acta 1769: 295–307. poising and activation of the phaseolin promoter. Plant Cell Chua, Y. L., A. P. Brown and J. C. Gray, 2001 Targeted histone acet- 18: 119–132. ylation and altered nuclease accessibility over short regions of the Offermann, S., T. Danker,D.Dreymu¨ller,R.Kalamajka,S. pea plastocyanin gene. Plant Cell 13: 599–612. To¨psch et al., 2006 Illumination is necessary and sufficient to Clayton,A.L.,C.A.Hazzalin andL.C.Mahadevan, 2006 Enhanced induce histone acetylation independent of transcriptional activ- histone acetylation and transcription: a dynamic perspective. Mol. ity at the C4-specific phosphoenolpyruvate carboxylase promoter Cell 23: 289–296. in maize. Plant Physiol. 141: 1078–1088. Danker, T., B. Dreesen,S.Offermann,I.Horst and C. Peterhansel, Pfluger, J., and D. Wagner, 2007 Histone modifications and dy- 2008 Developmental information but not promoter activity namic regulation of genome accessibility in plants. Curr. Opin. controls the methylation state of histone H3 lysine 4 on two pho- Plant Biol. 10: 645–652. tosynthetic genes in maize. Plant J. 53: 465–474. Reed, R., 2003 Coupling transcription, splicing and mRNA export. Delany, A. M., 2001 Measuring transcription of metalloproteinase Curr. Opin. Cell Biol. 15: 326–331. genes. Nuclear run-off assay vs analysis of hnRNA. Methods Mol. Rossi, V., S. Locatelli,S.Varotto,G.Donn,R.Pirona et al., Biol. 151: 321–333. 2007 Maize histone deacetylase hda101 is involved in plant de- Dion, M. F., S. J. Altschuler,L.F.Wu and O. J. Rando, velopment, gene transcription, and sequence-specific modula- 2005 Genomic characterization reveals a simple histone H4 tion of histone modification of genes and repeats. Plant Cell acetylation code. Proc. Natl. Acad. Sci. USA 102: 5501–5506. 19: 1145–1162. arley hook rower oland icks chaeffner heen E , K. W., M. S. S ,B.B -T ,L.H and C. S. S , A. R., and J. S , 1992 Maize C4 photosynthesis in- Pikaard, 2007 In vitro specificities of Arabidopsis co-activator volves differential regulation of phosphoenolpyruvate carboxyl- histone acetyltransferases: implications for histone hyperacetyla- ase. Plant J. 2: 221–232. tion in gene activation. Plant J. 52: 615–626. Shogren-Knaak, M., H. Ishii,J.M.Sun,M.J.Pazin,J.R.Davie et al., Fu, W., K. Wu and J. Duan, 2007 Sequence and expression analysis 2006 Histone H4–K16 acetylation controls chromatin structure of histone deacetylases in rice. Biochem. Biophys. Res. Commun. and protein interactions. Science 311: 844–847. 356: 843–850. Spencer, V. A., and J. R. Davie, 2001 Dynamically acetylated histone Haring, M., S. Offermann,T.Danker,I.Horst,C.Peterha¨nsel association with transcriptionally active and competent genes in et al., 2007 Chromatin immunoprecipitation: quantitative anal- the avian adult beta-globin gene domain. J. Biol. Chem. 276: ysis and data normalization. Plant Methods 2: 11. 34810–34815. Hazzalin, C. A., and L. C. Mahadevan, 2005 Dynamic acetylation Sugiharto, B., J. N. Burnell and T. Sugiyama, 1992 Cytokinin is of all lysine 4-methylated histone H3 in the mouse nucleus: anal- required to induce the nitrogen-dependent accumulation of ysis at c-fos and c-jun. PLoS Biol. 3: e393. mRNAs for phosphoenolpyruvate carboxylase and carbonic an- Imai, S.-I., C. M. Armstrong,M.Kaeberlein and L. Guarente, hydrase in detached maize leaves. Plant Physiol. 100: 153–156. 2000 Transcriptional silencing and longevity protein Sir2 is Suzuki,I.,C.Cretin,T.Omata and T. Sugiyama, 1994 Transcriptional an NAD-dependent histone deacetylase. Nature 403: 795–800. and posttranscriptional regulation of nitrogen-responding ex- Imhof, A., and A. P. Wolffe, 1998 Transcription: gene control by pression of phosphoenolpyruvate carboxylase gene in maize. targeted histone acetylation. Curr. Biol. 8: R422–R424. Plant Physiol. 105: 1223–1229. Jang, J. C., and J. Sheen, 1994 Sugar sensing in higher plants. Plant Taniguchi, M., K. Izawa,M.S.B.Ku, J.-H. Lin,H.Saito et al., Cell 6: 1665–1679. 2000 Binding of cell type-specific nuclear proteins to the 59- enuwein llis J , T., and C. D. A , 2001 Translating the histone code. flanking region of maize C4 phosphoenolpyruvate carboxylase Science 293: 1074–1080. gene confers its differential transcription in mesophyll cells. Kanai, R., and G. E. Edwards, 1999 The biochemistry of C4 photo- Plant Mol. Biol. 44: 543–557. synthesis, pp. 49–87 in C4 Plant Biology, edited by R. F. Sage and R. Tian,L.,M.P.Fong,J.J.Wang,N.E.Wei,H.Jiangetal.,2004 Reversible K. Monson. Academic Press, San Diego. histone acetylation and deacetylation mediate genome-wide, pro- Kausch, A. P., T. P. Owen,S.J.Zachwieja,A.R.Flynn and J. Sheen, moter-dependent and locus-specific changes in gene expression 2001 Mesophyll-specific, light and metabolic regulation of the during plant development. Genetics 169: 337–345. C4 PPCZm1 promoter in transgenic maize. Plant Mol. Biol. 45: 1–15. Tsuji, H., H. Saika,N.Tsutsumi,A.Hirai and M. Nakazono, Kouskouti, A., and I. Talianidis, 2005 Histone modifications de- 2006 Dynamic and reversible changes in histone H3-Lys4 meth- fining active genes persist after transcriptional and mitotic inac- ylation and H3 acetylation occurring at submergence-inducible tivation. EMBO J. 24: 347–357. genes in rice. Plant Cell Physiol. 47: 995–1003. Kouzarides, T., 2007 Chromatin modifications and their function. Turner, B. M., 2007 Defining an epigenetic code. Nat. Cell Biol. 9: 2–6. Cell 128: 693–705. Wu, J., S. H. Wang,D.Potter,J.C.Liu,L.T.Smith et al., Kurdistani,S.K.,S.Tavazoieand M. Grunstein, 2004 Mapping global 2007 Diverse histone modifications on histone 3 lysine 9 and histone acetylation patterns to gene expression. Cell 117: 721–733. their relation to DNA methylation in specifying gene silencing. Langdale, J. A., I. Zelitch,E.Miller and T. Nelson, 1988 Cell BMC Genomics 8: 131. position and light influence C4 versus C3 patterns of photosyn- Yanagisawa, S., and J. Sheen, 1998 Involvement of maize Dof zinc thetic gene expression in maize. EMBO J. 7: 3643–3652. finger proteins in tissue-specific and light-regulated gene expres- Langdale, J. A., W. C. Taylor and T. Nelson, 1991 Cell-specific ac- sion. Plant Cell 10: 75–89. cumulation of maize phosphoenolpyruvate carboxylase is corre- Yoshida, M., M. Kijima,M.Akita and T. Beppu, 1990 Potent and lated with demethylation at a specific site . 3 kb upstream of the specific inhibition of mammalian histone deacetylase both gene. Mol. Gen. Genet. 225: 49–55. in vivo and in vitro by trichostatin A. J. Biol. Chem. 265: 17174– Lee, J. S., and A. Shilatifard, 2007 A site to remember: H3K36 meth- 17179. ylation a mark for histone deacetylation. Mutat. Res. 618: 130–134. Zhang, K., V. V. Sridhar,J.Zhu,A.Kapoor and J. K. Zhu, Li, B., M. Gogol,M.Carey,D.Lee,C.Seidel et al., 2007 Combined 2007 Distinctive core histone post-translational modification action of PHD and chromo domains directs the Rpd3S HDAC to patterns in Arabidopsis thaliana. PLoS ONE 2: e1210. transcribed chromatin. Science 316: 1050–1054. 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. Communicating editor: T. P. Brutnell