Epigenetic changes at gene promoters in response to immune activation in utero

Brain, Behavior, and Immunity 30 (2013) 168–175

Contents lists available at SciVerse ScienceDirect

Brain, Behavior, and Immunity journal homepage: www.elsevier.com/locate/ybrbi

Epigenetic changes at gene promoters in response to immune activation in utero Bin Tang 1, Haiqun Jia 1, Ryan J. Kast, Elizabeth A. Thomas ⇑ Department of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Red., La Jolla, CA 92037, United States

a r t i c l e

i n f o

Article history: Received 24 November 2012 Received in revised form 21 January 2013 Accepted 29 January 2013 Available online 9 February 2013 Keywords: Histone Gene expression Schizophrenia Psychiatric Glutamate Immune Neurodevelopment

a b s t r a c t Increasing evidence suggests that maternal infection increases the risk of psychiatric disorders, such as schizophrenia and autism in offspring. However, the molecular mechanisms associated with these effects are unclear. Here, we have studied epigenetic gene regulation in mice exposed to non-specific immune activation elicited by polyI:C injection to pregnant dams. Using Western blot analysis, we detected global hypoacetylation of histone H3, at lysine residues 9 and 14, and histone H4, at lysine residue 8, in the cortex from juvenile (24 days of age) offspring exposed to polyI:C in utero, but not from adult (3 months of age) offspring, which exhibit significant behavioral abnormalities. Accordingly, we detected robust deficits in the expression of genes associated with neuronal development, synaptic transmission and immune signaling in the cortex of polyI:C-exposed juvenile mice. In particular, we found that several genes in the glutamate receptor signaling pathway, including Gria1 and Slc17a7, showed decreases in promoter-specific histone acetylation, and corresponding gene expression deficits, in polyI:C-exposed offspring at both juvenile and adult ages. In contrast, the expression of these same genes, in addition to Disc1 and Ntrk3, was elevated in the hippocampus of juvenile mice, in concordance with elevated levels of promoter-specific histone acetylation. We suggest that these early epigenetic changes contribute to the delayed behavioral abnormalities that are observed in adult animals after exposure to polyI:C, and which resemble symptoms seen in schizophrenia and related disorders. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction Complex neuropsychiatric disorders, such as schizophrenia, show high heritability (Giegling et al., 2008); however, family and twin studies have indicated that environmental factors also play important roles in the etiology of disease (Riley and Kendler, 2006; Ross et al., 2006). From a molecular perspective, it is thought that these genetic and environmental triggers alter the expression of important genes in the brain leading to the manifestation of psychiatric symptoms, including positive and negative symptoms, cognitive deficits, anxiety, etc. Microarray studies have revealed that several psychiatric disorders, including schizophrenia and autism, are associated with a wide range of gene expression changes, a majority of which are deficits (Horvath et al., 2011; Mirnics et al., 2006). Our previous studies have found that gene expression deficits, in particular, are more pronounced in young subjects (Narayan et al., 2008), and further that these expression decreases were associated with hypoacetylation of histones at gene promoters of specific schizophrenia-related genes (Tang et al., 2011). Hence, we have hypothesized that alterations in epigenetic gene ⇑ Corresponding author. Address: The Scripps Research Institute, Department of Molecular Biology, SP-2030, 10550 N. Torrey Pines Rd. La Jolla, CA 92037, United States. Fax: +1 858 784 2212. E-mail address: [email protected] (E.A. Thomas). 1 These authors contributed equally. 0889-1591/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbi.2013.01.086

regulation represent an early trigger, possibly occurring during important developmental stages which lead to abnormalities in postnatal brain development and ensuing behavioral phenotypes in affected individuals. Given that epigenetic-based mechanisms of gene regulation are known to occur during developmental stages, as well as in response to environmental events (Bale et al., 2010; Franklin and Mansuy, 2010), we chose to study this effect in a neurodevelopmental-environmental mouse model of psychiatric disease: the maternal immune activation model. In utero injection of the RNA viral mimicker, polyinosinic:polycytidylic acid (polyI:C), which elicits a non-specific immune response, results in pronounced neuropathological and neurochemical abnormalities and delayed behavioral deficits considered relevant to psychiatric disease, including prepulse inhibition deficits and several core symptoms of autism, in adult offspring (Geyer et al., 2001; Meyer et al., 2009; van den Buuse, 2010; Patterson, 2011; Malkova et al., 2012). This model has both face and construct validity for schizophrenia and autism and predictive validity for schizophrenia (Meyer and Feldon, 2010). In this study, we have measured the effects of maternal injection of polyI:C on epigenetic and gene expression changes in juvenile offspring (24 days of age), prior to the observation of behavioral changes, and in adult offspring (3 months of age), a time-point when offspring show robust psychiatric-like phenotypes. Our results reveal significant changes in

B. Tang et al. / Brain, Behavior, and Immunity 30 (2013) 168–175

histone acetylation, both on a global level and at specific promoter loci of schizophrenia- and development-related genes, in the cortex and hippocampus of juvenile offspring. Genes in the glutamate receptor signaling pathway were particular associated with epigenetic changes in response to prenatal immune activation. We propose that these events might parallel the mechanisms by which environmental insults contribute to the risk of neurodevelopmental disorders such as schizophrenia and autism in humans. Further, these findings may have therapeutic relevance, whereby the use of epigenetic drugs (inhibitors of histone modifying enzymes) might represent a relevant treatment option for psychiatric illnesses. 2. Methods 2.1. Mice Eight to 10 breeding trios were set up at the age of 8 weeks and allowed to mate overnight. Successful mating was verified the next morning by the presence of a vaginal plug and that day designated gestational day 0 (GD0). PolyI:C was prepared freshly before injection. Pregnant mice received a single i.p. injection polyI:C (5 mg/ kg) in 0.1 ml volume or saline (0.1 ml) at GD9. Mice were left undisturbed until weaning at 3 weeks of age. No differences in maternal care were observed between saline-treated and polyI:Ctreated dams. The lack of observed maternal neglect supported the rationale to refrain from cross-fostering pups. Offspring from these pregnant mice were tested in the open field and for thigmotaxis at 28–90 days of age, to verify behavioral deficits elicited by polyI:C exposure in utero. Groups of male offspring (at least n = 2 per litter from 3–5 different litters; n = 6–12 in total) were sacrificed at 24 days of age, (designated as ‘‘juvenile’’ mice), or 3 months of age, (designated as ‘‘adult’’ mice) brains removed and cortex, striatum and hippocampus dissected out for use in chromatin-immunoprecipitation or gene expression assays described below. For the Western blot experiments, juvenile mice were 28 days of age. 2.2. Behavioral testing Male mice were placed in the center of a square plexiglass chamber (27.3 cm  27.3 cm) (Med Associates INC) and allowed to freely explore. Horizontal and vertical activity was automatically recorded over a 30 min period using Med Associates software. Mice were not habituated to the chamber beforehand. The total distance traveled, rearing activity and the number of entries into the center of the arena (central 17 cm square) was recorded as a function of 10-min bins. 2.3. Western blotting Levels of acetylated histones H3 and H4 and total histone H3 were determined by Western blotting of isolated nuclei from cortical and hippocampal tissues of vehicle- and polyI:C-exposed male mice. Samples were homogenized in nuclear extraction buffer (0.32 M sucrose, 4 mM HEPES) with protease inhibitor cocktail (Roche) and then centrifuged at 800g for 15 min. The pellet was incubated for 2 h with nuclear extraction buffer containing 0.5% NP-40, then homogenized a second time followed by centrifugation at 800g for 15 min. The nuclear fractions were evaluated by the BCA protein assay (Pierce) to ensure equal loading. Protein aliquots were subjected to 4–12% SDS–PAGE and transferred onto nitrocellulose membranes using standard methods. Membranes were blocked with 5% non-fat milk in TBS-T (20 mM Tris, 500 mM NaCl, and 0.1% Tween 20). and incubated overnight at 4 °C with the both anti-ac-H3K9K14 (Upstate, Billerica, MA) and

169

anti-ac-H4K8 (Upstate, Billerica, MA), at the same time, given that their target sizes are different (observed molecular wts of 17 kDa and 11 kDa, for ac-H3K9/K14 and ac-H4K8, respectively; see http://www.Millipore.com and Fig. 2). After washing with TBS-T 3 times for 5 min each, membranes were incubated with horseradish-peroxidase-conjugated secondary antibodies in block solution at room temperature for 1–2 h. Blots were then stripped using standard protocols, and re-probed with an anti-an histone H3 (Abcam, Cambridge, MA). Chemiluminescent signal intensities were captured by Fluorchem E (Cell Biosciences) and quantified densitometrically using One-step AlphaView software (Cell Biosciences). The signal intensities of acetylated histones were normalized using the signal intensity of total histone H3. Statistically significant differences in protein expression were determined using Student’s t test (unpaired; two-tailed; GraphPad, San Diego, CA). 2.4. Chromatin-immunoprecipitation Chromatin-Immunoprecipitation (ChIP)-PCR was performed on cortex and hippocampus from vehicle- and polyI:C-injected mice (5 mg/kg) at 24 days of age or 12 weeks of age using an adaptation of a method previously described in detail (Luo et al., 1998). For cortex, only male mice were used, while for the hippocampus experiments, tissue was isolated from both males and females in equal ratios per vehicle or polyI:C group. Briefly, 60–100 mg of tissue was fixed with 1% of formaldehyde for 15 min at room temperature then homogenized to isolate nuclei. DNA was sonicated in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl [pH 8.0], 1 protease inhibitors cocktail (Roche, Germany)) to  2–8 kb in size of DNA fragments. 100 ll of precleared nuclear lysate was diluted with dilution buffer (1% Triton X 100, 2 mM EDTA, 20 mM Tris–HCl [pH 8.0], 150 mM NaCl, and 1 protease inhibitors cocktail), and incubated with 3 lg of histone ac-H3K9K14 (Upstate, Billerica, MA), 3 lg of histone ac-H4K8 (Upstate, Billerica, MA), 3 lg rabbit control IgG (Cell Signaling Technology, Danvers, MA) or total histone H3 (Abcam, Cambridge, MA) antibodies overnight at 4 °C. 60 ll of Protein A agarose beads (Millipore, CA) were added and incubated for 2 h to capture the immune complexes. The proteinDNA complexes were washed and eluted in elution buffer (1% SDS and 0.1 M NaHCO3) at 65 °C for 20 min. The proteins were digested by proteinase K, and the cross-linking reaction was reversed at 65 °C overnight. DNA was purified with phenol/chloroform and ethanol precipitation, and analyzed by real-time PCR analysis. 2.5. Real-time qPCR analysis Real-time qPCR analysis was performed using the StepOnePlus™ Real-Time PCR System (Life Technologies) on the recovered DNA from the ChIP experiments using primers directed against the proximal promoter regions of the selected genes (Suppl. Table 1) or on cDNA prepared from RNA from the same samples using the primers designed in the exonic regions of selected genes (Suppl. Table 1) as described previously (Desplats et al., 2006; Thomas et al., 2008). The proximal promoter region (1 kb upstream from transcription start site (TSS)) of each gene was obtained from UCSC browser (http://genome.ucsc.edu/cgi-bin/hgGateway). Primers were designed to generate amplicons of 80–150 nucleotides with similar melting temperatures (64 °C) using Invitrogen’s Primer Designer and their specificity for binding to the desired sequences was searched against the NCBI database. We analyzed the ChIP-qPCR data using the Percent Input Method (Invitrogen). Briefly, the amplification efficiency (AE) of the qPCR reaction for each primer pair and sample was determined by the Input DNA using the formula AE = 10^( 1/slope). The threshold cycle (Ct) value of Input which is 1% of the IP reaction was adjusted to 100% by subtracting 6.644 cycles (log2 of 100), and then the percent input

B. Tang et al. / Brain, Behavior, and Immunity 30 (2013) 168–175

Real-time qPCR analysis for the PCR arrays was performed using the RT2 SYBR green qPCR Master Mix (SABiosciences) on a StepOnePlus™ Real-Time PCR System (Life Technologies). cDNA from groups of male offspring (n = 3 per vehicle- and n = 3 polyI:C-exposed mice) at 4 weeks of age was tested on the NFjBmediated signal transduction pathway PCR array (Cat#: PAMM025), which contains 84 genes related to inflammatory pathways and the immune response. A set of 5 housekeeping genes (HKG) were chosen as internal loading controls for standardization between samples: Gusb, Hprt, Hsp90ab1, Gapdh and Actb. The relative gene expression of the genes was calculated as DCt sample = (Ct sample GENE) (Ct sample HKG). Then, the relative gene expression (RGE) = 2 power (DCt sample1 DCt sample 2). Results were analyzed using the PCR Array Data Analysis Web Portal (SABiosciences), followed by Benjami–Hochberg FDR correction.

3. Results 3.1. Behavioral changes resulting from polyI:C exposure in utero C57BL6/J mouse dams were injected with polyI:C or saline on gestation day 9. The offspring of these mice were weaned and sexed at postnatal day 28 and subjected to behavioral testing in an open field activity chamber. At 28 days of age, no differences in open field activity or exploratory behavior were detected in offspring exposed to polyI:C versus saline in utero. In contrast, significant differences in exploratory behavior (i.e. reduced time in the center) and hyperactivity (increased ambulatory distance and vertical activity) in the open field were observed in polyI:C-exposed adult offspring (3 months of age) compared to saline exposure (Fig. 1). This is consistent with other reports showing delayed abnormalities in adult offspring exposed to prenatal immune activation (Zuckerman and Weiner, 2005; Meyer and Feldon, 2009, 2011; Meyer et al., 2009; Connor et al., 2012).

3.2. Global hypoacetylation of histones in brains from juvenile mice exposed to immune activation in utero We next tested whether these behavioral abnormalities were associated with changes in global acetylation of histones at two different marks: lysines (K) 9 and 14 on histone H3 (acH3K9K14) and K8 on histone H4 (ac-H4K8), which are histone marks associated with active promoters (Heintzman et al., 2009; Ernst and Kellis, 2011). Using Western blotting, we detected global hypoacetylation of both histones H3 and H4 at these residues in the cortex of juvenile mice born to dams injected with polyI:C (Fig. 2). In contrast, histone acetylation levels were not significantly lower in the cortex of adult offspring (3 months of age) (Fig. 2). In the hippocampus, no significant changes in global histone acetylation were detected in either juvenile or adult mice (Fig. 2).

Because histone acetylation is known to alter chromatin structure and gene activity, we measured the effects of polyI:C exposure on gene expression changes in cortex and hippocampus from juvenile and adult offspring using real-time qPCR analysis. We picked 12 genes that have been previously implicated in the pathology of schizophrenia from genetic and/or microarray studies, with many known to play a role in neurodevelopment (Javitt, 2004; Yamaguchi et al., 2004; Hashimoto et al., 2005; Rapoport et al., 2005; Weickert et al., 2005; Akbarian and Huang, 2006; Otnaess et al., 2009; Potkin et al., 2009, 2010; Paul-Samojedny et al., 2010; Soumiya et al., 2011; Gonda et al., 2012). These include: glutamate receptor, ionotropic, AMPA1 (Gria1), glutamate receptor, ionotropic, AMPA2 (Gria2), Solute carrier family 17 (sodiumdependent inorganic phosphate cotransporter), member 7 (Slc17a7), roundabout homolog 1 (Drosophila) (Robo1), Rho GTPase activating protein 18 (Arhgap18), nuclear receptor subfamily 2, group F, member 1 (Nr2f1), neurotrophic tyrosine kinase, receptor,

A Ambulatory Distance (Units)

2.6. PCR arrays

3.3. Gene expression changes elicited by polyI:C exposure

1500

Vehicle

PolyI:C

** 1000

B Vertical Time (Sec)

was calculated by the formula 100  AE^(Adjusted Input Ct-IP Ct). For gene expression, the amount of cDNA in each sample was calculated using SDS2.1 software by the comparative Ct method and expressed as 2exp(Ct) using Hprt as an internal control. Significant differences in gene expression and histone acetylation were determined using Student’s t tests (unpaired; one or two-tailed) using GraphPad Prism Software (Prism GraphPad, San Diego).

500

4

6

60

8 Age (wks)

Vehicle

10

12

PolyI:C

***

40

20

0 4

6

8

10

12

Age (wks)

C Time in center area (sec)

170

200

Vehicle

PolyI:C

150

* 100 50 0

4 wks

8 wks

12 wks

Fig. 1. The offspring of mice given injections of polyI:C exhibit abnormalities in exploratory behavior. Pregnant dams received a single injection of polyI:C (5 mg/kg; i.p.) or saline on gestation day 9. Offspring (n = 10–12) were tested for open field exploratory behavior at 4, 8 and 12 weeks of age. Panel A shows ambulatory distance over a 10 min test period; Panel B shows vertical time over a 10 min period. Panel C shows time spent in the center area of the open field chamber (10 min period). Significant differences determined by two-way ANOVA in A and B and student’s t test in C. ⁄p < 0.05; ⁄⁄p < 0.005; ⁄⁄⁄p < 0.0001.

171

B. Tang et al. / Brain, Behavior, and Immunity 30 (2013) 168–175

Ac-H3K9K14 Ac-H4K8

10 kDa 20 kDa

Histone H3

Control

Ratio of Ac histone/total H3

Cortex – juvenile mice: 20 kDa

1.2

Control PolyI:C

1.0

**

0.8 0.6

***

0.4

PolyI:C

Ac-H3K9/14

20 kDa

Ac-H3K9K14 Ac-H4K8

10 kDa 20 kDa

Histone H3

Ac-H3K9K14 Ac-H4K8

10 kDa 20 kDa

Histone H3

Ratio of Ac histone/total H3

20 kDa

Control

1.0

0.5

Ac-H3K9/14

Cortex – adult mice:

20 kDa

1.0

0.5

0.0

Ac-H3K9/K14

Ac-H3K9K14 Ac-H4K8

10 kDa 20 kDa

Histone H3

Ac-H4K8

Control PolyI:C

2.0 1.5 1.0 0.5 0.0

Ac-H3K9/K14

Control

Ac-H4K8

Control PolyI:C

PolyI:C

Hippocampus – adult mice:

Ac-H4K8

Control PolyI:C

1.5

PolyI:C

Ratio of Ac histone/total H3

Control

Ratio of Ac histone/total H3

Hippocampus – juvenile mice:

Ac-H4K8

PolyI:C

Fig. 2. Levels of acetylated histone H3 (H3K9K14) and H4 (H4K8) in the brains of offspring of polyI:C injected dams. Western blot analysis was performed on cortex and hippocampus from vehicle- and polyI:C-exposed offspring at 28 days and 3 months of age. Each lane represents a brain sample from an individual animal, for blots from the juvenile mice, n = 5 vehicle-exposed mice and n = 6 polyI:C-exposed mice were analyzed. For adult mice, n = 4 vehicle-exposed mice and n = 5 polyI:C-exposed mice were analyzed. Bar graphs show quantification of the protein intensity of the bands in the Western blots using the One-step AlphaView software in the Fluorchem E Bioanalysis System. Asterisks, ⁄⁄denotes significant difference in expression at p < 0.01 and ⁄⁄⁄p < 0.001, using Student’s t test (unpaired; two-tailed).

type 3 (Ntrk3), apolipoprotein D (apoD), glutamic acid decarboxylase 1 (Gad1), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (Kras), disrupted in schizophrenia 1 (Disc1) and interleukin 6 (Il6). In juvenile offspring exposed to polyI:C, we detected significant decreases in expression for eight of these genes, Gria1, Gria2, Robo1, NR2f1, Ntrk3, Slc17a7, Arhgap18 and Disc1, in the cortex (Fig. 3). These findings are consistent with our Western blot data demonstrating significant histone hypoacetylation in juvenile, but not adult offspring, in response to polyI:C exposure. Only three genes, Robo1, Gria1 and Slc17a1, showed significantly lower expression in the cortex of adult offspring (3 months of age) (Fig. 3). In contrast, polyI:C exposure elicited increases in the expression of 9 of the 12 genes in hippocampus of juvenile mice (Fig. 3). These increases in expression were not detected at 3 months of age, with the exception of Il6, which was persistently elevated in expression in adult mice. Further, Arhgap18 and Gria1 were found to be significantly decreased in expression in the hippocampus of 3-month old polyI:C-exposed offspring. We also measured gene expression changes in the striatum, and no changes in the expression of most

of these genes were detected at either time point, with the exception of Gria2, whose expression was significantly decreased at 3 months of age in this region (Suppl. Fig. 1). 3.4. Altered acetylation patterns of histones at specific loci We further measured histone acetylation at specific gene promoters using chromatin immunoprecipitation (ChIP)-qPCR assays. ChIP was performed using ac-H3K9K14 or ac-H4K8 antibodies in the cortex and hippocampus followed by real-time qPCR analysis using primers directed against the proximal promoter regions of the same genes from our qPCR analyses. We found that levels of histone H3K9K14 were hypoacetylated specifically at the promoter regions of several genes that were downregulated in expression in the cortex of juvenile offspring, such as Gria1, Robo1, Arhgap18 and Ntrk3 (Fig. 4A), although no changes in acetylation levels of H4K8 were detected at these promoter loci (Suppl. Fig. 2). Trends towards decreased in histone acetylation were observed for Gad1 and Gria2, however, other genes, such as Disc1

172

B. Tang et al. / Brain, Behavior, and Immunity 30 (2013) 168–175

Fig. 3. Real-time qPCR analysis showing the effects of polyI:C on the expression of the indicated schizophrenia-related genes in the cortex and hippocampus of exposed offspring at 24 days and 3 months of age. Values shown are the mean ± S.E.M. expression values (n = 6 mice per group). Significant differences were determined by Student’s t-tests for the indicated genes. ⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001; #p < 0.08.

Fig. 4. Histone H3K9K14 acetylation levels at the promoter regions of the indicated genes in the cortex (A) and hippocampus (B) of juvenile offspring exposed to polyI:C in utero. Histone H3K9K14 acetylation levels were determined by chromatin-immunoprecipitation (ChIP)-qPCR assays (n = 6–12 mice per treatment group) at the promoter regions of the indicated genes (UniGene IDs). Open bars depict data from offspring of vehicle-treated dams, and gray or black bars depict data from polyI:C-exposed offspring. Asterisks denote significant differences in ac-H3K9K14 levels, as determined by Student’s t tests: ⁄p < 0.05; ⁄⁄p < 0.01.

and Nr2f1 which showed decreased gene expression in juvenile offspring did not show corresponding decreases in histone acetylation at either H3K9K14 or H4K8 marks (Fig. 4A). In the cortex of adult offspring, promoter regions of only three genes, Robo1, Gria1 and Slc17a1, showed hypoacetylation of histone H3K9K14 (Suppl. Fig. 3). In the hippocampus, histone acetylation was elevated at the promoter regions of several genes that were upregulated in expression in the hippocampus of juvenile offspring, such as Disc1, Nr2f1, Ntrk3, Gria1 and Gria2 (Fig. 4B). Trends towards increases in promoter histone acetylation were observed for Robo1 and Gad1, which were both elevated in expression in this region (Fig. 4B). These findings suggest that histone acetylation is an important epigenetic regulator of gene expression in this context.

3.5. PolyI:C effects on immune activation/inflammation-related gene expression in juvenile offspring Previous studies have demonstrated elevated levels of IL6 shortly after acute injection of polyI:C in the blood of pregnant dams and in fetal brain tissue (Samuelsson et al., 2006; Smith et al., 2007; Hsiao and Patterson, 2011; Connor et al., 2012). To test whether polyI:C elicits sustained activation of immune/inflammation factors in the brains of polyI:C-exposed offspring, we utilized a PCR array focusing on inflammatory- and immune responserelated gene expression in cortex from juvenile polyI:C-exposed offspring, the time point in which we observed substantial changes in gene expression (Fig. 2). This array assessed the expression changes for 84 genes related to inflammatory- and immune

B. Tang et al. / Brain, Behavior, and Immunity 30 (2013) 168–175

responses, including Il6, Il1a, Il1b, Il10, Tnf, Lta, and the Toll-like receptor genes, Tlrs1–6 (see Suppl. Table 2). In general, we did not detect robust changes in the expression of these genes in the cortex, with only a few genes showing small changes in expression due to polyI:C exposure (Suppl. Fig. 4), and none of the expression changes remaining significant after multiple tests correction (Benjami–Hochberg). Decreases in expression were observed for Tlr3, Tlr4 and Rel ( 1.2 to 1.37-fold changes) and the gene, Tnfrsf10b, showed an increased expression of 1.64-fold (Suppl Fig. 4).

4. Discussion Using a maternal immune activation mouse model of psychiatric disease, we demonstrate that significant epigenetic changes occur in response to polyI:C exposure in utero. Specifically, we find that: (1). Differential abnormalities in histone acetylation occur in the cortex and hippocampus in response to polyI:C exposure; (2). A majority of the observed abnormalities occured in juvenile mice, prior to the onset of behavioral phenotypes; and (3). Genes in the glutamate receptor signaling pathway were particular associated with epigenetic changes in response to prenatal immune activation. In this study, we detected hypoacetylation of histones at H3K9K14 and H4K8 in the cortex, both on a global level as well as at specific promoter loci, in juvenile offspring exposed to prenatal immune activation. It would be predicted that low levels of acetylation at these marks would result in reduced gene activity, which is consistent with our gene expression findings showing highly significant decreases in the expression of genes associated with neuronal development, synaptic transmission and immune signaling in the cortex of juvenile offspring. A recent study has also reported a wide range of gene expression changes in the whole brain from fetuses exposed to polyI:C, suggesting that indeed gene expression changes are occurring at an early stage of development (Garbett et al., 2012). In this study, we have defined the juvenile period of development as approximately postnatal day 24. Data from our ChIP-PCR assays also included offspring at 17 days of age, however we did not observe differences in promoter acetylation between 17 and 24 day-old offspring with the exception of the Gad1 promoter, which showed significant hypoacetylation at 17 days of age, but not 24 days of age (data not shown). In contrast, we did not detect histone acetylation deficits, nor robust deficits in gene expression, in the cortex of adult (3 month old) mice. These findings are consistent with previous microarray studies performed on cortex from adult mice that were exposed to polyI:C in utero, where minimal expression changes were detected on a global level at this age (Smith et al., 2007; Connor et al., 2012). Further, in the Connor et al. paper, genome-wide changes in another epigenetic mark, trimethylation of histone H3 at lysine 4 (H3K4me3), were also measured in the cortex of adult offspring and minimal alterations in H3K4me3 were detected in response to polyI:C exposure (Connor et al., 2012). In contrast to our findings from the cortex, we detected increases in histone acetylation at specific promoter loci in the hippocampus of juvenile polyI:C-exposed offspring, although such increases were not detected on the global level. These findings were consistent with the elevations in gene expression we detected in the hippocampus of juvenile mice for several genes, including Gad1, which is consistent with other studies that have found increased GAD67 expression in the hippocampal stratum oriens in juvenile brains (postnatal day 28) from mice prenatally treated with poly I:C (Harvey and Boksa, 2012). These findings highlight the important differences in epigenetic gene regulation between different brain regions.

173

While the activity of several genes related to neurodevelopment, such as Disc1 and Robo1, were found to be altered in this study, genes encoding proteins in the glutamate receptor signaling pathway were particularly notable as being epigenetically regulated. Gria1 and Gria2, which encode glutamate receptors, and Slc17a7, which encodes a glutamate transporter, showed decreases in gene expression in the cortex at 24 days of age, with corresponding decreases in promoter-specific H3K9K14 acetylation, as demonstrated by ChIP assays. Further, the gene Arhgap18, which encodes a RhoGAPs, GTPase-activating protein, has been implicated in mechanisms of axonal outgrowth, dendrogenesis, and cell migration during neural development at glutamatergic synapses (Tomoyuki Furuyashiki 1999), and is thought to play a role in the functional and structural synaptic plasticity triggered by the activation of glutamate receptors (Ponimaskin et al., 2007). These findings are of particular interest given that hypofunction of the glutamatergic system has been widely proposed as a pathogenic mechanism of schizophrenia (Seeman, 2009; Lin et al., 2011). Further, modulation of the glutamate signaling pathway is thought to have therapeutic benefit in schizophrenia (Javitt, 2004; Moghaddam and Javitt, 2011). Additionally, previous studies have shown that polyI:C-treated mice showed a significant increase in the basal level of glutamate in the hippocampus compared with the level in the saline-treated control group (Ibi et al., 2009), which in turn could cause a down-regulation of glutamate receptor subunits in the cortex, as we detected. Importantly, decreased expression and promoter acetylation of these genes were also detected in the adult cortex (3 months of age) whereby expression levels of the other genes tested were not significantly decreased, consistent with the lack of histone hypoacetylation observed in adult mice. These findings highlight the potential importance of the cortical glutamate receptor signaling pathway in the ensuing phenotypes. Substantial evidence implicates IL6 as a key mediator in the development of schizophrenic and autistic phenotypes in rodents (Samuelsson et al., 2006; Smith et al., 2007; Hsiao and Patterson, 2011; Connor et al., 2012). Specifically, significant increases in pro-inflammatory cytokines, including IL6, are detected in fetal brains shortly after maternal immune activation in rodents (Meyer et al., 2006, 2008; Connor et al., 2012) and notably, many of the behavioral effects caused by polyI:C injection can be mimicked by IL6 injection and blocked with an anti-IL6 antibody (Smith et al., 2007). Therefore, we tested whether increased Il6 gene expression was sustained in the brains of post-natal animals, and again detected brain region-specific effect of Il6 expression. Significant increases in Il6 expression were detected in the hippocampus at both juvenile and adult time points, but not in the cortex at either time point, although Il6 expression levels were slightly lower compared to vehicle-exposed mice. This is consistent with recent studies that have investigated cytokine protein levels in different ages of mice exposed to polyI:C at gestational age E12.5 and in different brain regions (Garay et al., 2012). In that study (Garay and colleagues), the authors reported lower levels of IL6 in the frontal cortex at post-natal day 30 (P30) in mice exposed to polyI:C, but elevated IL6 levels in hippocampus at P0, P14, and P60 time-points, (although IL6 levels were lower at the P30 time point) (Garay et al., 2012). Hence, it appears that IL6 is upregulated as an early event in response to polyI:C, but also that IL6 elevation represents a sustained effect, at least in the hippocampus, a brain region showing developmental abnormalities due to polyI:C exposure (Ito et al., 2010; Oh-Nishi et al., 2010; Wolf et al., 2010), and that this may contribute to developmental abnormalities. Garay and colleagues also reported a wide range of changes in other cytokines in different brain regions from postnatal mice exposed to polyI:C in utero, including decreases in IL1B and IL10 in the cortex of P30 mice. We did not detect changes in the expression of the genes encoding these cytokines, Il1b and Il10, in the

174

B. Tang et al. / Brain, Behavior, and Immunity 30 (2013) 168–175

cortex at 24 days of age in our study (Suppl Fig. 4). This could be due to the difference in the embryonic day of polyI:C exposure (E9 in our study and E12.5 in the other study) or due to differences in the time of expression of mRNA vs. protein levels. Alternatively, it is possible that our sample size for the PCR Array experiments were not large enough to detect changes. Due to cost issues, we was only able to process groups of n = 3 for the PCR Arrays; however, from our ChIP-PCR and qPCR experiments, we know that gene activity responses are highly variable in offspring from polyI: C-exposed mice, as also reported earlier (Garay et al., 2012). Despite demonstration of the critical role of IL6 in mouse maternal immune activation models and in human schizophrenia and autism, the mechanism underlying how IL6 acts to disrupt early brain development is not clear. A study dating back to 2001 suggested that inflammatory cytokines such as IL6 may exert epigenetic changes in cells via the regulation of a methyltransferase gene (Hodge et al., 2001). Since then, several additional studies have confirmed that IL6 can induce expression of the DNA maintenance methylation enzyme, DNMT-1 (Braconi et al., 2010; Foran et al., 2010), and can elicit changes in DNA methylation at the promoter regions of genes associated with tumor suppression, adhesion, apoptosis resistance, and growth-regulatory pathways (Hodge et al., 2005; Wehbe et al., 2006; Braconi et al., 2010; Foran et al., 2010; D’Anello et al., 2011; Thaler et al., 2011). Therefore, it is possible that IL6 acts as a mediator between polyI:C exposure and ensuing epigenetic changes in gene regulation and that this chain of events can provide a mechanism for how polyI:C exposure during critical developmental windows can lead to delayed detrimental effects on behavior. In the maternal immune activation model, we suggest that injection of polyI:C during gestational time points (i.e. E9) elicits a cascade of epigenetic changes leading to gene deregulation and altered expression of important developmental genes (Fig. 5). We suggest that these gene expression changes occur in early post-natal ages and lead to disruptions in the development of proper cortical circuitry and/or brain region connectivity. Although these changes in gene expression are not robustly sustained in adult ages, the delay or shift in neurodevelopment is already underway. These effects ultimately result in the behavioral and phenotypic abnormalities that are observed in the adult offspring of polyI:C-exposed mice (Fig. 5).

Our studies demonstrate that prenatal immune activation elicits alterations the epigenetic control of gene activity, and that these events occur in a temporal and region-specific pattern in the developing brain. These findings have translational relevance, whereby, we have hypothesized that gene expression deficits in schizophrenia begin early in life, i.e. much prior to symptom onset, and that they are due, in part, to epigenetic changes elicited by critical triggers sustained early in life (Tang et al., 2011). This is consistent with our previous studies on prefrontal cortex from human postmortem brain, in which we reported a hypoacetylation of histone H3 at important genomic loci in the prefrontal cortex of young subjects with schizophrenia, but not in cortical samples from older subjects (Tang et al., 2011). These findings suggest that early treatment with epigenetic drugs might represent a potential therapeutic strategy for psychiatric illnesses. For example, the global decreases in histone acetylation that we detected in the cortex (and the trends towards decreases in the hippocampus) might implicate the use of histone deacetylase inhibitors to restore normal chromatin structure on a global scale; however, one must consider that the local chromatin environment in other brain regions might be adversely affected. Nonetheless, given the enormous need for new therapeutics for mental illness, such epigenetic therapeutic approaches deserve further consideration.

Financial disclosures All authors report no financial disclosures related to this work Acknowledgment We thank Joel M. Gottesfeld for providing the ac-H4K8 antibody.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbi.2013.01.086.

Altered histone acetylation/ gene expression

Behavioral abnormalities

References Neurodevelopment delay Maternal Immune activation

E9

birth

30 days

60 days

90 days

Epigenetic changes

Robo1, Disc1, Ntrk3, Gria1, Gria2

Fig. 5. Schematic depiction of the epigenetic hypothesis of maternal immune activation. The X-axis shows a timeline of the effects of behavioral deficits (upper yaxis) and the effects on gene regulation (gene expression deficits and histone hypoacetylation in the cortex and gene expression elevation and histone hyperacetylation in the hippocampus) (lower y-axis). We hypothesized that injection of polyI:C during gestational time points (i.e. E9) elicits a cascade of alterations in epigenetic gene regulation. Altered gene expression patterns result in abnormalities in post-natal neuronal development (‘‘neurodevelopment delay’’) causing delayed behavioral deficits that are observed in adult animals, even though the molecular abnormalities are no longer present during this stage.

Akbarian, S., Huang, H.S., 2006. Molecular and cellular mechanisms of altered GAD1/GAD67 expression in schizophrenia and related disorders. Brain Res. Rev. 52, 293–304. Bale, T.L., Baram, T.Z., Brown, A.S., Goldstein, J.M., et al., 2010. Early life programming and neurodevelopmental disorders. Biol. Psychiatry 68, 314–319. Braconi, C., Huang, N., Patel, T., 2010. MicroRNA-dependent regulation of DNA methyltransferase-1 and tumor suppressor gene expression by interleukin-6 in human malignant cholangiocytes. Hepatology 51, 881–890. Connor, C.M., Dincer, A., Straubhaar, J., Galler, J.R., et al., 2012. Maternal immune activation alters behavior in adult offspring, with subtle changes in the cortical transcriptome and epigenome. Schizophr. Res. 140, 175–184. D’Anello, L., Sansone, P., Storci, G., Mitrugno, V., et al., 2011. Epigenetic control of the basal-like gene expression profile via Interleukin-6 in breast cancer cells. Mol. Cancer 9, 300. Desplats, P.A., Kass, K.E., Gilmartin, T., Stanwood, G.D., et al., 2006. Selective deficits in the expression of striatal-enriched mRNAs in Huntington’s disease. J. Neurochem. 96, 743–757. Ernst, J., Kellis, M., 2011. Discovery and characterization of chromatin states for systematic annotation of the human genome. Nat. Biotechnol. 28, 817–825. Foran, E., Garrity-Park, M.M., Mureau, C., Newell, J., et al., 2010. Upregulation of DNA methyltransferase-mediated gene silencing, anchorage-independent growth, and migration of colon cancer cells by interleukin-6. Mol. Cancer Res. 8, 471– 481. Franklin, T.B., Mansuy, I.M., 2010. Epigenetic inheritance in mammals: evidence for the impact of adverse environmental effects. Neurobiol. Dis. 39, 61–65. Garay, P.A., Hsiao, E.Y., Patterson, P.H., McAllister, A.K., 2012. Maternal immune activation causes age- and region-specific changes in brain cytokines in offspring throughout development. Brain Behav. Immun. (Epub ahead of print). Garbett, K.A., Hsiao, E.Y., Kalman, S., Patterson, P.H., et al., 2012. Effects of maternal immune activation on gene expression patterns in the fetal brain. Transl. Psychiatry 2, e98.

B. Tang et al. / Brain, Behavior, and Immunity 30 (2013) 168–175 Geyer, M.A., Krebs-Thomson, K., Braff, D.L., Swerdlow, N.R., 2001. Pharmacological studies of prepulse inhibition models of sensorimotor gating deficits in schizophrenia: a decade in review. Psychopharmacology 156, 117–154. Giegling, I., Hartmann, A.M., Genius, J., Benninghoff, J., et al., 2008. Systems biology and complex neurobehavioral traits. Pharmacopsychiatry 41 (Suppl. 1), S32– S36. Gonda, Y., Andrews, W.D., Tabata, H., Namba, T., et al., 2012. Robo1 regulates the migration and laminar distribution of upper-layer pyramidal neurons of the cerebral cortex. Cereb. Cortex (Epub ahead of print). Harvey, L., Boksa, P., 2012. A stereological comparison of GAD67 and reelin expression in the hippocampal stratum oriens of offspring from two mouse models of maternal inflammation during pregnancy. Neuropharmacology 62, 1767–1776. Hashimoto, T., Bergen, S.E., Nguyen, Q.L., Xu, B., et al., 2005. Relationship of brainderived neurotrophic factor and its receptor TrkB to altered inhibitory prefrontal circuitry in schizophrenia. J. Neurosci. 25, 372–383. Heintzman, N.D., Hon, G.C., Hawkins, R.D., Kheradpour, P., et al., 2009. Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature 459, 108–112. Hodge, D.R., Peng, B., Cherry, J.C., Hurt, E.M., et al., 2005. Interleukin 6 supports the maintenance of p53 tumor suppressor gene promoter methylation. Cancer Res. 65, 4673–4682. Hodge, D.R., Xiao, W., Clausen, P.A., Heidecker, G., et al., 2001. Interleukin-6 regulation of the human DNA methyltransferase (HDNMT) gene in human erythroleukemia cells. J. Biol. Chem. 276, 39508–39511. Horvath, S., Janka, Z., Mirnics, K., 2011. Analyzing schizophrenia by DNA microarrays. Biol. Psychiatry 69, 157–62. Hsiao, E.Y., Patterson, P.H., 2011. Activation of the maternal immune system induces endocrine changes in the placenta via IL-6. Brain Behav. Immun. 25, 604–615. Ibi, D., Nagai, T., Kitahara, Y., Mizoguchi, H., et al., 2009. Neonatal polyI:C treatment in mice results in schizophrenia-like behavioral and neurochemical abnormalities in adulthood. Neurosci. Res. 64, 297–305. Ito, H.T., Smith, S.E., Hsiao, E., Patterson, P.H., 2010. Maternal immune activation alters nonspatial information processing in the hippocampus of the adult offspring. Brain Behav. Immun. 24, 930–941. Javitt, D.C., 2004. Glutamate as a therapeutic target in psychiatric disorders. Mol. Psychiatry 9 (984–997), 79. Lin, C.H., Lane, H.Y., Tsai, G.E., 2011. Glutamate signaling in the pathophysiology and therapy of schizophrenia. Pharmacol. Biochem. Behav. 100, 665–677. Luo, R.X., Postigo, A.A., Dean, D.C., 1998. Rb interacts with histone deacetylase to repress transcription. Cell 92, 463–473. Malkova, N.V., Yu, C.Z., Hsiao, E.Y., Moore, M.J., et al., 2012. Maternal immune activation yields offspring displaying mouse versions of the three core symptoms of autism. Brain Behav. Immun. 26, 607–616. Meyer, U., Feldon, J., 2009. Prenatal exposure to infection: a primary mechanism for abnormal dopaminergic development in schizophrenia. Psychopharmacology 206, 587–602. Meyer, U., Feldon, J., 2010. Epidemiology-driven neurodevelopmental animal models of schizophrenia. Prog. Neurobiol. 90, 285–326. Meyer, U., Feldon, J., 2012. To poly(I:C) or not to poly(I:C): advancing preclinical schizophrenia research through the use of prenatal immune activation models. Neuropharmacology 62, 1308–1321. Meyer, U., Feldon, J., Fatemi, S.H., 2009. In-vivo rodent models for the experimental investigation of prenatal immune activation effects in neurodevelopmental brain disorders. Neurosci. Biobehav. Rev. 33, 1061–1079. Meyer, U., Murray, P.J., Urwyler, A., Yee, B.K., et al., 2008. Adult behavioral and pharmacological dysfunctions following disruption of the fetal brain balance between pro-inflammatory and IL-10-mediated anti-inflammatory signaling. Mol. Psychiatry 13, 208–221. Meyer, U., Nyffeler, M., Engler, A., Urwyler, A., et al., 2006. The time of prenatal immune challenge determines the specificity of inflammation-mediated brain and behavioral pathology. J. Neurosci. 26, 4752–4762. Mirnics, K., Levitt, P., Lewis, D.A., 2006. Critical appraisal of DNA microarrays in psychiatric genomics. Biol. Psychiatry 60, 163–176. Moghaddam, B., Javitt, D., 2011. From revolution to evolution: the glutamate hypothesis of schizophrenia and its implication for treatment. Neuropsychopharmacology 37, 4–15.

175

Narayan, S., Tang, B., Head, S.R., Gilmartin, T.J., et al., 2008. Molecular profiles of schizophrenia in the CNS at different stages of illness. Brain Res. 1239, 235–248. Oh-Nishi, A., Obayashi, S., Sugihara, I., Minamimoto, T., et al., 2010. Maternal immune activation by polyriboinosinic–polyribocytidilic acid injection produces synaptic dysfunction but not neuronal loss in the hippocampus of juvenile rat offspring. Brain Res. 1363, 170–179. Otnaess, M.K., Djurovic, S., Rimol, L.M., Kulle, B., et al., 2009. Evidence for a possible association of neurotrophin receptor (NTRK-3) gene polymorphisms with hippocampal function and schizophrenia. Neurobiol. Dis. 34, 518–524. Patterson, P.H., 2011. Maternal infection and immune involvement in autism. Trends Mol. Med. 17, 389–394. Paul-Samojedny, M., Kowalczyk, M., Suchanek, R., Owczarek, A., et al., 2010. Functional polymorphism in the interleukin-6 and interleukin-10 genes in patients with paranoid schizophrenia–a case-control study. J. Mol. Neurosci. 42, 112–119. Ponimaskin, E., Voyno-Yasenetskaya, T., Richter, D.W., Schachner, M., et al., 2007. Morphogenic signaling in neurons via neurotransmitter receptors and small GTPases. Mol. Neurobiol. 35, 278–287. Potkin, S.G., Macciardi, F., Guffanti, G., Fallon, J.H., et al., 2010. Identifying gene regulatory networks in schizophrenia. Neuroimage 53, 839–847. Potkin, S.G., Turner, J.A., Fallon, J.A., Lakatos, A., et al., 2009. Gene discovery through imaging genetics: identification of two novel genes associated with schizophrenia. Mol. Psychiatry 14, 416–428. Rapoport, J.L., Addington, A.M., Frangou, S., Psych, M.R., 2005. The neurodevelopmental model of schizophrenia: update 2005. Mol. Psychiatry 10, 434–449. Riley, B., Kendler, K.S., 2006. Molecular genetic studies of schizophrenia. Eur. J. Hum. Genet. 14, 669–680. Ross, C.A., Margolis, R.L., Reading, S.A., Pletnikov, M., et al., 2006. Neurobiology of schizophrenia. Neuron 52, 139–153. Samuelsson, A.M., Alexanderson, C., Molne, J., Haraldsson, B., et al., 2006. Prenatal exposure to interleukin-6 results in hypertension and alterations in the reninangiotensin system of the rat. J. Physiol. 575, 855–867. Seeman, P., 2009. Glutamate and dopamine components in schizophrenia. J. Psychiatry Neurosci. 34, 143–149. Smith, S.E., Li, J., Garbett, K., Mirnics, K., et al., 2007. Maternal immune activation alters fetal brain development through interleukin-6. J. Neurosci. 27, 10695– 10702. Soumiya, H., Fukumitsu, H., Furukawa, S., 2011. Prenatal immune challenge compromises development of upper-layer but not deeper-layer neurons of the mouse cerebral cortex. J. Neurosci. Res. 89, 1342–1350. Tang, B., Dean, B., Thomas, E.A., 2011. Disease- and age-related changes in histone acetylation at gene promoters in psychiatric disorders. Transl. Psychiatry 1, e64. Thaler, R., Agsten, M., Spitzer, S., Paschalis, E.P., et al., 2011. Homocysteine suppresses the expression of the collagen cross-linker lysyl oxidase involving IL-6, Fli1, and epigenetic DNA methylation. J. Biol. Chem. 286, 5578–5588. Thomas, E.A., Coppola, G., Desplats, P.A., Tang, B., et al., 2008. The HDAC Inhibitor, 4b, ameliorates the disease phenotype and transcriptional abnormalities in Huntington’s disease transgenic mice. Proc. Natl. Acad. Sci. USA 105, 15564– 15569. van den Buuse, M., 2010. Modeling the positive symptoms of schizophrenia in genetically modified mice: pharmacology and methodology aspects. Schizophr. Bull. 36, 246–270. Wehbe, H., Henson, R., Meng, F., Mize-Berge, J., et al., 2006. Interleukin-6 contributes to growth in cholangiocarcinoma cells by aberrant promoter methylation and gene expression. Cancer Res. 66, 10517–10524. Weickert, C.S., Ligons, D.L., Romanczyk, T., Ungaro, G., et al., 2005. Reductions in neurotrophin receptor mRNAs in the prefrontal cortex of patients with schizophrenia. Mol. Psychiatry 10, 637–650. Wolf, S.A., Melnik, A., Kempermann, G., 2010. Physical exercise increases adult neurogenesis and telomerase activity, and improves behavioral deficits in a mouse model of schizophrenia. Brain Behav. Immun. 25, 971–980. Yamaguchi, H., Zhou, C., Lin, S.C., Durand, B., et al., 2004. The nuclear orphan receptor COUP-TFI is important for differentiation of oligodendrocytes. Dev. Biol. 266, 238–251. Zuckerman, L., Weiner, I., 2005. Maternal immune activation leads to behavioral and pharmacological changes in the adult offspring. J. Psychiatr. Res. 39, 311– 323.