Molecular Psychiatry (2014), 1–13 © 2014 Macmillan Publishers Limited All rights reserved 1359-4184/14 www.nature.com/mp

ORIGINAL ARTICLE

Epigenetic modulation of chronic anxiety and pain by histone deacetylation L Tran1, J Schulkin2, CO Ligon1 and B Greenwood-Van Meerveld1,3,4 Prolonged exposure of the central amygdala (CeA) to elevated corticosteroids (CORT) facilitates long-term anxiety and pain through activation of glucocorticoid receptors (GRs) and corticotropin-releasing factor (CRF). However, the mechanisms maintaining these responses are unknown. Since chronic phenotypes can be sustained by epigenetic mechanisms, including histone modifications such as deacetylation, we tested the hypothesis that histone deacetylation contributes to the maintenance of chronic anxiety and pain induced by prolonged exposure of the CeA to CORT. We found that bilateral infusions of a histone deacetylase inhibitor into the CeA attenuated anxiety-like behavior as well as somatic and visceral hypersensitivity resulting from elevated CORT exposure. Moreover, we delineated a novel pathway through which histone deacetylation could contribute to CORT regulation of GR and subsequent CRF expression in the CeA. Specifically, deacetylation of histone 3 at lysine 9 (H3K9), through the coordinated action of the NAD+-dependent protein deacetylase sirtuin-6 (SIRT6) and nuclear factor kappa B (NFκB), sequesters GR expression leading to disinhibition of CRF. Our results indicate that epigenetic programming in the amygdala, specifically histone modifications, is important in the maintenance of chronic anxiety and pain. Molecular Psychiatry advance online publication, 7 October 2014; doi:10.1038/mp.2014.122

INTRODUCTION The secretion of glucocorticoids (CORT) is fundamental to the endocrine response to stress1 and supports coordination of behavioral adaptations including anxiety and nociception.2–4 However, prolonged exposure of brain regions, such as the amygdala, to CORT can precipitate chronic conditions that can be detrimental to mental and physical health.5,6 For example, in animals, prolonged elevation of CORT in the central nucleus of the amygdala (CeA) was found to induce anxiety-like behaviors,7,8 somatic allodynia and visceral hyperalgesia.9,10 Evidence from preclinical studies suggests that the effects of CORT on anxiety and nociception involve the activation of glucocorticoid receptors (GRs) in the CeA.11,12 Activation of GR enhances a distinct neural network within the CeA, which is mediated via release of corticotropin-releasing factor (CRF).5,13,14 Several studies report that sustained elevations of CORT in the CeA by either chronic stress or repeated pharmacological CORT treatment increase the expression of CRF in the CeA.7,8,15–21 Moreover, the activation of the amygdala by CRF has been directly implicated in the pathophysiology of anxiety,22 somatic allodynia23 and visceral hyperalgesia.24 Previously, we demonstrated a concomitant decrease in GR and increase in CRF expression following a period of prolonged elevation of CORT in the CeA that persisted despite depletion of the source of CORT.21 Both anxiety-like behaviors and enhanced nociception induced by acute exposure of the CeA to CORT were found to be long term and persisted for at least 28 days beyond depletion of the source of CORT.25 Although the precise mechanism maintaining the observed behaviors was unclear, recent studies have implicated an important role of the epigenome in

sustaining extrinsic phenotypes.26 One key permutation of the epigenome that can subsequently result in sustained abnormal behaviors is histone deacetylation, a chemical modification that leads to sequestering of gene expression by the histones.27,28 The primary goal of the present study was to test the hypothesis that histone deacetylation in the CeA contributes to the chronicity of the elevated anxiety-like behavior, somatic allodynia and visceral hyperalgesia observed following elevation of CORT within the CeA. A secondary goal was to provide evidence supporting a molecular mechanism connecting histone deacetylation to chronic stress-induced behaviors. We specifically investigated acetylation at lysine 9 on histone 3 (H3K9), which has been shown to regulate the GR promoter.27,29,30 We also explored sirtuin-6 (SIRT6) as a candidate deacetylase involved in this process because of the established link to H3K9 deacetylation at nuclear factor kappa B (NFκB)-controlled genes such as GR.31–33

METHODS Animals Experiments were performed on male Fischer 344 rats, 250–320 g on arrival (Charles Rivers Laboratory, Wilmington, MA, USA). Upon arrival animals were group-housed and maintained on a 12-h light/dark cycle (lights on at 0530 h) with ad libitum access to food and water except during fasting 12–18 h before colorectal distension (CRD) procedures. Animals were acclimated to the animal facility for 1 week before experimentation. Following surgery, all animals were single-housed to prevent postsurgical complications. All procedures were approved by the Oklahoma City Veterans Affairs Medical Center Animal Care and Use Committee (IACUC; protocol #0807-004) in accordance with standards established by the Guide for Care and Use of Laboratory Animals (1996). All

1 Oklahoma Center for Neuroscience, University of Oklahoma Health Science Center, Oklahoma City, OK, USA ; 2Department of Neuroscience, Georgetown University, Washington, DC, USA; 3V.A. Medical Center, University of Oklahoma Health Science Center, Oklahoma City, OK, USA and 4Department of Physiology, University of Oklahoma Health Science Center, Oklahoma City, OK, USA. Correspondence: Dr B Greenwood-Van Meerveld, V.A. Medical Center, Research Admin, Rm. 151G, 921 N.E. 13th Street, Oklahoma City, OK 73104, USA. E-mail: [email protected] Received 13 May 2014; revised 1 August 2014; accepted 21 August 2014

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2 experiments were carried out in accordance with the International Association for the Study of Pain (IASP) recommended guidelines of the study of pain.

to the treatment protocol. The procedure was repeated three times using the same point on the same paw with 5 min intervals between each measurement.

Surgical procedures

Visceral sensitivity assessment

Stereotaxic implantation of micropellets. Implantation of micropellets on the dorsal margin of the CeA was performed as previously described.9,12,21,25,34 Rats were anesthetized with intraperitoneal injection of Ketamine (100 mg kg − 1; Hospira, Lake Forest, IL, USA)/Xylazine (10 mg kg − 1; Ben Venue Laboratories, Bedford, OH, USA). Using aseptic technique, a small incision was made to reveal the skull, and 1 mm holes were drilled at − 2.5 mm posterior to bregma and lateral ± 4.2 mm. Bilateral micropellets of either CORT (30 μg) or cholesterol (CHOL) (30 μg) were lowered − 7.0 mm from dura. The concentration of CORT and CHOL was selected based on our previous studies.9,12

Instrumentation. Instrumentation for visceral sensitivity assessment was performed as previously described.9,12,25,34–36 In brief, rats were fasted for 16–18 h before visceral sensitivity assessment to allow for insertion of the colonic balloon. On the morning of the experiment, rats were transported to the laboratory and anesthetized with 2% isoflurane. A colonic balloon (6 cm) was inserted 11 cm past the anal canal into the colon and secured to the base of the tail with medical tape. Following cessation of anesthesia, the rats were placed in their home cage and recovered for 30 min before CRD series.

Stereotaxic implantation of cannula for CeA infusions. Implantation of cannula was performed as previously described.24 Custom indwelling cannula, injector and dummy cannula were purchased from Plastics One (Roanoke, VA, USA). Each cannula (25-gauge surgical steel), which extended 7.0 mm below the threaded pedestal, was placed flush to the skull at the same coordinates as micropellet placement. Two stainless steel mounting screws (Plastics One) were placed on opposite sides of the cannula, which was held to the skull with Cerebond Adhesive (Plastics One). The incision was closed, and the rats recovered undisturbed for 1 week. Trichostatin A (TSA) (100 ng μl − 1; Sigma-Aldrich, St Louis, MO, USA), suberoylanilide hydroxamic acid (SAHA) (100 ng μl − 1; Sigma-Aldrich), or vehicle (VEH) (0.1% dimethyl sulfoxide/saline) was administered once daily for a total of 7 days. Concentrations and treatment period were based on previous experiments.27,35 Rats were anesthetized with 2% isoflurane (Aerrane, Baxter Healthcare, Deerfield, IL, USA) and positioned within the anesthesia mask so that the cannula was accessible. The dummy cannula was unscrewed and the matched injector was placed into the cannula. A total volume of 0.5 μl of TSA, SAHA or VEH, previously demonstrated to restrict diffusion to the CeA,24 was administered through each cannula at a rate of 0.1 μl min − 1 for a total of 5 min using a microsyringe and injection pump (Hamilton, Reno, NV, USA). The injection cannula was left in place for an additional 5 min to ensure complete diffusion of infusates. The injector was then removed and replaced by the dummy cannula.

Verification of micropellet and cannula placement Postmortem verification of micropellet and cannula placement was conducted as previously described.36 Animals were briefly anesthetized with 5% isoflurane, decapitated, and whole brains were rapidly removed and flash-frozen with isopentane chilled to − 80 °C. Frozen brains were subsequently sectioned at 50 μm on a cryostat (Bright-OTF; Hacker Instruments and Industries, Fairfield, NJ, USA), with sequential sections thaw-mounted onto glass slides. Sections were then Nissl-stained using cresyl violet. The exact localization of the micropellet and cannula placement was confirmed with the aid of a stereotaxic atlas.37

Assessment of anxiety-like behavior Anxiety-like behavior was assessed on the elevated plus-maze (EPM) 24 h following the final drug treatment as previously described.7,9,12,34 Rats were allowed to acclimate for 30 min to the experimental procedure room, and were then placed in the center of the EPM facing an open arm. Behavior was recorded by video camera for 5 min, and an investigator blind to treatment analyzed the video footage. The percentage of time spent in the open arms and the number of open arm entries were used to quantify anxiety-like behavior, with decreased open arm exploration and entries indicating higher anxiety. Additional parameters observed included scanning and rearing behaviors.

Somatic sensitivity assessment Somatic mechanical threshold was determined using an electronic von Frey (IITC, Woodland Hills, CA, USA) 24 h following the EPM.10,34,36 Animals were acclimated to the procedure room for 30 min before unrestrained placement on an elevated mesh floor (12 mm × 12 mm grid) in a clear plastic enclosure apparatus (21 × 27 × 15 cm) for an additional 30 min. The apparatus probe (10 μl plastic tip, U.S. Scientific, Orlando, FL, USA) was applied to the plantar surface of the hind-paw, and the force required to elicit withdrawal of the hind limb was recorded by an experimenter blind Molecular Psychiatry (2014), 1 – 13

Colorectal distension. Colorectal sensitivity was measured in conscious, freely moving rats in response to graded colonic distension as previously described.9,12,25,34–36 Briefly, the colonic balloon catheter was attached to a Distender Series IIR Barostat (G & J Electronics, Toronto, Ontario, Canada). Each constant pressure distension series consisted of a 10-min recording period at 0 mm Hg, followed by randomized 10-min inflation at 20, 40 and 60 mm Hg separated by 10-min rest periods (0 mm Hg). The visceromotor response was quantified and recorded as the total number of abdominal contractions in response to distension pressures.35,38 To prevent experimental bias, the observing investigator was blind to the treatment protocol.

In situ hybridization Detection of CRF expression was performed as previously described.21,36 Animals were sedated briefly with isoflurane and decapitated. Whole brains were extracted, flash frozen in prechilled 2-methylbutane, and were stored in a plastic container at − 80 °C for cryosectioning. Tissue sections (10 μm) containing the CeA (bregma − 2.5 mm) were collected from each brain. Sections were fixed in 4% paraformaldehyde/PBS and incubated with 0.1% active diethylpyrocarbonate (DEPC; Sigma-Aldrich). Samples were blocked using a streptavidin/biotin blocking kit (Vector Laboratories, Burlingame, CA, USA) and prehybridized at 58 °C in prehybridization buffer (50% formamide, 5xSSC, 40 μg ml − 1 salmon sperm DNA). The biotinylated CRF probe (10 μM; 5′-TGT-GTG-CTA-AAT-GCA-GAA-TCGT-3′) synthesized by Exiqon (Wobum, MA, USA) was then added to the buffer. The hybridization reaction was carried out overnight at 58 °C and then detected using streptavidin-conjugated with Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA). Slides were coverslipped and imaged using a Zeiss LSM 510 confocal microscope (Carl Zeiss MicroImaging, LLC, Thornwood, NY, USA) as previously described.21,36 Analysis of the optical density was made using the ImageJ software (NIH, Bethesda, MD, USA).

Immunofluorescence Immunofluorescence was carried out as previously described.21 Sections (10 μm) containing the CeA were fixed in equal parts of acetone:methanol and blocked with 10% normal goat serum (Invitrogen). Following blocking, sections were incubated with one of the following: anti-acetyl-H3K9 (07352; Millipore, Billerica, MA, USA), anti-c-Fos (ab7963; Abcam, Cambridge, MA, USA), anti-SIRT6 (NB100-2522; Novus Biologicals, Littleton, CO, USA), anti-NFκB p50 (sc-114; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CRF (ab8901; Abcam), anti-GR (h-300; Santa Cruz Biotechnology) or anti-GR (ab9918; Millipore) for co-localization experiments with SIRT6, and incubated overnight at 4 °C. Negative controls were incubated with 10% Normal Goat Serum. Slides were then washed in TBS-T and incubated with either Alexa Fluor 488-conjugated anti-mouse antibodies or Alexa Fluor 594-conjugated anti-rabbit antibodies (Life Technologies, Carlsbad, CA, USA) for 2 h at room temperature. Following incubation, the sections were washed in TBS-T and the nucleus was counterstained with 4,6-diamidino-2phenylindole. Slides were coverslipped using ProLong Gold Anti-Fade mounting medium (Life Technologies) and visualized using confocal microscopy.

Chromatin immunoprecipitation Chromatin immunoprecipitation (ChIP) assays were performed according to Weaver et al.,27 adapted to the MAGnify ChIP assay protocol (Life Technologies). Tissue samples were micro-dissected from each rat brain using 1 mm hole punches, and each sample predominantly contained the © 2014 Macmillan Publishers Limited

Histone regulation of anxiety and pain L Tran et al

3 CeA. Chromatin was immunoprecipitated using one of the following antibodies: rabbit polyclonal anti-acetyl-H3K9 (Millipore), rabbit polyclonal anti-GR (Abcam), or normal non-immune rabbit IgG antibody (Millipore). One tenth of the lysate was reserved as an input control. The rat GR exon 17 promoter region (GenBank accession number AJ271870) of uncrosslinked DNA was subjected to real-time PCR amplification (forward primer: 5′-TGTGACACACTTCGCGCA-3′; reverse primer: 5′-GGAGGGAAACCGAGT TTC-3′) using RT2 PCR Mastermix (Qiagen, Valencia, CA, USA) on a StepOne Plus System (Life Technologies). The rat CRF promoter (GenBank accession number M54987.1) was amplified using forward primer: 5′-TTC-CATTTT-AGG-GCT-CGT-TG-3′ and reverse primer: 5′-CGA-CCC-TCT-TCA-GAAAGC-AC-3′, designed to include two conserved glucocorticoid response elements in the amplicon.39 The thermocycler protocol involved an initial denaturation cycle (5 min, 95 °C), 40 cycles of denaturation (1 min, 95 °C), annealing (1 min, 56 °C) and extension (1 min, 72 °C), followed by a final extension cycle (10 min, 72 °C) and held at 4 °C. All reactions were carried out in triplicate. Relative quantification of binding was calculated by normalizing the immunoprecipitated DNA C(t) values to the input DNA C(t) values [ΔC(t)] and transformed [2^ΔC(t)] to show relative quantities.

Sequential ChIP The sequential ChIP (seq-ChIP) was performed using Dynabeads (Life Technologies) coated with Protein A/G. For the first round ChIP, the Dynabeads were coupled to anti-NFκB p50, rabbit polyclonal anti-c-Jun (Abcam), or anti-c-Fos antibodies according to the manufacturer’s protocol, and a separate set of Dynabeads were coupled to anti-SIRT6 or anti-GR antibodies for the second round of ChIP. Chromatin was prepared similar to the single ChIP assay and one-tenth of the lysate was reserved as an input control. The remainder of the lysate was incubated with the Dynabead–NFκB p50 complex at 4 °C overnight with rotation. Following incubation, the complex was washed and the chromatin was eluted with 1 M glycine (pH 2.7). The buffer was desalted using an UltraCruz Micro G-25 spin column (Santa Cruz Biotechnology) and the product was incubated with the Dynabead–SIRT6 complex at 4 °C overnight with rotation. Crosslinking was reversed using Proteinase K, and the DNA was extracted using the Dynabead DNA Direct Universal system. The purified DNA was then subjected to real-time PCR to analyze GR exon 17 promoter binding. Additional controls include lysate immunoprecipitated with two rounds of non-immune rabbit IgG, immunoprecipitation (IP) with anti-NFκB and nonimmune rabbit IgG, and IP with non-immune rabbit IgG and anti-SIRT6.

RNA and protein extraction RNA and protein were extracted from the same tissue preparation with the SurePrep Purification Kit (Fischer BioReagents, Fair Lawn, NJ, USA) using the protocol for RNA and protein extraction. Protein was quantified using the Experion Pro260 system (Bio-Rad, Hercules, CA, USA) and RNA was quantified using the Experion RNA StdSens system (Bio-Rad). The samples were aliquoted and stored at − 80 °C for subsequent quantitative reversetranscription PCR (qRT-PCR) and western blot analysis.

Nuclear protein extraction Nuclear proteins were extracted from the tissue samples using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce Biotechnology, Rockford, IL, USA) in the presence of protease and phosphatase inhibitor cocktails (Pierce Biotechnology) according to the manufacturer’s protocol. Protein quantification of the extraction product was performed using the Experion Pro260 system. Following quantification, the samples were aliquoted and stored at − 80 °C for subsequent analysis.

Immunoprecipitation IP was performed using the Dynabead Protein A/G system (Life Technologies). Dynabeads were washed twice in PBS pH 7.4 containing 0.1% bovine serum albumin and incubated with 1 μg antibody for 10 min with rotation at room temperature. After washing the Dynabead–antibody complex twice, Dynabeads were incubated with nuclear extract for 10 min at room temperature with rotation. The Dynabead–antibody–protein complex was washed two times and transferred to a clean tube. The complex was resuspended in 20 μl of elution buffer and incubated for 2 min at room temperature. The supernatant was then collected and stored at − 80 °C for subsequent analysis. Visualization and quantification of the IP products were performed using the Experion Pro260 system. © 2014 Macmillan Publishers Limited

Quantitative reverse-transcription PCR Quantification of CRF mRNA was performed using qRT-PCR as previously described.35 Extraction of total RNA was followed by cDNA synthesis using RT2 First Strand cDNA Kit and qPCR using SYBR Green qPCR Mastermix in a total reaction volume of 25 μl (Qiagen). Samples were run in triplicates and ‘no template’ conditions served as a negative control. The primers were acquired from Life Technologies and included forward primer: 5′-CGGACC-GCC-TCT-CCC-TCT-CC-3′; reverse primer: 5′-GCT-GTC-CCC-CAA-CTCCAC-GC-3′, and the housekeeping gene 28S was used to normalize the samples (forward primer: 5′-GAA-GGC-AAG-ATG-GGT-CAC-CA-3′; reverse primer: 5′-GAA-CTT-CCG-TGG-GTG-ACT-CC-3′). The relative quantity of GR and CRF mRNA from each sample was calculated as the difference in C(T) for CRF mRNA minus C(T) for 28S rRNA [ΔC(T)], and calibrated to C(T) of CHOL-treated control sample [ΔΔC(T)]. Fold change in transcription is expressed as 2[ − ΔΔC(T)].

Western blot Quantification of specific proteins and identification of protein products from IP were made via western blot. Approximately 30 μg of total protein extract, nuclear protein extract or IP product was solubilized in Laemmli buffer supplemented with 2-mercaptoethanol and denatured at 95 °C for 5 min. The samples were then resolved on a 4–20% gradient Tris-Glycine polyacrylamide gel (Bio-Rad) using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Millipore). The membranes were blocked with 1% casein in TBS for 1 h. Blots were then incubated for 2 h with primary antibodies, which were the same antibodies used for immunofluorescence, and anti-GAPDH (SigmaAldrich) was used for normalization. Following antibody incubation, the blots were washed in three changes of TBS-T and incubated for 1 h with HRP-conjugated secondary anti-rabbit or anti-mouse antibodies (Millipore). After three more washes in TBS-T, bands were visualized with ECL Western Blot Detection Kit (Amersham, Piscataway, NJ, USA), and imaged using an Omega 12iC chemiluminescent imager (UltraLum, Claremont, CA, USA). Densitometry was performed using the ImageJ software (NIH, Bethesda, MD, USA).

Electrophoretic mobility shift assay A putative NFκB binding site was predicted adjacent to the 5’ end of the 17 GR promoter by the AliBaba2.1 software (BIOBASE Biological Databases, Beverly, MA, USA) using the Transfac 6.0 library of mononucleotide weight matrices. Single-stranded DNA oligonucleotides containing the site were synthesized (Life Technologies) and annealed at 95 °C in annealing buffer (10 mM Tris, 1 mM EDTA, and 50 mM NaCl; pH 8.0). The sequences of the oligonucleotides were as follows: sense, 5′-TGC-AGT-CCT-GCC-CCGAGA-GCA-AGC-GGC-CAG-GGC-TCT-GCG-GCA-CCG-TTT-CC-3′ and anti-sense, 5′-GGA-AAC-GGT-GCC-GCA-GAG-CCC-TGG-CCG-CTT-GCT-CTC-GGG-GCA-GGACTG-CA-3′. For competition assay, a truncated sequence was designed as follows: sense, 5′-CCG-AGA-GCA-AGC-GGC-CAG-GGC-TCT-G-3′ and antisense, 5′-CAG-AGC-CCT-GGC-CGC-TTG-CTC-TCG-G-3′. Shift assays were performed using reagents from the LightShift EMSA kit (Pierce Biotechnologies) and adapted to the Experion 1 kD DNA system (Bio-Rad). Binding reactions, run in triplicates, were performed at room temperature at a final volume of 20 μl. The following were added to each reaction in order: water, binding buffer (10 mM Tris, 50 mM KCl, 1 mM DTT, pH 7.5), 1 μg Poly (dI·dC), 4 μg nuclear protein extract, and 100 ng of annealed probe. For competition, 200 nmol excess of truncated probe was used. Upon binding to protein, the truncated probe is undetectable by the Experion autoelectrophoresis system. Supershift assays included 1 μg of anti-NFκB antibody, which was added 10 min into the binding reaction. The reaction continued for an additional 10 min for a total of 20 min. Experion 1 kD DNA chips were prepared according to the manufacturer’s instructions and 1 μl of each binding reaction was loaded into the wells. The chips were then run on the electrophoresis system using the 1 kD DNA program.

Experimental design A schematic diagram is provided in Figure 1. All animals (n = 142) received bilateral stereotaxic micropellet implants containing either inert CHOL (n = 58) or CORT (n = 84) onto the dorsal margin of the CeA. A subset of animals within the CHOL (n = 32) or the CORT (n = 32) did not receive any further experimental manipulations, and the postmortem tissue collected from these animals was used for gene interaction studies (A). The Molecular Psychiatry (2014), 1 – 13

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Figure 1. Experimental design. Animals were divided into three protocols (A–C). Animals in group A were implanted with CHOL/ CORT micropellets onto the central amygdala (CeA) and were used for gene interaction experiments. Group B received CHOL/CORT micropellets and cannula implants localized to the CeA. Following an infusion regimen, animals were used for behavioral experiments. Animals in group C followed an identical protocol to group B, but were not tested for behavior. CHOL, cholesterol; CORT, corticosteroid.

remaining animals CHOL (n = 58) or CORT (n = 20) received bilateral stereotaxic cannula implants localized to CeA. Following a 1-week surgical recovery period, the animals received daily bilateral infusions via the CeA cannulae with either the histone deacetylase inhibitor TSA, vorinostat (SAHA) or VEH for 7 days, resulting in the following groups: CHOL+VEH (n = 14), CHOL+TSA (n = 6), CHOL+SAHA (n = 6), CORT+VEH (n = 26), CORT +TSA (n = 18) and CORT+SAHA (n = 8). One day following the final infusion, a subset of animals from each group underwent behavioral testing, with each test separated by at least 24 h to prevent carryover effects (B). The remaining animals were not exposed to any behavioral assessments, and tissue samples were collected for gene expression studies (C). Three animals were excluded from analysis due to surgical complications and were originally assigned to CHOL+VEH, CHOL+TSA and CORT+TSA. One animal (CORT+VEH) was excluded due to complications with CRD instrumentation. Eight of the CHOL+VEH and CORT+VEH were included to supplement the colonic sensitivity experiments and were not exposed to any other behavioral tests.

Data analysis Replicates for von Frey, ChIP/seqChIP and qRT-PCR experiments were averaged into a single n-value. Means for gene expression and ChIP experiments were analyzed by Student’s unpaired t-test for significance. A Spearman’s rho correlation coefficient was calculated to determine the strength of relationship between GR expression and SIRT6 expression. A two-way analysis of variance (ANOVA) was used to determine the significance of main effects and interaction terms for EPM and von Frey test, and a two-way ANOVA with repeated measures (RM-ANOVA) was used to correct for multiple distension pressures in the visceromotor response experiments. A Bonferroni’s post hoc test was used to compare individual means between groups or treatments. GraphPad Prism 6.0b software (La Jolla, CA, USA) was used for all analyses and Po 0.05 was considered as significant. Data are presented as mean ± s.e.m.

RESULTS TSA treatment attenuates anxiety-like behavior, somatic allodynia and visceral hyperalgesia Localization of micropellet placement and verification of the cellular integrity of central amygdaloid neurons were performed following Nissl staining of the brain tissue sections (Figures 2a Molecular Psychiatry (2014), 1 – 13

and b). In response to micropellet implantation and drug infusions, there is no evidence of damage to the CeA, including no changes in cell density and no evidence of necrosis, apoptosis or microglia infiltration. Overall there was a main effect for CORT implant (F1,42 = 9.738, Po 0.01) and histone deacetylase inhibitor infusion (F2,42 = 3.132, P = 0.05) on open arm time. Treatment with CORT+VEH resulted in a significant decrease (22.9 ± 6.15%) in the time spent exploring the open arms of the EPM (Figure 2d; P o 0.01) compared with CHOL+VEH, which was attenuated by TSA treatment (P = 0.01; 21.3 ± 8.02%). There was no effect of interaction for open arm time (F2,42 = 1.07, P = 0.35). When comparing the effect of SAHA, there was no significant difference between CORT+VEH and CORT +SAHA (P = 0.08). There was a main effect of CORT implantation (F1,30 = 7.317; P = 0.01) and histone deacetylase inhibitor infusion treatment on somatic threshold (F2,30 = 18.47; P o0.001) as well as a significant interaction (F2,30 = 14.75, P o0.001). Implants of CORT onto the CeA significantly lowered mechanical somatic threshold (Figure 2c; Po 0.001) from 69.3 ± 4.45 g to 44.7 ± 2.54 g, which was attenuated by TSA treatment (P o 0.001; 67.5 ± 2.81 g). Likewise, treatment with SAHA significantly increased threshold to 74.5 ± 2.11 g (P o 0.001). Results of the visceromotor response following CRD showed that CORT implant (F1,42 = 15.74; P o0.001), histone deacetylase inhibitor infusion (F2,42 = 14.65; P o0.001) had an overall significant effect, and there was significant interaction (F2,42 = 17.58; P o 0.001). There was a significant increase in the number of contractions (14.2 ± 1.92) at the highest distension pressure of 60 mm Hg pressure (Figure 2e; P o 0.001) in CORTtreated animals compared with CHOL, indicative of visceral hyperalgesia. The increase in the number of abdominal contractions in response to CRD was attenuated by TSA treatment (P o 0.001; 16.4 ± 2.33), or SAHA treatment (P o0.01; 9.61 ± 2.17). Exposure of the CeA to CORT decreases histone acetylation and alters GR/CRF expression Histone acetylation in the CeA was specifically assessed at the lysine 9 residue of histone 3 (H3K9). Animals receiving CORT implantation showed 45.2 ± 8.2% less total acetyl-H3K9 compared with CHOL-implanted animals (t10 = 4.074; P o 0.001). As illustrated in Figure 3a, many neurons in the CeA of CORT-treated animals lacked positive staining for H3K9 acetylation. There was no change in total H3 expression (Supplementary Figure S1A) when quantified by immunofluorescence (t10 = 0.3221; P = 0.377) or western blot (t10 = 0.1331; P = 0.898). GR, c-Fos and CRF expression was quantified by immunofluorescence and in situ hybridization, respectively. Following CORT implantation onto the dorsal margin of the CeA, there was a reduction (48.2 ± 5.5%) in GR expression within the CeA (Figure 3b; t10 = 4.773; P o0.001). Overall neuronal activity in the CeA was determined by quantifying immunoreactivity for the immediateearly gene c-Fos following implantation of CORT or CHOL. Animals treated with CORT exhibited a higher level (71.7 ± 3.9%) of c-Fos immunoreactivity in the CeA compared with CHOL controls (Figure 3c; t10 = 4.297; P o0.01), indicating that the prolonged elevation of CORT induced a hyperactivation of the CeA. The results showed that animals stereotaxically implanted with micropellets of CORT exhibited a 1.2 ± 0.3-fold higher expression of CRF when compared with CHOL-implanted controls (Figure 3d; t10 = 4.455; P o 0.001). Further quantification of acetylated H3K9 and GR expression via western blot confirmed the blunted expression levels (Figure 3e). There was a 78.1 ± 0.03% decrease in acetylated H3K9 (t10 = 2.493; P = 0.02) and a 66.0 ± 0.06% decrease in GR expression (t10 = 2.040; P = 0.04). Quantification of CRF mRNA expression using real-time qRT-PCR supported in situ hybridization experiments showing an upregulation (Figure 3f). Results indicate an overall 5.02 ± 1.91-fold © 2014 Macmillan Publishers Limited

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Figure 2. Infusions of a histone deacetylase inhibitor attenuates behaviors induced by elevating corticosteroids (CORT) in the central amygdala (CeA). (a) Summary of the micropellet and cannula placements based on postmortem histological verification. (b) Nissl-stained coronal section containing the CeA. (c) Evaluation of somatic mechanical threshold using an electronic von Frey. (d) Anxiety-like behavior was assessed on the elevated plus-maze for percent of time exploring the open arms in addition to total arm entries, scanning and rearing behavior (table). (e) Visceral sensitivity quantified as the total number of abdominal contractions in response to graded distensions of the colon (left column) and specifically at 60 mm Hg (right column). Data shown are means ± s.e.m.; n = 13 CHOL+VEH, 11 CORT+VEH, 5 CHOL +TSA, 5 CORT+TSA, 6 CHOL+SAHA, and 8 CORT+SAHA; *P o0.05, **Po0.01, ***P o0.001 compared with CHOL+VEH; +P o0.05, ++Po 0.01, +++ P o0.001 compared with CORT+VEH by 2-way analysis of variance (ANOVA) or repeated measures (RM-ANOVA) and Bonferonni posttest. CHOL, cholesterol; SAHA, suberoylanilide hydroxamic acid; TSA, trichostatin A; VEH, vehicle.

(t10 = 2.9807; P = 0.01) increase in CRF mRNA levels in CORTimplanted animals compared with CHOL-implanted controls. TSA attenuates CORT-induced changes in gene expression Further substantiating the role of histone acetylation in GR and CRF gene expression, a group of animals that received no © 2014 Macmillan Publishers Limited

behavioral assessments were treated with TSA or VEH following CORT implants. TSA treatment significantly enhanced H3K9 acetylation (Figure 4a; t10 = 4.074; P o 0.001) 60.9 ± 12.7% compared with VEH without changing total H3 expression (Supplementary Figure S1B; t10 = 0.0172; P = 0.49). Animals treated with TSA also showed greater GR expression (84.6 ± 9.0%) in the CeA compared Molecular Psychiatry (2014), 1 – 13

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Figure 3. Corticosteroid (CORT) elevation in the central amygdala (CeA) decreases histone acetylation and alters gene expression. (a) Representative confocal images from immunofluorescent staining for total acetylated H3K9 (red) and nuclear counterstained with 4,6diamidino-2-phenylindole (DAPI) (blue) with densometric quantification shown (right). (b) Expression of glucocorticoid receptor (GR) quantified by immunofluorescence. (c) Neuronal activation assessed by quantification of c-Fos immunofluorescence. (d) Corticotropinreleasing factor (CRF) mRNA quantified by in situ hybridization. Images were captured at × 40 magnification and scale bar represents 50 μm. (e) Western blot quantification of acetylated H3K9 (~17 kDa) and GR α/β expression (~95/90 kDa) normalized to GAPDH (~36 kDa). (f) Real-time quantitative reverse-transcription PCR (qRT-PCR) quantification of CRF mRNA. Data shown are means ± s.e.m.; n = 6 animals/group; *Po 0.05, **P o0.01, ***P o0.001 compared with cholesterol (CHOL) by Student’s unpaired t-test.

with animals treated with VEH (Figure 4b; t10 = 4.736; P o0.001), and protection of histone acetylation reduced c-Fos expression (164.1 ± 38.0%) in the CeA (Figure 4c; t10 = 2.787; P = 0.02). Following treatment with TSA, there was an overall 50.5 ± 6.6% decrease in the expression of CRF in the CeA (Figure 4d; t10 = 2.742; P = 0.01). Acetylated H3K9 levels and GR expression in response to TSA treatment were also assessed via Western blot (Figure 4e). Results confirmed a significant increase in H3K9 acetylation (185.6 ± 78.5%; t8 = 2.276; P = 0.03) and a significant increase (108.9 ± 52.6%) in GR expression (t8 = 1.970; P = 0.04). CRF mRNA expression levels were confirmed using real-time qRTPCR (Figure 4f). Following CORT+TSA treatment, there was a Molecular Psychiatry (2014), 1 – 13

0.45 ± 0.10-fold (t10 = 2.9807; P = 0.01) decrease in CRF mRNA expression compared with CORT+VEH treatment. Elevated amygdala CORT removes H3K9 acetylation at the GR promoter To determine whether elevating CORT in the CeA influences GR regulation, acetyl-H3K9-ChIP was used followed by qRT-PCR with primers directed toward the 17 GR promoter region (Figure 5a). Our results revealed a significant decrease (Figure 5b; t8 = 2.7872; P = 0.02) in association of actyl-H3K9 with the 17 GR promoter, which strengthens histone masking of the promoter and may © 2014 Macmillan Publishers Limited

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Figure 4. Effect of inhibiting histone deacetylation on corticosteroid (CORT)-induced gene expression. Gene expression in CORT-implanted animals with either infusions of vehicle (VEH) or trichostatin A (TSA) into the central amygdala (CeA) were compared using immunofluorescence. (a) Representative confocal image for total acetylated H3K9 (red) and nuclear counterstained with 4,6-diamidino-2phenylindole (DAPI) (blue) with densometric quantification shown (right). (b) Expression of glucocorticoid receptor (GR) quantified by immunofluorescence. (c) Neuronal activation assessed by quantification of c-Fos immunofluorescence. (d) Corticotropin-releasing factor (CRF) mRNA quantified by in situ hybridization. Scale bar represents 50 μm and images were captured at × 40. (e) Western blot quantification of acetylated H3K9 (~17 kDa) and GR α/β expression (~95/90 kDa) normalized to GAPDH (~36 kDa). (f) Real-time quantitative reverse-transcription PCR (qRT-PCR) quantification of CRF mRNA. Data shown are means ± s.e.m.; n = 6 or 5 animals/group; *Po 0.05, **P o0.01, ***P o0.001 compared with CORT+VEH by Student’s unpaired t-test.

contribute to the long-term silencing of GR transcription. The consequences of diminished GR expression on CRF promoter binding in the amygdala following localized elevation of CORT were assessed by GR-ChIP followed by real-time PCR with primers designed to include the conserved GR response elements (Figure 5c). Elevating amygdala CORT reduced binding of GR to the CRF promoter (Figure 5d; t8 = 2.6221; P = 0.02). There was no significant difference between input DNA for acetylated H3K9 binding experiments (t8 = 1.8595; P = 0.10) or GR binding experiments (t8 = 0.0518; P = 0.96). © 2014 Macmillan Publishers Limited

CORT modulation of the CeA influences specific histone deacetylase expression The histone deacetylase SIRT6, from the family of nicotinamide adenine dinucleotide-dependent protein deacetylases, was assessed as a potential candidate mediating the increased H3K9 deacetylation. Following CORT treatment, there was a 51.9 ± 6.0% increase in SIRT6 in the CeA compared with CHOL-treated animals (Figure 6a; t10 = 2.564; P = 0.01). When the tissue sections containing the CeA were analyzed with both anti-SIRT6 and anti-GR Molecular Psychiatry (2014), 1 – 13

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8 antibodies, there was co-localization of the two gene products, supporting the potential for interaction. The increase in SIRT6 strongly correlated with the decrease in GR expression (P o 0.001; r = − 0.95). Additionally, results following treatment with TSA showed decreased expression of SIRT6 (Figure 6b; t10 = 7.620; Po 0.001). Quantifications of the optical density and correlation analysis are illustrated in Figure 6c. Histone deacetylases can be targeted to the GR promoter The NFκB was investigated as a possible intermediate that localized SIRT6 to the GR promoter. Following CORT treatment, there was no change in overall expression of NFκB compared with CHOL (t10 = 1.3032; P = 0.22); however, there was a qualitative increase in nuclear localization of NFκB (Figure 6d). The increase in nuclear localization (105.4 ± 46.4%; t8 = 2.5665; P = 0.03) was confirmed and quantified using western blot following extraction of nuclear proteins (Figure 6e). As shown in Figure 6f, IP with either anti-SIRT6 or NFκB produces the same banding pattern, indicating that the two proteins can be found in the same protein complex. The presence of NFκB and SIRT6 in the IP product was confirmed via western blot. Electrophoretic mobility shift assay

with probes corresponding to a putative NFκB binding site proximal to the exon 17 of the GR promoter was used to determine whether NFκB could localize and bind to the GR promoter (Figure 6g). Nuclear protein extracts were combined with the probes resulting in a band shift, indicating positive protein–DNA interaction. The shifted band was no longer present when the reaction was challenged with a sequence competitor, confirming that binding was exclusive to the kB response element. To identify the binding protein, anti-NFκB p50 antibodies were added to the binding reaction, resulting in a loss of the shifted band. As previously demonstrated,40 the anti-NFκB p50 antibody used in this study binds to a critical site on the transcription factor, inhibiting the probe-NFκB complex formation, resulting in an absence of the shifted band. To strengthen the potential role of SIRT6 in catalyzing histone deacetylation at the GR promoter, a seq-ChIP analysis of NFκB/SIRT6 binding to the GR promoter was performed (Figure 6h). Our results demonstrated that the NFκB/ SIRT6 complex can bind to the GR promoter in the CeA and that the interaction is increased following CORT implantation (11.36 ± 6.46-fold; t8 = 3.2397; P o0.01) compared with CHOLimplanted animals. There was no significant difference in the input DNA (t8 = 0.3958; P = 0.70).

Figure 5. Analysis of the relationship between acetylated H3K9, glucocorticoid receptor (GR) and corticotropin-releasing factor (CRF). Acetylated H3K9 was evaluated as a marker for chromatin structure. Gene regions analyzed are shown for (a) the 17 region of the GR promoter and for (c) the CRF promoter. The probe sequence used in the electrophoretic mobility shift assay (EMSA) is denoted (*), and the κB response element and GR response element are underlined, respectively. Chromatin was immunoprecipitated with antibodies for (b) acetylated H3K9 followed by real-time PCR quantification of bound 17 GR promoter, or (d) GR followed by real-time PCR analysis of CRF promoter binding. Left: Normalization to input DNA loading control. The inset line shows background binding activity determined by control IgG immunoprecipitation. Right: baseline quantification of input DNA. Data shown are means ± s.e.m.; n = 5 animals/group; *P o0.05, ***P o0.001 compared with CHOL by Student’s unpaired t-test. CeA, central amygdala.

Figure 6. Elevated central amygdala (CeA) corticosteroid (CORT) induces histone deacetylation and involves sirtuin-6 (SIRT6). (a) Representative confocal fluorescent micrographs of the histone deacetylase SIRT6 (green) and glucocorticoid receptor (GR) (red) immunoreactivity in the central amygdala (CeA) followed by 4,6-diamidino-2-phenylindole (DAPI) (blue) nuclear counterstaining. Images for cholesterol (CHOL) (top row) and CORT (middle row) were captured at × 40 magnification and lower image was captured at × 100 for colocalization visual. The × 100 image does not represent any treatment group and is shown for localization of SIRT6 and GR only. (b) Representative fluorescent micrograph for CORT-treated animals in addition to either infusions of vehicle (VEH) (top row) or trichostatin A (TSA) (bottom row). (c) Densometric quantification of SIRT6 immunoreactivity following CORT or CHOL treatment (left), correlation analysis of SIRT6 and GR expression (middle), and SIRT6 immunoreactivity following CORT treatment in addition to VEH or TSA infusions into the CeA (right). (d) Representative immunofluorescent image of nuclear factor kappa B (NFκB) reactivity. Red arrows show nuclear localization. (e) Western blot analysis of nuclear protein extract from the CeA from CHOL-treated animals (left lane) or CORT (right lane) for NFκB, SIRT6 and H3 for loading control. Quantification of the optical densities is shown on the right. (f) Digital gel generated by the Experion autoelectrophoresis system (top) visualizing products from NFκB or SIRT6 immunoprecipitation. Western blot analysis of immunoprecipitated products using antibodies for NFκB and SIRT6. (g) Digital gel visualization from the Experion autoelectrophoresis electrophoretic mobility shift assay (EMSA) using probes corresponding to a portion of the GR promoter sequence. (h) Binding of NFκB/SIRT6 complex was assessed using seqChIP followed by real-time PCR quantification of the 17 region of the GR promoter. (i) Binding of c-Jun (left) and c-Fos (right) to the corticotropin-releasing factor (CRF) promoter. (j) Subsequent ChIP for GR using chromatin from the c-Jun ChIP to show co-binding of GR/AP-1. Data shown are means ± s.e.m.; n = 6 or 5 animals/group; *P o0.05, **P o0.01, ***P o0.001, compared with CHOL, and +++Po0.001 compared with CORT+VEH by Student’s unapired t-test. Molecular Psychiatry (2014), 1 – 13

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Figure 7. Model summary. The nuclear factor kappa B (NFκB) and AP-1 (formed from c-Fos/c-Jun dimerization) pathways are activated and facilitate the early responses to stress, including corticotropinreleasing factor (CRF) expression. Corticosteroid (CORT) is then released to activate glucocorticoid receptor (GR), which localizes to the nucleus and suppresses CRF through interaction with AP-1. NFκB recruits sirtuin-6 (SIRT6) to the GR promoter and mediates SIRT6 deacetylation of H3K9. Deacetylated H3K9 sequesters GR expression, relieving the negative effects of GR on AP-1 induction of gene expression. The overall result is a sustained potentiation of CRF expression.

GR represses AP-1 action on the CRF promoter The specific mechanistic role of GR in regulating the CRF promoter was of interest considering the downregulation of GR in the CeA in response to prolonged CORT exposure. A seq-ChIP analysis was used to determine whether downregulation of GR influenced AP-1/GR interaction with the CRF promoter. In the first ChIP sequence depicted in Figure 6i, there was a significant increase in binding of the c-Jun (t8 = 4.352; P o 0.01) and c-Fos (t8 = 2.850; P = 0.01) subunits of AP-1 in CORT-implanted animals (65.4 ± 14.7and 44.74 ± 14.55-fold) compared with CHOL (1.22 ± 0.41- and 2.98 ± 1.70-fold). Subsequent IP of the captured chromatin from the c-Jun ChIP assay with anti-GR antibodies revealed a significant decrease (t8 = 4.049; P o0.01) in the proportion of GR co-binding to the CRF promoter with AP-1 (Figure 6j). There was a 0.03 ± 0.01 fractional recovery in animals implanted with CORT micropellets compared with 0.50 ± 0.14 in CHOL-implanted animals. DISCUSSION The present study revealed the impact of chromatin modifications, specifically acetylation of H3K9, on chronic anxiety-like behavior and nociception following a prolonged elevation of CORT in the CeA (summarized in Figure 7). One of our key findings was that inhibiting histone deacetylases in the CeA attenuated anxiety-like behavior, somatic allodynia and visceral hyperalgesia induced by exposing the CeA to CORT. Results indicated that elevating CORT in the CeA induced robust H3K9 deacetylation, specifically at the 17 GR promoter, concomitant with a decrease in GR expression and binding. Loss of GR activity was observed in conjunction with enhanced c-Fos and CRF expression in the CeA. Further evidence suggested that the CORT-induced H3K9 deacetylation was catalyzed by SIRT6 through NFκB activation. The molecular processes observed following prolonged CORT activation in the CeA were reversed by treatment with histone deacetylase inhibitors, highlighting the importance of histone deacetylation in the long-term maintenance of anxiety-like behavior and nociception induced by elevated amygdala CORT. Our previous study demonstrated that a prolonged elevation of CORT in the CeA induced anxiety-like behaviors, somatic allodynia and visceral hyperalgesia that were sustained up to 28 days © 2014 Macmillan Publishers Limited

despite depletion of the source of CORT.25 The persistence of the phenotypes suggested that remodeling of the epigenome may have been involved, but the precise mechanisms remained to be elucidated. More recently, we demonstrated that chronic nociception induced by repeated psychological stress involved histone deacetylation.35 In the present study, we found that the exposure to elevated amygdala CORT caused widespread changes in histone acetylation, particularly at H3K9. Treatment with a histone deacetylase inhibitor not only reversed the effects of global acetylation, but also the pain phenotypes, suggesting that histone deacetylation is involved in the behavioral response to elevated CORT in the CeA. An important caveat to note is that our previous studies indicated CORT implants onto the CeA do not alter basal diurnal rhythm, but the predominant physiological effects are seen following a stressor.7,8 Accordingly, since epigenetic mechanisms can be activated by chronic exposure to external stressor,35 persistent environmental cues may also contribute to the overall phenotypes observed. Although treatment with a histone deacetylase inhibitor also decreased the anxiety-like behaviors observed, our results indicate that the treatment reduced basal levels of anxiety in addition to those induced by elevated CORT in the CeA. Moreover, our behavioral experiments using animals treated with SAHA revealed identical results to TSA in the somatic and visceral sensitivity assays, but the effect on anxiety did not reach statistical significance. Our data suggested that the effects of TSA on anxiety may include mechanisms beyond inhibition of histone deacetylase activity, such as modulation of NfκB activity,41–43 which contrasts SAHA treatment,44–47 or increasing neuropeptide Y expression in the CeA, reported to simultaneously prevent anxiety-like behaviors.48 Despite the effects on anxiety possibly being collateral, the results from the present study bridged our previous findings by highlighting the important role of histone deacetylation, specifically in the CeA, in maintaining the chronic nociception. The findings also demonstrated that histone deacetylation is a consequence of prolonged CORT exposure, such as during periods of chronic stress. It is important to note that our previous study also showed differential DNA methylation, another epigenetic mechanism, in the CeA following repeated psychological stress. However, since the interaction between DNA methylation and histone acetylation has not been fully established, the mechanisms were independently studied. Histone acetylation at H3K9 is a well-established marker of active chromatin,49,50 and our data showed that following elevated CORT exposure there was a significant decrease in H3K9 acetylation in the CeA. However, in light of the fact that a loss of H3K9 acetylation can influence a large array of genes due to the non-specificity of histones, we focused our investigation on the role of histone deacetylation on GR gene expression. Regulation of GR has not only been reported to be under epigenetic regulation of H3K9 acetylation, but also been shown to mediate CORTinduced anxiety-like behavior and nociception.12,25 Since our experiments in the present study indicated that TSA attenuated anxiety, but may not be specific to the CORT-induced anxiety, investigating the effect of TSA particularly on GR regulation was important in determining whether TSA can influence CORTinduced anxiety. Consistent with other studies investigating the long-term regulation of GR transcription, we found a decrease in H3K9 acetylation at the 17 GR promoter region, which was predicted to potentiate histone masking of the GR promoter. Consequently, there was a sustained reduction in GR expression reported in this study and others.27 Thus, the data indicated that one role for histone deacetylation following prolonged CORT activation of the CeA is sustaining a decrease in GR expression. In contrast to GR, CRF expression in the CeA following prolonged elevation of CORT has been examined by several studies.7,8,15–21 Similar to previous findings, in the present study CORT exposure increased CRF in the CeA, and persisted despite Molecular Psychiatry (2014), 1 – 13

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12 depletion of the source of CORT. Since the heightened CRF expression was reversed by histone deacetylase inhibitor treatment, the findings suggested that long-term regulation of CRF could be influenced by histone deacetylation, at least indirectly via GR regulation. We found that GR associated with conserved glucocorticoid response element consensus sequences on the CRF promoter, and that the binding as well as expression of GR was decreased following CORT treatment. Functional genomic studies have indicated that the glucocorticoid response elements overlap with AP-1 response elements, and have suggested that GR may regulate CRF expression by interacting with AP-1.39,51,52 Thus, the decrease in GR following CORT implantation onto the CeA is likely responsible for our observed increase in c-Fos and c-Jun binding to the CRF promoter. Although the interface between GR and AP-1 has been reported in various other genes and cells throughout the body,53,54 the precise mechanism of interaction between GR and AP-1 in the CeA is unclear since the relationship is tissue dependent.55 For instance, the co-binding of GR and transcription factors to DNA is often necessary to induce a gene effect,56 which has been reported for GR and AP-1.54,57 In contrast, others have shown that GR and AP-1 interaction does not require DNA binding, but rather protein–protein interactions.58–60 In the present study, we observed co-binding of AP-1/GR, and the proportion of co-binding was decreased following CORT implantation onto the CeA. Our data support the possibility that regulation of CRF expression in the CeA involves an interaction between GR and AP-1, and the effect of elevating CORT levels is a shift in CRF transcription that is sustained via epigenetic regulation of GR. Identification of the specific histone deacetylase responsible for the removal of H3K9 acetylation was of significance since histone deacetylase inhibitors, such as TSA, influence a wide range of deacetylases. Of the known histone deacetylases directly antagonized by TSA, none have been identified to specifically deacetylate at H3K9. However, a recent study demonstrated that TSAresistant type III histone deacetylase inhibitors could be indirectly downregulated by TSA, including SIRT6.61 Importantly, SIRT6 is a potent deacetylase that has been shown to associate specifically at the H3K9 site,33 and a SIRT6 homolog, OsSRT1, was discovered to specifically target genes related to stress and metabolism.62 Upon investigating the potential of SIRT6 to mediate CORTinduced H3K9 deacetylation, we found an increased expression of SIRT6 resulting from elevated CORT exposure. SIRT6 also colocalized with GR and the expression of SIRT6 was negatively correlated with GR expression, supporting the novel possibility that CORT-induced deacetylation of H3K9 is mediated by a sirtuin deacetylase. Another critical component of the present study was to delineate a mechanism facilitating SIRT6 recruitment specifically to the GR promoter, as histone deacetylases can influence multiple targets. Previous studies have demonstrated that SIRT6 can be shuttled to specific DNA sites by NFκB.32 Furthermore, NFκB can be activated in response to psychological stress,63 and has been known to associate with the human GR promoter.64 Therefore, we hypothesized that NFκB may facilitate localization of SIRT6 to the GR promoter in the CeA. In support of our hypothesis, we demonstrated that, following elevated CORT treatment, there was an increase in nuclear localization of NFκB. Moreover, SIRT6 and NFκB co-immunoprecipitated from nuclear extracts of the amygdala, and the SIRT6/NFκB complex bound specifically to the kB consensus sequence proximal to exon 17 GR promoter in the vicinity of H3K9 association. Overall, the presented data revealed a mechanism allowing localized targeting of SIRT6 to histones at the GR promoter. Although specific SIRT6 antagonists are not available to confirm the role of SIRT6 in regulating anxiety and nociception, future studies utilizing specific gene knockdown to determine the precise role of SIRT6 may be possible. Another caveat to note is that while we provide evidence supporting SIRT6-mediated Molecular Psychiatry (2014), 1 – 13

histone deacetylation involvement, our results do not preclude involvement of other histone modulations and gene targets that may also be involved in long-term regulation of anxiety and nociception. Our results provide novel evidence to support the involvement of histone deacetylation in the maintenance of the anxiety-like behavior and nociception induced by prolonged elevation of CORT in the CeA. The data in the present study also highlight putative molecular pathways that are induced by chronic CORT exposure and sustained by histone deacetylation, suggesting that epigenetic mechanisms such as histone deacetylation are important contributing factors involved in chronic anxiety and pain. Since epigenetic programming is a dynamic process that can be reversed as we have shown, our findings may have further clinical relevance. A better understanding of the underscored mechanisms could facilitate improved therapies targeted at reprogramming inappropriate adaptations. Thus, reversing stress-induced epigenetic mechanisms may be the key to attenuating chronic symptomatology. CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGMENTS BG-VM would like to acknowledge the generous funding support for her Research Career Scientist and Merit Review Awards from the Department of Veterans Affairs. We would also like to acknowledge the Oklahoma Medical Research Foundation Imaging Core for their assistance and use of their confocal microscope.

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