Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2006 Nov 15;103(48):18267–18272. doi: 10.1073/pnas.0608702103

Enhanced anxiety and stress-induced corticosterone release are associated with increased Crh expression in a mouse model of Rett syndrome

Bryan E McGill *,†, Sharyl F Bundle ‡, Murat B Yaylaoglu §, James P Carson §,, Christina Thaller §, Huda Y Zoghbi *,‡,‖,**,††,‡‡
PMCID: PMC1636379  PMID: 17108082

Abstract

Rett syndrome (RTT), a postnatal neurodevelopmental disorder, is caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Children with RTT display cognitive and motor abnormalities as well as autistic features. We studied mice bearing a truncated Mecp2 allele (Mecp2308/Y mice) and found evidence of increased anxiety-like behavior and an abnormal stress response as evidenced by elevated serum corticosterone levels. We found increased corticotropin-releasing hormone (Crh) gene expression in the paraventricular nucleus of the hypothalamus, the central amygdala, and the bed nucleus of the stria terminalis. Finally, we discovered that MeCP2 binds the Crh promoter, which is enriched for methylated CpG dinucleotides. In contrast, the MeCP2308 protein was not detected at the Crh promoter. This study identifies Crh as a target of MeCP2 and implicates Crh overexpression in the development of specific features of the Mecp2308/Y mouse, thereby providing opportunities for clinical investigation and therapeutic intervention in RTT.

Keywords: autism, corticotropin-releasing hormone, methyl-CpG-binding protein 2

Rett syndrome (RTT, MIM 312750) is an X-linked dominant neurological disorder that affects ≈1 in 10,000 females (1). The clinical features of RTT include mental retardation, ataxia, hand stereotypes, seizures, and breathing irregularities (1). In addition, behavioral abnormalities, most prominently social behavior deficits and autistic features, are prominent (1, 2). Mutations in the methyl-CpG-binding protein 2 (MECP2) gene, which encodes the MeCP2 protein, account for the vast majority of RTT cases (1, 3). MeCP2 binds methylated CpG dinucleotides in close proximity to an A/T-rich motif (4), and it controls chromatin architecture by recruiting proteins that assist in gene silencing, including histone deacetylases (5, 6), histone methyltransferases (7), DNA methyltransferases (8), and CoREST (9). Additionally, MeCP2 regulates RNA splicing via an interaction with the YB-1 protein (10).

Based on its role in transcriptional repression, mutations that disrupt the normal function of MeCP2 are predicted to result in the misexpression of its targets (11). To date, transcriptional profiling of Mecp2-null mice has uncovered only three target genes, Sgk (12), Fkbp5 (12), and Uqcrc1 (13). Additionally, candidate gene approaches have identified BDNF (14, 15) and DLX5 (16) as MeCP2 targets. Notably, the mechanism(s) by which altered expression of these genes contributes to the RTT neurobehavioral phenotype remains elusive.

Previously, we generated the Mecp2308 mouse, which carries a hypomorphic Mecp2 allele in which a truncating mutation is placed after amino acid 308 (17). Mecp2308 male mice are viable up to 1 year of age and replicate many aspects of human RTT, including cognitive and motor impairments, seizures, and social behavior deficits (17–19). Initial behavioral analysis of Mecp2308/Y mice on a highly mixed background indicated that they have increased anxiety in the open field assay (17). Moreover, we noticed a tremor in Mecp2308/Y mice that becomes more severe with handling, suggesting that they are easily stressed (B.E.M. and H.Y.Z., unpublished observation). These observations are consistent with findings from clinical investigations of the behavior of RTT patients, which indicate that episodes of heightened anxiety occur more frequently in RTT (up to 75% of cases) than in other forms of mental retardation (2, 20). Anxiety is also reported in cases in which MECP2 mutations cause additional neurobehavioral phenotypes (21, 22). These features of RTT, together with preliminary observations of the behavior of Mecp2308/Y mice, led us to propose that anxiety is an important component of the behavioral phenotype of RTT and that MeCP2 regulates key molecule(s) relevant to this behavior. To test this hypothesis, we performed behavioral and physiologic analyses on Mecp2308/Y mice and investigated the effect of Mecp2 mutation on a molecular pathway that contributes to anxiety-related behaviors.

Results

Mecp2308/Y Mice Display Enhanced Levels of Anxiety-Like Behavior.

We evaluated the anxiety-like behavior of Mecp2308/Y mice by using the open field (23), the elevated plus maze (24), and the light/dark box (25). In the open field, Mecp2308/Y mice explored the center of the arena far less than their WT counterparts (P = 0.005) (Fig. 1A). In the elevated plus maze, Mecp2308/Y mice spent less time in the open arms than WT animals (P = 0.005) (Fig. 1B). However, there was no difference in the number of total arm entries between genotypes (P = 0.258) (Fig. 1C), suggesting that reduced open arm activity by Mecp2308/Y mice was due to increased anxiety, and not hypoactivity or motor impairment. In the light/dark box, Mecp2308/Y mice spent significantly less time in the lit side of the apparatus (P = 0.010) (Fig. 1D) and made fewer transitions than WT animals (P = 0.010) (Fig. 1E). Overall, the results of these assays indicate that Mecp2308/Y mice display increased anxiety-like behavior compared with WT animals.

Fig. 1.

Fig. 1.

Open in a new tab

Mecp2308/Y mice display increased levels of anxiety behavior. (A) Mecp2308/Y mice explored the center of the open field less than WT mice. (B and C) Mecp2308/Y mice spent less time exploring the open arms of the elevated plus maze than WT mice (B), but both groups made a similar number of total arm entries (C). (D and E) In the light/dark box, Mecp2308/Y spent less time in the lit side of the apparatus (D) and made fewer transitions between the two sides of the box than WT mice (E). Values represent mean ± SEM. The asterisks indicate significant genotype differences (∗, P < 0.05; ∗∗, P < 0.01). NS, nonsignificant effects.

Mecp2308/Y Mice Have an Enhanced Corticosterone Response to Restraint Stress.

Besides anxiety, the mammalian stress response includes the activation of the hypothalamic-pituitary-adrenal (HPA) axis, which leads to glucocorticoid release from the adrenal cortex into the circulation (26). To determine whether Mecp2308/Y mice have a hyperactive HPA axis, we analyzed serum levels of the predominant murine glucocorticoid, corticosterone, in these animals under basal conditions and after 5, 15, and 30 min of acute restraint, as well as 30 min after 30 min of restraint. Both morning and evening basal levels were similar between genotypes (P = 0.884 and P = 0.995, respectively) (Fig. 2A). However, we found a significant effect of treatment (F4,50 = 29.5, P = 1.29e−12) and a significant genotype × treatment interaction effect on corticosterone levels after restraint (F4,50 = 3.03, P = 0.026). Specifically, Mecp2308/Y mice experienced elevated peak corticosterone levels (786 ± 68 ng/ml) >1.5 times (P = 0.010) as high as those measured in WT animals (478 ± 85 ng/ml) (Fig. 2B). These results indicate that Mecp2308/Y mice have an enhanced physiologic response to stress, characterized by increased HPA axis activity, in addition to an abnormal behavioral response to stress.

Fig. 2.

Fig. 2.

Open in a new tab

Mecp2308/Y mice have an enhanced corticosterone response to stress. (A) Basal morning and evening corticosterone levels are similar between Mecp2308/Y and WT mice. (B) Mecp2308/Y mice (black line) have an enhanced corticosterone response to stress compared with WT mice (gray line), which is evident after 30 min of restraint. Values represent mean ± SEM. The asterisk indicates a significant post hoc t test at the 30-min time point (∗∗, P < 0.01). NS, nonsignificant effects.

Mecp2308/Y Mice Overexpress the Corticotropin-Releasing Hormone (Crh) Transcript.

Corticotropin-releasing hormone (CRH), a neuropeptide encoded by the Crh gene, coordinates the behavioral and physiologic response to stress (27). The combination of increased anxiety and elevated stress-induced corticosterone release in Mecp2308/Y mice led us to hypothesize that these animals might also have enhanced Crh expression. To answer this question, we used in situ hybridization (ISH) to localize areas of Crh transcription and analyze Crh expression levels in the brains of Mecp2308/Y and WT mice. We focused our attention on three brain regions: the paraventricular nucleus of the hypothalamus (PVN) (Fig. 3A), from which CRH release activates the HPA axis (28); the central amygdala (CeA) (Fig. 3B), where CRH promotes a behavioral response akin to conditioned fear; and the bed nucleus of the stria terminalis (BNST) (Fig. 3C), where CRH elicits anxiety-like behavior (29). We used custom-designed imaging software (30) to count Crh-expressing cells from tissue sections that spanned the entirety of these regions in multiple animals. Using this method, we identified a significant effect of genotype on the number of cells strongly expressing Crh in the PVN (P = 1.84e−4) (Fig. 3D), the CeA (P = 0.017) (Fig. 3E), and the BNST (P = 0.018) (Fig. 3F). We confirmed these results by performing quantitative real-time RT-PCR (QRTPCR) for Crh on cDNA derived from the PVN and the CeA of another group of Mecp2308/Y and WT mice. Again, we found a significant effect of genotype on Crh expression in the PVN (P = 0.020) (Fig. 3G) and the CeA (P = 0.022) (Fig. 3H). Collectively, these studies indicate that Mecp2308/Y mice overexpress Crh in brain regions critical for the behavioral and physiologic responses to stress. Importantly, we assayed expression levels of glucocorticoid receptor and mineralocorticoid receptor RNA, as well as pharmacologic suppression of corticosterone release in Mecp2308/Y mice, to evaluate the negative feedback arm of the HPA axis and found that it functions normally (Fig. 7, which is published as supporting information on the PNAS web site). These data suggest that Crh overexpression is the primary source of HPA axis dysfuntion in these animals.

Fig. 3.

Fig. 3.

Open in a new tab

Mecp2308/Y mice have enhanced Crh expression in brain regions linked to corticosterone release and fear/anxiety behavior. ISH was performed for Crh. Here, we show representative sections from the PVN (A), the CeA (B), and the BNST (C), along with pseudocolored versions of each section demonstrating enhanced Crh expression in Mecp2308/Y mice compared with WT controls. Quantification of strongly expressing cells in the entire PVN, CeA, or BNST identified by ISH provided evidence of increased Crh expression in Mecp2308/Y mice (D, E, and F, respectively). QRTPCR confirms enhanced Crh expression in the PVN (G) and the CeA (H) of Mecp2308/Y mice. Values represent mean ± SEM. The asterisks indicate significant genotype differences (∗, P < 0.05; ∗∗, P < 0.01).

MeCP2 Binds to a Conserved, Methylated Region of the Crh Promoter.

Our finding of Crh overexpression in Mecp2308/Y mice raised the possibility that MeCP2 might play a role in regulating the transcription of Crh. To investigate this possibility, we used bisulfite sequencing (31) to determine whether hypothalamic DNA contains methylated CpG sites at the Crh promoter and whether such methylation differs between Mecp2308/Y and WT mice. We examined the 337 bp closest to the start site; among these 337 bp were nine CpG sites that are conserved between mice and humans (Fig. 8, which is published as supporting information on the PNAS web site). We found a significant effect of CpG site on methylation frequency (F8,72 = 6.15, P = 4.88e−6), indicating that, indeed, some CpG sites were more frequently methylated than others. However, we did not detect an effect of genotype (F1,72 = 0.465, P = 0.498) or a genotype × CpG site interaction effect (F8,72 = 0.448, P = 0.888) on methylation frequency (Fig. 4). This finding assured us that the increase in Crh expression is not secondary to methylation differences at the promoter.

Fig. 4.

Fig. 4.

Open in a new tab

The Crh promoter contains methyl-CpG dinucleotides. Bisulfite sequencing was used to analyze the methylation status of nine CpG sites in the Crh promoter of Mecp2308/Y and WT mice. Several methylated CpG sites were identified, but there was no effect of genotype on their methylation status. Values represent mean ± SEM.

Next, to determine whether MeCP2 binds the Crh promoter, we performed ChIP on brain tissue from WT, Mecp2308/Y, and Mecp2-null mice with an antibody specific to the N terminus of MeCP2, a region of MeCP2 that is present in both the WT MeCP2 and the MeCP2308 truncated protein. The DNA fragments recovered were used as template for quantitative PCR (QPCR) employing primer sets that spanned the region from 3.5 kb upstream to 1.7 kb downstream of the +1 site of Crh, an area that includes the conserved promoter, the sole intron, and nearly the entire coding sequence of the gene (Fig. 5A). Compared with ChIP on Mecp2-null chromatin, ChIP on WT chromatin with this antibody enriched for the methylated domain of the Crh promoter, but ChIP on Mecp2308/Y chromatin did not enrich for any region of Crh analyzed (Fig. 5 B and D). Statistical analysis of the QPCR data using a two-way ANOVA revealed a significant effect of genotype (F1,36 = 223.9, P = 4.99e−17) and region (F5,36 = 2.56, P = 0.045), as well as a significant genotype × region interaction (F5,36 = 2.87, P = 0.028). Similar results were obtained when we performed ChIP with an antibody specific to the C terminus of MeCP2, a region of MeCP2 that is present in WT MeCP2, but not MeCP2308 protein (Fig. 5 C and D). Again, a two-way ANOVA revealed a significant effect of genotype (F1,36 = 150.1, P = 2.1e−14) and region (F5,36 = 2.89, P = 0.027), as well as a significant genotype × region interaction (F5,36 = 2.97, P = 0.024). Collectively, these results point to a specific association between WT MeCP2 and the methylated promoter region of Crh in vivo. In contrast, the MeCP2308 protein was not detected at the Crh promoter.

Fig. 5.

Fig. 5.

Open in a new tab

The Crh promoter is bound by MeCP2, but not MeCP2308, in vivo. (A) Crh consists of two exons (Ex. 1 and Ex. 2) flanking a single intron. The second exon contains the coding region of Crh (shaded part of Ex. 2). A putative CpG island (CpG) lies in the promoter. Approximate regions of chromatin-immunoprecipitated DNA amplified by QPCR are identified by arrows. ChIP was performed on whole brain lysate from Mecp2308/Y and WT mice by using antibody specific to the N terminus (B) or the C terminus (C) of MeCP2, and QPCR was used to quantify Crh DNA in each immunoprecipitate. (D) We also performed nonquantitative PCR on these samples. The PCR products were run on agarose gels and visualized by ethidium bromide staining. Shown here are results representative of PCR using primers that covered the same regions of Crh as the QPCR primers. (B and C) Asterisks indicate an overall effect of genotype on the amount of DNA recovered by ChIP (∗∗, P < 0.01). Values represent mean ± SEM.

MeCP2 suppresses transcription by recruiting corepressors to DNA (5–9). Given our observation that MeCP2 binds the Crh promoter, we sought to confirm the repressive function of MeCP2 at this locus. To this end, we performed sequential ChIP (seqChIP) on WT mouse brain tissue. The first round (primary) ChIP was conducted with antibodies against acetylated histone H3, a mark of transcriptionally active chromatin, or dimethyl-histone H3 Lys-9, a mark of transcriptionally inactive chromatin (32). We used anti-C-terminal MeCP2 for the second round (secondary) ChIP, which we performed on half of the product of the primary ChIP; the remaining sample was saved for analysis of the primary ChIP. DNA recovered from both ChIP steps was analyzed by QPCR for the presence of the Crh promoter. In this way, we were able to simultaneously evaluate the activity state of Crh as well as MeCP2 binding to the Crh promoter.

When we performed the primary ChIP, we recovered more Crh promoter with anti-acetyl histone H3 than when we used anti-dimethyl histone H3 Lys-9 (Fig. 6A, P = 0.006), suggesting that the majority of cells express some baseline level of Crh. However, when we performed the second round ChIP with anti-MeCP2, we recovered significantly more Crh from chromatin that had been initially immunoprecipitated with anti-dimethyl-histone H3 Lys-9 than from chromatin that had been initially immunoprecipitated with anti-acetyl histone H3 (Fig. 6B, P = 0.029). Thus, the results of seqChIP indicate that MeCP2 preferentially associates with a transcriptionally inactive, dimethyl-histone H3 Lys-9-rich form of the Crh promoter in mice.

Fig. 6.

Fig. 6.

Open in a new tab

Under basal conditions in vivo, MeCP2 is bound to a repressed form of the Crh promoter. SeqChIP was performed on hypothalamic chromatin from WT mice. (A) More Crh was recovered when the primary ChIP was conducted with anti-acetyl histone H3 than when it was conducted with anti-dimethyl-histone H3 Lys-9. (B) Secondary ChIP with anti-C-terminal MeCP2 on the samples recovered in A indicates that MeCP2 preferentially associates with dimethyl-histone H3 Lys-9 at the Crh promoter. Values represent mean ± SEM. The asterisks are used to indicate significant differences (∗, P < 0.05).

Discussion

In this study, we identify Crh as a target of MeCP2 and we describe behavioral and physiologic phenotypes in Mecp2308/Y mice that are consistent with enhanced CRH signaling. Behaviorally, CRH induces anxiety (27). For example, intracerebroventricular delivery of CRH into the central nervous system of rodents is anxiogenic (27). Likewise, transgenic mice overexpressing Crh have enhanced anxiety (27). Conversely, pharmacologic antagonists of CRH receptor-1 signaling are anxiolytic, and Crhr1 knockout mice have reduced anxiety (27). CRH signaling in the CeA and the BNST are particularly critical to producing these behavioral effects (29). Given the established role of CRH in anxiety, it is likely that the anxiety phenotype that we observed in Mecp2308/Y mice is linked to enhanced Crh expression in the BNST and the CeA.

Like the behavioral effects of CRH, the effects of CRH on the physiologic response to stress are well known. CRH produced in the PVN initiates HPA axis activation and elicits glucocorticoid release from the adrenal cortex (28). Thus, exogenous administration of CRH in the PVN of rodents transiently increases circulating corticosterone, and transgenic overexpression of Crh chronically elevates serum glucocorticoid levels (27). Conversely, CRH antagonists block glucocorticoid release, and both CRH and CRH receptor-1 knockout mice have blunted post-stress serum glucocorticoid concentrations (27). We found evidence of elevated stress-induced serum corticosterone levels and increased Crh expression in the PVN of Mecp2308/Y mice compared with controls. Given the role of PVN CRH in stimulating the HPA axis (28), it is likely that enhanced Crh expression in the PVN of Mecp2308/Y mice is behind the hypercorticosteronemia phenotype that we identified. Thus, enhanced Crh expression in Mecp2308/Y mice seems to explain both the physiologic and the behavioral phenotypes of these animals that we describe here.

Interestingly, increased anxiety-like behavior and HPA axis hyperactivity are not limited to Mecp2308/Y mice but seem to be features that are shared by other mouse models of RTT as well as humans with RTT. Gemelli et al. observed increased anxiety-like behavior in conditional Mecp2 knockout mice (33), whereas constitutive Mecp2-null mice demonstrate a trend toward increased serum glucocorticoid levels and have enhanced expression of two glucocorticoid-inducible genes, Sgk and Fkbp5 (12). We analyzed Crh expression in Mecp2-null mice, but we did not detect any significant abnormalities, possibly because of the time at which we tested them or other features of this mutant (data not shown). With regard to humans with RTT, careful surveys indicate that anxiety-like behaviors occur frequently (2, 20). Furthermore, there is evidence of increased urinary cortisol excretion in girls with RTT (34), suggesting that humans with MECP2 mutations also experience elevated serum glucocorticoid levels. Given these data, the results of our analysis of Mecp2308/Y mice support the conclusion that anxiety and HPA axis hyperactivity are components of the phenotype produced by MECP2 mutations.

The behavioral and physiologic data show that Mecp2308/Y mice are exposed to high levels of CRH and corticosterone. Interestingly, some of the chronic effects of these compounds resemble characteristic aspects of RTT. Specifically, decreased dendritic branching, reduced synaptic plasticity, and memory impairment are features observed in the context of repeated glucocorticoid exposure (35), exposure to high concentrations of CRH (36, 37), and Mecp2/MECP2 mutation (19, 38–40). These similarities suggest that an overabundance of CRH and/or glucocorticoids might contribute to other aspects of the RTT phenotype beyond anxiety-like behaviors. In the future, clinical studies should examine the HPA axis in children with RTT and determine whether misregulation of CRH and/or glucocorticoids contributes to additional phenotypes associated with MECP2 mutations.

MeCP2 modulates the transcriptional activity of Crh in WT mice, but Crh transcriptional regulation is impaired in Mecp2308/Y mice. Our results indicate that CpG methylation at the Crh promoter is unaltered in the Mecp2308/Y mice, suggesting that enhanced Crh expression in Mecp2308/Y mice is due to a functional defect in the MeCP2308 protein itself. Whereas the exact biochemical mechanism that prevents the MeCP2308 protein from properly regulating Crh transcription remains unidentified, the simplest explanation is either that the MeCP2308 protein is unable to bind the Crh promoter or that it binds with reduced affinity. However, we cannot exclude the possibility that the MeCP2308 protein is not pulled down under our assay conditions.

It has been hypothesized that RTT phenotypes are due to the misexpression of MeCP2 target genes (11). Here, we show that WT MeCP2 binds to the Crh promoter. We also describe enhanced Crh expression in Mecp2308/Y mice together with behavioral and physiologic phenotypes consistent with heightened levels of CRH. Additionally, we do not detect the MeCP2308 protein at the Crh promoter. This observation provides both a plausible explanation for enhanced Crh expression in Mecp2308/Y mice and a viable connection between MeCP2 dysfunction and the behavioral and physiologic phenotypes of these animals. Based on our findings, we propose that impaired regulation of CRH expression contributes to the anxiety behavior and increased cortisol levels observed in patients with RTT. Thus, in this study, we identify Crh as an MeCP2 target gene to which specific, quantifiable components of the RTT phenotype can be attributed. Importantly, our understanding of the basic biology of Crh regulation in a mouse model of RTT will allow us to evaluate pathway-specific pharmacologic and genetic interventions aimed at relieving the behavioral and physiologic abnormalities that we describe here. Such studies will provide a first step toward mechanism-based translational studies in RTT.

Materials and Methods

Animals.

We used littermate pairs of WT and Mecp2308/Y animals that were the F1 offspring of 129SvEv females heterozygous for the Mecp2308 allele crossed with WT C57BL/6J males. Mecp2308/Y males were used to avoid the confounding effects of X chromosome inactivation (41).

Behavioral Studies.

Behavioral tests were performed on 4-month-old male mice during the light period (0900–1300; lights on 0700–1900). We maintained lighting at 50 lux and used a white-noise generator (Lafayette Instruments, Lafayette, IN) to maintain ambient background noise at 60 dB. For the open field assay, mice (n = 29 per genotype) were placed in the center of a 40 × 40 × 30-cm arena, and their activity was quantified over a 30-min period by a computer-operated Digiscan optical animal activity system (Acuscan, Columbus, OH). The elevated plus maze consists of two opposing closed arms (25 × 7.5 × 15.5 cm) and two opposing open arms (25 × 7.5 × 0.5 cm) connected to a center platform (7.5 × 7.5 cm) and elevated 50 cm above the floor. Mice (n = 28 per genotype) were placed in the center of the maze, and their activity was observed for 10 min. The light/dark box consists of a clear plastic chamber (36 × 20 × 26 cm) separated from a covered black plastic chamber (15.5 × 20 × 26 cm) by a black plastic divider with a 10.5 × 5-cm opening. Mice (n = 23 per genotype) were placed at the far end of the lit side, and their activity was observed for 10 min.

Corticosterone Studies and Stress-Induction Protocol.

Morning and evening basal corticosterone levels (collected between 0700 and 0900 and between 1700 and 1900, respectively) were obtained from animals (n = 5–9 per genotype) that were undisturbed for 12 h. Stress-induced corticosterone levels were obtained from animals (n = 5–10 per genotype) that were restrained in 50-ml conical tubes. Mice were killed by rapid decapitation, and trunk blood was collected in prechilled tubes containing EDTA. The blood was centrifuged at 0.8 × g for 10 min, and serum was collected and frozen at −80°C until it was analyzed. Serum corticosterone levels were measured by using an enzyme-linked immunoassay (IDS Inc., Fountain Hills, AZ).

Crh Expression Analysis.

We generated an ISH probe for Crh by using reverse-transcribed mouse cDNA as a template. Primer sequences are published in Supporting Text, which is published as supporting information on the PNAS web site. ISH was performed on brain sections from Mecp2308/Y and WT mice (n = 6 per genotype) by using a robotic platform as described (42). We performed quantitative analysis of the ISH signal on sections spanning the entire PVN, CeA, and BNST by using the Celldetekt protocol to determine cellular gene expression strengths (30). QRTPCR on cDNA generated from Mecp2308/Y and WT mouse brain regions of interest (n = 8–12 per genotype) was performed with TaqMan primers on an ABI 7300 Real Time PCR System (Applied Biosystems, Foster City, CA). Primer sequences for Crh and glyceraldehyde-3-phosphate dehydrogenase (Gapd), our internal control, are provided in Supporting Text.

Sodium Bisulfite Sequencing.

We performed sodium bisulfite sequencing on hypothalamic DNA isolated from five WT and Mecp2308/Y mice as described (31). Primers and PCR conditions can be found in Supporting Text. The PCR product was cloned into the pCR4-TOPO vector (Invitrogen, Carlsbad, CA) and transformed. We prepared 30 minipreps per sample and sequenced 17–27 of these per sample.

ChIP.

Our procedure for chromatin isolation and immunoprecipitation, along with TaqMan primer sets used for QPCR,can be found in Supporting Text. We analyzed ChIP and seqChIP QPCR data by normalizing to the appropriate input sample and subtracting out background (defined as the normalized quantity recovered by ChIP with nonspecific IgG). For data in Fig. 5, we calculated the fold enrichment over the normalized quantity recovered by ChIP on Mecp2-null chromatin. Means were calculated from four independent ChIPs with each antibody on WT and Mecp2308/Y chromatin, as well as from six independent seqChIP experiments.

Statistics.

Two-way ANOVA was used to analyze the time course of corticosterone release after restraint (genotype × time interval); the results of bisulfite sequencing (genotype × CpG site); and the results of QPCR mapping of the MeCP2-binding site in the Crh promoter (genotype × primer site). We used post hoc t tests with Bonferroni correction to compare genotype differences in corticosterone release at discrete time points after restraint. We used Student's t test to analyze measures recorded in the open field assay, elevated plus maze, and the light/dark box; basal corticosterone levels; gene expression ratios obtained by QRTPCR; and the number of cells strongly expressing Crh by ISH. A nonparametric Mann–Whitney test was used to analyze the results of the seqChIP experiment. We consistently used two-sided tests and α = 0.05 to determine statistical significance. Results are presented as mean ± SEM.

Supplementary Material

Supporting Information
pnas_0608702103_index.html (13.9KB, html)

Acknowledgments

We thank Gregor Eichele for his advice regarding in situ hybridization, Marwan Shinawi for controls and protocols for bisulfite sequencing, Juan Young for advice on the restraint stress protocol, E. O'Brian Smith and Mariella DeBiasi for helpful discussions, and the Zoghbi laboratory members for their critical input. This work was supported by National Institutes of Health (NIH) Fellowship F30 MH068996 (to B.E.M.); National Library of Medicine Grant 5T15LM07093 from the Keck Center for Computational and Structural Biology, and National Center for Research Resources Grant P41 RR02250 (to J.P.C.); NIH Grant R01 HD40301 (to H.Y.Z.); and Core Laboratories of the Mental Retardation and Developmental Disabilities Research Center Grant P30 HD024064. H.Y.Z. is an investigator with the Howard Hughes Medical Institute.

Abbreviations

RTT

Rett syndrome

MECP2

methyl-CpG-binding protein 2

HPA

hypothalamic-pituitary-adrenal

CRH

corticotropin-releasing hormone

ISH

in situ hybridization

PVN

paraventricular nucleus of the hypothalamus

CeA

central amygdala

BNST

bed nucleus of the stria terminalis

QRTPCR

quantitative real-time RT-PCR

QPCR

quantitative PCR

seqChIP

sequential ChIP.

Footnotes

The authors declare no conflict of interest.

References

  • 1.Moretti P, Zoghbi HY. Curr Opin Genet Dev. 2006;16:276–281. doi: 10.1016/j.gde.2006.04.009. [DOI] [PubMed] [Google Scholar]
  • 2.Mount RH, Charman T, Hastings RP, Reilly S, Cass H. J Child Psychol Psychiatry. 2002;43:1099–1110. doi: 10.1111/1469-7610.00236. [DOI] [PubMed] [Google Scholar]
  • 3.Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. Nat Genet. 1999;23:185–188. doi: 10.1038/13810. [DOI] [PubMed] [Google Scholar]
  • 4.Klose RJ, Sarraf SA, Schmiedeberg L, McDermott SM, Stancheva I, Bird AP. Mol Cell. 2005;19:667–678. doi: 10.1016/j.molcel.2005.07.021. [DOI] [PubMed] [Google Scholar]
  • 5.Jones PL, Veenstra GJ, Wade PA, Vermaak D, Kass SU, Landsberger N, Strouboulis J, Wolffe AP. Nat Genet. 1998;19:187–191. doi: 10.1038/561. [DOI] [PubMed] [Google Scholar]
  • 6.Nan X, Ng HH, Johnson CA, Laherty CD, Turner BM, Eisenman RN, Bird A. Nature. 1998;393:386–389. doi: 10.1038/30764. [DOI] [PubMed] [Google Scholar]
  • 7.Fuks F, Hurd PJ, Wolf D, Nan X, Bird AP, Kouzarides T. J Biol Chem. 2003;278:4035–4040. doi: 10.1074/jbc.M210256200. [DOI] [PubMed] [Google Scholar]
  • 8.Kimura H, Shiota K. J Biol Chem. 2003;278:4806–4812. doi: 10.1074/jbc.M209923200. [DOI] [PubMed] [Google Scholar]
  • 9.Lunyak VV, Burgess R, Prefontaine GG, Nelson C, Sze SH, Chenoweth J, Schwartz P, Pevzner PA, Glass C, Mandel G, Rosenfeld MG. Science. 2002;298:1747–1752. doi: 10.1126/science.1076469. [DOI] [PubMed] [Google Scholar]
  • 10.Young JI, Hong EP, Castle JC, Crespo-Barreto J, Bowman AB, Rose MF, Kang D, Richman R, Johnson JM, Berget S, Zoghbi HY. Proc Natl Acad Sci USA. 2005;102:17551–17558. doi: 10.1073/pnas.0507856102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Caballero IM, Hendrich B. Hum Mol Genet. 2005;14(Spec No 1):R19–R26. doi: 10.1093/hmg/ddi102. [DOI] [PubMed] [Google Scholar]
  • 12.Nuber UA, Kriaucionis S, Roloff TC, Guy J, Selfridge J, Steinhoff C, Schulz R, Lipkowitz B, Ropers HH, Holmes MC, Bird A. Hum Mol Genet. 2005;14:2247–2256. doi: 10.1093/hmg/ddi229. [DOI] [PubMed] [Google Scholar]
  • 13.Kriaucionis S, Paterson A, Curtis J, Guy J, Macleod N, Bird A. Mol Cell Biol. 2006;26:5033–5042. doi: 10.1128/MCB.01665-05. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Chen WG, Chang Q, Lin Y, Meissner A, West AE, Griffith EC, Jaenisch R, Greenberg ME. Science. 2003;302:885–893. doi: 10.1126/science.1086446. [DOI] [PubMed] [Google Scholar]
  • 15.Martinowich K, Hattori D, Wu H, Fouse S, He F, Hu Y, Fan G, Sun YE. Science. 2003;302:890–893. doi: 10.1126/science.1090842. [DOI] [PubMed] [Google Scholar]
  • 16.Horike S, Cai S, Miyano M, Cheng JF, Kohwi-Shigematsu T. Nat Genet. 2005;37:31–40. doi: 10.1038/ng1491. [DOI] [PubMed] [Google Scholar]
  • 17.Shahbazian M, Young J, Yuva-Paylor L, Spencer C, Antalffy B, Noebels J, Armstrong D, Paylor R, Zoghbi H. Neuron. 2002;35:243–254. doi: 10.1016/s0896-6273(02)00768-7. [DOI] [PubMed] [Google Scholar]
  • 18.Moretti P, Bouwknecht JA, Teague R, Paylor R, Zoghbi HY. Hum Mol Genet. 2005;14:205–220. doi: 10.1093/hmg/ddi016. [DOI] [PubMed] [Google Scholar]
  • 19.Moretti P, Levenson JM, Battaglia F, Atkinson R, Teague R, Antalffy B, Armstrong D, Arancio O, Sweatt JD, Zoghbi HY. J Neurosci. 2006;26:319–327. doi: 10.1523/JNEUROSCI.2623-05.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Sansom D, Krishnan VH, Corbett J, Kerr A. Dev Med Child Neurol. 1993;35:340–345. doi: 10.1111/j.1469-8749.1993.tb11646.x. [DOI] [PubMed] [Google Scholar]
  • 21.Cohen D, Lazar G, Couvert P, Desportes V, Lippe D, Mazet P, Heron D. Am J Psychiatry. 2002;159:148–149. doi: 10.1176/appi.ajp.159.1.148-a. [DOI] [PubMed] [Google Scholar]
  • 22.Couvert P, Bienvenu T, Aquaviva C, Poirier K, Moraine C, Gendrot C, Verloes A, Andres C, Le Fevre AC, Souville I, et al. Hum Mol Genet. 2001;10:941–946. doi: 10.1093/hmg/10.9.941. [DOI] [PubMed] [Google Scholar]
  • 23.Paylor R, Nguyen M, Crawley JN, Patrick J, Beaudet A, Orr-Urtreger A. Learn Mem. 1998;5:302–316. [PMC free article] [PubMed] [Google Scholar]
  • 24.Pellow S, File SE. Pharmacol Biochem Behav. 1986;24:525–529. doi: 10.1016/0091-3057(86)90552-6. [DOI] [PubMed] [Google Scholar]
  • 25.Crawley J, Goodwin FK. Pharmacol Biochem Behav. 1980;13:167–170. doi: 10.1016/0091-3057(80)90067-2. [DOI] [PubMed] [Google Scholar]
  • 26.Koob GF. Biol Psychiatry. 1999;46:1167–1180. doi: 10.1016/s0006-3223(99)00164-x. [DOI] [PubMed] [Google Scholar]
  • 27.Bale TL, Vale WW. Annu Rev Pharmacol Toxicol. 2004;44:525–557. doi: 10.1146/annurev.pharmtox.44.101802.121410. [DOI] [PubMed] [Google Scholar]
  • 28.Bruhn TO, Plotsky PM, Vale WW. Endocrinology. 1984;114:57–62. doi: 10.1210/endo-114-1-57. [DOI] [PubMed] [Google Scholar]
  • 29.Walker DL, Toufexis DJ, Davis M. Eur J Pharmacol. 2003;463:199–216. doi: 10.1016/s0014-2999(03)01282-2. [DOI] [PubMed] [Google Scholar]
  • 30.Carson JP, Eichele G, Chiu W. J Microsc (Oxford) 2005;217:275–281. doi: 10.1111/j.1365-2818.2005.01450.x. [DOI] [PubMed] [Google Scholar]
  • 31.Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, Paul CL. Proc Natl Acad Sci USA. 1992;89:1827–1831. doi: 10.1073/pnas.89.5.1827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Fischle W, Wang Y, Allis CD. Curr Opin Cell Biol. 2003;15:172–183. doi: 10.1016/s0955-0674(03)00013-9. [DOI] [PubMed] [Google Scholar]
  • 33.Gemelli T, Berton O, Nelson ED, Perrotti LI, Jaenisch R, Monteggia LM. Biol Psychiatry. 2006;59:468–476. doi: 10.1016/j.biopsych.2005.07.025. [DOI] [PubMed] [Google Scholar]
  • 34.Motil KJ, Schultz RJ, Abrams S, Ellis KJ, Glaze DG. J Pediatr Gastroenterol Nutr. 2006;42:419–426. doi: 10.1097/01.mpg.0000189370.22288.0c. [DOI] [PubMed] [Google Scholar]
  • 35.McEwen BS. Annu Rev Neurosci. 1999;22:105–122. doi: 10.1146/annurev.neuro.22.1.105. [DOI] [PubMed] [Google Scholar]
  • 36.Brunson KL, Eghbal-Ahmadi M, Bender R, Chen Y, Baram TZ. Proc Natl Acad Sci USA. 2001;98:8856–8861. doi: 10.1073/pnas.151224898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Chen Y, Bender RA, Brunson KL, Pomper JK, Grigoriadis DE, Wurst W, Baram TZ. Proc Natl Acad Sci USA. 2004;101:15782–15787. doi: 10.1073/pnas.0403975101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Armstrong DD. J Child Neurol. 2005;20:747–753. doi: 10.1177/08830738050200090901. [DOI] [PubMed] [Google Scholar]
  • 39.Hagberg B, Aicardi J, Dias K, Ramos O. Ann Neurol. 1983;14:471–479. doi: 10.1002/ana.410140412. [DOI] [PubMed] [Google Scholar]
  • 40.Nelson ED, Kavalali ET, Monteggia LM. Curr Biol. 2006;16:710–716. doi: 10.1016/j.cub.2006.02.062. [DOI] [PubMed] [Google Scholar]
  • 41.Young JI, Zoghbi HY. Am J Hum Genet. 2004;74:511–520. doi: 10.1086/382228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Yaylaoglu MB, Titmus A, Visel A, Alvarez-Bolado G, Thaller C, Eichele G. Dev Dyn. 2005;234:371–386. doi: 10.1002/dvdy.20441. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information
pnas_0608702103_index.html (13.9KB, html)
pnas_0608702103_1.pdf (51.1KB, pdf)
pnas_0608702103_2.pdf (406.7KB, pdf)

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES