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Research Report Epigenetic chromatin modifications in the cortical spreading depression Diego Passaro a,1 , Gina Rana a,1 , Marina Piscopo a , Emanuela Viggiano b , Bruno De Luca b , Laura Fucci a, a Dipartimento di Biologia Strutturale e Funzionale, Università di Napoli Federico II, Via Cinthia, 80126, Napoli, Italy b Dipartimento di Medicina Sperimentale, II Università di Napoli, Via Costantinopoli 16, 80138 Napoli, Italy ARTICLE INFO ABSTRACT Article history: Accepted 1 March 2010 Available online 6 March 2010 Preconditioning with Cortical Spreading Depression induces a sort of tolerance to a subsequent episode of ischemia. The mechanism of this tolerance is not clear. We studied if such treatment induces epigenetic chromatin modifications on the hemispheres of rats preconditioned by Cortical Spreading Depression. The contralateral hemispheres were used as control. We determined the level of H3K4 and H3K9 methylation and the mRNA amounts for the two well known H3K4 methyltransferases (MLL and SET7) in rats 24°h after the Cortical Spreading Depression induction. Western blotting experiments have been performed using three different types of primary antibodies against mono-, di- and tri- methyl H3K4 and primary antibody anti-dimethyl H3K9. In the same samples we checked if the H3 histones were replaced by the H3.3 histone variants that could be an additional marker of chromatin modifications. The level of mono- and di-methyl H3K4 was significantly lower in samples of the treated hemispheres than those of the contralateral hemispheres (40% and about 60%, respectively) while the level of tri-methylation remained unchanged. The level of di-methyl H3K9 was almost 60% higher in the treated hemispheres than the contralateral hemispheres. The treatment for Cortical Spreading Depression affected also the level of expression of H3K4 histone methyltransferase MLL and SET7 that decreased in the treated hemispheres in comparison to the control hemispheres (80% and 40%, respectively). The treatment for Cortical Spreading Depression instead had no effects on the overall amounts of mRNA for H3 and H3.3 histones. In conclusion epigenetic chromatin modifications are evident in rats 24°h after the Cortical Spreading Depression induction. © 2010 Elsevier B.V. All rights reserved. Keywords: Histone Histone-methyltransferase Methylation Cortical Spreading Depression Chromatin BRAIN RESEARCH 1329 (2010) 1 9 Corresponding author. Fax: + 39 081 679233. E-mail address: [email protected] (L. Fucci). Abbreviations: BDNF, brain-derived neurotrophic factor; CSD, Cortical Spreading Depression; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; H3K4, H3 histone lysine 4; HIF-1, hypoxia inducible factor-1; H3K4me3, H3 histone lysine 4 trimethylation; HMT, histone methyl-transferase; iNOS, inducible nitric oxide synthase; MLL, mixed lineage leukemia; NET, not exposed or treated; nNOS, neuronal nitric oxide synthase; RT, reverse transcription; SET, Su(var)3-9, Enhancer-of-zeste, Trithorax 1 These authors contributed equally to this work. 0006-8993/$ see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.03.001 available at www.sciencedirect.com www.elsevier.com/locate/brainres

Epigenetic chromatin modifications in the cortical spreading depression

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Research Report

Epigenetic chromatin modifications in the corticalspreading depression

Diego Passaroa,1, Gina Ranaa,1, Marina Piscopoa, Emanuela Viggianob,Bruno De Lucab, Laura Fuccia,⁎aDipartimento di Biologia Strutturale e Funzionale, Università di Napoli Federico II, Via Cinthia, 80126, Napoli, ItalybDipartimento di Medicina Sperimentale, II Università di Napoli, Via Costantinopoli 16, 80138 Napoli, Italy

A R T I C L E I N F O

⁎ Corresponding author. Fax: +39 081 679233.E-mail address: [email protected] (L. Fucci).Abbreviations: BDNF, brain-derived neuro

dehydrogenase; H3K4, H3 histone lysine 4; Hmethyl-transferase; iNOS, inducible nitric oxnitric oxide synthase; RT, reverse transcripti1 These authors contributed equally to this

0006-8993/$ – see front matter © 2010 Elsevidoi:10.1016/j.brainres.2010.03.001

A B S T R A C T

Article history:Accepted 1 March 2010Available online 6 March 2010

Preconditioning with Cortical Spreading Depression induces a sort of tolerance to asubsequent episode of ischemia. Themechanism of this tolerance is not clear. We studied ifsuch treatment induces epigenetic chromatin modifications on the hemispheres of ratspreconditioned by Cortical Spreading Depression. The contralateral hemispheres were usedas control. We determined the level of H3K4 and H3K9 methylation and the mRNA amountsfor the two well known H3K4 methyltransferases (MLL and SET7) in rats 24°h after theCortical Spreading Depression induction. Western blotting experiments have beenperformed using three different types of primary antibodies against mono-, di- and tri-methyl H3K4 and primary antibody anti-dimethyl H3K9. In the same samples we checked ifthe H3 histones were replaced by the H3.3 histone variants that could be an additionalmarker of chromatin modifications. The level of mono- and di-methyl H3K4 wassignificantly lower in samples of the treated hemispheres than those of the contralateralhemispheres (40% and about 60%, respectively) while the level of tri-methylation remainedunchanged. The level of di-methyl H3K9 was almost 60% higher in the treated hemispheresthan the contralateral hemispheres. The treatment for Cortical Spreading Depressionaffected also the level of expression of H3K4 histone methyltransferase MLL and SET7 thatdecreased in the treated hemispheres in comparison to the control hemispheres (80% and40%, respectively). The treatment for Cortical Spreading Depression instead had no effectson the overall amounts of mRNA for H3 and H3.3 histones. In conclusion epigeneticchromatin modifications are evident in rats 24°h after the Cortical Spreading Depressioninduction.

© 2010 Elsevier B.V. All rights reserved.

Keywords:HistoneHistone-methyltransferaseMethylationCortical Spreading DepressionChromatin

trophic factor; CSD, Cortical Spreading Depression; GAPDH, glyceraldehyde 3-phosphateIF-1, hypoxia inducible factor-1; H3K4me3, H3 histone lysine 4 trimethylation; HMT, histoneide synthase; MLL, mixed lineage leukemia; NET, not exposed or treated; nNOS, neuronalon; SET, Su(var)3-9, Enhancer-of-zeste, Trithoraxwork.

er B.V. All rights reserved.

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1. Introduction

Cortical spreading depression (CSD) is characterized by self-propagating depolarization associated by depression ofspontaneous electrical activity for several minutes (Leao,1944) and can be induced by different stimuli (Bureš andLánský, 2004). CSD waves spread unilaterally across onehemisphere from the region stimulated in all directions atthe velocity of 2–3 mm/min, involving subsequently distantparts of the cerebral cortex (Leao, 1944). CSD is a reversiblephenomenon, accompanied by changes in cerebral bloodflow, metabolism and cellular ion balance (Somjen, 2001),but it is not associated with tissue injury in normoxic brain(Nedergaard and Hansen, 1988). CSD is probably involved inthe pathogenesis of several neurological disorders as stroke(Hossmann, 1996; Fabricius et al., 2006), migraine aura(Ebersberger et al., 2001; Lauritzen, 1994), epilepsy (Fabriciuset al., 2008; Gorji, 2001).

It has been reported that CSD enhances the damageinduced by ischemia in rat in the peri-infarction area (Iijimaet al., 1992; Mies et al., 1993) because it increases glucose andATP utilization, and O2 consumption (Mayevsky and Weiss,1991). Interestingly, preconditioning with CSD induces a sortof tolerance to a subsequent episode of ischemia (Kobayashiet al., 1995; Matsushima et al., 1998; Taga et al., 1997;Yanamoto et al., 1998; Otori et al., 2003). The mechanism ofthis tolerance is not clear. Several studies demonstrated thatCSD induces the expression of neuroprotective genes as HIF-1,iNOS (Viggiano et al., 2008), nNOS (Caggiano and Kraig, 1998;Shen and Gundlach, 1999), brain-derived neurotrophic factor(BDNF) (Matsushima et al., 1998; Urbach et al., 2006).

Recently mitotically heritable epigenetic modificationshave been shown to affect transcription regulation and toplay a key role in human pathologies, including inflammatoryand neoplastic disorders (van der Maarel, 2008).

Epigenetic modifications of genomes involve two majormechanisms such as post-translational modification of his-tone proteins in chromatin and methylation of DNA itself,which are regulated by distinct, but coupled, pathways.Histone methylation is a process of adding one, two or threemethyl groups to certain amino acids on histone tails.

Each level of modification can have different biologicaleffects depending on the precise residues and contexts. Forexample, trimethylation of lysine 4 in histone H3 (H3K4me3)occurs virtually in all active geneswhereas H3K9me3 occurs incompact pericentromeric heterochromatin which is transcrip-tionally inert (Völkel and Angrand, 2007).

Moreover high levels of H3K4 trimethylation appeared to beassociated with the 5′ regions and showed a strong positivecorrelation with transcription rates, active polymerase IIoccupancy and histone acetylation (Ruthenburg et al., 2007).In vertebrates the majority of H3K4 dimethylation colocalizeswith H3K4 trimethylation in discrete zones (Bernstein et al.,2005). Recently several interactors of methylated H3K4,including the basal transcription factor TFIID, have beenidentified and it has been demonstrated that they bind tothe target via different domains (Ruthenburg et al., 2007;Vermeulen et al., 2007).

Several enzymes are involved in the H3K4 methylation. Todate with the exception of Dot1/DOT1 L all known lysine

histonemethyltransferases contain a conservedmethyltrans-ferase domain, termed SET (Su(var)3-9, Enhancer-of-zeste,Trithorax) domain of about 130 amino acids. The lysinemethyltransferase activity has been implicated in carcino-genesis, probably because these SET-domain proteins will joinseveral other families of chromatin modifying enzymes(acetylases, deacetylases and kinases) that have alreadyimplicated in cancer (Kouzarides, 1999).

Set1 is the only H3K4 specific histone methyl-transferase(HMT) present in yeast and is directly involved in geneexpression; the Set1 family has evolved into more complexsystems in higher eukaryotes (Ansari et al., 2008). In mam-mals, six Set1 family members have been characterized:Set1A, Set1B and four MLL (mixed lineage leukemia)-familyof HMTs. All these HMTs possess H3K4 HMT activity and playrole in gene activation (Ansari et al., 2008). Similarly to yeastSet1, MLL1, MLL2, MLL3 and human Set1 (hSet1) are alsopresent in distinct multi-protein complexes sharing threecommon subunits Ash2, Rbbp5, Wdr5 along with severalspecific components (Ansari et al., 2008).

A number of HKMTs have a SET domain not preceded by acysteine-rich region and not followed by a post-SET-domain;this class includes SET7/9 that has a methyltransferaseactivity not limited to lysine of H3 histones but also tumoursuppressor p53 or TAF (Völkel and Angrand, 2007). Thisenzyme in human generates exclusively histone H3 mono-methyl-lysine 4, probably because its lysine-binding channelexcludes the lysine side chain with methyl groups.

It was originally thought that histone methylation was apermanent modification. This theory prevailed until therecent discovery of lysine-specific demethylase 1, whichspecifically removes mono- and di- but not tri-methyl groupsfrom lysine residues, and Jumonji domain-containing, whichcan demethylate all the three status of methylated lysineresidue (Cheng and Zhang, 2007). Therefore, similar to histoneacetylation, histone methylation is also a regulated dynamicprocess.

In this paper we studied if CSD causes epigenetic chromatinmodifications in rats preconditioned by the treatment. To thisaimwedetermined the level of H3K4 andH3K9methylation andSET7 and MLL1 mRNA amounts in rats treated 24°h earlier forCSD. In the same samples we checked if the H3 histones werereplaced by theH3.3 histone variants that could be an additionalmarker of chromatin modifications.

2. Results

We have experimentally analyzed the level of mono-, di- andtri-methylation of histone H3 lysine 4 (H3K4) and the level ofdimethylation of histone H3 lysine 9 (H3K9) in proteinextracts derived from hemisphere of rats whose brain havebeen treated for CSDwith KCl. The contralateral hemisphereshave been treated with NaCl and are therefore used ascontrol. The EEG and DC recording confirmed that the KClapplication caused several waves of spreading depression asexpected.

The histone proteins were extracted and isolated from thebrain of rats by treatment with acids, according to the protocolreported in Experimental methods. Determination of protein

Fig. 1 – SDS-PAGE of proteins extracted by the ipsilateral andcontralateral hemispheres of rats treated for CSD. Lanes 1–2,5–6: proteins extracted by the KCl-treated hemispheres oftwo different rats (in duplicate). Lanes 3–4, 7–8: proteinsextracted by the NaCl-treated hemispheres of two differentrats (in duplicate). M: LowMolecularWeight Marker (BioRad).The molecular weights of markers are reported on the left.

Fig. 3 – Panels A–B: Western blotting experiments andrelative histograms on proteins extractedfrom ipsilateral (lanes 1–2) and contralateral hemispheres(lanes 3–4), neither exposed nor treated (NET). PanelsC–D: Western blotting experiments and relative histogramson proteins extracted from hemispheres (lanes 1–2) notexposed or treated (NET) and contralateralhemispheres (lanes 3–4) treated with NaCl. (R): hemispheresof rats treated with KCl. (L) Hemispheres of rats treated withNaCl. p value>0.05 (not significant) in both cases incomparison to contralateral hemispheres.

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concentration has been performed using BioRad protein assaye checked by PAGE in presence of SDS. No clear differencesbetween samples are evident in Fig. 1.

Western blotting experiments have been performed usingthree different types of primary antibodies against mono-, di-and tri-methyl H3K4 and primary antibody anti-dimethyl H3K9.

The mono- and di-methyl H3K4 antibodies revealed onlybands of the expected molecular weight in each samplewhile the tri-methyl H3K4 antibodies revealed a band of highmolecular weight and, in some cases, multiple bands of lowermolecular weights (Fig. 2). These data suggest the ability ofH3K4me3 to form complex/polymer in the used experimentalconditions.

After scanning intensity of bands was quantified with theBioRad software Quantity One. Level of histone methylationwas expressed as a percentage by comparing the intensities of

Fig. 2 – Western blotting experiments on acid-soluble proteins extracted from the brains of rats treated for CSD, usingantimono-, di- and tri-methyl H3K4 and antidimethyl H3K9. Lanes 1–2: proteins extracted by KCl-treated hemispheres. Lanes3–4: proteins extracted by NaCl-treated hemispheres.

Fig. 5 – Panels A–B: agarose gel electrophoresis and relativehistograms of RT-PCR amplification products of MLL1, SET7and GAPDH on total RNAs extracted from brains of rats notexposed or treated (NET). Lanes 1–2, 6–7, 11–12: hemispheresnot exposed or treated (R). Lanes 3–4, 8–9, 13–14: hemispheresnot exposed or treated (L). Lanes 5, 10, 15: negative controls.M: Molecular Weight Markers 100 bp. p value>0.05 (notsignificant) in comparison to the contralateral hemispheres.

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the bands in the samples of the treated hemispheres withthose of the contralateral hemispheres. Samples were ana-lyzed in duplicate on each gel and each experiment was re-peated three times. Data obtained on two out of eight ratswere not reported because the CSD was not very efficient.

As first control the level of H3K4me2 was determined onsamples corresponding to the contralateral hemispheres of thecerebral cortex of rats, neither exposed nor treated (N.E.T.). Asshown in Fig. 3 (panels A and B), there are no differencesbetween the two samples, suggesting that the two hemispheresin native conditions have the same total level of H3K4 di-methylation.

In order to exclude any role of the stress caused by exposureof the hemispheres on the levels of H3K4 di-methylation,Western blotting experiments were performed on samples ofthe hemispheres treated with NaCl and contralateral hemi-spheres not exposed. As shown in Fig. 3 (panels C and D) thereare no differences between the two samples suggesting that thestress caused by exposure has no effects on the levels of di-methylation in H3K4.

Results obtained with H3K4me1, H3K4me2, H3K4me3 andH3K9me2 antibodies are shown in Fig. 4. The level of mono-and di-methyl H3K4 was significantly lower in samples of thehemispheres than that of the contralateral hemispheres (40%and about 60%, respectively) while the level of tri-methylationremains unchanged. On the contrary the level of the di-methylH3K9 is almost 60% higher in samples of the hemisphere thanthe contralateral hemisphere.

Changes in the total level of H3K4 methylation after CSDtreatment may be caused by changes in the activities of H3K4methyltransferase. Then we have analyzed by RT-PCR thelevel of expression of two well-known H3K4 methyltrans-

Fig. 4 – Levels of mono-, di- and tri-methylated H3K4 and di-methemispheres of 6 rats treated for CSD. (R) Hemispheres of rats trp values<0.001 for H3K4me1, H3K4me2, H3K9me2 in comparisosignificant) for H3K4me3 in comparison to the hemispheres trea

ferases (MLL and SET7) in the brain of rats after treatment forthe CSD. Total RNA was extracted from brain tissue takenfrom the two hemispheres and the concentration of RNAwas

hylated H3K9 measured on protein extracted from theeated with KCl. (L) Hemispheres of rats treated with NaCl.n to the hemispheres treated with NaCl. p value>0.05 (notted with NaCl.

Fig. 6 – Panels A–B: agarose gel electrophoresis and relativehistograms of RT-PCR amplification products of MLL1, SET7and GAPDH on total RNAs extracted from brains of ratstreated for CSD. Lanes 1–2, 6–7, 11–12: hemispheres treatedwith KCl (R). Lanes 3–4, 8–9, 13–14: hemispheres treated withNaCl (L). Lanes 5, 10, 15: negative controls. M: MolecularWeight Markers 100 bp. p value<0.001 for all the data incomparison to the hemispheres treated with NaCl.

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determined with the Nanodrop Spectrophotometer andverified by electrophoresis on agarose gel in denaturingconditions. GAPDH (Glyceraldehyde 3-phosphate dehydroge-nase) was used as an internal control in the RT-PCRexperiment. The amplified DNA was analyzed by electropho-resis on 1% agarose gel in order to verify the length of theamplification product and to measure its amount. Afterscanning the intensity of the bands was determined usingthe BioRad “Quantity One” software.

It is important to note that the two hemispheres of a ratneither exposed nor treated (N.E.T.) have the same levels ofmRNA for MLL and SET7, as shown in Fig. 5.

CSD instead affects the level of expression of histonemethyltransferase MLL e SET7 that decrease in the hemi-spheres if compared to the contralateral hemispheres used ascontrol (80% and 40%, respectively). Data are reported in Fig. 6.

Moreover CSD has no effects on the amounts of mRNA forH3 and H3.3 histones (Fig. 7).

Fig. 7 – Panels A–B: agarose gel electrophoresis and relativehistograms of RT-PCR amplification products of H3, H3.3 andGAPDH on total RNAs extracted from brains of rats treated forCSD. Lanes 1–2, 6–7, 11–12: hemispheres treated with KCl (R).Lanes 3–4, 8–9, 13–14: hemispheres treated with NaCl (L).Lanes 5, 10, 15: negative controls. M: Molecular WeightMarkers 100 bp. p value>0.05 (not significant) for all the datain comparison to the hemispheres treated with NaCl.

3. Discussion

Epigenetic modifications of our genome involve DNA methyl-ation, covalent modifications of the histone tails, nucleosomeoccupancy and turnover and higher chromatin folding. Thesemitotically heritable epigenetic modifications can affecttranscription regulation and are increasingly recognized tobe causally involved in a broad spectrumof human conditions,ranging frommonogenic to multifactorial disorders, includinginflammatory and neoplastic disorders (van der Maarel, 2008).

In chromatin brain the delicate balance of different histonemethylation marks seems to be essential for orderly develop-ment and function (Akbarian and Huang, 2009; Shen andCasaccia-Bonnefil, 2008). In addition recent discoveriesshowed that the deletion syndrome associated with severemental retardation encompasses the H3K9-specific histonemethyltransferase (EHMT1) (Kleefstra et al., 2006) gene andmutations in the gene that encodes an H3K4me3-specificdemethylase results in mental retardation and autism (Adeg-bola et al., 2008; Iwase et al., 2007).

Then it is reasonable that histone methylation processmight play some important role in the tolerance to subsequentepisode of ischemia in rats preconditioned by CSD.

In order to study if CSD causes alterations in histonemethylation we determined the global level of mono-, di- andtri-methylation H3K4 and di-methylation H3K9 in rats 24°hafter the CSD induction.

The present study demonstrates that CSD affects methyl-ation of histone H3; in particular, the level of mono and di-methylation of H3K4 in protein extracts, derived from thehemispheres of rats treated for CSD, decreases significantlywhile the level of tri-methylation remains unchanged and theamount of di-methyl H3K9 decreases.

Obviously the global change in the level of H3K4 and H3K9methylation suggests an overall alteration of the chromatinstructure in the nuclei of the brain cells treated for CSD butcannot give any indication about the structure of genomicregion containing specific genes. In order to get informationabout H3K4 and H3K9 methylation at individual loci we areperforming experiments of ChIP with specific anti-methyl-histone antibodies. Preliminary results show that genesswitched on by the CSD treatment are more dimethylated in

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H3K4 and less dimethylated in H3K9 indicating that overallalteration of the chromatin structure are affected mainly bythe methylation of repeated sequences in the genome. In ourexperiments the global level of H3K4 dimethylation changedwhile trimethylation is unaffected; these data are unexplain-able on the basis of the results reported in literature. In fact, invertebrates, the majority of H3K4 dimethylation co-localizeswith H3K4 trimethylation in discrete zones about 5–20 nucleo-somes in length proximate to highly transcribed genes(Schneider et al., 2004). In more active genes, H3K4 di/trimethylation may facilitate higher transcription ratesthrough the maintenance of greater chromatin accessibility(Nishioka et al., 2002; Zegerman et al., 2002; Pray-Grant et al.,2005). Thus, H3K4me2/H3K4me3 may be part of a positivefeedback loop facilitating transcription and providing amolecular memory of transcriptional activity (Gerber andShilatifard, 2003). Clearly all the data reported in literaturerefer to specific genes that are transcribed while our studiesrefer to the entire genome containing also repeated sequences.It is to note that alsoMishra et al. (2009) demonstrated that theglobal level of H3K4 trimethylation is not affected during thecell cycle of HELA cells but there is fluctuation of trimethyla-tion at specific promoters of homeobox-containing genes.

Only recently some evidence suggested that Set-1-depen-dent histone methylation might be involved in gene silencing.Pinskaya et al. (2009) showed that GAL1 promoter is attenu-ated by the H3K4me2/me3 deposited by cryptic transcription.

The opposite behaviour of the H3K4 dimethylation, thatincreases, and H3K9 di-methylation, that decreases, is insteadin agreement with the data reported in literature.

If the CSD affects the global levels of H3K4 methylation theenzymes that are implicated in such modifications have to bereduced or in the activity or in the total amount. For thisreason we measured by PCR the mRNAs for the two commonHMTs, MLL and SET7, in the hemispheres comparing with thecontralateral hemispheres of rats treated for CSD. It isimportant to note that in the control rats where the hemi-spheres were not stressed in any way (neither exposed nortreated with KCl) the amounts of the mRNAs were similar inthe ipsilateral and contralateral hemispheres.

Methylation of histone lysines is catalyzed mainly byseveral histone-specific methyltransferases containing theSET domain. SET1p is considered the fundingmember of SET1class of SET proteins. In mammals, six SET1 family membershave been identified: SET1a and SET1b and four mixed lineageleukemia (MLL) family H3K4 methyltransferases, MLL1, MLL2,MLL3 and MLL4. The best-characterized mammalian SET1family member is MLL1 that exists in human as multiproteincomplexes containing Ash2, Wdr5, Rbbp5, CpG-binding pro-tein (CGBP) and Dpy30 components (Ansari et al., 2008;Steward et al., 2006; Hughes et al., 2004; Goo et al., 2003; Douet al., 2006; Pavri et al., 2006). As suggested by Patel et al. (2009)MLL1 core complex is predominantly an H3K4 dimethyltrans-ferase. Instead the SET7/9, that has a relatively limited activesite volume, is predominantly monomethyltransferase.

As expected from the decreased global level of mono anddimethylated H3K4 in the hemispheres of rats treated for CSD,the amounts of the mRNAs for the MLL1 and Set7 methyl-transferases also decrease suggesting a more extensivemolecular change in brain after CSD treatment.

An additional mechanism of change in chromatin structureis linked to the replacement of histone H3 by its variant H3.3.According to recent studies, euchromatic regions transcription-ally active are rich in histone H3.3. Hake and Allis (2006)proposed that the H3 variants play a major role in celldifferentiation and cell lineage restriction and proposed aspeculative hypothesis, the H3 barcode hypothesis. The mam-malian genome is indexed by histone H3 variants in a nonrandom fashion that reflects the assembly mechanisms and“personalized” chaperons and exchange factors. The histoneH3.3 is incorporated into transcriptionally active regions,whereas the histone H3.2 is deposited in transcriptionally silentareas that can be reversibly activated, depending on cellularneeds (facultative heterochromatin) and the histoneH3.1mightthen be localized to genes that are constitutively silent. Thetranscriptionally active euchromatic regions are rich in H3.3variants, that are indeed more frequent dimethylated in lysineK4 while the variant H3.1 is more susceptible to H3-K9methylation (McKittrick et al., 2004).

Then we tested the effect of CSD also on the total amount ofthe transcripts for H3 andH3.3 histones, butwe did not find anyevident global changes. These results suggest that CSD affectsthe structure of the chromatin through methylation of the H3histone tails but not through the substitution of H3 variants.

In conclusion CSD can cause heritable epigenetic modifica-tions such as histone tail methylation that can affect thetranscription rate of repeated sequences and genomic locicontaining specific genes.

4. Experimental procedures

4.1. Animals

Adult male Wistar rats (280–300 g) were housed in pairs atcontrolled temperature (22±1°), and humidity (70%) with12:12 h light–dark cycle from 7.00 to 19.00. The experimentswere in accordance with the European Communities CouncilDirective of 24 November 1986 (86/609/EEC).

Data were obtained on six rats treated for CSD. Further-more, we analyzed as control two rats not exposed or treated(NET) and two rats in which one hemisphere was treated withNaCl and contralateral hemisphere was not exposed.

4.2. CSD

The rats were anaesthetized with chloral hydrate intraperito-neal (i.p.) (0.6 g/kg bw). A thermistorwas inserted into the colon,and body temperature was maintained at 37 °C using a heatingpad. The head was fixed in a stereotaxic frame and after skullexposure a round hole 3 mm in diameter was centered 5 mmfrom midline and 4mm caudal from bregma. After removal ofthedura, CSDwas inducedbyplacinga filterpaper soaked in1 MKCl (30 μl) (or 0,9 % NaCl in the sham operated rats) for 15 min.CSD waves and EEG were recorded with 2 Ag/AgCl electrodes(1 mmdiameter): onewas applied on the surface of the exposedparietal cortex throughasmallhole (3 mmlateral to themedlineand 2 mm posterior to bregma) and the other was fixedsubcutaneously in the neck skin (reference electrode). Theelectrodes were connected to a polygraph (Dynograph;

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Beckman). After 24 h the animalswere sacrificed and the brainswere quickly removed; the cortex of both hemispheres wassectioned and frozen in liquid nitrogen.

4.3. RT-PCR

mRNA amounts in specimens were determined by RT-PCR.Total RNAs were extracted from prefrontal cortex using TrizolReagent (Invitrogen, Paisley, UK). RNA was quantified spec-trophotometrically at the Nanodrop Apparatus and the RNAintegrity was checked by agarose gel electrophoresis. TotalRNA was reverse transcribed into cDNA using primers specificfor rat H3 and H3.3 histones, SET7 and MLL1 (H3K4 methyl-transferase). RT reactionmixtures contained 1 μg RNA sample,25 pmol primers random, 10 U of AMV reverse transcriptase(Promega, Milan, Italy) in a final volume of 25 μl RT buffer(50 mM Tris–HCl pH 8.3, 50 mM KCl, 50 mM MgCl2, 0.5 mMspermidine, 10 mM DTT). All chemicals were from Promega.Reverse transcription was performed at 42 °C for 1 h. Sampleswere then heated to 70 °C for 10 min to denature the reversetranscriptase and stored at −20 °C. Control samples in whichRNA was replaced by DEPC-treated water were prepared.

Preliminary PCR experiments have been performed inorder to ensure that the amounts of the amplified productswere in a linear relationship with the initial cDNA amount. Inthe case of rat H3, H3.3, SET7 and MLL1 4 µl of cDNA wasamplified by PCR using 1 U TaqDNApolymerase (Promega) andspecific primers; 2 μl of cDNAwere used in the amplification ofthe control glyceraldehyde-3-phosphate dehydrogenase(GAPDH). PCR was performed in a thermal cycler (BioRad)and consisted of an initial denaturation step of 3 min at 94 °Cfollowed by 35 cycles of 60 s at 94 °C, 30 s at 60 °C and then 60 sat 72 °C. At the end of the amplification samples wereincubated for a further 10 min at 72 °C. PCR was performedusing 1.5 mM MgCl2, 0.2 μM mix dNTP, 1 U of GoTaq DNAPolymerase (Promega, Milan, Italy), 25 pmol forward andreverse primers, in a final volume of 50 μl PCR buffer.Sequences of primers are reported below.

Primers

Sequence Product size Genebank accession no.

H3

Forward 5′-CACCTGGTACTGTGGCATTG-3′ 147 bp XM220509 Reverse 5′-CCAGAGCTCGGCTGTAATG-3′

H3.3

Forward 5′-AGGCCCGGGACCGTGGCTCTA-3′ 147 bp X73683 Reverse 5′-TCAAAGTGCAGCCATCGGT-3′

SET7

Forward 5′-GTCGGGGAAGTCAATGAAGA-3′ 350 bp NM001109558 Reverse 5′-GCCGACTCTCTCATCTCCAG-3′

MLL1

Forward 5′-AGCTCTGCTTTGTATCCTGTGGGT-3′ 599 bp XM236194 Reverse 5′-GCCTTACCGTGGGAAGAACG-3′

GAPDH

Forward 5′-TCCTGCACCACCAACTGCTTAGCC-3′ 376 bp BC087743 Reverse 5′-TAGCCCAGGATGCCCTTTACTGGG-3′

9 μl of each PCR reaction was analysed by electrophoresison 1% agarose gel stainedwith ethidium bromide. The Gel Doc1000 system (BioRad) and Quantity One softwarewere used forthe quantification of the PCR products.

4.4. Proteins extraction

The rat cerebral cortex provided by Dr E. Viggiano of the IIUniversity of Napleswas dissected, frozen in liquid nitrogen andstored at −80 °C. After unfreezing, seven volumes of lysis buffer(10 mM Hepes, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM spermidine,12% glycerol, 0.5 mM DTT, 0.1 mM EGTA) were added to 1mg oftissue togetherwith theRochecocktail ofprotease inhibitors (1×).Tissues were manually dismembered with a sterile pestle andcentrifugedat 800g for 15 ' at 4 °C inEppendorf Centrifuge; pelletswerewashed three timeswith 2ml of lysis buffer. 3.5 volumes of0.2 NHClwere added to pellets and the sampleswere kept on icefor 30 min; suspensions were centrifuged at 4 °C for 10 min at14,000 g. Protein concentration of the supernatant was deter-mined by BioRad assay and verified by Coomassie staining ofsodium dodecyl sulphate-polyacrylamide gels (SDS-PAGE).

4.5. Western blotting analysis

Total proteins were extract from prefrontal cortex of rat(ipsilateral and contralateral hemispheres), the concentrationwas determined according to Bradford.

SDS-PAGE was performed on 20 μg protein samples using18 % polyacrylamide gels (BioRad). For immunoblotting, theproteins were transferred from unstained gels to nitrocellulosemembrane (GeHealthcare) in the experiments with anti-H3K4antibody and PVDF membrane (Millipore) in those with anti-H3K9 antibody using a Trans-Blot SD semi-dry electrophoretictransfer cell (BioRad) overnight. Each sample was analysed induplicate and the analysis was repeated three times. Thedifferent antibodies were used in accordance to the manufac-turer. The membranes were blocked in non-fat dry milk bufferand then were incubated with 1:500, 1:10.000, 1:500 and 1:500

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dilutions of rabbit polyclonal anti-H3K4me1, H3K4me2, H3K4me3and H3K9me2 antibodies (Upstate), respectively, at room temper-ature with constant shaking. After three washes in water, themembraneswere incubatedwith a 1:5.000 dilution of peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) at room temperaturewith constant shaking. Protein signal was visualized via chemi-luminescence using the ECL Western Blotting Detection Systemand Hyperfilm ECL autoradiography film (Amersham PharmaciaBiotech, Inc.). Signals were quantified using the Gel Doc 1000system (BioRad) and Quantity One software.

4.6. Statistical analysis

Statistical analysis on Western blot and RT-PCR data wascarried by a t-test using the software GraphPad Prism. Resultsof the analysis with a value of p<0.05 were considered to bestatistically significant.

Acknowledgments

This work was supported by the Regione Campania (L.R. n. 5,28/3/2003; year 2005).

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