Epigenetic malleability at core promoter initiates tobacco PR-1a expression post salicylic acid treatment

Tobacco’s PR-1a gene is induced by pathogen attack or exogenous application of salicylic acid (SA). Nucleosome mapping and chromatin immunoprecipitation assay were used to delineate the histone modifications on the PR-1a promoter. However, the epigenetic modifications of the inducible promoter of the PR-1a gene are not fully understood yet. Southern approach was used to scan the promoter of PR-1a to identify presence of nucleosomes, ChIP assays were performed using anti-histones antibodies of repressive chromatin by di- methylated at H3K9 and H4K20 or active chromatin by acetylated H3K9/14 and H4K16 to find epigenetic malleability of nucleosome over core promoter in uninduced or induced state post SA treatment. Class I and II mammalian histone deacetylase (HDAC) inhibitor TSA treatment was used to enhance the expression of PR-1a by facilitating the histone acetylation post SA treatment. Here, we report correlated consequences of the epigenetic modifications correspond to disassembly of the nucleosome (spans from − 102 to + 55 bp, masks TATA and transcription initiation) and repressor complex from core promoter, eventually initiates the transcription of PR-1a gene post SA treatment. While active chromatin marks di and trimethylation of H3K4, acetylation of H3K9 and H4K16 are increased which are associated to the transcription initiation of PR-1a following SA treatment. However, in uninduced state constitutive expression of a negative regulator (SNI1) of AtPR1, suppresses AtPR1 expression by six-fold in Arabidopsis thaliana. Further, we report 50-to-1000-fold increased expression of AtPR1 in uninduced lsd1 mutant plants, up to threefold increased expression of AtPR1 in uninduced histone acetyl transferases (HATs) mutant plants, SNI1 dependent negative regulation of AtPR1, all together our results suggest that inactive state of PR-1a is indeed maintained by a repressive complex. The study aimed to reveal the mechanism of transcription initiation of tobacco PR-1a gene in presence or absence of SA. This is the first study that reports nucleosome and repressor complex over core promoter region maintains the inactivation of gene in uninduced state, and upon induction disassembling of both initiates the downstream gene activation process.


Introduction
PR-1a (Pathogenesis Related-1a) gene is a major defenserelated gene of the PR family of tobacco (Nicotiana tabacum). Linker scanning mutagenesis of the PR-1a promoter identified two as-1 elements and one W-box in the activator region as strong positive, weak negative, and strong negative cis-elements respectively [1]. The core promoter region of the PR gene family has a conserved TATA, initiator (INR), and downstream promoter element (DPE)-like elements, located about + 28 to + 33 nucleotides downstream of the transcription start site and plays an important role in the initiation of gene transcription [2,3]. The detailed chromatin This manuscript is published online by BioRxiv (CSHL) pre-print platform and DOI is https:// doi. org/ 10. 1101/ 2021. 07. 24. 453639. There is no license agreement between authors and pre-print press. 1 3 modifications of PR gene promoter especially in core promoter sequences during the induction have not been reported yet. Basic amino acids (lysine, arginine and histidine) of N-terminus of histone proteins are marks for post-translational modifications including acetylation, methylation, phosphorylation and ubiquitination, these dynamic modifications are reversible and control the chromatin structure [4,5]. Later, these modifications termed as 'histone code' that determine the hetero or active chromatin state and transcriptional silencing or activation of genes. The histone acetylation reversibly modified by histone acetyltransferases (HATs) and histone deacetylases (HDACs). In Arabidopsis, there are 12 histone acetyltransferases and 18 histone deacetylases. Histone acetyltransferases are classified in four families: (a) the GNAT/HAG, (b) the MYST or HAM, c) the p300/CBP and d) the TAFII250 or HAF families [6]. Arabidopsis has three (HAG1, HAG2 and HAG3) GNAT family members, two (HAM1 and HAM2) MYST or HAM family genes, five (HAC1, HAC2, HAC4, HAC5 and HAC12) CBP/ p300-family genes [7], and two (HAF1 and HAF2) TAFII250 or HAF family genes respectively [8].
Until 2004, histone methylation was thought to be irreversible, first discovered LSD1 removes stable histone methylation through histone exchange therefore histone methylation is considered as reversible as well [9]. It is a dynamic process that regulates not only the methylation of histones but also the interaction of histones with other functional proteins. Later, histone demethylases such as LSD1 (also known as KDM1A) which demethylates mono-and di-methylated lysine, specifically histone 3, lysine 4 and 9 (H3K4 and H3K9) [10][11][12] and Jumonji C (JmjC domain-containing) proteins [13][14][15] were also identified. Four LSD1 like proteins have been reported in A. thaliana based on conserved domains (amine oxidase and SWIRM) found on the human LSD1 [11]. From Saccharomyces pombe to humans, histone demethylase LSD1 family is conserved and regulates histone methylation by both histone methylases and demethylases. Unlike LSD1, which can only remove mono and dimethyl lysine modifications, JmjC domain-containing histone demethylases (JHDMs) can remove all three-histone lysine-methylation states.
Acetylation of histones H3 and H4 is mostly associated with transcriptionally active euchromatin, while methylation is associated with either active or inactive chromatin depending on the methylated amino acid residue [16]. Methylation at H3K4, H3K36, and H3K79 is the hallmark of active transcription, whereas methylation at H3K9, H3K27, and H4K20 is associated with transcriptionally inert heterochromatin [12]. Lysine can be monomethylated, di-methylated, or trimethylated and each methylation state may have a unique biological function, further increasing the complexity of the 'histone code'. Overall, histone methylation and acetylation are important for almost all stages of development by ensuring proper regulation of coordinated gene expression from plants to humans and aberrant histone methylation or acetylation cause several developmental disease implications [17][18][19].
Epigenetic regulation of genes widely studied in higher eukaryotes, however in plants fewer studies are published focusing on the role of histone modifications (acetylation/ deacetylation or methylation/demethylation) in the gene expressions including FLC gene, PR genes of defense pathway, pea plastocyanin gene and wheat TaPR1 [20][21][22][23][24][25][26]. Other than histone modifications, none of the studies reported yet detailed mechanisms of transcription initiation or any presence of nucleosomes on the genes and their fate after getting the induction signal. Histone repression marks remodel in to active marks and nucleosome over core promoter region either slides upstream/downstream or disassembles for accession of transcription machinery before [27] or concurrently [28] with transcriptional activation.
In plant system, our study first time reports the underneath mechanism of tobacco PR-1a silencing in resting condition or induction in presence of SA. We scanned the promoter region, identified the presence of six nucleosomes on promoter region and more importantly, nucleosome over core promoter region. Moreover, we identified presence of a LSD1 like-CoREST-HDAC1 repressor complex over core promoter region with nucleosome in resting condition, play a major role in the gene silencing. However, more study needs to be done for further validation. Active chromatin marks on core promoter's nucleosome upon SA treatment, initiate the changes in chromatin organization on PR-1a locus, eventually open the chromatin by disassembling the nucleosome. We showed that fine tunes of histone acetylation, methylation and expression of negative regulators (Arabidopsis AtSNI1) tightly control the PR genes expression, it is important to provide the defense response and preparation for SAR.

Plant materials and growth condition
Nicotiana tabacum cv. Petite Havana, used as the wild type, was grown in the greenhouse at 22 °C ± 1 in long-day conditions (16 h light-8 h dark). Arabidopsis thaliana Col-0 was used as the wild type. All the mutants were in Col-0 background and Arabidopsis LSD1 mutants [12]

Plasmid constructions and plant transformation
The PR-1a promoter was amplified from the genomic DNA of tobacco (Nicotiana tabacum) by using forward PRF and reverse PRR primers (Table S1) and fused to gusA gene in pBluescript SK + as in [3]. Agrobacterium tumefaciens mediated plant transformation was performed comprising construct containing PR-1a promoter to examine the expression in stable transgenic lines of Nicotiana tabacum cv. Petit Havana.

SA and TSA treatments of plant leaves
The effect of SA (Sigma-Aldrich, Cat # S7401) and TSA (Sigma-Aldrich, Cat # T8552) on promoter expression were studied on discs. Ethyl alcohol (Sigma-Aldrich, Cat # E7023) was used as solvent for both SA and TSA. For SA, we made 50 mg/ml stock solution in ethyl alcohol (aliquoted in small vials and used one vial at a time to avoid freeze and thaw), added desired amount of stock solution in reverse osmosis (RO) water to get final 2 mM final SA concentration [3]. Acidic pH of solution was neutralized with potassium hydroxide (KOH) [29]. For TSA, we made 10 mM stock solution in ethyl alcohol, added desired amount of stock solution in RO water to get final 300 µM final TSA concentration.
We performed solvent control experiment in the beginning of any experiments to test the SA induction (Supplementary 5S). Discs of three cm diameter were excised from expanded leaves of transgenic tobacco plants and floated on water or 2 mM SA in petri-dish. For inhibition of histone deacetylases, the leaves were treated with 300 µM TSA. The leaves were incubated for 12 h in light at 25 ± 2 °C. In the case of Arabidopsis, 100 mg intact 21-days old plantlets were floated on water or SA or SA and TSA.

DNA sequence mapping of nucleosome's border
The 10 g leaves were treated with water or 2 mM SA for 12 h with gentle agitation in light. After 12 h, the samples were subjected to cross-linking in NIB1 buffer (0.5 M hexylene glycol, 20 mM KCl, 20 mM PIPES at pH 6.5, 0.5 mM EDTA, 0.1% Triton X-100, 7 mM 2-mercaptoethanol) in the presence of 1% formaldehyde for 10 min. The crosslinking was stopped by adding glycine to a final concentration of 0.125 M for 5 min at room temperature. The leaves were then rinsed with water, grounded to powder in liquid nitrogen, and treated with nuclei isolation buffer NIB1. The extract was filtered through 4 layered muslin cloth and finally filtered sequentially through 80, 60, 40, and 20 μm mesh sieves. The filtrate was centrifuged at 3000×g at 4 °C for 10 min. The pellet was suspended in NIB2 (NIB1 without Triton X-100) and centrifuged again. The pellet was suspended in 5% percoll, loaded on 20-80% percoll (U.S. Biologicals, USA) step gradient, and centrifuged. The nuclei were removed from the 20-80% percoll interface, washed in NIB2, and resuspended in NIB1 buffer. The nuclear preparation equivalent to A 260 of 100 was incubated with micrococcal nuclease (300 units/μl) (Fermentas, USA) in a buffer containing 25 mM KCl, 4 mM MgCl 2 , 1 mM CaCl 2 , 50 mM Tris-Cl at pH 7.4 and 12.5% glycerol at 37 °C for 10 min. The reaction was stopped by adding an equal volume of 2% SDS, 0.2 M NaCl, 10 mM EDTA, 10 mM EGTA, 50 mM Tris-Cl at pH 8 and treated with proteinase K (100 μg/ml) (Ambion, USA) for 1 h at 55 °C. The crosslink was reversed by heating at 65 °C overnight. The DNA was extracted by phenol: chloroform and precipitated in ethanol. The DNA was separated on 1.5% agarose gel and fragments of an average size of 150 bp were purified, denatured, and hybridized with 20 ng of end-labelled forward PF3 and reverse NR1 primers of region 1. Primer extension was performed at 37 °C using 13 units of sequenase (U.S. Biologicals, USA) in 1× sequenase buffer containing 0.01 M DTT and 0.1 mM dNTPs according to manufacturer's protocol including ladders of all four nucleotides. The products were analyzed in 8% sequencing gels. The sequences of primers used in primer extension are given in Table S1.

Detection of nucleosomes on tobacco PR-1a promoter using a ChIP DNA template
PCR was used to locate nucleosomes in the upstream, downstream, and core promoter regions. MNase digested mononucleosome DNA precipitated with H3 was used as a template to detect the amplicon in uninduced, induced state and TSA treated leaves. Mono-nucleosomes were purified using Hydroxyapetite (HAP) protocol [30]. The forward primers (PF3, NPAF1, NPAF5, and NPCF1) and the reverse primers (NR1, NPAR1, NPAR5, and NPCR1) were used to analyze the protection of the core promoter (− 102 to + 55 bp), the upstream (− 362 to − 213 and − 262 to − 102 bp) and downstream (+ 59 to + 208 bp) regions respectively of the PR-1a promoter against micrococcal nuclease digestion in the uninduced and induced states. The sequences of all primers are given in Table S1. To do native ChIP, 1.5-2 g leaf discs of tobacco excised from 8-9-week-old plants were floated on water or 2 mM SA and 300 µM TSA for 12 h with gentle agitation in the light. After 12 h the samples were rinsed with water and ground into powder in liquid nitrogen. Nuclei were extracted and washed with 1 ml of buffer N (15 mM Trizma base, 15 mM NaCl, 60 mM KCl, 250 mM sucrose, 5 mM MgCl 2 , 1 mM CaCl 2, pH 7.5, 7 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 50 μl/ml plant protease inhibitor cocktail) (Sigma chemicals, USA). Thereafter nuclei were suspended in 100 μl buffer N. DNA content was estimated in a 10 μl aliquot and MNase treatment were given using 1unit/μg DNA for 10 min at 37 °C., and finally eluted in 300 μl of HAP elution buffer (500 mM NA 2 PO4 pH7.2, 100mMNaCl, 1 mM EDTA) and was diluted with 1700 μl of ChIP dilution buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8, 167 mM NaCl, and 50 μl/ml protease inhibitor cocktail). The diluted chromatin solution was then subjected to 1 h of precleaning treatment at 4 °C with 80 μl of salmon sperm DNA/ protein agarose (Upstate; ). An aliquot of 50 μl was removed for the total input DNA control. Immunoprecipitation was performed overnight (18 h) at 4 °C using 600 μl chromatin solution with histone H3 antibodies (typically at 1:150 final dilutions) or without antibodies (mock control). Immunoprecipitates were collected after incubation with 40 μl of salmon sperm DNA/protein agarose (50% suspension in dilution buffer) at 4 °C for 1 h. The protein A agarose beads bearing immunoprecipitate were then subjected to sequential washes and eluted twice with 250 μl elution buffer each time (1% SDS and 0.1 M NaHCO 3 ). For the input DNA control (50 μl), 450 μl elution buffer was added. Protein was removed by 1.1 μl proteinase K (20 mg/ml) at 45 °C for 1 h and RNA by 2 μl of RNaseA (1 mg/ml) digestion at 37 °C for 1 h. The DNA was purified by phenol: chloroform extraction and ethanol precipitation, and purified DNA was resuspended in 50 μl TE buffer for PCR analysis.

Southern hybridization to detect nucleosomes in the promoter region of tobacco PR-1a
Twenty micrograms of purified MNase-digested DNA were analyzed to find out the position of nucleosomes in the PR-1a promoter. Eight probes of 200 bp from the core promoter region were designed (R1-R8). For positive control, 10 pg PR-1a promoter (PCR amplified) and for negative control 10 pg of sonicated calf thymus DNA was used. The entire DNA was transferred on to nylon membrane and incubated at 42 °C overnight with a probe.

ChIP PCR using precipitated DNA with different antibodies
The leaf discs (1.5-2 g) of tobacco excised from 8-9-weekold plants were floated on water or 2 mM SA for 12 h with gentle agitation in light. After 12 h the samples were subjected to 1% formaldehyde cross-linking in a cross-link buffer (0.4 M sucrose, 10 mM Tris-HCl, pH 8, and 1 mM EDTA) under vacuum for 10 min. Formaldehyde cross-linking was stopped by adding glycine to a final concentration of 0.125 M and incubating for 5 min at room temperature. The leaf pieces were then rinsed with water and ground to powder in liquid nitrogen. Nuclei were extracted and lysed with 300 μl of lysis buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, 1% SDS, 1 mM PMSF, 10 mM Sodium butyrate, 1 mM benzamidine, and 50 μl/ml protease inhibitor cocktail) (Sigma Chemicals, USA). The resulting chromatin was subjected to pulse sonication (six pulses, 95% power output for eight times) using a Bransonic M3210 (Danbury, USA) to obtain DNA fragments with sizes ranging from 500 to 1000 bp. After sonication, a 25 μl aliquot was removed for the total input DNA control, and the rest of the chromatin solution was diluted 10 times with ChIP dilution buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8, 167 mM NaCl, and 50 μl/ml protease inhibitor cocktail). The diluted chromatin solution was then subjected to 1 h of precleaning treatment at 4 °C with 40 μl of salmon sperm DNA/protein agarose (Upstate; 16-157) (50% suspension in dilution buffer without Sodium butyrate and protease inhibitor cocktail) to reduce nonspecific interactions between protein-DNA complexes and the agarose beads. Immunoprecipitation was performed overnight (18 h) at 4 °C using 600 μl chromatin solution with antibodies (typically at 1:150 final dilutions) or without antibodies (mock control). Immunoprecipitates were collected after incubation with 40 μl of salmon sperm DNA/protein agarose (50% suspension in dilution buffer) at 4 °C for 1 h. The protein A agarose beads bearing immunoprecipitate were then subjected to sequential washes and eluted twice with 250 μl elution buffer (1% SDS and 0.1 M NaHCO 3 ). Samples were then reverse cross-linked at 65 °C under high salt (0.2 M NaCl) for 6 h. For the input DNA control (25 μl), 275 μl TE buffer (10 mM Tris-HCl, pH 8, and 1 mM EDTA) was added and reverse cross-linked. After reversing the crosslinks, the protein was removed by 1.1 μl of proteinase K (20 mg/ml) at 45° C for 1 h and RNA by 2 μl of RNaseA (1 mg/ml) (Qiagen) digestion at 37 °C for 1 h. The DNA was purified by phenol: chloroform extraction and ethanol precipitation. Purified DNA was resuspended in 40 μl TE buffer for PCR analysis.
For ChIP PCR the target region of the PR-1a promoter was -102 to + 55 with reference to the transcription start site. Forward primer PF3 and reverse primer NR1 were used for amplifying the core promoter. Tobacco ACTIN promoter was taken as an internal control for active chromatin, using forward AGF and reverse AGR primers for PCR Table S1. For testing the enrichment of various modifications on the R8 promoter at different time points, forward primer PF3 and reverse primer NR1 were used for qPCR. Reactions were placed in 25 μl volume in triplicate according to the manufacturer's instruction (Invitrogen SYBR green ER) on ABI PRISM 7500.

Transcript detection of different defense-related genes of Arabidopsis using qPCR
To compare the transcript levels of AtPR1, AtSNI1, AtPDF1.2, and AtASN1, leaves of wild type A. thaliana plants (Col-0) were treated with water, 2 mM SA, 300 µM TSA alone or TSA and SA both. After 12 h, total RNA was isolated by Tri-reagent (Sigma) and treated with RNase-free DNase (Invitrogen). For temporal expression of tobacco PR-1a, the total RNA was isolated from the leaf discs which were floated on SA for different periods. The first-strand cDNA was synthesized, using 2 μg RNA, as per manufacturer's instructions (Invitrogen, USA). qPCR was used to determine the expression of AtPR1 in uninduced and induced states, using forward ATPRF and reverse ATPRR primers. Tobacco PR-1a expression at different time points was followed by PCR by using forward NPRF and reverse NPRR primers. The AtACTIN7 and UBIQUITIN genes were used as an internal control. The sequences of primers used are given in Table S1.

Results
Nucleosome over core promoter of PR-1a spans from − 102 to + 55 bp in the uninduced state Earlier, we reported a distinct nucleosome over the core promoter region of PR-1a in the uninduced state disassembles upon SA induction to initiate the transcription [3]. In the present study, we reported the mapping of nucleosomes using a primer extension method. It was performed after confirming the presence of nucleosome as well as on the entire promoter of PR-1a by southern hybridization. We performed by dividing the entire length of the PR-1a promoter (1.5 Kb) into eight distinct regions of around 200 bp (R1-R8). The region encompassing the core promoter and transcription start site (TSS) was designated as R8 (supplementary Fig. 1SA). The mono-nucleosome template from uninduced tobacco plants was prepared by digesting with micrococcal nuclease (MNase) enzyme (digest the linker region). Probes from different regions of the PR-1a promoter were used in southern hybridization with the MNase digested mono-nucleosome template. Southern hybridization reveals the presence of nucleosomes over five regions including R1, R2, R4, R5, and R8 on the promoter (supplementary Fig. 1SB). Nucleosome boundaries of R8 nucleosome (over core promoter) were mapped using the primer extension method with forward (PF3) and reverse (NR1) primers (Fig. 1a is showing the sequencing with one primer). The boundaries of the nucleosome were found to be spanning from − 102 to + 55 bp (with respect to the TSS) in PR-1a (Fig. 1b). The nucleosome over R8 masked the TATA region, transcription initiation site (+ 1), and downstream promoter region (− 102 to + 55 bp) in the uninduced state of PR-1a (supplementary Fig. 1SC).

Histone acetylation (H3K9/14Ac, H4K16Ac and trimethylation of H3-K4) marks associated with the temporal transcript activation of PR-1a followed by SA treatment
It was demonstrated that the PR-1a induction coincided with the disappearing or disassembly of the nucleosome over region 8 (R8) [3]. Here, we further examined the epigenetic changes in chromatin responsible for the disassembly of the nucleosome. Since histone acetylation associated with transcriptional activation and histone acetylation of H3K9/14 and H4K16 has been demonstrated in the activation of genes [31]. We checked the acetylation status of the H3K9 and H4K16 of R8 nucleosome using the ChIP approach in a time-dependent manner. ChIP results showed that the onset of transcription of PR-1a strongly correlated with the H3K9/14 and H4K16 acetylation, and trimethylation of H3-K4. Acetylation of these lysine residues increased -102 gradually from 3 to 9 h post-SA treatment reaching a maximum at 9 h (Fig. 2a-c). We performed the solvent control experiment for SA and TSA to see induction of PR-1a due to indeed effect of SA or TSA and result shows that there was no induction in the controls while SA or SA + TSA samples were induced (supplementary Fig. 5S).
The PR-1a is a late inducible promoter, its expression was noticed 9 h post SA treatment (Fig. 2d) which is well correlate to nucleosome occupancy on core promoter region (R8, Group 3), its occupancy reduced from 9 to 12 h while nucleosome occupancy remains same on pollen specific NTP303 core promoter. The core promoter of NTP303 was protected by a nucleosome [3] since NTP303 is not related to SA induction, position of the nucleosome was not altered by the SA treatment, establishing specificity of the SA treatment and the nucleosomal response ( Supplementary Fig. 2S). To further understand the correlation between PR-1a transcription and acetylation of its promoter, we studied the temporal regulation of PR-1a in response to SA and performed ChIP with acetylated H3K9/14 and tri-methylated H3K4 antibodies on the core promoter at different time points. The onset of transcription of PR-1a was correlated strongly with the acetylation of H3K9/14 (Fig. 2c). The H3K9/14 was highly acetylated at 9 h, remained so till 12 h post-SA treatment, and then declined. The results indicated that during 9 to 12 h post-SA treatment, there was a sharp, though the transient increase in acetylation of H3 in the nucleosome of the core promoter, whereas a slight increase in tri-methylation of H3K4 from 6 to 9 h post-SA treatment.

GNAT family member AtHAG1 Arabidopsis mutant show reduced induction of AtPR1 followed by SA treatment
To examine, whether the PR-1a locus genetically interacted with histone acetyltransferases, we performed experiments in Arabidopsis thaliana (Ws ecotype) because histone acetyltransferase mutants' plants of tobacco were not available and also assuming histone acetyltransferases are conserved in Arabidopsis as well. Therefore, we decided to examine three HAT-related RNAi mutants of A. thaliana (in Ws ecotype background) i.e., hag3, hac1, and hxa1 were examined ( Supplementary Fig. 4S). Arabidopsis HAG3 mutant plant (cs3983) belongs to GNAT family member of HATs, negatively regulates the expression of DNA repair enzymes after UV-B exposure [32]. HAC1 (cs30904) belongs to CBP/ p300 family of HATs [9] and HXA1 (cs30992) or HAC9 from CBP/p300 family of HATs. However, mutant plants of HAC1 and HXA1 are not well studied yet in Arabidopsis. The AtPR1 transcript in the mutants in uninduced states showed a significant increase of transcript as compared to the wild type Ws ecotype. An increase in the uninduced expression of AtPR1 suggested the loss of stringent regulation in the uninduced condition. Therefore, histone acetyltransferases HAG3, HAC1, and HXA1 are mediate the acetylation of histone marks on the nucleosomes to regulate the AtPR1 transcription.

Nucleosome disassembly from PR-1a core promoter is essential for transcriptional activation
The disappearance of the nucleosome could be either due to nucleosome sliding or complete disassembly. To further understand the fate of nucleosome remodelling at the PR-1a core promoter, we addressed the histone H3 occupancy either on Group 3 (R8) or on flanking upstream Group 1 or 2 and downstream promoter region group 4 of PR-1a by ChIP using the anti-H3 antibody in uninduced and SA treated leaves. We observed distinct nucleosome over group 3 (R8, − 102 to + 55) as evident PCR amplified product in case of uninduced control ( Fig. 1 and supplementary Fig. 1S).
Since SA treatment affects histone acetylation of the nucleosome over PR-1a core promoter region upon the induction (Fig. 2), the histone H3 occupancy was also studied in TSA, an inhibitor of histone deacetylase) treated tobacco leaf discs in the presence or absence of SA (Fig. 3a, b). The nucleosome over group 3 disappeared with SA induction, however, treatment with TSA in the presence or absence of SA inhibited nucleosome disappearance (Fig. 3a). We did not observe nucleosome protection over group 2 (− 213 to − 102) in any of the conditions tested indicating the lack of nucleosome over this region (Fig. 3b).
Multiple sets of primer pairs and ChIP template DNA were used to detect nucleosomes associated with different regions of the core promoter and flanking promoter of PR-1a (Fig. 3). The promoter flanking region in group 1 (− 362 to − 213) and group 4 (+ 59 to + 208) also have distinct nucleosomes, however, these nucleosomes did not show any change post-SA or TSA treatment.

TSA enhances early expression of tobacco PR-1a followed by SA treatment
The effect of HDAC inhibitor TSA was examined on the expression of PR-1a. The leaves were treated with SA for 4 h (to get the induction signal) and then shifted to either water or TSA. The expression of the PR-1a promoter was examined by assaying the GUS reporter gene fused to it. The analysis of three independent transgenic lines showed a clear effect of TSA as shown in average representative Fig. 4. We randomly selected three transgenic lines of PR-1a: GUS (1329-2, 4 and 10) and leaf discs were treated with SA followed by transfer in water or TSA. The expression was higher in the TSA-treated leaves till 25 h in comparison to the control leave discs (transferred in water) (Fig. 4). Higher expression correlated well with the H3K9/14 acetylation (Fig. 2a, c). After 25 h, there was no difference in expression in the two cases. The results indicated that short exposure to SA leads to transcription of PR-1a which was vulnerable to suppression by HDACs. However, after 25 h in water or TSA, stable H3K9/14 acetylation-insensitive expression was noticed.

Histone methylation plays a dual role in the transcriptional regulation of PR-1a
The role of histone methylation of nucleosome over the core promoter in PR-1a expression was also examined, using ChIP-qPCR with antibodies specific to mono-, di-or tri-methylated H3K4, H3K9 and H4K20. A gradual increase in H3K4 me1 (Fig. 5a) H3K4 me2 (Fig. 5b), H3K4me3 (Fig. 5c), and H3K9 me3 (Fig. 5f) were observed till 9 h post-SA treatment coinciding with transcription activation of PR-1a (Fig. 2c) and removal of nucleosome from the core promoter (Fig. 3). In contrast, H3K9 me1 (Fig. 5d) and me2 (Fig. 5e) were found to be enriched in the uninduced conditions and decreased subsequently post the SA treatment. H3K4 mono-methylation increased gradually up to 9 h accompanies the transcriptional activation at 9 h post-SA treatment (Fig. 5a). Increased trimethylation of lysine residues of H3K9 showed a dual role of histone methylation (activation and repression) in the transcriptional regulation of PR-1a. The methylation state of H4K20 was studied further, mono-, di-and tri-methylation of H4K20 (Fig. 5g-i) showed significantly low signals.

The human lysine specific demethylase 1 (LSD1) like gene causes silent state of PR-1a in an uninduced state
To examine whether the PR1 locus genetically interacted with LSD1 like genes, we performed experiments in Arabidopsis thaliana (Ws) because LSD1 like mutants of tobacco plants were not available. Four putative homologs (1-4) of LSD1 have been reported in A. thaliana viz. At3G13682, At3G10390, At1G62830 and At4G16310 [10]. We carried out quantitative real-time PCR of the AtPR1 transcript in these lsd1 like mutants in the uninduced state. In all the four mutants, a high level of AtPR1 was noticed in the uninduced state in contrast to a very low level of uninduced AtPR1 in wild type (Fig. 6a). The mutations in LSD1 like genes (At3G10390, At1G62830, and At4G16310) led to nearly constitutive expression of AtPR1. The results established that the lysine-specific demethylase family was involved in giving repressed chromatin conformation to the AtPR1 region in A. thaliana in the uninduced state. The results on lsd1 mutants encouraged us to determine the recruitment of LSD1 on the core promoter region of PR-1a.

TSA enhances the expression of AtSNI1, a negative regulator of AtPR1
Tobacco PR-1a promoter was not induced in the presence of TSA alone (Supplementary Fig. 3SA), we performed ChIP using anti-H3 antibody to detect the presence of nucleosome over core promoter region (R8, Group 3) post SA, TSA or SA + TSA treatment. Results show histone H3 was enriched in TSA treatment, it suggests the occupancy of nucleosome, which explains the PR-1a suppression in presence of TSA ( Supplementary Fig. 3SAB). To address, why TSA prevents the induction of the PR-1a, we carried out experiments on Arabidopsis thaliana (Columbia ecotype) because the regulators of the PR-1a gene in tobacco have not been identified. In A. thaliana, a negative regulator gene of AtPR1, called AtSNI1 has earlier been reported [33]. The expression of the regulatory genes was examined after treatment with SA and TSA. The AtSNI1 gene was not activated by SA treatment but was induced by TSA (Fig. 6b). The jasmonic acid (JA) inducible AtPDF1.2 gene was repressed by SA [34], while TSA did not affect its expression. The TSA inducible AtGDAS was used as a positive control and AtACTIN7 as an internal control in the experiments.

The LSD1-CoREST-HDAC1 complex associates with the silent state of PR-1a
Our results suggest the LSD1 maintains the silent state of PR-1a in the uninduced condition. In other studies, LSD1 was reported to be a part of the LSD1-CoREST-HDAC1 suppressor complex of neuronal genes in non-neural cells [35]. We examined whether this repressor complex was involved in maintaining the silent state of PR-1a also in the uninduced state. First, we checked the presence of LSD1 like protein on the core promoter region in an uninduced state. ChIP analysis of PR-1a locus was carried out in uninduced and induced states using custom-made (Supplementary information 1) anti-LSD1 specific antibody. ChIP qPCR result suggested that LSD1 like protein was indeed present on the core promoter region in the uninduced state (Fig. 6c). Next, we looked for the LSD1-CoREST-HDAC1 Complex on the PR1-a locus. We performed ChIP using again anti-LSD1 like, anti-CoREST, and anti-HDAC1 specific antibodies. The results indicate the presence of CoREST and HDAC1 in the uninduced state of PR-1a chromatin similar to noticed for LSD1. The CoREST and HDAC1 were reduced when PR-1a was activated by SA (Fig. 6d).

Discussion
SA is the key signal molecule for the establishment of SAR [36]. Transcripts of tobacco PR-1a or AtPR1 are accumulated in response to SA signalling, which is a marker for the establishment of SAR [37,38]. Several efforts were made to elucidate the molecular mechanism of transcriptional regulation of PR genes [39]. In our present study, we focused on the epigenetic regulation core promoter nucleosome of the PR-1a gene and identified five nucleosomes over the promoter region of PR-1a in the uninduced condition spanning from the TATA-box and transcription initiation site to an upstream region (as-1 like element) (Fig. 1a, b; Supplementary Fig. 1S). The nucleosome over the TATA-box is responsible for the silent state of PR-1a transcription in the uninduced condition [3] and the unmasking of the TATAbox region is crucial to establish the pre-initiation complex and recruitment of RNA polymerase II [40,41]. The mechanism involving masking of the TATA-box by the nucleosome and suppression of transcription has been reported in several eukaryotic promoters [1,42]. The nucleosome over region 8 (R8) (Supplementary Fig. 2SB and 3SB) disappears post-SA treatment ( Fig. 3; group 3 (R8) and coincides with the PR-1a transcription (Fig. 4, Supplementary Fig. 2SA).
The disappearance of the nucleosome over the R8 could be either because of nucleosome sliding [43,44] or nucleosome disassembly [45][46][47] both mechanisms have been demonstrated in detail in different eukaryotic promoters [45]. Our native ChIP experiment using an anti-H3 antibody (Fig. 2a,  b, Supplementary Fig. 3SB) establishes that the disappearance of the nucleosome over the R8 (group 3) could not be possible because of sliding since the region immediately downstream of the core promoter (group 4) was occupied by a nucleosome and region immediately upstream (group 2) is always free of the nucleosome. It further confirms the lack of core histone from the R8 (group 3) in the SA-induced condition (Fig. 4). Thus, our results strongly support that the disappearance of the nucleosome over the R8 post-SA treatment is due to complete nucleosome disassembly. The Anti-Silencing Function1 gene (Asf1) is reported to disassemble the nucleosome in budding yeast [46,48]. Homologs of Asf1 have been reported from A. thaliana as well, suggested the possibility that nucleosome over the core region of tobacco PR-1a is disassembled by homologs of such genes. Following SA induction of PR-1a, acetylation of H3K9/14 increased 9 h post-SA treatment (Fig. 2a Histone methylation status on R8 was analyzed by ChIP assay using antibodies against mono-, di-and tri-methyl H3-K4, H3-K9 and H4-K20 (a-i). ChIP assay was per-formed using these antibodies on tobacco leaves treated with water (uninduced) or SA (induced) up to 24 h. The immunoprecipitated DNA was analyzed by qPCR. The histogram represents the % input (Y-axis) at different time points (X-axis) with SD 1 3 induction (Fig. 5) [3]. A rapid transient increase in acetylation of H3K9/14 at 9 h and a slight increase in tri-methylation of H3K4 in the activation of PR-1a transcription at the same time (Fig. 2c) indicate that the H3-K9/K14 acetylation is linked for the active state of PR-1a core promoter. The acetylation of H3-K9/14 has been reported in the activation of RBCS-1A and IAA3 genes [28,49]. Microarray analyses in tobacco and A. thaliana seedlings show that TSA induces changes in gene expression and affects histone acetylation in specific genes [11,50]. In A. thaliana, histone deacetylase AtHD1 (also called HDA19) is involved in the regulation of pathogen response genes [51]. We observed TSA-mediated suppression of AtPR1 transcription (Fig. 6b) and also inhibition in nucleosome modeling at the core promoter (Fig. 4) when TSA was provided along with SA. These results were surprising in the context of the importance of H3K9/14 and H4K16 acetylation required for PR-1a activation (Fig. 2a, b). Presence of nucleosome on core promoter region explains TSA-mediated suppression of PR-1a ( Supplementary  Fig. 3SAB).
Modification of the histone H3K4 di-and tri-methylation also enrich till 9 h post-induction and positively correlate transcriptional activation (Fig. 5b, c). Mono methylation of H3K4 is initially very little enrichment and its transient mild enrichment till 9 h at the PR-1a promoter. Earlier reports also suggest that the presence of H3K4me2 and H3K4me3 in plants is usually correlated with the active transcription of the highly expressed genes, whereas H3K4me1 is distributed within transcribed regions [52]. Our results also suggested that histone modification such as mono and di-methylation at lysine 9 and 20 of H3 and H4 respectively were found increased in the uninduced state of PR-1a (Fig. 5d-h). This agrees with the earlier reports that H4K20 methylation results in the repression of genes, which is associated with silent chromatin and inhibits acetylation of H4K16 [53,54]. Following SA induction, a decrease in H3K9 mono-and di-methylation suggested internal control. c Presence of LSD1 on chromatin of core promoter region of PR-1a was analysed by ChIP assay using anti-LSD1 antibody. The immunoprecipitated DNA was analyzed by PCR. Input DNA was used as ChIP control. d Detection of LSD1-like complex at core promoter region of PR-1a in uninduced state by ChIP PCR. ChIP assay was performed by using antibodies against LSD1, CoR-EST and HDAC1. The representative PCR products indicate the presence of LSD1, CoREST and HDAC1, in uninduced state. Input DNA was used as ChIP control their involvement in repressing the locus in the uninduced state, also reported by several other studies [5,55,56]. This decrease may be their conversion to the trimethylated state as shown by the H3K9 trimethylation enrichment, which is a mark for transcriptional activation [57]. Lack of H4K20 methylation in transcriptionally active regions has also been reported in the Drosophila male X chromosome as the methylation of H4K20 precludes acetylation of the neighboring H4K16, both processes being competitive [58]. However, ORC1-dependent gene activation in plants is associated with an increase in H4 acetylation and H4K20 trimethylation [59]. Moreover, monomethylated H4K20 is associated with heterochromatin, and di-and tri-methylated H4K20 are associated with euchromatin in Arabidopsis [60].
It is conceivable that the loss in di-and tri-methylation of H4K20 and di-methylation of H3K4 in PR-1a in the induced state results from enzymatic demethylation. A human LSD1 that demethylates mono and di-methylated H3K4 has been identified [10], suggesting the involvement of LSD1 like genes in tobacco or Arabidopsis for demethylation of the di-methylated H3K4 [61] (Fig. 6c,  d). Full enzymatic activity of LSD1 requires its association with other proteins, such as CoREST (restin corepressor) Fig. 7 Probable Model suggesting the sequential events and ordered modifications of chromatin over the PR-1a promoter in tobacco leaf. Histone modifications associated with various PR-1a promoter states are shown. The promoter region has six distinct nucleosomes including downstream nucleosome in the repressed state, as shown in (a). The nucleosome over core promoter has repressive histone marks (mono, di and trimethylated H4-K20 and H3-K9) and LSD1-CoREST-HDAC1 repressor complex (a). Following SA mediated activation (b) of PR-1a promoter, the repressor complex is dissociated from the core promoter region, possibly through the recruitment of histone acetyltransferase, resulting in H3K9ac and H4K16ac. Active histone methylation marks (mono, di and trimethylated H3-K4) also increase. Acetylation at H3-K9/14 and H4-K12 lead to decrease in histone-DNA interactions eventually nucleosome disappears from the core promoter (c) region, leading to the recruitment of pre-initiation complex (PIC Core promoter complex, indicating that regulatory subunits can have a role in modulating demethylase activity [10,62,63]. Our study showed that five nucleosomes cover the promoter region of PR-1a including a nucleosome over the downstream region (core promoter) or upstream activator region (covers as-1-like element responsible for induction) [64]. After induction, the nucleosome over the core promoter disassembles and provides the access to transcription initiation machinery on the nucleosome-free core promoter region. In conclusion, we suggest nucleosome association with LSD1-CoREST-HDAC1 suppressor-like complex correlate the silent state of PR-1a locus (Fig. 7).