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Neuroscience Letters
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elatonin induces histone hyperacetylation in the rat brain
ennard P. Niles ∗, Yi Pan, Sean Kang, Ayush Lacoulepartment of Psychiatry & Behavioural Neurosciences, McMaster University, HSC-4N77, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5
i g h l i g h t s
Novel evidence that melatonin increases histone acetylation in rat brain.Significant increases in histone H3 and H4 acetylation in hippocampus and striatum.Significant increases in phospho-ERK 1/2 observed in hippocampus and striatum.Findings implicate chromatin remodeling and epigenetic regulation in melatonin action.
r t i c l e i n f o
rticle history:eceived 12 September 2012eceived in revised form 22 January 2013ccepted 25 January 2013
eywords:elatonin
a b s t r a c t
We have reported that melatonin induces histone hyperacetylation in mouse neural stem cells, suggestingan epigenetic role for this pleiotropic hormone. To support such a role, it is necessary to demonstratethat melatonin produces similar effects in vivo. Histone acetylation, following chronic treatment withmelatonin (4 �g/ml in drinking water for 17 days), was examined by western blotting in selected ratbrain regions. Melatonin induced significant increases in histone H3 and histone H4 acetylation in thehippocampus. Histone H4 was also hyperacetylated in the striatum, but there were no significant changes
in histone H3 acetylation in this brain region. No significant changes in the acetylation of either histoneH3 or H4 were observed in the midbrain and cerebellum. An examination of kinase activation, whichmay be related to these changes, revealed that melatonin treatment increased the levels of phospho-ERK(extracellular signal-regulated kinase) in the hippocampus and striatum, but phospho-Akt (protein kinaseB) levels were unchanged. These findings suggest that chromatin remodeling and associated changes in
of ge
the epigenetic regulation
he pineal indoleamine, melatonin, induces widespread phys-ological effects in mammals via at least two high-affinity
protein-coupled MT1 and MT2 receptors, which influenceultiple signaling pathways through Gi, Gq, Gs and other G pro-
eins [7,10]. In addition to inhibition of adenylate cyclase (AC)ctivity, with a consequent decrease in cAMP signaling, mela-onin has been shown to activate the mitogen-activated proteininase (MAPK)-extracellular signal-regulated kinase (ERK) path-ay [34,41]. Signaling studies in a model of acute ischemic stroke
uggest that the acute neuroprotective effect of melatonin involvesctivation of the phosphatidyl inositol-3-kinase (PI-3-K)/Akt (pro-ein kinase B) pathway, whereas ERK-1/2 and c-Jun N-terminalinase (JNK)-1/2, in addition to Akt signaling, appear to be involved
n its long-term effects [19]. There is also evidence that melatoninan interact with other cellular targets including protein kinase C,almodulin and quinone reductase 2 [6,28]. It has been shown to
potentiate a GABAA receptor-mediated current in the hypothala-mus via the MT1 receptor subtype, while inhibiting this currentin the hippocampus through the MT2 subtype [40]. Since both ofthe melatonin receptor subtypes are linked to inhibition of cAMPproduction, the mechanisms generating the differences in GABAAreceptor responses presumably involve other divergent signalingpathways. In accordance with this, transfection studies with humanmelatonin receptors in HEK cells indicate that the MT2, but notthe MT1, receptor is coupled to inhibition of the cGMP pathway[30]. Thus, melatonin can interact with multiple cellular targets toproduce its diverse effects [13]. Moreover, recent studies indicatethat physiological concentrations of melatonin increase histone H3acetylation in mouse C17.2 neural stem cells, suggesting a rolein epigenetic regulation for this hormone [37]. In order to deter-mine the potential significance of this in vitro observation, we haveexamined whether melatonin induces similar modifications of his-tone H3 and H4 proteins in vivo.
Male Sprague-Dawley rats (3–4 weeks old) were maintained
under a 12:12 lighting cycle with lights on at 07:00 h and allowedto acclimatize for one week before the start of treatment. Animalswere randomly assigned to control or melatonin treatment groupsand they had free access to food and water. Control animals
ig. 1. Hyperacetylation of histone H3 and/or histone H4 in rat hippocampus and str3 (AcH3), acetylated H4 (AcH4), total H3 and total H4 histones in the hippocampus
C, D) and striatum (G, H), as a percentage of total H3 and total H4, respectively. Da
eceived the melatonin vehicle (0.04% ethanol in drinking water)nd the treatment group received melatonin (4 �g/ml) in theirrinking water, as previously reported [36]. After 17 days, ani-als were decapitated and the entire hippocampus, both striata,idbrain and cerebellum were rapidly dissected on ice, using
n established procedure [15]. Brain tissues were snap-frozenn dry ice/ethanol and stored at −80 ◦C, until used for proteinnalysis. All experiments were carried out according to the guide-ines set by the McMaster University Animal Research Ethicsoard.
Brain tissue samples were homogenized in ice-cold buffer (pH.4) containing 10 mM Tris–HCl, 5 mM NaF, 1 mM Na3VO4, 1 mMDTA, 1 mM EGTA and 320 mM sucrose. The homogenates werencubated on ice for 10 min and then centrifuged (800 × g) for0 min at 4 ◦C. Following removal of the cytosolic fraction, theuclear pellet was resuspended in 500 �L of 0.4 N H2SO4 and incu-ated on ice for 30 min. The supernatant containing nuclear proteinas collected by centrifugation (14,000 × g) for 10 min at 4 ◦C and
ransferred to a fresh tube. Nuclear proteins were precipitated with00% trichloroacetic acid containing 4 mg/mL deoxycholic acid.he pellet was washed with ice-cold acetone and resuspended in0 mM Tris–HCl (pH 8.0). Protein concentrations were determinedith the DC Protein Assay Kit (Bio-Rad Laboratories, Mississauga,N).
For the assessment of histone acetylation, 10 �g of nuclearroteins were electrophoresed on 15% sodium-docecyl-sulfateolyacrylamide gels (SDS-PAGE) for approximately 1 h at 200 V4 ◦C). To detect the phosphorylation of ERK1/2 and Akt, 20 and
0 �g (respectively) of cytoplasmic proteins were separated onhe gel. The proteins were then transferred to polyvinylidene fluo-ide (PVDF) membranes (EMD Millipore Corporation, Billerica, MA)vernight at 25 V (4 ◦C). Membranes were blocked with 5% skim
following chronic melatonin treatment. Representative immunoblots of acetylated) and striatum (E, F), as indicated. Quantification of AcH3 and AcH4 in hippocampuswn are means ± S.E.M. (n = 3). *p < 0.05; **p < 0.01 vs control.
milk in Tris Buffered Saline (TBS) for 1 h and then incubated withthe following primary antibodies, at the indicated dilutions: acetyl-histone H3 (K9/18) (1:2500; EMD Millipore Corporation, Billerica,MA), histone H4 pan-acetyl antibody (1:5000; Active Motif, Carls-bad, CA), phospho-p44/42 (ERK1/2) (Thr202/Tyr204) (1:2000) andphospho-Akt (Ser473) (1:2000 dilution; Cell Signaling Technology,Danvers, MA). Subsequently, membranes were stripped and re-probed with primary antibodies against total histone H3 (1:5000),total histone H4 (1:5000; Active Motif, Carlsbad, CA), ERK1/2(p44/p42) clone MK12 (1:5000; EMD Millipore Corporation, Biller-ica, MA), and Akt (1:1000; Cell Signaling Technology, Danvers, MA).Following incubation with primary antibodies, membranes wereincubated with an HRP- conjugated goat anti-rabbit IgG (histoneH3, histone H4, Akt) or a goat anti-mouse IgG (ERK1/2) in TBS with5% skim milk for 2 h at room temperature. Following treatment withenhanced chemiluminescence (ECL) reagents (Amersham Phar-macia Biotech, Piscataway, NJ), proteins were detected by filmfluorography and optical density analysis performed with AlphaIm-age 2200 software. Western data for acetylated or phosphorylatedproteins were normalized with respect to total histone H3, totalhistone H4, total ERK1/2 or total Akt. After conversion to per-centage values, data were analyzed by unpaired Student’s t test,with p ≤ 0.05 taken as the level of significance. Data shown aremeans ± S.E.M.
There were no differences in either body weight (control:290 ± 6 g, n = 4; melatonin: 289 ± 10 g, n = 5) or weekly waterconsumption (control: 223 ± 13 ml, n = 4; melatonin: 240 ± 12 ml,n = 5). Chronic treatment with melatonin caused a significant
increase in histone H3 (Fig. 1A and C; p < 0.05) and histone H4(Fig. 1B and D; p < 0.01) acetylation in the hippocampus. Simi-larly, histone H4 acetylation was increased (p < 0.05) in the striatum(Fig. 1F and H), but there was no significant change in histone H3
ig. 2. Acetylation levels of histone H3 and H4 in rat midbrain and cerebellum followcetylated H4 (AcH4), total H3 and total H4 histones in the midbrain (A, B) and cerebellum (G, H), as a percentage of total H3 and total H4, respectively. Data show
Fig. 1E and G) acetylation in this brain region, following melatoninreatment. A trend toward increased acetylation of histone H3 wasetected in the midbrain (Fig. 2A and C; p = 0.07), but histone H4cetylation was unchanged (Fig. 2B and D). Neither of the two his-one proteins was altered in the cerebellum (Fig. 2E–H). Melatoninaused significant (p < 0.01) increases in the levels of phospho-RK1/2 in the hippocampus (Fig. 3A and C) and striatum (Fig. 3End G), but there were no significant changes in phospho-Akt levelsn either region (Fig. 3B, D, F, H).
We have reported that melatonin induces a concentration-ependent increase in histone H3 acetylation in mouse C17.2 neuraltem cells [37]. Increases in the mRNA expression of both class I andlass II histone deacetylase (HDAC) isoforms were also observed,uggesting a compensatory increase in HDAC expression [37], asas been reported following histone hyperacetylation by HDAC
nhibitors including sodium butyrate, trichostatin and valproic acid2,9]. In keeping with the foregoing, we now report for the first time,hat chronic treatment with melatonin caused significant changesn histone acetylation in the rat brain. Histone H3 and/or histone4 hyperacetylation, as observed in the hippocampus and striatum,re typically associated with chromatin remodeling and enhancedene transcription [20], suggesting that these brain regions areargets for the epigenetic effects of melatonin. The MAPK/ERKathway, which has been linked to histone acetylation [12,21],as activated by melatonin in the hippocampus, suggesting its
nvolvement in the hyperacetylation of both H3 and H4 histones,s observed in this brain region. Phospho-ERK levels were alsoncreased in the striatum following melatonin treatment, althoughistone H4 hyperacetylation in this area was accompanied by a
hronic melatonin treatment. Representative immunoblots of acetylated H3 (AcH3),llum (E, F), as indicated. Quantification of AcH3 and AcH4 in midbrain (C, D) andmeans ± S.E.M. (n = 3).
trend toward a decrease in histone H3 acetylation. An examinationof the PI-3-K-Akt pathway, which can be activated by melatonin[19] and which has been linked to protein acetylation via phos-phorylation of the histone acetyltransferase, p300 [22], revealedno significant changes in phospho-Akt levels in the hippocampusor striatum. These preliminary findings suggest that activation ofERK but not Akt may be involved in chromatin remodeling bymelatonin. However, given the time-dependent and differentialactivation of both ERK and Akt in the rat brain [25], further studiesare required to determine whether the observed changes in kinasephosphorylation are causally related to histone acetylation bymelatonin.
The G protein-coupled melatonin MT1 and/or MT2 recep-tors are expressed in all the rat brain areas examined in thisstudy [18,26,36]. The detection of significant melatonin-inducedincreases in histone H3 or H4 acetylation in the hippocampus andstriatum but not in the midbrain and cerebellum could involveregional differences in melatonin signaling. The expression lev-els of the MT1 and MT2 subtypes, differential coupling to Gprotein isoforms, differences in receptor subtype phosphoryla-tion and receptor oligomerization [8,42], may all contribute tocell/tissue-specific variations in melatonin signaling, with conse-quent changes in downstream effects. In vitro studies with CHOcells transfected with MT1 or MT2 melatonin receptors have shownthat melatonin activates the MAPK/ERK pathway via the MT1 recep-
tor [41]. Similarly, melatonin was reported to activate this pathwayin a mouse (GT1–7) neuronal cell line, which expresses both MT1and MT2 receptors, but the subtype(s) involved was not determined[34]. More recently, melatonin was found to phosphorylate the
Fig. 3. Phosphorylation levels of ERK 1/2 and Akt in rat hippocampus and striatum following chronic melatonin treatment. Representative immunoblots of phospho-ERK1 mpush kt, re
isiaMP[mbMrrthh
dhsihatHggmgm
/2 (p-ERK 1/2), phospho-Akt (p-Akt), total ERK 1/2 and total Akt, in the hippocaippocampus (C, D) and striatum (G, H), as a percentage of total ERK 1/2 and total A
nsulin receptor �-subunit tyrosine kinase, with subsequent insulinubstrate 1 (IRS-1) activation and association with PI-3-K, resultingn downstream phosphorylation of Akt [3]. Based on the block-de of melatonin-induced insulin receptor phosphorylation by theT2-selective antagonist, 4-phenyl-2-propionamidotetraline (4P-
DOT), the authors suggested the involvement of this receptor3]. Although the mechanism(s) underlying histone acetylation by
elatonin await clarification, the foregoing suggests that one oroth of its G protein-coupled MT1 and MT2 receptors are involved.elatonin has been reported to interact with members of the
etinoid Z receptor (RZR)/retinoid acid receptor-related orphaneceptor (ROR) subclass of nuclear receptors [1,5,29]. It is possiblehat the epigenetic effects of melatonin also involve RORs, whichave been shown to recruit chromatin remodeling complexes withistone acetylase activity [4,11].
Histone hyperacetylation, following treatment with histoneeacetylase inhibitors, such as trichostatin A, suberoylanilideydroxamic acid and MS-27-275, induces or suppresses the tran-cription of about 2–10% of the genes examined by microarrayn lymphoid and tumor cell lines [14,39]. Recent in vivo studiesave shown a strong correlation between histone H3 acetylationt gene loci in the rodent brain and gene transcription [24]. Thus,he ability of melatonin to induce hyperacetylation of histone3 and H4 proteins with associated chromatin remodeling andene transcription, dramatically increases the potential gene tar-
ets for this pleiotropic indoleamine hormone, in keeping with itsultiple physiological effects. There is increasing evidence that epi-
enetic mechanisms are involved in regulating diverse aspects ofammalian physiology, including circadian, neuronal, endocrine,
(A, B) and striatum (E, F), as indicated. Quantification of p-ERK 1/2 and p-Akt inspectively. Data shown are means ± S.E.M. (n = 3). **p < 0.01 vs control.
immune and cardiovascular functions [35]. Since these physiologi-cal activities are all influenced by melatonin [13,28], which induceshistone marks associated with gene transcription, as shown pre-viously [37] and in the present study, it is likely that epigeneticmodulation is involved. Evidence that histone hyperacetylation canprotect against oxidative stress [38], suggests that this mechanismis linked to the antioxidant [33], anti-aging [31] and pro-survivaleffects of melatonin in animal models of Alzheimer’s disease [23].Chromatin remodeling, which plays a central role in neurode-velopment and neurogenesis [16,17], may also contribute to theneurogenic effects of melatonin observed in the hippocampus[32]. Similarly, the beneficial effects of melatonin, in various mod-els of neurodegeneration, may involve its induction of histonehyperacetylation, which has been linked to the expression of sev-eral neuroprotective proteins including brain derived neurotrophicfactor (BDNF), glial cell line-derived neurotrophic factor (GDNF),cerebral dopamine derived neurotrophic factor (CDNF) and mes-encephalic astrocyte derived neurotrophic factor (MANF) [27,43].Interestingly, chronic treatment with an identical dose of mela-tonin, as used in the present study, preserved striatal dopaminergicintegrity in a 6-hydroxydopamine model of Parkinson’s disease[36], suggesting the involvement of epigenetic mechanisms in theneuroprotective effects of melatonin.
Acknowledgement
This work was supported by the Canadian Institutes of HealthResearch (CIHR), Canada.
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L.P. Niles et al. / Neurosci
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