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First published online April 27, 2007; 10.1104/pp.107.099473 Plant Physiology 144:1508-1519 (2007) © 2007 American Society of Plant Biologists Down-Regulation of a SILENT INFORMATION REGULATOR2-Related Histone Deacetylase Gene, OsSRT1, Induces DNA Fragmentation and Cell Death in Rice1,[C],[W]National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China (L.H., Q.S., F.Q., C.L., Y.Z.); Department of Quartermaster, Military Economy Academy, Wuhan 430035, China (L.H.); and Institut de Biotechnologie des Plantes, Université Paris Sud 11, 91405 Orsay, France (D.-X.Z.)
The SILENT INFORMATION REGULATOR2 (SIR2) family proteins are NAD+-dependent histone deacetylases. Sir2 is involved in chromatin silencing at the mating-type loci, rDNA, and telomeres in yeast and is associated with lifespan extension in yeast, worms, and flies, but also in a broader range of additional functions. In this work, we investigated the role of OsSRT1, one of the two SIR2-related genes found in rice (Oryza sativa). We show that OsSRT1 is a widely expressed nuclear protein with higher levels in rapidly dividing tissues. OsSRT1 RNA interference induced an increase of histone H3K9 (lysine-9 of H3) acetylation and a decrease of H3K9 dimethylation, leading to H2O2 production, DNA fragmentation, cell death, and lesions mimicking plant hypersensitive responses during incompatible interactions with pathogens, whereas overexpression of OsSRT1 enhanced tolerance to oxidative stress. Transcript microarray analysis revealed that the transcription of many transposons and retrotransposons in addition to genes related to hypersensitive response and/or programmed cell death was activated. Chromatin immunoprecipitation assays showed that OsSRT1 down-regulation induced histone H3K9 acetylation on the transposable elements and some of the hypersensitive response-related genes, suggesting that these genes may be among the primary targets of deacetylation regulated by OsSRT1. Our data together suggest that the rice SIR2-like gene is required for safeguard against genome instability and cell damage to ensure plant cell growth, but likely implicates different molecular mechanisms than yeast and animal homologs.
Histone acetylation involves the transfer of acetyl groups from acetyl-CoA to Lys residues of histones. Hyperacetylation of histones leads to relaxation of chromatin structure and is associated with transcriptional activation, whereas hypoacetylation of histones induces chromatin compaction and gene repression (Carrozza et al., 2003  ). Histone acetylation is catalyzed by histone acetyltransferases, whereas histone deacetylation is catalyzed by histone deacetylases (HDACs). Plant HDACs can be grouped into four subclasses. Three of them have primary homology to the three classes of HDACs (RDP3, HDA1, and SIR2) found in yeast and animal cells (Pandey et al., 2002). The fourth class of plant HDACs (known as the HD2 class) is found only in plants (Lusser et al., 1997; Pandey et al., 2002).
The SILENT INFORMATION REGULATOR2 (SIR2) family proteins, known also as sirtuins, are NAD+-dependent protein deacetylases. They contain a 200-amino acid domain (Pfam designation PF02146) conserved from bacteria to humans (Frye, 2000
Yeast has four additional Sir2 homologs, termed Hst1 to Hst4, in addition to the founding member. All of the yeast members belong to class I of the Sir2-related proteins (Frye, 2000
Plant genomes seem to contain relatively fewer SIR2 homologs than the other eukaryotes. In Arabidopsis (Arabidopsis thaliana), only two SIR2 family gene sequences (named atSRT1 and atSRT2) have been identified. Phylogenetic analysis of identified plant SIR2 homologs shows that they belong to only two of the four classes of the family, classes that have only plant and animal members (Pandey et al., 2002
Rice Genome Contains Two SIR2-Related Genes
Sequence analysis of the rice genome revealed two SIR2-related genes, named OsSRT1 and OsSRT2. OsSRT1 and other plant SRT1 homologs are found in the same class (class IV), whereas OsSRT2 belongs to class II of the SIR2-related genes (Pandey et al., 2002
Down-Regulation of OsSRT1 by RNAi Induced Programmed Cell Death in Rice
To study the physiological function of OsSRT1, a 412-bp segment of the 3'-untranslated region of the gene (Fig. 3A
), which was not conserved with OsSRT2, was inserted in inverted repeats to build a construct for RNAi. The construct was used to transform an indica rice variety ‘Minghui63’. About 20 independent transgenic lines were produced and analyzed for OsSRT1 expression during the root regeneration stage. Three of them showed either reduced or no expression of the endogenous gene, suggesting an effect of RNAi (Fig. 3B). To further analyze whether there was any effect of OsSRT1 RNAi on histone modification, we did western-blot analyses using antibodies raised specifically against acetylated histone H3 and acetylated H3K9, because several nuclear SIR2 proteins in yeast and animal cells have been shown to be mainly involved in histone H3 and H3K9 deacetylation (Blander and Guarente, 2004
The RNAi lines were selected for phenotype observation and further analysis. The RNAi plants at the two-leaf stage (about 14 d after germination) began to produce brown dots on leaves, which became larger at latter stages, leading to precocious leaf senescence (Fig. 4 ). Only two of the three RNAi lines could produce seeds. The severest line died before getting into maturity. The transgenic lines showing no alteration of OsSRT1 expression or histone modification did not manifest the phenotype (data not shown), suggesting that the lesion mimic phenotype was induced by OsSRT1 down-regulation. The lesions were reminiscent of cell death induced by hypersensitive responses during plant pathogen infections, suggesting that OsSRT1 RNAi might have induced programmed cell death (PCD). To test this hypothesis, young leaf sheaths (T1 generation) were incubated with 3,3'-diaminobenzidine (DAB) to detect H2O2 (Thordal-Christensen et al., 1997 ). H2O2 production was detected in cells of OsSRT1 RNAi leaf sheaths at day 7 after germination (before appearance of the symptom). At day 21 after germination, more cells produced H2O2 at higher levels (Fig. 5A
). H2O2 production is an indicator of plant cells undergoing hypersensitive PCD during incompatible plant-pathogen interactions (Brodersen et al., 2002). Therefore, the lesion mimic phenotype of OsSRT1 RNAi plants suggested that the down-regulation of OsSRT1 induced PCD in rice. To confirm whether OsSRT1 RNAi induced PCD, the second leaves of 2-week-old wild-type and OsSRT1 RNAi (T1 generation) plants were fixed, sectioned, and processed for terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). TUNEL can sensitively detect DNA fragmentation, one of the hallmarks of PCD, by labeling exposed 3' hydroxyl ends of DNA fragments using fluorescein-dUTP. The same leaf sections were simultaneously stained with propidium iodide to reveal all nuclei (red) in each section. None of the nuclei in wild type was TUNEL positive but, in contrast, most nuclei in the OsSRT1 RNAi leaf section were TUNEL positive (green; Fig. 5B), indicating that DNA damage was generally induced in the OsSRT1 RNAi leaves.
Overexpression of OsSRT1 Enhanced Tolerance to Oxidative Stress To further study the function of OsSRT1, the cDNA was inserted in an overexpression vector under the control of the maize ubiquitin promoter. More than 30 independent lines were obtained. Northern-blot analysis showed that most of the transgenic plants overexpressed OsSRT1 (Fig. 6A ). Western-blot analysis of enriched histone fractions detected a decrease of H3K9 acetylation in overexpression plants (Fig. 6B). The overexpression plants showed no particular visible or morphological phenotype. However, when treated with paraquat (1,1'-dimethyl-4,4'-bipyridylium), an herbicide that induces oxidative stresses in plants, the overexpression plants showed an enhanced tolerance compared to the wild type, as demonstrated by fewer and smaller lesions observed on the overexpression plants than the wild type (Fig. 6, C and D), whereas no clear difference was seen between wild-type and transgenic siblings without overexpression of OsSRT1 (Supplemental Fig. S1). This suggests that the increased tolerance to paraquat was induced by OsSRT1 overexpression.
Transcriptomic Analysis Revealed Activation of Many Transposon and PCD-Related Genes To study whether the down-regulation of OsSRT1 affected gene expression, we compared the transcripts of the RNAi to the wild-type plants by microarray analysis (Affymetrix). RNAs were isolated from young leaves of 11-d-old plants (before appearance of lesions in the RNAi plants). Analysis of data from three biological repeats revealed that 521 genes were up-regulated and 213 down-regulated (with q value at 5%). (The data are under the accession no. GSE7197 at http://www.ncbi.nlm.nih.gov/geo.) Gene ontology classification of the deregulated genes revealed that most categories had more up- than down-regulated genes, in agreement with the global up/down ratio (Table I ). For instance, the transposon categories (both DNA and retroelements) had 40 members activated in the RNAi leaves, but only four DNA elements were repressed. Most of the activated transposons listed in Table II were not expressed in wild-type rice as revealed by background signals of the microarray hybridizations. In addition, a relatively large number of stress-responsive and stress-related (i.e. phenylpropanoid metabolism, defense response, DNA repair) genes were deregulated (Table I). To confirm the microarray data, we performed semiquantitative reverse transcription (RT)-PCR analysis of RNA isolated from OsSRT1 RNAi young leaves harvested at 7, 11, and 21 d after germination to compare with wild-type and OsSRT1 overexpression plants. As shown in Figure 7A , the expression of tested DNA and retroelements was induced early in 7-d-old RNAi leaves. Overexpression of OsSRT1 had a negative effect on the expression of the DNA elements, but seemed to have some positive effect on the two tested retroelements (Fig. 7A). It was not known at this stage whether the induction of the retroelement was directly related to the OsSRT1 overexpression or a consequence of an indirect effect induced by the overexpression.
We compared the expression of two hypersensitive response (HSR201 and HSR203J) marker genes (Czernic et al., 1996 ) and a cytochrome P450 gene (called APO) that is closely related to wheat (Triticum aestivum) CYP709C1 and CYP709C3v2, both of which are suggested to be involved in wheat defense to pathogens (Kandel et al., 2005; Kong et al., 2005). HSR201 and APO, but not HSR203J, were found to be induced by OsSRT1 RNAi in the microarray data (Table III
). Consistently, the RT-PCR results showed that HSR201 and APO, but not HSR203J, were activated early in 7-d-old RNAi plants (Fig. 7B). In contrast, HSR203J was repressed by OsSRT1 overexpression (Fig. 7B).
The microarray data revealed that the OsSRT1 RNAi induced SAG12 but not SAG13 (Table III), both of which are senescence-associated genes (Pontier et al., 1999 ; Brodersen et al., 2002). RT-PCR revealed that SAG12 was induced 11 d after germination. OsSRT1 overexpression repressed the SAG12 at day 21 after germination. The expression of SAG13 was not significantly altered by the deregulation of OsSRT1 (Fig. 7B). The microarray data also showed moderate induction of PATHOGENESIS-RELATED (PR) genes that are activated during hypersensitive responses (Table III). The RT-PCR results showed the induction of the tested PR genes 11 d after germination, later than that of HSR201 and APO (Fig. 7B), in agreement with the fact that PR gene induction is a downstream event of hypersensitive responses. OsSRT1 overexpression showed induction of PR1a and PR1b at day 7 after germination, but repression of the tested PR genes at day 21 after germination. These observations suggested that PR genes might be not the direct targets of OsSRT1 and that the deregulation of OsSRT1 might have an indirect impact on PR gene expression.
To study whether the activation of the transposable elements and PCD marker gene expression by down-regulation of OsSRT1 was linked to alterations in histone acetylation, we performed chromatin immunoprecipitation (ChIP) assays. Chromatin fragments isolated from 11-d-old leaves of wild-type and OsSRT1 RNAi plants were immunoprecipitated with antibodies against acetylated histone H3 or acetylated histone H3K9. The precipitated chromatin DNA was analyzed by real-time PCR to test for enrichment relative to nonprecipitated (input) genomic DNA. The enrichment of promoter fragments relative to input chromatin DNA in the wild type (arbitrarily assessed as 100%) was compared to that found for the transgenic plants. As shown in Figure 8
, H3K9 acetylation was induced on both the tested DNA and retroelements. H3K9 acetylation was clearly induced on HSR201 and APO, in agreement with the expression data. In addition, the overall H3 acetylation was significantly induced on the DNA elements and HSR201. This suggested an induction of acetylation of other H3 Lys residues by H3K9 acetylation in the chromatin regions, as there is an agonistic relationship between different Lys residues for acetylation (Strahl and Allis, 2000
Compared to other eukaryotes, plants have relatively fewer SIR2-related genes. This would suggest that the plant members may have a larger spectrum of functions compared to their yeast or animal counterparts. For instance, human SIRT1, SIRT6, and SIRT7 are localized to nucleus, but SIRT1 has been shown to regulate nonhistone proteins involved in apoptosis, cell survival, transcription, and metabolism. Our data showed that OsSRT1 is nuclear localized, suggesting that the rice protein may function mainly in the nucleus. Alternatively, the smaller number of SIR2-related genes found in plants may be compensated by other plant HDAC genes, as plants possess an additional class of HDAC genes, namely, HD2 (Lusser et al., 1997 ; Pandey et al., 2002). This plant-specific class of HDACs lacks sequence similarity with other classes of HDACs that are conserved from plants to humans (Pandey et al., 2002). Arabidopsis HD2-related proteins (named HDT1–HDT4) are nucleolus localized. Analysis of the Arabidopsis family members identifies HDT1 as a gene important for silencing of one parental set of ribosomal RNA genes in a genetic hybrid, an epigenetic phenomenon called nucleolar dominance (Lawrence et al., 2004). In addition, it has recently been shown that Arabidopsis HDA6, a member of the RPD3-type of HDACs, is also involved in the rDNA silencing (Earley et al., 2006).
Both of the identified plant SIR2-related genes were significantly divergent from yeast SIR2, as they were found in different subclasses. Deletion of yeast SIR2 caused increases of histone acetylation within the rDNA region. However, we did not detect any changes of histone acetylation on rice rDNA in the OsSRT1 RNAi plants, in which overall rDNA expression seemed not to be affected (data not shown). Accordingly, the OsSRT1 protein seemed not to be confined or enriched in the nucleolus (Fig. 2B). Instead, our data showed clear increases of H3K9 acetylation on the tested DNA transposable elements and retroelements in the OsSRT1 RNAi plants. The increases of H3K9 acetylation were in agreement with the transcriptional activation of the DNA and retroelements. In addition, our data showed that the down-regulation of OsSRT1 also affected H3K9 acetylation and expression of hypersensitive response and PCD marker genes, and induced apoptotic cell death on leaves. In agreement with the data, overexpression of OsSRT1 decreased H3K9 acetylation and exhibited enhanced tolerance to an oxidative agent. OsSRT1 is found in the same class (IV) as human nucleus-localized HsSIRT6 and HsSIRT7 proteins, but OsSRT1 is more closely related to HsSIRT6 than HsSIRT7. HsSIRT6 has a weak in vitro HDAC activity. SIRT6 knockout mice display a deficiency in DNA repair and genomic instability (Mostoslavsky et al., 2006
We showed that OsSRT1 was widely expressed in rice, with highest levels in active cell dividing organs/tissues. Down-regulation of OsSRT1 by RNAi induced lesion mimic cell death and precocious senescence, whereas overexpression showed tolerance to oxidative stress. These data suggest that OsSRT1 is involved in the safeguard against genome instability and/or oxidative stress, required for plant cell growth. Histochemical staining, TUNEL assays, and molecular marker gene analysis demonstrated that cell death was induced in OsSRT1 RNAi plants. The TUNEL positive signals detected in nuclei of the RNAi leaf cells were indicative of DNA fragmentation, supporting the occurrence of apoptotic PCD in the RNAi plants. However, the production of H2O2 and activation of HSR201 suggested that the cell death in the OsSRT1 RNAi plants also resembled hypersensitive response-mediated PCD. Either both types of PCD were induced by OsSRT1 down-regulation, or different triggers of PCD may be interdependent in plants and the downstream effectors of PCD may be shared among different pathways. In plants, it is not known whether PCD occurs in response to DNA damage as a defense mechanism, as DNA damage-induced PCD in mammalian cells requires the activation of p53 (Chowdhury et al., 2006 Molecular analysis showed an increase of H3K9 acetylation and a decrease of H3K9 dimethylation in OsSRT1 RNAi leaves. Since H3K9 dimethylation is found to be mainly associated with inactive chromatin, these data suggest that OsSRT1 is needed for deacetylation and subsequent dimethylation of H3K9 to inactivate chromosomal domains. The early activation of transposable elements and two of some PCD marker genes, along with the increase of H3K9 acetylation on these genes, suggests that both transposable elements and protein-coding genes may be among the primary targets of OsSRT1. Whether OsSRT1 is directly involved in the repression of the targets requires further analysis.
The activated transposable elements were silent in wild-type plants as judged by the hybridization signals that were at near background levels, suggesting these transposable elements might be within silent heterochromatin domains that are known to be associated with DNA methylation (Tariq et al., 2003
Gene Cloning and Sequence Analysis The cDNA fragments of OsSRT1 were amplified from rice (Oryza sativa L. sp. indica) ‘Minghui63’ by RT-PCR. Two micrograms of total RNA from young panicles were reverse transcribed in a total volume of 20 µL with 0.5 µg oligo(dT)15, 0.75 mM dNTPs, 10 mM dithiothreitol, and 100 units SuperScript II RNase H– reverse transcriptase (Invitrogen). The following PCR primers were designed: SRT1-F (5'-GGGGGTACCGAGAGATGTCACTTGGCTATGC-3'; a KpnI site was introduced and is underlined) and SRT1-R (5'-GGGGGATCCCCAGCTTTCACATGCACTAG-3'; a BamHI site was introduced and is underlined). ExTaq DNA polymerase (TaKaRa) was used to amplify with the following cycling profile: 94°C for 3 min; 30 cycles of 94°C for 1 min, 57°C for 1 min, and 72°C for 2 min; and extension at 72°C for 10 min. The PCR product was cloned into the pGEM-T vector to obtain the pT237 clone (Promega) and confirmed by sequencing from both ends.
For sequence analysis, all of the SRT family sequences that were used for sequence alignment and phylogenetic analysis were downloaded from the plant ChromDB database (http://www.chromdb.org/). The sequences of the Sir2 domain (Pfam accession no. PF02146) were searched in the Pfam database (http://pfam.janelia.org/). Phylogeny reconstruction of all Sir2 domain sequence alignments was performed by MEGA 3.1 (Kumar et al., 2004
The nuclear localization vector was constructed by replacing the GUS fragment of pCAMBIA1391Xb (CAMBIA) with a ubiquitin promoter-GFP cassette. The coding region of OsSRT1 cDNA was amplified using the following primer pair: FU-F 5'-GGGGAATTCTCGGGAGAAGCTTACTTGATTGAG-3' (an EcoRI site was introduced and is underlined) and FU-R 5'-GGGGGATCCACTGATCGAAGAAATGGCAAAGG-3' a BamHI site was introduced and is underlined). The amplified fragment was inserted upstream to and in frame with GFP. The procedure of bombarding onion (Allium cepa) epidermal cells was as described (Dai et al., 2007
A 412-bp cDNA fragment of OsSRT1 was amplified using primers RNAi-F (5'-GGGACTAGTGGTACCAGTCCTGCAAGAGTTGCAAC-3' with a SpeI site, bold letters, and a KpnI site, underlined integrated) and RNAi-R (5'-GGGGAGCTCGGATCCCCAGCTTTCACATGCACTAG-3' with a SacI site and a BamHI site). PCR products were digested with KpnI/BamHI and SacI/SpeI, respectively, and inserted into pDS1301 (Chu et al., 2006
The overexpression vector was constructed by directionally inserting the full cDNA sequence (digested with BamHI/KpnI) into the binary vector pU1301, which was modified based on pCAMIA1301 (CAMBIA) and contained a maize (Zea mays) ubiquitin promoter. Agrobacterium tumefaciens (strain EHA105)-mediated transformation of rice plants was conducted according to a published protocol (Lin and Zhang, 2005
For northern-blotting analysis, 15 µg of total RNA samples extracted from tissues or organs harvested from field-grown rice plants was separated in 1.2% (w/v) formamide-denaturing agarose gels, before being transferred to nylon membranes. Gene-specific probes were labeled with 32P-dCTP using the Random Primer kit (Invitrogen) and hybridized to the RNA blots. The probe of OsSRT1 was digested from pT237 plasmid with KpnI and SpeI, a fragment of 635 bp of the cDNA.
For microarray analysis, transgenic and wild-type seedlings were grown in half-strength Murashige and Skoog medium under a 16-h-light/8-h-dark cycle at 25°C for 11 d, for three biological repeats. RNA samples were extracted using TRIzol (Invitrogen) as described by the manufacturer. Hybridization with Affymetrix GeneChip Rice Genome Arrays was performed at CapitalBio Corporation. The dataset was normalized with the option of all probe sets scaled to the target signal of 100. The genes with expression calls as absent from at least 11 arrays were filtered, resulting in 17,806 genes for further analyses. The significance analysis of microarrays (SAM) Excel add-in (Tusher et al., 2001 For semiquantitative RT-PCR analysis, 2 µg of total RNA was reverse-transcribed in a total volume of 20 µL with 0.5 µg oligo(dT)15, 0.75 mM dNTPs, 10 mM dithiothreitol, and 100 units of SuperScript II RNase H– reverse transcriptase (Invitrogen). PCR was performed in a total volume of 20 µL with 1 µL of the RT reactions, 0.2 µM gene-specific primers, and 1 unit of rTaq (TaKaRa). Twenty-five to 30 cycles were performed. Rice actin cDNA was used as internal control. The sequences of the used primers are listed Supplemental Table S1.
The DAB uptake method (Thordal-Christensen et al., 1997
The TUNEL assays were performed using the In Situ Cell Death Detection Kit-Fluorescein (Roche Diagnostics). Leaf tissues from wild-type and RNAi plants were fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (pH 7.2) containing 0.1% (v/v) Triton X-100 and Tween 20 at 4°C overnight and embedded in paraplasts. Eight-micrometer sections on glass slides were dewaxed in xylene, rehydrated, and then pretreated with 20 mg/mL proteinase K in 10 mM Tris-Cl, pH 7.5, for 20 min at room temperature. Two slides, treated with 1,500 units/mL DNase I in 50 mM Tris-Cl, pH 7.5, 1 mM MgSO4, and 1 mg/mL bovine serum albumin for 20 min at room temperature, served as positive controls. Two slides, labeled in the absence of the terminal deoxyribonucleotidyl transferase enzyme, served as negative controls. Vectashield mounting medium (Vector Laboratories) and protease inhibitors (1 mg/mL; Sigma) were used to mount the slides before they were viewed and photographed with a Leica microscope.
For paraquat treatments, leaves of 14-d-old plants (three replications, 50 per treatment) were exposed to a surface application of 10 µM paraquat (Sigma) in a 0.1% solution of the nonionic surfactant Ortho X77 (Valent USA) 5 h during the light period. The controls were treated with 0.1% surfactant alone (Donahue et al., 1997
Rice leaf histone protein extraction was performed as described (Tariq et al., 2003
ChIP assays were performed as described (Benhamed et al., 2006
Genomic DNA (1 µg) was digested for 6 h at 37°C with 30 units of HpaII, MspI, or HaeIII. Five percent of the digested and input (undigested) DNA was analyzed by PCR (Onodera et al., 2005 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number XP_471492.
The following materials are available in the online version of this article.
We thank Li Xianhua for technical assistance, Xu Caiguo for rice field management, and Song Huazhi for assistance with confocal microscopy. We acknowledge Qiu Deyun and Xie Weibo for help in microarray data analysis. Received March 13, 2007; accepted April 17, 2007; published April 27, 2007.
1 This work was supported by grants from the National Special Key Program of Rice Functional Genomics and the National Natural Science Foundation of China. 
2 These authors contributed equally to the article. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Dao-Xiu Zhou (dao-xiu.zhou{at}u-psud.fr).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.107.099473 * Corresponding author; e-mail dao-xiu.zhou{at}u-psud.fr; fax 33–1–69153424.
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ABSTRACT
RESULTS
-mercaptoethanol), then separated on a 16% SDS-PAGE and transferred to an Immobilon-P PVDF transfer membrane (Millipore). The membrane was blocked with 2% bovine serum albumin in phosphate-buffered saline (pH 7.5), and incubated overnight with primary antibodies, such as anti-acetylated histone H3, anti-dimethyl-histone H3K9, and anti-acetyl-histone H3K9 (from Upstates; catalog nos. 06–599, 07–441, and 07–352, respectively) in a 1:5,000 dilution at room temperature. After three washes (30 min each), the secondary antibody (goat anti-rabbit IgG [SouthernBiotech]) at 1:10,000 dilution was used. Visualization was performed using the SuperSignal West Pico kit (Pierce) according to the manufacturer's instructions. 
