FGF2 is a target and a trigger of epigeneticmechanisms associated with differences inemotionality: Partnership with H3K9me3Sraboni Chaudhurya,1, Elyse L. Aurbacha, Vikram Sharmaa, Peter Blandino, Jr.a, Cortney A. Turnera, Stanley J. Watsona,b,and Huda Akila,b,1

aMolecular and Behavioral Neuroscience Institute and bDepartment of Psychiatry, University of Michigan, Ann Arbor, MI 48109

Contributed by Huda Akil, June 30, 2014 (sent for review March 26, 2014; reviewed by Ronald DeKloet and Johannes M. H. M. Reul)

Posttranslational modifications of histone tails in chromatin tem-plate can result from environmental experiences such as stress andsubstance abuse. However, the role of epigenetic modifications aspotential predisposing factors in affective behavior is less wellestablished. To address this question, we used our selectively bredlines of high responder (bHR) and low responder (bLR) rats thatshow profound and stable differences in affective responses, withbLRs being prone to anxiety- and depression-like behavior andbHRs prone to addictive behavior. We first asked whether thesephenotypes are associated with basal differences in epigeneticprofiles. Our results reveal broad between-group differencesin basal levels of trimethylated histone protein H3 at lysine 9(H3K9me3) in hippocampus (HC), amygdala, and nucleus accum-bens. Moreover, levels of association of H3K9me3 at GlucocorticoidReceptor (GR) and Fibroblast growth Factor 2 (FGF2) promotersdiffer reciprocally between bHRs and bLRs in these regions,consistent with these genes’ opposing levels of expression androles in modulating anxiety behavior. Importantly, this basal epi-genetic pattern is modifiable by FGF2, a factor that modulatesanxiety behavior. Thus, early-life FGF2, which decreases anxiety,altered the levels of H3K9me3 and its binding at FGF2 and GR pro-moters of bLRs rendering them more similar to bHRs. Conversely,knockdown of HC FGF2 altered both anxiety behavior and levels ofH3K9me3 in bHRs, rendering them more bLR-like. These findingsimplicate FGF2 as a modifier of epigenetic mechanisms associatedwith emotional responsiveness, and point to H3K9me3 as a keyplayer in the regulation of affective vulnerability.

MR | H3K9me2 | H4K20me3 | plus maze

Chromatin remodeling is a mediator of lasting neural changes inresponse to experience, such as exposure to stress and drugs of

abuse (1–7). Indeed, the interaction of certain modified histoneswith specific gene promoters has been shown to be an importantmechanism of experience-dependent neuroplasticity (8–13). Al-though numerous studies have examined the impact of the envi-ronment on neural epigenetic profiles, relatively few studies havefocused on preexisting differences in epigenetic profiles as po-tential predisposing factors in emotional reactivity. Such studiesrequire the availability of an animal model where difference in“temperament” or propensity for specific affective responses canbe reliably predicted and altered. Our laboratory has generatedsuch an animal model that captures vulnerability for “internalizingdisorders” vs. “externalizing disorders.” Selectively bred high re-sponder (bHR) rats exhibit greater responsiveness to novelty andto drug seeking behavior (externalizing behaviors), whereas se-lectively bred low responders (bLR) exhibit greater anxiety anddepression-like responses (internalizing behaviors) (14, 15). Thesegenetically bred phenotypes amplify behavioral traits observed inoutbred animals (16–20).Several genes have been implicated in modifying these phe-

notypes, both in the bred and outbred lines (14). In particular,a key gene in stress regulation, the glucocorticoid receptor (GR)

showed higher levels of mRNA expression in the hippocampus(HC) of outbred LRs relative to outbred HRs, and has beenimplicated in increased anxiety behavior in these animals. Thus,administration of a GR antagonist into the HC reduced anxietybehavior in outbred LR rats. Importantly, bLRs also exhibitsignificantly higher levels of hippocampal GR mRNA com-pared with bHRs, whereas the mineralocorticoid receptor (MR)showed no differences between the lines (17). Moreover, ourmouse genetic studies have demonstrated that GR is an early-lifemodifier of emotional reactivity, with its overexpression beforeweaning enhancing anxiety throughout life (21). This then sug-gests that GR a critical molecular organizer of stable differencesin affective reactivity.A countervailing modifier of anxiety behavior is the Fibroblast

Growth Factor-2 (FGF2). This molecular organizer plays a criticalrole in brain development and hippocampal neurogenesis (22, 23).Moreover, FGF2 has been proposed as an endogenous anxiolyticand antidepressant that is depleted in the brain of depressedhumans (24, 25). Its direct chronic administration was anxiolyticand antidepressant in rodents (25) and the silencing of endoge-nous hippocampal FGF2 using short-hairpin RNA increasedanxiety-like behavior in outbred rats (26, 27). Our bLRs, whichexhibit higher anxiety and depression behaviors, have lower basallevels of FGF2 mRNA in HC and nucleus accumbens (NAcc) (28,29), and an environmental manipulation during adulthood thatdecreases anxiety behavior induces FGF2 expression selectively inthese bLRs (30). Moreover, early-life FGF2 administration se-lectively decreases anxiety in bLRs throughout life (28).In the present study, we evaluated the basal levels of various

modified histone proteins (H3 and H4) in the HC, amygdala,and NAcc in the bHR and bLR rats. We then focused on a re-pressive trimethylated histone protein H3 at lysine 9 (H3K9me3)which is one of the most widely studied repressed modified

Significance

This study investigates the role of epigenetics as a predisposingfactor that modifies inborn emotional reactivity and the vul-nerability or resilience to affective disorders, such as majordepression or substance abuse. It tests the hypothesis thatspecific modified histones contribute to basal differences intemperament that bias emotional responses to the environ-ment. It further describes the role of a growth factor that canmodify these epigenetic patterns as it alters affective behavior.

Author contributions: S.C., E.L.A., C.A.T., S.J.W., and H.A. designed research; S.C., E.L.A.,V.S., P.B., and C.A.T. performed research; S.C. contributed new reagents/analytic tools;S.C., E.L.A., and V.S. analyzed data; and S.C., V.S., and H.A. wrote the paper.

Reviewers: R.D., Leiden University; and J.M.H.M.R., University of Bristol.

The authors declare no conflict of interest.1To whom correspondence may be addressed. Email: sraboni@umich.edu or akil@umich.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1411618111/-/DCSupplemental.

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histones (31), and which showed reliable bHR vs. bLR differ-ences. Using chromatin immunoprecipiation (ChIP) assays, weasked whether some of the basal variations in GR and FGF2expression between the bred lines might be the result of differ-ences in the association of this histone at their promoter. Finally,we asked whether manipulating FGF2 either via early-life ad-ministration or via virally mediated knockdown can modify theepigenetic patterns observed in the bred lines.

ResultsEndogenous Levels of Methylated Repressive Histone Protein H3K9me3Are Altered in bHR-LR Rats. Because HC, amygdala, and NAcc arebrain regions implicated in anxiety and addiction, we evaluatedrepressive modified H3 in these areas. In general, the levels ofH3K9me3 differed across the three regions studied (Fig. 1A).More importantly, within each of the three brain areas, we ob-served a decrease in the levels of H3K9me3 (P < 0.001; Fig. 1C)in bHRs relative to bLRs. Because modified histone H4K20me3has been identified to play a role in responsiveness to stress andaddiction, we tested its levels in the same three brain regions andfound no differences in the levels of H4K20me3 between bHRsand bLRs (Fig. 1 B and D). Thus, decreased levels of the re-pressive H3K9me3 are distinctive and may play a role in regu-lation of stress and anxiety-related genes in the bLRs comparedwith bHRs. Therefore, we further investigated the association ofH3K9me3 with the promoters of specific genes implicated in ourbehavioral phenotypes.

Association of Methylated Repressive Histones at Stress- and Anxiety-Related Genes Are Altered. GR and MR are both ligand-modu-lated transcription factors that transduce the actions of the cir-culating stress hormones corticosterone in rodents and cortisol inhumans (32) and modulate anxiety behavior (17, 33, 34). Giventhe differences in anxiety and stress responsiveness betweenbHR and bLR rats (15, 35), we evaluated the association of thesegenes with the modified forms of histones using ChIP assays. Theassociation levels of H3K9me3 at the GR promoter were sig-nificantly increased in bHRs HC (P < 0.001), amygdala, andNAcc (P < 0.002) compared with bLRs (Fig. 2 A and C), con-sistent with the lower level of GR expression in the less anxiousbHRs relative to the more anxious bLRs. By contrast, the asso-ciation of H3K9me3 at the MR promoter did not show anyalterations in HC, amygdala, and NAcc of bHR versus bLR rats(Fig. 2 A and D), indicating that the differences between the bredlines is specific to particular stress genes.

Because FGF2 plays a key role as an endogenous anxiolyticand shows higher expression levels in the less anxious bHRs (25,28), we used a ChIP assay to investigate the association levelsof various modified histone at the FGF2 gene. Compared withbLRs, negligible association was observed for the repressiveH3K9me3 (P < 0.001) at the FGF2 gene promoter in bHR ratHC, amygdala, and NAcc (Fig. 2 A and B). This decreased as-sociation of the repressive methylated histone with FGF2 in HCmay be critical for the up-regulated gene expression of FGF2 inHC of bHRs.

Effects of Early-Life FGF2. Differential alteration of the basal levels ofmodified histone and its association at FGF2 promoter in bHR rats. Wehave previously shown that the injection of a single dose of FGF2(20 ng/g, s.c.) on the day after birth (PND2) produced a lifelongeffect on emotionality, enhanced drug self-administration inoutbred rats (36) and decreased anxiety-like behavior especiallyin bLR rats (28). In essence, early-life FGF2 shifts the behavioralphenotype from bLR toward bHR. To determine the possiblemediation of this behavioral effect by epigenetic mechanisms, weused early-life FGF2 administration and evaluated the levels ofrepressive methylated histone protein and its association withFGF2 promoter in HC, amygdala, and NAcc. Detailed statisticalanalyses are provided in SI Results. In HC and NAcc, we ob-served that there is a significant decrease in the overall level ofH3K9me3 in the bLRs following FGF2 injection compared withthe bLR vehicle treated group (P < 0.001; post hoc analysis). Bycontrast, in the amygdala, the bLRs showed no alterations inH3K9me3 levels, whereas in the bHRs neonatal FGF2 increasedH3K9me3 levels relative to vehicle-treated controls only inamygdala (Fig. 3 A and B).Likewise, the association of H3K9me3 with the FGF2 pro-

moter also significantly decreased (P < 0.001; two-way ANOVAwith post hoc analysis) in HC and NAcc in bLRs following early-life FGF2 injection and this was not observed in the amygdala ofbLRs. By contrast, the bHRs showed no impact of the FGF2neonatal treatment on the H3K9me3 association to FGF2 inany of the brain regions. This result demonstrates that early-life FGF2 reduced the difference in association of repressive

Fig. 1. Differential regulation of repressive methylated histones in variousbrain regions of bLR and bHR rats. (A and C) bHRs showeda significant decreasein the levels of repressive methylated histone protein H3K9me3 in the HC,amygdala, and NAcc in comparison with bLR (***P < 0.001, n = 6 per group). (BandD) However, the levels of repressivemethylated histone protein H4K20me3did not differ between bHRs and bLRs (***P < 0.001, n = 6 per group).

Fig. 2. bLR and bHR rats show differential association of methylated his-tone H3K9me3 at FGF2, GR, and MR promoters in various brain regions.(A and B) bHRs showed a significant (n = 6) decrease in the levels of asso-ciation of repressive methylated histone protein H3K9me3 at FGF2 (***P <0.001, n = 6 per group) (A and C) and a significant increase at GR in the HC,amygdala, and NAcc compared with bLRs (***P < 0.001, **P < 0.002, n = 6per group). (A and D) Note the unaltered association of histone protein ofH3K9me3 at MR promoter in both bHR and bLRs.

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methylated histone H3K9me3 at FGF2 promoter between bHRand bLR in HC and NAcc (Fig. 4 A and B). Conversely, theassociation of H3K9me3 with GR promoter was increased in HC(P < 0.001; post hoc analysis) in bLRs following early-life FGF2injected without altering the NAcc or amygdala (Fig. 4 A and C)and without affecting the bHRs. Thus, the early-life interventionby FGF2 regulates modified histones differentially in variousbrain regions related to anxiety and addiction, primarily modi-fying H3K9me3 levels and associations in the bLRs to renderthem more similar to bHRs.Alteration of methylation level of the FGF2 promoter.DNA methylationtypically leads to transcriptional silencing and H3K9 methylationhas been linked with DNA methylation (37, 38), with H3K9me3inducing DNA methylation via activation of various DNAmethyltransferases (39, 40). As we have observed an alteration inthe association of H3K9me3 at the FGF2 promoter, we furtherassessed the methylation levels at that promoter following neo-natal FGF2 exposure. In untreated animals, we observed a sig-nificant increase in the methylation levels of the FGF2 promoterin bLR hippocampus relative to bHRs (P < 0.001; post hocanalysis) consistent with the lower expression level of FGF2 inthe bLRs. However, following early-life FGF2 the methylationlevels of FGF2 significantly decreases in bLRs compared withvehicle treated bLRs (P < 0.001; post hoc analysis) (Fig. S1).Thus, in the FGF2-treated groups, we observed no differences inthe levels of methylation in the hippocampus between bHR andbLR rats. These results demonstrated that the basal repressionof FGF2 in bLRs can be manipulated by early-life FGF2 inter-ventions, with neonatal FGF2 modifying the epigenetic signatureat its own promoter into adulthood.

FGF2 Knockdown in Adult Hippocampus Alters the Behavior andLevels of Modified Histone (H3K9me3) in the bHRs. Our previouswork in outbred rats demonstrated that the knockdown of FGF2in the hippocampus results in an anxiogenic effect (26). In thepresent study, we compared the selectively bred lines and ob-served that in the elevated plus-maze, FGF2 knockdown in thehippocampus eliminated differences between bHR and bLR be-havior (Fig. 5A). Specifically, bHRs administered a lentiviralvector (LV) expressing a short-hairpin RNA containing a non-silencing control sequence, LVshNS, spent significantly moretime in the open arms than the bLRs administered LVshNS (posthoc Bonferroni test: P < 0.001; Fig. 5A). However, administrationof a lentiviral vector expressing a short-hairpin sequence targetedto FGF2, LVshFGF2, abolished these differences in anxiety be-havior between the bred lines (Fig. 5B, P = 0.38). Interestingly,hippocampal FGF2 knockdown impacted open arm behaviordifferentially: bHRs administered LVshFGF2 spent significantlyless time in the open arms of the maze than did bHRs adminis-tered LVshNS (P = 0.016); by contrast there were no differencesbetween bLRs administered LVshNS and LVshFGF2 (P =0.454). Thus, FGF2 knockdown selectively enhanced anxiety be-havior in the typically less anxiety-prone bHRs.We then assessed the levels of repressive modified histone

H3K9me2 and H3K9me3 in both the HC and the amygdala ofbHR/bLR rats from this study. Following LVshFGF2 in the HC,there was an increase in the levels of H3K9me3 in bHRs com-pared with bLRs LVshNS or compared with the handled animalswithout any lentivirus in the HC (Fig. 6 A and B; P < 0.001;detailed statistical analyses are described in the SI Results). Inother words, the basal difference between the bHRs and thebLRs was significantly reduced in the HC following FGF2knockdown. The alteration in epigenetic signature was not ob-served in the amygdala of the same brain. Moreover, the level ofanother repressive modified histone, H3k9me2, was not alteredby the lentivirus either in HC or amygdala, demonstrating boththe regional and target specificity of the FGF2 knockdown (Fig.6 A and C).

DiscussionThis series of studies demonstrates three phenomena relating tothe role of chromatin modifications in the control of affectivebehavior: (i) There are basal and pervasive differences in thelevels of the modified histone protein H3K9me3 between twolines of rats that were derived from common outbred stocks andselectively bred for differences in emotional reactivity. Overall,levels of H3K9me3 are higher in the more behaviorally inhibitedanimals, the bLRs. (ii) Specific genes implicated in respon-siveness to stress and anxiety, namely GR and FGF2, show dif-ferential association with H3K9me3 that are in line with differencesin their gene expression levels across the two lines. Thus, the as-sociation of the inhibitory H3K9me3 with GR is lower in bLRs,consistent with the higher levels of GR expression in these moreanxiety-prone animals; by contrast, the association of H3K9me3with FGF2 in bLRs is higher, consistent with the low expression ofFGF2 in these animals. (iii) FGF2 is not only a target of epigenetic

Fig. 3. Early-life FGF2 alters the levels of H3K9me3in various brain regions of bLR rats. Immunoblot (A)and graph (B) show a significant (n = 6) decrease inthe levels of H3K9me3 in HC and NAcc of bLR ratsand increased in the levels of H3K9me3 in amygdalaof bHR rats treated with FGF2 compared with vehi-cle treatment (***P < 0.001, n = 6 per group).

Fig. 4. Early-life FGF2 alters the association of H3K9me3 at FGF2 and GRpromoters. (A and B) The association of H3K9me3 at FGF2 decreased sig-nificantly in HC and NAcc bLR rats following neonatal FGF2 exposure (***P <0.001, n = 6 per group). (A and C) However, the association of H3K9me3 atGR increased significantly only in HC following neonatal FGF2 exposure.Note that the amygdala remained unaltered in the association pattern ofH3K9me3 at FGF2 and GR (***P < 0.001, n = 6 per group).

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modification by H3K9me3, it is also a trigger of epigenetic changesthat can impact this modified histone protein and its associationwith various gene promoters.These results are summarized in Fig. S2, and their implications

for the roles of FGF2, GR, and H3K9me3 are depicted in Fig. 7.Together, they implicate H3K9me3 as a key player in the controlof affective responsiveness, especially anxiety behavior. Theyalso demonstrate that FGF2 partners with H3K9me3 to modu-late the neural phenotype at the epigenetic level, both broadly(overall level of modified histone across several brain regions)and very specifically (opposing patterns of association with spe-cific promoters that exert opposing control on anxiety). Below,we discuss each of these three principal findings.

Basal Differences in the Posttranslational Modifications at H3K9me3,a Histone Associated with Transcriptional Repression. Relative tobLRs, the more active bHRs exhibit an overall decreased level ofthe repressive H3K9me3. This decrease was observed in all threebrain regions examined: the HC, NAcc, and amygdala. It is no-table that this epigenetic signature was stable across generations,as rats from three generations of our selectively bred lines(generations 31, 34, and 35) were used in the various studies andexhibited similar basal differences in levels of H3K9me3.Both H3K9me3 and H4K20me3 are associated with transcrip-

tional repression and silencing (41, 42). It is therefore notable thatthe differential pattern in modified histones is selective, as weobserved no basal differences in levels of H4K20me3 betweenbHRs and bLRs in the three brain regions studied. However, thisdoes not preclude differences in other modified histone proteinsacross the lines that play a role in transcriptional activationor inhibition. The functional role of this global difference inH3K9me3 is not currently well understood, but it is notable thatmanipulations that alter anxiety behavior also altered the globallevels of this histone, as discussed below. It will be of interest toascertain whether other animal models of basal differences inanxiety behavior reveal similar differences in H3K9me3. If so, thiswould implicate this modified histone as a target for altering thepropensity for excessive anxiety.

Association of Modified Histones at the Promoters of FGF2 and GR.Alteration in the association of modified histones at promotersites is crucial for the regulation of transcriptional activity ofspecific genes, thereby regulating their expression levels. Despitethe overall difference in the global pattern of H3K9me3 levels inthe two selectively bred lines, the association with specific geneswas quite specific and not necessarily reflective of the overall levelsof the modified histone. Thus, GR, which we and others haveimplicated in greater basal anxiety (17, 21, 43) and which iselevated basally in the bLRs (17), exhibited a lower level of

association with H3K9me3 which is consistent with the higherexpression levels of that gene. This result was observed in all threebrain regions studied: the HC, amygdala, and NAcc. Once again,these marks were specific: We saw no differences in the associa-tion of other histones, e.g., H4k20me3 with the GR gene pro-moter. Moreover, the effect was selective to GR but did not affecta closely related gene, MR, which showed no differences in toeither modified histone across the two lines (data not shown).By contrast, the association of H3K9me3 with FGF2 was higher

in the bLRs, which was more reflective of the overall elevation ofthis modified histone in these animals. This finding is also con-sistent with the decrease in FGF2 mRNA levels in the bLR ratsacross several brain regions (28, 29). This set of results suggeststhat the differential levels of association of the modified histoneH3 with susceptibility genes (FGF2 and GR) correlate with thedifferences in gene expression in HC and amygdala, along withdifferences in stress responsiveness and anxiety behavior.Although this series of studies has focused on basal differences

in H3K9me3, work by others has demonstrated the respon-siveness of this modified histone to environmental manipu-lations, such stress and addiction further supporting its role inthe control of affective behavior. In particular, acute cocaineexposure increased H3K9me3 levels in NAcc (44) and has anadaptive role in animal models of stress (7). In the NAcc, cocainealso alters the association between the modified histone H3(acetylation, methylation, or phosphorylation) with various genesincluding long interspersed nuclear element-1 (44), H-Ras1 (7),mitogen and stress-activated protein kinase-1 MSK1, c-fos, andc-jun (45). Our findings that basal differences in H3K9me3 as-sociation affect regulating genes in animals with differing emo-tional reactivity, together with the responsiveness of the H3histone to various manipulations that modify affective behavior,converge to suggest a critical role of this modified histone inregulating emotional behavior.

Role of FGF2 in Altering the Levels of Repressive H3K9me3 and ItsAssociation at FGF2 and GR Promoters. An emerging literature thatbegan with studies in human depressed brains and was followedup by a range of studies in animals models strongly implicatesFGF2 in the modulation of affect, especially in decreasing anx-iety behavior (25, 27). Our own work has shown that neonatalFGF2 resulted in a long-term decrease in anxiety-like behavior inbLR which naturally exhibit high anxiety behavior (28). Studieshave demonstrated that intracerebroventricular infusion of chronic

Fig. 5. Hippocampal knockdown of FGF2 eliminates characteristic differ-ences in anxiety-like behavior between bHR and bLR rats. (A) Nonsilencingcontrol virus-injected animals show prototypical behavioral differences onthe elevated plus maze: bHRs spent significantly more time on the openarms and less time in the closed arms than do bLRs (*P < 0.05, n = 6 pergroup). (B) In contrast, hippocampal knockdown of FGF2 eliminated thebehavioral differences between bHR and bLR animals (n = 6 per group).

Fig. 6. Hippocampal knockdown of FGF2 differentially regulates repressivemethylated histones various brain regions of bLR and bHR rats. Immunoblot(A) and graph (B) show that knockdown FGF2 significantly (n = 6) increasedthe levels of H3K9me3 only in the bHR HC (***P < 0.001, n = 6 per group).Immunoblot (A) and graph (C) show that the levels of H3K9me2 remainedunaltered in both the HC and the amygdala of bHRs and bLRs followinghippocampal knockdown of FGF2.

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FGF-2 can inhibit anhedonia-like behavior following chronicunpredictable stress. Also selective infusion of FGF-2 in thePFC, but not into dorsal striatum, shows antidepressant-likeand anxiolytic-like effects following forced swim test and noveltysuppressed feeding test (46). Moreover, knockdown of FGF2using single microinjection in the hippocampus resulted in ananxiety-like effect in outbred animals (26). However, the mech-anism whereby FGF2 exerts its long lasting effects (e.g., betweenneonatal administration and adulthood) remains unknown. Thecurrent study tested the possibility that chromatin remodelingwas one of the mechanisms of these lasting effects of FGF2.Indeed, we show here that a brief exposure to FGF2 in earlylife resulted in a long-term decrease in the expression levels ofH3k9me3 in adult bLRs relative to controls. This effect was notobserved in the bHRs who exhibit high basal levels of FGF2.Other studies have also identified epigenetic factors to be in-fluential in early developmental period for adaptation in un-derstanding vulnerability and resilience (47). It is notable thatthis selectivity in H3K9me3 modulation parallels the behavioraleffects of early-life FGF2, which decreased anxiety behavior inbLRs without affecting bHRs (28).Moreover, DNA methylation is often associated with tran-

scriptional repression, possibly via the repression of retro-transposons and other foreign elements (48). Interestingly, weobserved that methylation at FGF2 was basally higher in bLRhippocampus suggesting increased repression of FGF2 in bLRs,consistent with our H3K9me3 findings. However, early-lifeFGF2 decreased the methylation of FGF2 selectively in bLRhippocampus. This finding is consistent with the view that neo-natal administration of exogenous FGF2 to animals with lowbasal levels of this growth factor (bLRs) resulted in epigeneticchanges that enhanced their endogenous FGF2 function, resultingin an anxiolytic effect (28).Because our previous FGF2 knockdown study was carried out

in outbred animals (26) we asked whether the effect of knockingdown FGF2 specifically in hippocampus would produce a dif-ferential effect on anxiety behavior between the selectively bredlines, and whether this differential behavioral effect would beassociated with differences in H3K9me3. FGF2 knockdown, inthe hippocampus increased anxiety in the bHRs who have higherbasal levels of FGF2 and are typically less prone to anxiety. Thismanipulation led to a highly selective change in the levels ofH3K9me3, only in the hippocampus and only in the bHRs.

Taken together, our findings suggest that FGF2 exerts its effectson anxiety behavior, at least in part, by modulating H3K9me3 toinfluence both its overall levels and its association with specifictarget genes such as GR as well as its own (FGF2) promoter. Theimpact on a critical modified histone protein likely representsa key feature of FGF2’s ability to trigger long-term changes inneuroplasticity and modulation of emotional reactivity.It is notable that most of our effects were consistently seen in the

hippocampus, but not necessarily in the amygdala. This may bedue to the fact that this study has focused on unlearned, sponta-neous anxiety behavior, which is regulated by the hippocampus,with a particular role of the ventral dentate gyrus (49). Other teststhat rely on learned fear responses (which also differ betweenbHR and bLR animals- unpublished) and other manipulationsthat modify the reactivity to fear might uncover a role of theepigenetic signature of the amygdala in their mediation.Given that the FGF system is altered in major depression (25)

and given the current findings demonstrating that FGF2 is botha target and a trigger of changes in H3K9me3, it is reasonable tohypothesize that this modified histone protein is a key player inmood disorders, and that the H3K9me3-FGF2 interaction couldrepresent an important target for treatment or prevention ofmajor depression.In sum, we have observed the existence of basal epigenetic

differences that may function during development to determinelong-term resilience or vulnerability to anxiety. However, theseepigenetic differences are also intrinsically sensitive to inter-vention, thus enabling preventive strategies in vulnerable indi-viduals. As importantly, we propose a particular molecule, FGF2,as being able to orchestrate epigenetic changes that affect a rangeof targets, including the FGF system itself and its functional op-ponent the GR/stress system. The bidirectional relation betweenFGF2 and epigenetic mechanisms has the potential to providea number of novel molecular targets for resilience enhancement.

Materials and MethodsSelectively Bred Rats (bHR and bLR). The bLR-HR rats were generated from ourin-house breeding colony, where the bLR/bHR lines have beenmaintained forseveral generations (35). On the basis of the locomotor activity, bHR and bLRrats of our breeding colony were screened as described (30). Peripheralneonatal FGF2 administrations were performed as previously described onboth bHRs and bLRs (28). Knockdown of FGF2 in the hippocampus was alsoperformed as described (26) on bHRs and bLRs. Adult rats from both bHR/bLR lines were killed and several brain regions (HC, amygdala, and NAcc)were processed to be used for different experiments.

Western Blot Analysis. The detailed procedure forWestern blot is presented inSI Materials and Methods. Briefly, the tissues from three brain regions (HC,amygdala, and NAcc) were homogenized and resolved in 12.5% (wt/vol)SDS/PAGE and transferred to PVDF membrane (Millipore). The membraneswere blocked in 10% (wt/vol) nonfat milk and incubated with various pri-mary antibodies. Enhanced chemiluminescence (GE Healthcare) was usedfollowing manufacturer’s instructions for visualization of antigen-antibodybinding. Quantification of the autoradiographs was done using Image J (NIHSoftware) after normalization.

Chromatin Immunoprecipitation Assay. The brain regions (HC, amygdala, andNAcc) were dissected from the frozen bHR-LR rat brain by a punchingmethod. The detailed procedure has been mentioned in SI Materials andMethods. Briefly, the dissected regions were cross-linked, homogenized andcentrifuged. The pellets were suspended in the nuclear lysis buffer and usingthe Sonicator (Branson), the extracted chromatin was sheared to 200–300 bp.The chromatin was then subjected to immunoprecipitation using anti-bodies against anti-H3K9me3 (Abcam) overnight at 4 °C. Protein–DNA–antibody complexes were precipitated with Dynabeads@protein A (Invitrogen)for 2 h at 4 °C. The precipitated protein–DNA complexes were eluted fromthe antibody and then reversed-cross-linking was done in 0.3 M NaCl at 65 °Covernight. Proteins were digested with proteinase K for 1 h at 45 °C. TheDNA were extracted, purified (Qiagen), and quantified using PCR. Primersdirected to the gene promoter used for amplification are listed in SIMaterials and Methods.

Fig. 7. FGF2 is both a target and a modifier of H3K9me3: Overall re-lationship between molecular organizers and anxiety behavior. In general,higher overall levels of H3K9me3 are associated with increased anxietybehavior. Moreover, FGF2 enhancement is anxiolytic and GR activation isanxiogenic. In high anxiety states, H3K9me3 shows lower association withGR and higher association with FGF2. Enhancing FGF2 decreases H3K9me3levels, decreases its association with the FGF2 promoter, and increases itsassociation with GR.

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DNA Methylation. The brain regions (HC, amygdala, and NAcc) were dissectedfrom the frozen bHR-LR rat brain using a 19-gauge syringe (C310GA/TW/SP).The genomic DNA was isolated from the dissected regions using PureLinkGenomic DNA kit manufacturer’s protocol (Life Technologies). The isolatedgenomic DNA was further processed to extract unmethylated and methyl-ated DNA using CpG MethylQuest DNA Isolation kit manufacturer’s protocol(Millipore). The detailed protocol for genomic DNA extraction and isolationof methylated DNA is presented in the SI Materials and Methods. The PCRproduct for total, methylated and unmethylated DNA was compared. Signalintensity was quantified using Image J (NIH Software).

FGF2 Knockdown Experiments. All adult bLR-HR rats were housed 2–3 animalsper cage for the duration of the study (35). Lentivirus (1 μL) containing eithera short-hairpin RNA targeted against FGF2 (LVshFGF2) or a scrambled ver-sion of the targeted sequence (LVshNS) was infused (26). After 4 wk fromsurgery, animals were subjected to elevated plus maze test under dim light(30 lx). Detailed protocol has been illustrated in SI Materials and Methods for

the knockdown study. After 24 h of the behavioral test, animals were killedand brains were stored at −80°C for Western blotting.

Statistical Analysis. For Western blot and CHIP data analysis, a Student t testwas used to analyze basal differences between bHR and bLR rats. Two-wayANOVAs were performed for Figs. 3–6. A three-way ANOVA was performedfor Fig. S1. All detailed statistical analyses are presented in SI Materials andMethods and SI Results, and the most interesting significant findings arepresented in the main text.

ACKNOWLEDGMENTS. We thank Angela Koelsch for assistance in thebreeding and maintenance in the bHR/bLR rat lines. This work wassupported by National Institute on Drug Abuse Grant 5 P01 DA021633,National Institutes of Health Grant R01MH104261, National Institute ofMental Health Grant R01-MH-104261, Office of Naval Research GrantsN00014-09-1-0598 and N00014-12-1-0366, the Pritzker NeuropsychiatricDisorders Research Consortium, and the Hope for Depression ResearchFoundation.

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