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Research report
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Histone modifications of the Crhr1 gene in a rat model of depression following chronic stress
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Qirong Wan a , Kai Gao a , Han Rong a , Min Wu b , Huiling Wang a , Xiaoping Wang a , Gaohua Wang a,c,∗ , Zhongchun Liu a,c,∗ a
Department of Psychiatry, Renmin Hospital, Wuhan University, Jiefang Road 238#, Wuhan 430060, PR China College of Life Sciences, Wuhan University, Wuhan 430072, PR China c Institute of Neuropsychiatry, Wuhan University, Wuhan 430060, PR China b
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h i g h l i g h t s • We examine changes of CRHR1 expression in the hypothalamus of stressed rats. • Stress may change the level of H3K9 tri-methylation at CRHR1 gene promoter. • CRHR1 expression of stressed rats may correlate with H3k9 tri-methylation.
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Article history: Received 3 March 2014 Received in revised form 13 May 2014 Accepted 16 May 2014 Available online xxx
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Keywords: Depression CRHR1 Epigenetic regulation Hypothalamus Histone methylation
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1. Introduction
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Multiple lines of evidence suggest a link between depression and changes in hypothalamic-pituitaryadrenal (HPA)-axis hormone dynamics, including altered regulation of the corticotrophin-releasing hormone (CRH) and its main receptor, corticotrophin-releasing hormone receptor 1 (CRHR1). However, the precise molecular mechanisms underlying depression remain poorly understood. In this study, we employed a model of depression in rats by subjecting animals to 21 days of chronic unpredictable mild stress (CUMS). Real-time PCR and western blotting were used to study the mRNA and protein expression levels of CRHR1 in the hypothalamus. In addition, chromatin immunoprecipitation assays were used to detect histone methylation at the Crhr1 gene promoter; the levels of histone H3 trimethylation at lysines 4 (H3K4) and 9 (H3K9) reflect active transcription and transcriptional repression, respectively. Rats exposed to CUMS exhibited significant reduction in locomotion and sucrose preference. These behavioral alterations were associated with elevated expression levels of CRHR1 mRNA and protein in the hypothalamus of rats in the CUMS group. We also found that the levels of H3K9 trimethylation at the Crhr1 gene promoter in the CUMS group were significantly lower than those in the control group, whereas H3K4 trimethylation levels were the same for both groups. Taken together, our findings suggest that the increase in CRHR1 expression in the hypothalamus of stressed rats correlates with a decrease in the repressive chromatin state caused by reduced H3K9 trimethylation levels. These data are the first in vivo evidence of a role for chromatin modifications in the regulation of Crhr1 gene expression in the hypothalamus, and may provide novel insight into therapeutic approaches to treat depression. © 2014 Published by Elsevier B.V.
Major depression is a devastating illness characterized primarily by low mood, apathy, loss of appetite, loss of libido, and sometimes
∗ Corresponding authors at: Department of Psychiatry, Renmin Hospital, Wuhan University, Jiefang Road 238#, Wuhan 430060, PR China. Tel.: +86 88041911 81399; fax: +86 27 88072022. E-mail addresses: [email protected], [email protected] (G. Wang), [email protected] (Z. Liu).
by suicidal thoughts and high mortality and disability rates. Recent studies show that the incidence of depression is about 3.1% worldwide and 3% to 5% in China [1]. It has also been predicted that in 2020 depression will become the second-largest contributor to the global burden of disease [2]. Although depression is the most common psychiatric disorder, its pathophysiology is poorly understood and there is an urgent need to design new drugs for the treatment of this debilitating disease. The hypothalamus–pituitary–adrenal (HPA) axis plays an important role in the pathogenesis of depression. Several studies have shown that about 70% of depressed patients exhibit
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Please cite this article in press as: Wan Q, et al. Histone modifications of the Crhr1 gene in a rat model of depression following chronic stress. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.031
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dysfunction in the HPA axis [3]. When subjected to strong, persistent stimulation, the body’s basic response includes neuroendocrine changes, such as changes in the levels of corticotrophin-releasing hormone (CRH) and its main receptor, corticotrophin-releasing hormone receptor 1 (CRHR1). Depressed patients exhibit elevated concentrations of CRH in the blood, cerebrospinal fluid and urine [4,5]. CRHR1 belongs to the G-proteincoupled receptor family and is considered to play a pivotal role in mediating the CRH-elicited effects in depression and anxiety [6]. Using CRHR1 antagonists in patients with major depression and in a stress-related animal model of depression showed clear antidepressant and anxiolytic effect [7]. Consequently, the Crhr1 gene is a candidate gene for the study of antidepressant pharmacogenetics [8,9]. The Crhr1 gene is located on chromosome 17q21–22, spanning 20 kb of genomic DNA and containing 14 exons. It has three known isoforms arising from alternative splicing [10]. Epigenetics is the study of heritable changes in gene expression that occur without a change in DNA sequence. The main forms of epigenetic modifications are DNA methylation, histone acetylation and histone methylation. The epigenetic hypothesis suggests that these chromatin modifications may provide another pathogenic mechanism that is mediated by internal and external environmental factors (such as toxins, hormones, nutrition). The hypothesis helps to explain why the rate of various complex diseases increases significantly with age. It also explains the discordance in disease incidence among monozygotic twins [11]. Recent studies suggest that early life events can have long-term behavioral effects on individuals that are mediated by epigenetic mechanisms. High levels of pup licking, grooming and nursing behaviors exhibited by rat mothers correlated with a higher degree of methylation on some sites of the GR17 promoter region and NGFI-A binding sequence in their offspring, compared to offspring of mothers that exhibited low levels of these behaviors. These differences appear in the first week after birth and may continue into adulthood [12]. However, it is too early to confirm that epigenetic mechanisms play a decisive role in the onset of depression. Further exploration of depression-related genes, brain regions, neural circuits and their interaction is the future direction of this field of research. Here, we use the chronic unpredictable mild stress (CUMS) model of depression in rats to study the epigenetic mechanisms that mediate changes in Crhr1 gene expression in the hypothalamus, with the aim of providing the experimental and theoretical basis for designing related drugs for the treatment of depression. 2. Materials and methods 2.1. Animals 30 adult male Sprague-Dawley rats (weighing 190–210 g) were obtained from the Limited Company of Hunan Slack King Experimental Animals. One week before testing, all animals were housed in groups of four in a room with standard laboratory conditions (12/12-h light/dark cycle: lights on 8:00, off 20:00, 22 ± 2 ◦ C, food and water ad libitum). All animal experiments were carried out in accordance with the instructions for laboratory animal care issued by the Ministry of Science and Technology of the People’s Republic of China in 2006. 2.2. Chronic unpredictable mild stress We employed the CUMS model of depression according to the procedure described by Luo et al. [13] with some modification. The stressors included cage tilting (45◦ ) for 24 h, damp sawdust (200 ml water in a cage) for 24 h, swimming in 25 ◦ C water for 15 min, 24 h
of food deprivation, 24 h of water deprivation, nip tail for 1 min, shaking for 15 min (120 rpm rocking bed), immobilizing for 1 h in a 25 cm × 8 cm cylindrical plastic rodent restrainer, and alterations of the light/dark cycle with one period of continuous overnight illumination. Animals were individually exposed to stressors for 3 weeks. Control animals with free access to water and food were undisturbed. 2.3. Sucrose preference test The sucrose preference test was used to assess anhedonia on the 1st and 22nd day. All rats were fed 7 days to adapt to drinking sucrose. Training consisted of initial 48 h sucrose solution exposure without any other food or water available, and five additional consecutive 1 h periods per day of sucrose availability [14]. As described by Willner et al. [15], before each test, the animals were deprived of water for 23 h. Rats were individually exposed to a bottle of 1% sucrose solution and a bottle of water for 1 h and consumption of sucrose intake and water intake were measured by comparing the weight of the bottle before and after the test. Sucrose preference (%) = (sucrose intake/total fluid intake) × 100%. 2.4. Open field test The rats were placed in the center of a dimly illuminated rectangular cage (120 cm × 90 cm × 35 cm) and observed for 10 min. Locomotor activity was measured using an automated video tracking system (Ethovision 3.0, Noldus, the Netherlands). The frequency of rearing (standing upright on the hind legs, while forepaws were free) was recorded manually. The cage was cleaned thoroughly before the next animal was tested, and the room was kept quiet. 2.5. Measuring mRNA levels by real-time PCR Rats were sacrificed by cervical dislocation after the open field test. The hypothalamus was collected quickly for RNA quantification. RNA was extracted using a commercial kit (Yuanping Hao, China) and mRNA was reverse transcribed to cDNA using a first-strand synthesis kit (Tiangen, China). The amount of cDNA was quantified using real-time PCR. The following primers were used to amplify specific cDNA regions of the transcripts of interest: -actin (5 -cgttgacatccgtaaagacctc-3 and 5 -taggagccagggcagtaatct-3 ) and CRHR1 (5 -gctttcatcctacgcaacg3 , and 5 -tagcagccctcaccgaac-3 ). -Actin quantification was used as an internal control for normalization. Fold differences of mRNA levels over control values were calculated using the Ct method (Applied Biosystems manual). PCRs were run in triplicate for each brain sample, and at least three independent sample pairs were used for each statistical analysis. 2.6. Western blot analysis of CRHR1 protein levels The hypothalamus was dissected and directly transferred into RIPA buffer (50 mM Tris–HCl pH 8.0, 50 mM HEPES pH 8.0, 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 0.5 mM PMSF, 1 g/ml aprotinin, 1 g/ml leupeptin, 1 g/ml pepstatin, 1 mM EDTA, 10 M Na3VO3, 10 M NaF). Tissues were homogenized with a glass homogenizer. Supernatant was collected after centrifugation at 800 × g for 5 min at 4 ◦ C. Protein concentration was measured using BCA protein assay reagents (Thermo; Reagent A: 2 ml, Reagent B: 40 l, 1× PBS: 95 l, Protein: 5 l). Equal amounts of protein were loaded into SDS-PAGE gels (Invitrogen) and electrophoresis was performed at 200 V for 50 min. Proteins were transferred to Polyvinylidene Fluoride (PVDF) membrane (Invitrogen) and the membrane was blocked with 5% non-fat dry milk in Phosphate Buffered Saline Tween-20 (PBST). The membrane
Please cite this article in press as: Wan Q, et al. Histone modifications of the Crhr1 gene in a rat model of depression following chronic stress. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.031
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Fig. 1. Effects of CUMS on sucrose preference (a) and open field test (b, c, d) in rats. Data are represented as the means ± S.D. **P < 0.01. (a) 1 h 1% sucrose consumption of rats after 21 days of exposure to stress. (b) Total distance traveled by rats in the open field test. (c) Velocity of rats in the open field test. (d) Frequencies of rearing of rats in the open field test.
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was incubated with primary goat polyclonal CRHR1 (Santa Cruz) antibodies (1:1000 dilution) and rabbit actin (Sigma) antibodies (1:1000 dilution), overnight at 4 ◦ C on a rocking platform. The blotting membrane was washed with PBST and incubated with secondary antibody (1:5000 dilution) for 1 h at room temperature on a rocking platform. The membrane was washed with PBST, developed using Chemiluminescent HRP substrate following the manufacturer’s instructions (ECL: A + B equal volume in RT) and exposed to X-ray film for an appropriate time period. Band densities were measured using Kodak 1D Scientific Imaging System and Image software.
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2.7. Chromatin immunoprecipitation assays (CHIP)
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Chromatin was extracted from hypothalamic tissue by following published protocols with minor modifications [16]. Whole hypothalamus was minced to 1 mm-sized pieces, and immediately crosslinked in 270 l 37% formaldehyde for 15 min at room temperature. We added glycine to a final concentration of 0.125 M to stop the crosslinking reaction while shaking for 10 min at 4 ◦ C. The tissue was washed three times in cold 1× PBS containing protease inhibitors (1 mM PMSF, 1 l/ml apoprotin, and 1 l/ml leupeptin). Tissue was homogenized in 1 ml cell lysis buffer (10 mM Tris, 10 mM Nacl, 0.2% NP-40, PMSF, apoprotin, leupeptin), and incubated on ice for 15 min, followed by centrifugation for 5 min at 5000 rpm at 4 ◦ C. The supernatant was discarded and the pellet was incubated in 850 l nuclear lysis buffer (50 mM Tris–Cl, pH 8.1, 10 mM EDTA, 1% SDS, PMSF, apoprotin, leupeptin) for 30 min
on ice. Next, the extracted chromatin was sheared to 300–500 bp using the Xinzhi Dismembrator 550 (Ningbo, China). Each sample was sonicated for 8 min on ice, at 40% of maximum power, with a 1 min pause every 2 min, and the degree of DNA fragmentation was observed. The chromatin solution was pre-cleared with protein G (GE, USA) for 2 h. It was then immunoprecipitated overnight at 4 ◦ C with 4 g of antibodies directed against H3 methylated on Lys4 (Millipore, USA) or Lys9 (Millipore, USA). As a control, samples were immunoprecipitated with 4 g non-immune rabbit IgG and rabbit polyclonal anti-histone H3 antibodies (Abcam, England). The beads were sequentially washed once with low salt, high salt, and LiCl buffers and washed twice with 1× TE. The DNA–histone complex was then eluted from the beads with elution buffer and protein K. DNA and histones were dissociated overnight at 65 ◦ C. DNA was purified using a commercial kit (Shenggong, China) and quantified using real-time PCR. The levels of specific histone modifications at the gene promoter were determined by measuring the amount of DNA associated with methylated histone by quantitative real-time PCR (ABI Prism 7500; Applied Biosystems, Foster City, CA). Specific primers were designed to amplify proximal promoter regions (5 -ccgctgtctccacttatctt-3 and 5 -tccctcgttcgttcactcat-3 ). Tubulin (5 -tagaaccttcctgcggtcgt-3 and 5 -ttttcttctgggctggtctc-3 ), and globin (5 -tgaccaatagtctcggagtcctg3 and 5 -aggctgaaggcctgtccttt-3 ) were used as controls. Input and immunoprecipitated DNA amplification reactions were run in triplicate in the presence of SYBRGreen (Ferments, Canada), and at least three independent sample pairs were used for each statistical analysis. The comparative CT values were used to determine relative
Please cite this article in press as: Wan Q, et al. Histone modifications of the Crhr1 gene in a rat model of depression following chronic stress. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.031
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Fig. 2. The increase of CRHR1 mRNA expression was induced by chronic unpredictable mild stress in the hypothalamus. CUMS-exposed rats were subjected to various stressors for 21 days. mRNA levels of CRHR1 was assayed by RT-PCR in the hypothalamus, values are means ± S.D. (N = 4 in each group). Expression of hypothalamic CRHR1 mRNA in CUMS group was significantly increased compared to the control group (P < 0.01).
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expression compared to input or total histone H3. Fold differences (depression group ChIP relative to control group ChIP) were then determined by raising 2 to the Ct power.
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2.8. Statistical analysis
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Values reported in the text and figures are represented as mean ± S.D. The significance of the differences between groups was determined using one-way ANOVA. P-value < 0.05 was considered to indicate statistical significance.
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depression. Stressors were introduced to the animals in an unpredictable order and repeated throughout the experiment. The rats in the control group were housed in normal conditions without any disturbances. The behavioral effects of CUMS were examined, and the data are shown in Fig. 1: sucrose preference (Fig. 1a) and the indices of open field test, including total distance moved (Fig. 1b), velocity (Fig. 1c), and frequency of rearing (Fig. 1d). In our study, there was no statistical difference between the two groups before CUMS (data not shown). At the end of CUMS, the rats displayed a significant reduction in the sucrose preference compared with control rats (mean ± S.D., 84.53 ± 4.95 for control group, 60.07 ± 9.86 for CUMS group, P < 0.01). Moreover, stressors had significantly different effects on the performance of the two groups in the open field test: total moved distance (1949.37 ± 164.44 cm and 1081.06 ± 117.50 cm, respectively for control group and CUMS group, P < 0.01) and velocity (4.13 ± 0.46 cm/s and 2.09 ± 0.45 cm/s, respectively for control group and CUMS group, P < 0.01). There was also a significant difference in the frequency of rearing between CUMS group (3.12 ± 1.83) and control group (12.60 ± 2.58) (P < 0.01). Reduced sucrose consumption implies animals exposed to CUMS had decreased responsiveness to rewarding stimuli. The results of the open field test suggest that animals exposed to CUMS exhibit reduced activity.
3.2. CRHR1 mRNA expression Based on strong evidence implicating CRHR1 in the pathophysiology of depression and anxiety [18], we performed RT-PCR analysis to investigate the level of CRHR1 mRNA expression in the hypothalamus after CUMS. As shown in Fig. 2, one-way ANOVA of the RT-PCR data revealed that mRNA expression of hypothalamic CRHR1 in CUMS group was elevated when compared to the control group.
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3.1. Effects of CUMS on sucrose preference tests and open field tests Many studies have provided evidence that the CUMS model simulates the core symptom of human depression: anhedonia [17]. The sucrose preference test is used to validate animal models of
A representative photomicrograph is shown in Fig. 3a to illustrate CRHR1 protein analysis using Western blot. Quantification of these data showed that the levels of CRHR1 in the CUMS group were significantly higher than those in the control group (Fig. 3b, P < 0.05).
Fig. 3. Gray-scale value of hypothalamic CRHR1 in control group and CUMS group (P < 0.05). Representative western blot bands (a) and densitometric analyses of the bands (b). Data are represented as the mean ± S.D. *P < 0.01 (one-way ANOVA).
Please cite this article in press as: Wan Q, et al. Histone modifications of the Crhr1 gene in a rat model of depression following chronic stress. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.031
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Fig. 4. CHIP assay to quantify the abundance of H3 lysine-4-trimethylation, and H3 lysine-9-trimethylation on CRHR1 gene promoter in hypothalamus after 21 days chronic unpredictable mild stress. (a) The level of histone H3K4 tri-methylation between two groups was not significantly different (P > 0.05). (b) Relative fold enrichment of histone H3K9 tri-methylation at CRHR1 gene promoters in CUMS rats versus control rats, demonstrating that CUMS leads to a significant decrease of H3K9 tri-methylation. *P < 0.05.
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3.4. Quantification of stress-induced histone modifications at the Crhr1 promoter by CHIP assays Histone modifications have recently been associated with depression-like behavior [19]. To understand better the molecular mechanisms of depression, we studied histone modifications at the promoter of the Crhr1 gene in the rat hypothalamus after 21 days of exposure to chronic mild stress. Our data show that CUMS did not significantly alter the levels of trimethylated histone H3K4, which is implicated in the activation of gene expression. Meanwhile, we investigated the effect of CUMS on the methylation of histone H3K9, a marker of a silenced chromatin state. Consistent with the increase in expression of the of Crhr1 gene observed after CUMS, the levels of histone H3K9 tri-methylation were significantly reduced at the Crhr1 promoter in CUMS rats compared to control rats (Fig. 4b). Taken together, these data suggest that CUMS induces a remarkable increase in Crhr1 expression in the hypothalamus, which correlates with a significant decrease in H3-trimk9 levels at the Crhr1 promoter.
4. Discussion The importance of epigenetic mechanisms, shown in recent years to be a major determinant of gene regulation, has received much attention in the study of depression. Many studies suggest that the long-term regulation of gene expression through epigenetic mechanisms can mediate permanent changes in brain function and contribute to the pathogenesis of psychiatric disorders [20]. This conclusion has been confirmed in models of seizures [21], psychostimulant treatment [20], and depression [22–24]. Among many epigenetic modifications, DNA methylation of CpG dinucleotides, acetylation and methylation at the N-terminal tails of histones are the most prevalently studied modifications in molecular analyses of behavioral models [25]. The goal of the present study was to determine whether the expression of Crhr1 was regulated by chromatin modification in the CUMS model. The results showed that the exposure of rats to chronic mild stress can lead to depressive behavior that is associated with changes of CRHR1 expression in the hypothalamus. They further show that these changes in gene expression are mediated, at least partially, through chromatin modifications at the Crhr1 promoter. Depression is a common and persistent mood disorder characterized by despair, helplessness and social withdrawal. We used 21 days of CUMS to build a classical model of depression. Its theoretical basis was consistent with the occurrence and course of
exacerbation in depressed patients. Rasenick et al. [26] have suggested that depression in such a model was the result of many stress factors. Sucrose preference and open-field tests are regarded as useful and valid behavioral markers of depression in an animal paradigm. Anhedonia – the inability to enjoy pleasurable stimuli – is another symptom commonly associated with depression in humans. In our experiment, sucrose preference in the CUMS group was significantly reduced, compared to the control group. We presume that there may be three reasons for this reduction: (1) chronic stress damages nerve cells in the neural reward system and induces the reduced ability to experience happiness; (2) sucrose preference was decreased because the animals were tired after being deprived of food, water and subjected to other stressors; and (3) the decline in sucrose consumption was induced by metabolic changes resulting from fasting [27]. Furthermore, the results of the open-field test provided a measure of exploratory behavior and emotional responses in new environments. Our current data confirm that the CUMS group exhibited decreased distance moved, and slower velocity. The results indicate that stressed rats are less active. In addition, the frequency of rearing in the CUMS group was decreased, as compared to the control group. Decreased rearing frequency indicates that the exposure of rats to stress decreased their curiosity in a novel environment. In summary, the behavioral defects induced by CUMS, including decreased activity and waned interest, are reminiscent of several depressive-like symptoms in humans. We hypothesize that these behavioral changes are accompanied by changes in histone modifications at the promoter of the Crhr1 gene in the hypothalamus. Nestler and coworkers [16,19] have shown associations between histone modifications and changes in behavioral function in response to antidepressant treatment and chronic electroconvulsive seizures in rodents. Over the past 40 years, the most consistent physiological association in depression has been hyperactivity of the HPA axis [28]. However, it is still debatable whether the increased activity of the HPA axis is caused by depression, or itself is involved in the development of depression. Wasserman et al. [29] suggested that the Crhr1 gene was a marker for suicide in depressed males exposed to low stress. In our previous study, we tested whether the polymorphisms of three sites (rs1876828, rs242939 and rs242941) in the Crhr1 gene are related to the effect of 6 weeks of fluoxetine treatment in 127 Han Chinese patients with major depressive disorders (MDD). The results show that the rs242941G/G genotype and homozygous GAG haplotype of the three single-nucleotide polymorphisms are associated with the therapeutic response to fluoxetine in MDD patients with high-anxiety. However, there are several limitations in this study [30]. The potential pathological mechanisms of
Please cite this article in press as: Wan Q, et al. Histone modifications of the Crhr1 gene in a rat model of depression following chronic stress. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.031
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depression need to be further investigated, particularly the interaction between the Crhr1 gene and environmental factors, to explain the pathogenesis of depression. Our experiments show a marked increase in CRHR1 mRNA and protein expression in the hypothalamus of CUMS-exposed rats. The correlation between hypothalamus CRHR1 levels and behavioral response in our model further emphasizes the role of CRHR1 in the pathophysiology of mood disorders. Our findings also suggest that the levels of CRHR1 expression may be related to the changes in histone modifications. In recent years, epigenetics has become a popular field of investigation in translational medical research. Specifically, there has been greater effort in psychiatric research to explore the impact of DNA methylation and histone modifications on disease. Histone modifications affect the structural dynamics of the nucleosome that regulates chromatin regulation of gene expression through the recruitment of specific interacting proteins that recognize a single or conformational set of modifications. H3K4 methylation is considered to be associated with activation of gene expression, and H3K9 methylation is associated with gene repression. Our experiments show that CUMS induces a significant decrease in H3K9 trimethylation, concomitant with enhanced expression of the Crhr1 gene. Such long-lived changes in chromatin modifications may be one of the crucial mechanisms for stress-induced neuroendocrine changes in the hypothalamus. Recently, many researchers focused on chromatin remodeling that is associated with depression, in different brain regions and at different genes. Tsankova et al. presented findings to suggest that stress caused by chronic social defeat alters chromatin regulation of the Brain-derived neurotrophic factor (Bdnf) gene. They showed long-lasting increases in H3K27 dimethylation, a repressive modification, specifically at the promoter of the down-regulated Bdnf gene [22]. Chronic imipramine treatment was shown to reverse the repression of the Bdnf gene by inducing H3 acetylation, as well as H3K4 methylation [21]. These results are consistent with the histone code hypothesis: a combination of several histone modifications may ultimately determine the outcome of gene expression. Researchers believe that a variety of epigenetic mechanisms can be involved simultaneously in the regulation of gene expression. Taken together, our data suggest that CUMS induces a long-lasting increase in CRHR1 expression in the hypothalamus, which may correlates with a significant decrease of repressive chromatin state produced by the reduced H3k9 trimethylation levels at promoter of the Crhr1 gene. Meanwhile, we think that epigenetic modifications of the CRHR1 gene may be one reason for the CRHR1 expression in the CUMS model, and there may be any other regulatory pathways. More work need be further studied. Acknowledgments
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This study was supported by grants from the National Natural Science Foundation of China (30971040, 30900459, 81271496), the National Key Technology R&D Program during the 11th Five-Year of China (2007BAI17B05), the Nature Science Foundation of Hubei Province (2005ABA105) and the Youth Talent Foundation of Hubei Province Hygiene Department (QJX2008-23). We sincerely thank Dir. Wumin of the Life Sciences School of Wuhan University for technical advice. The funding sources played no role in the analysis and interpretation of data, in the writing of the report, or in the decision to submit the paper for publication.
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Genetic or epigenetic difference causing discordance between monozygotic twins as a clue to molecular basis of mental disorders. Mol Psychiatr 2005;7:622–30. [12] Weaver IC, Cervoni N, Champagne FA, D Alessio AC, Sharma S, Seckl JR. Epigenetic programming by maternal behavior. Nat Neurosci 2004;8:847–54. [13] Luo DD, An SC, Zhang X. Involvement of hippocampal serotonin and neuropeptide Y in depression induced by chronic unpredicted mild stress. Brain Res Bull 2008;77:8–12. [14] Harro J, Häidkind R, Harro M, Modiri AR, Gillberg PG, Pähkla R. Chronic mild unpredictable stress after noradrenergic denervation: attenuation of behavioural and biochemical effects of DSP-4 treatment. Eur Neuropsychopharmacol 1999;1:5–16. [15] Willner P, Towell A, Sampson D, Sophokleous S, Muscat R. Reduction of sucrose preference by chronic unpredictable mild stress and its restoration by a tricyclic antidepressant. Psychopharmacology 1987;3:358–64. [16] Tsankova NM, Kumar A, Nestler EJ. Histone modifications at gene promoter regions in rat hippocampus after acute and chronic electroconvulsive seizures. J Neurosci 2004;24:5603–10. [17] Willner P, Muscat R, Papp M. Chronic mild stress-induced anhedonia: a realistic animal model of depression. Neurosci Biobehav Rev 1992;4:525–34. [18] Holsboer F, Ising M. Central CRH system in depression and anxiety – evidence from clinical studies with CRH 1 receptor antagonists. Eur J Pharmacol 2008;583:350–7. [19] Tsankova NM, Berton O, Renthal W, Kumar A, Neve RL, Nestler EJ. Sustained hippocampal chromatin regulation in a mouse model of depression and antidepressant action. Nat Neurosci 2006;4:519–25. [20] Kumar A, Choi KH, Renthal W, Tsankova NM, Theobald DE, Truong HT. Chromatin remodeling is a key mechanism underlying cocaine-induced plasticity in striatum. Neuron 2005;2:303–14. [21] Wilkinson MB, Xiao G, Kumar A, LaPlant Q, Renthal W, Sikder D. Imipramine treatment and resiliency exhibit similar chromatin regulation in the mouse nucleus accumbens in depression models. J Neurosci 2009; 24:7820–32. [22] Tsankova N, Renthal W, Kumar A, Nestler EJ. Epigenetic regulation in psychiatric disorders. Nat Rev Neurosci 2007;5:355–67. [23] McGowan PO, Kato T. Epigenetics in mood disorders. Environ Health Prev Med 2008;1:16–24. [24] Wilkinson MB, Xiao G, Kumar A, LaPlant Q, Renthal W, Sikder D. Imipramine treatment and resiliency exhibit similar chromatin regulation in the mouse nucleus accumbens in depression models. J Neurosci 2009;24:7820–32. [25] Franklin TB, Russig H, Weiss IC, Gräff J, Linder N, Michalon A. Epigenetic transmission of the impact of early stress across generations. Biol Psychiat 2010;5:408–15. [26] Rasenick MM, Chaney KA, Chen J. G protein-mediated signal transduction as a target of antidepressant and antibipolar drug action: evidence from model systems. J Clin Psychiat 1996;13:49–55. [27] Forbes NF, Stewart CA, Matthews K, Reid IC. Chronic mild stress and sucrose consumption: validity as a model of depression. Physiol Behav 1996; 6:1481–4. [28] Nemeroff CB. The corticotropin-releasing factor (CRF) hypothesis of depression: new findings and new directions. Mol Psychiatr 1996;4:336–42. [29] Wasserman D, Sokolowski M, Rozanov V, Wasserman J. The CRHR1 gene: a marker for suicidality in depressed males exposed to low stress. Genes Brain Behav 2008;1:14–9. [30] Liu Z, Zhu F, Wang G, Xiao Z, Tang J, Liu W. Association study of corticotrophin-releasing hormone receptor1 gene polymorphisms and antidepressant response in major depressive disorder. Neurosci Lett 2007; 2:155–8.
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Research report
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Histone modifications of the Crhr1 gene in a rat model of depression following chronic stress
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Qirong Wan a , Kai Gao a , Han Rong a , Min Wu b , Huiling Wang a , Xiaoping Wang a , Gaohua Wang a,c,∗ , Zhongchun Liu a,c,∗ a
Department of Psychiatry, Renmin Hospital, Wuhan University, Jiefang Road 238#, Wuhan 430060, PR China College of Life Sciences, Wuhan University, Wuhan 430072, PR China c Institute of Neuropsychiatry, Wuhan University, Wuhan 430060, PR China b
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h i g h l i g h t s • We examine changes of CRHR1 expression in the hypothalamus of stressed rats. • Stress may change the level of H3K9 tri-methylation at CRHR1 gene promoter. • CRHR1 expression of stressed rats may correlate with H3k9 tri-methylation.
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Article history: Received 3 March 2014 Received in revised form 13 May 2014 Accepted 16 May 2014 Available online xxx
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Keywords: Depression CRHR1 Epigenetic regulation Hypothalamus Histone methylation
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1. Introduction
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Multiple lines of evidence suggest a link between depression and changes in hypothalamic-pituitaryadrenal (HPA)-axis hormone dynamics, including altered regulation of the corticotrophin-releasing hormone (CRH) and its main receptor, corticotrophin-releasing hormone receptor 1 (CRHR1). However, the precise molecular mechanisms underlying depression remain poorly understood. In this study, we employed a model of depression in rats by subjecting animals to 21 days of chronic unpredictable mild stress (CUMS). Real-time PCR and western blotting were used to study the mRNA and protein expression levels of CRHR1 in the hypothalamus. In addition, chromatin immunoprecipitation assays were used to detect histone methylation at the Crhr1 gene promoter; the levels of histone H3 trimethylation at lysines 4 (H3K4) and 9 (H3K9) reflect active transcription and transcriptional repression, respectively. Rats exposed to CUMS exhibited significant reduction in locomotion and sucrose preference. These behavioral alterations were associated with elevated expression levels of CRHR1 mRNA and protein in the hypothalamus of rats in the CUMS group. We also found that the levels of H3K9 trimethylation at the Crhr1 gene promoter in the CUMS group were significantly lower than those in the control group, whereas H3K4 trimethylation levels were the same for both groups. Taken together, our findings suggest that the increase in CRHR1 expression in the hypothalamus of stressed rats correlates with a decrease in the repressive chromatin state caused by reduced H3K9 trimethylation levels. These data are the first in vivo evidence of a role for chromatin modifications in the regulation of Crhr1 gene expression in the hypothalamus, and may provide novel insight into therapeutic approaches to treat depression. © 2014 Published by Elsevier B.V.
Major depression is a devastating illness characterized primarily by low mood, apathy, loss of appetite, loss of libido, and sometimes
∗ Corresponding authors at: Department of Psychiatry, Renmin Hospital, Wuhan University, Jiefang Road 238#, Wuhan 430060, PR China. Tel.: +86 88041911 81399; fax: +86 27 88072022. E-mail addresses: [email protected], [email protected] (G. Wang), [email protected] (Z. Liu).
by suicidal thoughts and high mortality and disability rates. Recent studies show that the incidence of depression is about 3.1% worldwide and 3% to 5% in China [1]. It has also been predicted that in 2020 depression will become the second-largest contributor to the global burden of disease [2]. Although depression is the most common psychiatric disorder, its pathophysiology is poorly understood and there is an urgent need to design new drugs for the treatment of this debilitating disease. The hypothalamus–pituitary–adrenal (HPA) axis plays an important role in the pathogenesis of depression. Several studies have shown that about 70% of depressed patients exhibit
http://dx.doi.org/10.1016/j.bbr.2014.05.031 0166-4328/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Wan Q, et al. Histone modifications of the Crhr1 gene in a rat model of depression following chronic stress. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.031
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dysfunction in the HPA axis [3]. When subjected to strong, persistent stimulation, the body’s basic response includes neuroendocrine changes, such as changes in the levels of corticotrophin-releasing hormone (CRH) and its main receptor, corticotrophin-releasing hormone receptor 1 (CRHR1). Depressed patients exhibit elevated concentrations of CRH in the blood, cerebrospinal fluid and urine [4,5]. CRHR1 belongs to the G-proteincoupled receptor family and is considered to play a pivotal role in mediating the CRH-elicited effects in depression and anxiety [6]. Using CRHR1 antagonists in patients with major depression and in a stress-related animal model of depression showed clear antidepressant and anxiolytic effect [7]. Consequently, the Crhr1 gene is a candidate gene for the study of antidepressant pharmacogenetics [8,9]. The Crhr1 gene is located on chromosome 17q21–22, spanning 20 kb of genomic DNA and containing 14 exons. It has three known isoforms arising from alternative splicing [10]. Epigenetics is the study of heritable changes in gene expression that occur without a change in DNA sequence. The main forms of epigenetic modifications are DNA methylation, histone acetylation and histone methylation. The epigenetic hypothesis suggests that these chromatin modifications may provide another pathogenic mechanism that is mediated by internal and external environmental factors (such as toxins, hormones, nutrition). The hypothesis helps to explain why the rate of various complex diseases increases significantly with age. It also explains the discordance in disease incidence among monozygotic twins [11]. Recent studies suggest that early life events can have long-term behavioral effects on individuals that are mediated by epigenetic mechanisms. High levels of pup licking, grooming and nursing behaviors exhibited by rat mothers correlated with a higher degree of methylation on some sites of the GR17 promoter region and NGFI-A binding sequence in their offspring, compared to offspring of mothers that exhibited low levels of these behaviors. These differences appear in the first week after birth and may continue into adulthood [12]. However, it is too early to confirm that epigenetic mechanisms play a decisive role in the onset of depression. Further exploration of depression-related genes, brain regions, neural circuits and their interaction is the future direction of this field of research. Here, we use the chronic unpredictable mild stress (CUMS) model of depression in rats to study the epigenetic mechanisms that mediate changes in Crhr1 gene expression in the hypothalamus, with the aim of providing the experimental and theoretical basis for designing related drugs for the treatment of depression. 2. Materials and methods 2.1. Animals 30 adult male Sprague-Dawley rats (weighing 190–210 g) were obtained from the Limited Company of Hunan Slack King Experimental Animals. One week before testing, all animals were housed in groups of four in a room with standard laboratory conditions (12/12-h light/dark cycle: lights on 8:00, off 20:00, 22 ± 2 ◦ C, food and water ad libitum). All animal experiments were carried out in accordance with the instructions for laboratory animal care issued by the Ministry of Science and Technology of the People’s Republic of China in 2006. 2.2. Chronic unpredictable mild stress We employed the CUMS model of depression according to the procedure described by Luo et al. [13] with some modification. The stressors included cage tilting (45◦ ) for 24 h, damp sawdust (200 ml water in a cage) for 24 h, swimming in 25 ◦ C water for 15 min, 24 h
of food deprivation, 24 h of water deprivation, nip tail for 1 min, shaking for 15 min (120 rpm rocking bed), immobilizing for 1 h in a 25 cm × 8 cm cylindrical plastic rodent restrainer, and alterations of the light/dark cycle with one period of continuous overnight illumination. Animals were individually exposed to stressors for 3 weeks. Control animals with free access to water and food were undisturbed. 2.3. Sucrose preference test The sucrose preference test was used to assess anhedonia on the 1st and 22nd day. All rats were fed 7 days to adapt to drinking sucrose. Training consisted of initial 48 h sucrose solution exposure without any other food or water available, and five additional consecutive 1 h periods per day of sucrose availability [14]. As described by Willner et al. [15], before each test, the animals were deprived of water for 23 h. Rats were individually exposed to a bottle of 1% sucrose solution and a bottle of water for 1 h and consumption of sucrose intake and water intake were measured by comparing the weight of the bottle before and after the test. Sucrose preference (%) = (sucrose intake/total fluid intake) × 100%. 2.4. Open field test The rats were placed in the center of a dimly illuminated rectangular cage (120 cm × 90 cm × 35 cm) and observed for 10 min. Locomotor activity was measured using an automated video tracking system (Ethovision 3.0, Noldus, the Netherlands). The frequency of rearing (standing upright on the hind legs, while forepaws were free) was recorded manually. The cage was cleaned thoroughly before the next animal was tested, and the room was kept quiet. 2.5. Measuring mRNA levels by real-time PCR Rats were sacrificed by cervical dislocation after the open field test. The hypothalamus was collected quickly for RNA quantification. RNA was extracted using a commercial kit (Yuanping Hao, China) and mRNA was reverse transcribed to cDNA using a first-strand synthesis kit (Tiangen, China). The amount of cDNA was quantified using real-time PCR. The following primers were used to amplify specific cDNA regions of the transcripts of interest: -actin (5 -cgttgacatccgtaaagacctc-3 and 5 -taggagccagggcagtaatct-3 ) and CRHR1 (5 -gctttcatcctacgcaacg3 , and 5 -tagcagccctcaccgaac-3 ). -Actin quantification was used as an internal control for normalization. Fold differences of mRNA levels over control values were calculated using the Ct method (Applied Biosystems manual). PCRs were run in triplicate for each brain sample, and at least three independent sample pairs were used for each statistical analysis. 2.6. Western blot analysis of CRHR1 protein levels The hypothalamus was dissected and directly transferred into RIPA buffer (50 mM Tris–HCl pH 8.0, 50 mM HEPES pH 8.0, 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 0.5 mM PMSF, 1 g/ml aprotinin, 1 g/ml leupeptin, 1 g/ml pepstatin, 1 mM EDTA, 10 M Na3VO3, 10 M NaF). Tissues were homogenized with a glass homogenizer. Supernatant was collected after centrifugation at 800 × g for 5 min at 4 ◦ C. Protein concentration was measured using BCA protein assay reagents (Thermo; Reagent A: 2 ml, Reagent B: 40 l, 1× PBS: 95 l, Protein: 5 l). Equal amounts of protein were loaded into SDS-PAGE gels (Invitrogen) and electrophoresis was performed at 200 V for 50 min. Proteins were transferred to Polyvinylidene Fluoride (PVDF) membrane (Invitrogen) and the membrane was blocked with 5% non-fat dry milk in Phosphate Buffered Saline Tween-20 (PBST). The membrane
Please cite this article in press as: Wan Q, et al. Histone modifications of the Crhr1 gene in a rat model of depression following chronic stress. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.031
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Fig. 1. Effects of CUMS on sucrose preference (a) and open field test (b, c, d) in rats. Data are represented as the means ± S.D. **P < 0.01. (a) 1 h 1% sucrose consumption of rats after 21 days of exposure to stress. (b) Total distance traveled by rats in the open field test. (c) Velocity of rats in the open field test. (d) Frequencies of rearing of rats in the open field test.
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was incubated with primary goat polyclonal CRHR1 (Santa Cruz) antibodies (1:1000 dilution) and rabbit actin (Sigma) antibodies (1:1000 dilution), overnight at 4 ◦ C on a rocking platform. The blotting membrane was washed with PBST and incubated with secondary antibody (1:5000 dilution) for 1 h at room temperature on a rocking platform. The membrane was washed with PBST, developed using Chemiluminescent HRP substrate following the manufacturer’s instructions (ECL: A + B equal volume in RT) and exposed to X-ray film for an appropriate time period. Band densities were measured using Kodak 1D Scientific Imaging System and Image software.
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2.7. Chromatin immunoprecipitation assays (CHIP)
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Chromatin was extracted from hypothalamic tissue by following published protocols with minor modifications [16]. Whole hypothalamus was minced to 1 mm-sized pieces, and immediately crosslinked in 270 l 37% formaldehyde for 15 min at room temperature. We added glycine to a final concentration of 0.125 M to stop the crosslinking reaction while shaking for 10 min at 4 ◦ C. The tissue was washed three times in cold 1× PBS containing protease inhibitors (1 mM PMSF, 1 l/ml apoprotin, and 1 l/ml leupeptin). Tissue was homogenized in 1 ml cell lysis buffer (10 mM Tris, 10 mM Nacl, 0.2% NP-40, PMSF, apoprotin, leupeptin), and incubated on ice for 15 min, followed by centrifugation for 5 min at 5000 rpm at 4 ◦ C. The supernatant was discarded and the pellet was incubated in 850 l nuclear lysis buffer (50 mM Tris–Cl, pH 8.1, 10 mM EDTA, 1% SDS, PMSF, apoprotin, leupeptin) for 30 min
on ice. Next, the extracted chromatin was sheared to 300–500 bp using the Xinzhi Dismembrator 550 (Ningbo, China). Each sample was sonicated for 8 min on ice, at 40% of maximum power, with a 1 min pause every 2 min, and the degree of DNA fragmentation was observed. The chromatin solution was pre-cleared with protein G (GE, USA) for 2 h. It was then immunoprecipitated overnight at 4 ◦ C with 4 g of antibodies directed against H3 methylated on Lys4 (Millipore, USA) or Lys9 (Millipore, USA). As a control, samples were immunoprecipitated with 4 g non-immune rabbit IgG and rabbit polyclonal anti-histone H3 antibodies (Abcam, England). The beads were sequentially washed once with low salt, high salt, and LiCl buffers and washed twice with 1× TE. The DNA–histone complex was then eluted from the beads with elution buffer and protein K. DNA and histones were dissociated overnight at 65 ◦ C. DNA was purified using a commercial kit (Shenggong, China) and quantified using real-time PCR. The levels of specific histone modifications at the gene promoter were determined by measuring the amount of DNA associated with methylated histone by quantitative real-time PCR (ABI Prism 7500; Applied Biosystems, Foster City, CA). Specific primers were designed to amplify proximal promoter regions (5 -ccgctgtctccacttatctt-3 and 5 -tccctcgttcgttcactcat-3 ). Tubulin (5 -tagaaccttcctgcggtcgt-3 and 5 -ttttcttctgggctggtctc-3 ), and globin (5 -tgaccaatagtctcggagtcctg3 and 5 -aggctgaaggcctgtccttt-3 ) were used as controls. Input and immunoprecipitated DNA amplification reactions were run in triplicate in the presence of SYBRGreen (Ferments, Canada), and at least three independent sample pairs were used for each statistical analysis. The comparative CT values were used to determine relative
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Fig. 2. The increase of CRHR1 mRNA expression was induced by chronic unpredictable mild stress in the hypothalamus. CUMS-exposed rats were subjected to various stressors for 21 days. mRNA levels of CRHR1 was assayed by RT-PCR in the hypothalamus, values are means ± S.D. (N = 4 in each group). Expression of hypothalamic CRHR1 mRNA in CUMS group was significantly increased compared to the control group (P < 0.01).
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expression compared to input or total histone H3. Fold differences (depression group ChIP relative to control group ChIP) were then determined by raising 2 to the Ct power.
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Values reported in the text and figures are represented as mean ± S.D. The significance of the differences between groups was determined using one-way ANOVA. P-value < 0.05 was considered to indicate statistical significance.
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depression. Stressors were introduced to the animals in an unpredictable order and repeated throughout the experiment. The rats in the control group were housed in normal conditions without any disturbances. The behavioral effects of CUMS were examined, and the data are shown in Fig. 1: sucrose preference (Fig. 1a) and the indices of open field test, including total distance moved (Fig. 1b), velocity (Fig. 1c), and frequency of rearing (Fig. 1d). In our study, there was no statistical difference between the two groups before CUMS (data not shown). At the end of CUMS, the rats displayed a significant reduction in the sucrose preference compared with control rats (mean ± S.D., 84.53 ± 4.95 for control group, 60.07 ± 9.86 for CUMS group, P < 0.01). Moreover, stressors had significantly different effects on the performance of the two groups in the open field test: total moved distance (1949.37 ± 164.44 cm and 1081.06 ± 117.50 cm, respectively for control group and CUMS group, P < 0.01) and velocity (4.13 ± 0.46 cm/s and 2.09 ± 0.45 cm/s, respectively for control group and CUMS group, P < 0.01). There was also a significant difference in the frequency of rearing between CUMS group (3.12 ± 1.83) and control group (12.60 ± 2.58) (P < 0.01). Reduced sucrose consumption implies animals exposed to CUMS had decreased responsiveness to rewarding stimuli. The results of the open field test suggest that animals exposed to CUMS exhibit reduced activity.
3.2. CRHR1 mRNA expression Based on strong evidence implicating CRHR1 in the pathophysiology of depression and anxiety [18], we performed RT-PCR analysis to investigate the level of CRHR1 mRNA expression in the hypothalamus after CUMS. As shown in Fig. 2, one-way ANOVA of the RT-PCR data revealed that mRNA expression of hypothalamic CRHR1 in CUMS group was elevated when compared to the control group.
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3.1. Effects of CUMS on sucrose preference tests and open field tests Many studies have provided evidence that the CUMS model simulates the core symptom of human depression: anhedonia [17]. The sucrose preference test is used to validate animal models of
A representative photomicrograph is shown in Fig. 3a to illustrate CRHR1 protein analysis using Western blot. Quantification of these data showed that the levels of CRHR1 in the CUMS group were significantly higher than those in the control group (Fig. 3b, P < 0.05).
Fig. 3. Gray-scale value of hypothalamic CRHR1 in control group and CUMS group (P < 0.05). Representative western blot bands (a) and densitometric analyses of the bands (b). Data are represented as the mean ± S.D. *P < 0.01 (one-way ANOVA).
Please cite this article in press as: Wan Q, et al. Histone modifications of the Crhr1 gene in a rat model of depression following chronic stress. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.031
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Fig. 4. CHIP assay to quantify the abundance of H3 lysine-4-trimethylation, and H3 lysine-9-trimethylation on CRHR1 gene promoter in hypothalamus after 21 days chronic unpredictable mild stress. (a) The level of histone H3K4 tri-methylation between two groups was not significantly different (P > 0.05). (b) Relative fold enrichment of histone H3K9 tri-methylation at CRHR1 gene promoters in CUMS rats versus control rats, demonstrating that CUMS leads to a significant decrease of H3K9 tri-methylation. *P < 0.05.
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3.4. Quantification of stress-induced histone modifications at the Crhr1 promoter by CHIP assays Histone modifications have recently been associated with depression-like behavior [19]. To understand better the molecular mechanisms of depression, we studied histone modifications at the promoter of the Crhr1 gene in the rat hypothalamus after 21 days of exposure to chronic mild stress. Our data show that CUMS did not significantly alter the levels of trimethylated histone H3K4, which is implicated in the activation of gene expression. Meanwhile, we investigated the effect of CUMS on the methylation of histone H3K9, a marker of a silenced chromatin state. Consistent with the increase in expression of the of Crhr1 gene observed after CUMS, the levels of histone H3K9 tri-methylation were significantly reduced at the Crhr1 promoter in CUMS rats compared to control rats (Fig. 4b). Taken together, these data suggest that CUMS induces a remarkable increase in Crhr1 expression in the hypothalamus, which correlates with a significant decrease in H3-trimk9 levels at the Crhr1 promoter.
4. Discussion The importance of epigenetic mechanisms, shown in recent years to be a major determinant of gene regulation, has received much attention in the study of depression. Many studies suggest that the long-term regulation of gene expression through epigenetic mechanisms can mediate permanent changes in brain function and contribute to the pathogenesis of psychiatric disorders [20]. This conclusion has been confirmed in models of seizures [21], psychostimulant treatment [20], and depression [22–24]. Among many epigenetic modifications, DNA methylation of CpG dinucleotides, acetylation and methylation at the N-terminal tails of histones are the most prevalently studied modifications in molecular analyses of behavioral models [25]. The goal of the present study was to determine whether the expression of Crhr1 was regulated by chromatin modification in the CUMS model. The results showed that the exposure of rats to chronic mild stress can lead to depressive behavior that is associated with changes of CRHR1 expression in the hypothalamus. They further show that these changes in gene expression are mediated, at least partially, through chromatin modifications at the Crhr1 promoter. Depression is a common and persistent mood disorder characterized by despair, helplessness and social withdrawal. We used 21 days of CUMS to build a classical model of depression. Its theoretical basis was consistent with the occurrence and course of
exacerbation in depressed patients. Rasenick et al. [26] have suggested that depression in such a model was the result of many stress factors. Sucrose preference and open-field tests are regarded as useful and valid behavioral markers of depression in an animal paradigm. Anhedonia – the inability to enjoy pleasurable stimuli – is another symptom commonly associated with depression in humans. In our experiment, sucrose preference in the CUMS group was significantly reduced, compared to the control group. We presume that there may be three reasons for this reduction: (1) chronic stress damages nerve cells in the neural reward system and induces the reduced ability to experience happiness; (2) sucrose preference was decreased because the animals were tired after being deprived of food, water and subjected to other stressors; and (3) the decline in sucrose consumption was induced by metabolic changes resulting from fasting [27]. Furthermore, the results of the open-field test provided a measure of exploratory behavior and emotional responses in new environments. Our current data confirm that the CUMS group exhibited decreased distance moved, and slower velocity. The results indicate that stressed rats are less active. In addition, the frequency of rearing in the CUMS group was decreased, as compared to the control group. Decreased rearing frequency indicates that the exposure of rats to stress decreased their curiosity in a novel environment. In summary, the behavioral defects induced by CUMS, including decreased activity and waned interest, are reminiscent of several depressive-like symptoms in humans. We hypothesize that these behavioral changes are accompanied by changes in histone modifications at the promoter of the Crhr1 gene in the hypothalamus. Nestler and coworkers [16,19] have shown associations between histone modifications and changes in behavioral function in response to antidepressant treatment and chronic electroconvulsive seizures in rodents. Over the past 40 years, the most consistent physiological association in depression has been hyperactivity of the HPA axis [28]. However, it is still debatable whether the increased activity of the HPA axis is caused by depression, or itself is involved in the development of depression. Wasserman et al. [29] suggested that the Crhr1 gene was a marker for suicide in depressed males exposed to low stress. In our previous study, we tested whether the polymorphisms of three sites (rs1876828, rs242939 and rs242941) in the Crhr1 gene are related to the effect of 6 weeks of fluoxetine treatment in 127 Han Chinese patients with major depressive disorders (MDD). The results show that the rs242941G/G genotype and homozygous GAG haplotype of the three single-nucleotide polymorphisms are associated with the therapeutic response to fluoxetine in MDD patients with high-anxiety. However, there are several limitations in this study [30]. The potential pathological mechanisms of
Please cite this article in press as: Wan Q, et al. Histone modifications of the Crhr1 gene in a rat model of depression following chronic stress. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.031
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depression need to be further investigated, particularly the interaction between the Crhr1 gene and environmental factors, to explain the pathogenesis of depression. Our experiments show a marked increase in CRHR1 mRNA and protein expression in the hypothalamus of CUMS-exposed rats. The correlation between hypothalamus CRHR1 levels and behavioral response in our model further emphasizes the role of CRHR1 in the pathophysiology of mood disorders. Our findings also suggest that the levels of CRHR1 expression may be related to the changes in histone modifications. In recent years, epigenetics has become a popular field of investigation in translational medical research. Specifically, there has been greater effort in psychiatric research to explore the impact of DNA methylation and histone modifications on disease. Histone modifications affect the structural dynamics of the nucleosome that regulates chromatin regulation of gene expression through the recruitment of specific interacting proteins that recognize a single or conformational set of modifications. H3K4 methylation is considered to be associated with activation of gene expression, and H3K9 methylation is associated with gene repression. Our experiments show that CUMS induces a significant decrease in H3K9 trimethylation, concomitant with enhanced expression of the Crhr1 gene. Such long-lived changes in chromatin modifications may be one of the crucial mechanisms for stress-induced neuroendocrine changes in the hypothalamus. Recently, many researchers focused on chromatin remodeling that is associated with depression, in different brain regions and at different genes. Tsankova et al. presented findings to suggest that stress caused by chronic social defeat alters chromatin regulation of the Brain-derived neurotrophic factor (Bdnf) gene. They showed long-lasting increases in H3K27 dimethylation, a repressive modification, specifically at the promoter of the down-regulated Bdnf gene [22]. Chronic imipramine treatment was shown to reverse the repression of the Bdnf gene by inducing H3 acetylation, as well as H3K4 methylation [21]. These results are consistent with the histone code hypothesis: a combination of several histone modifications may ultimately determine the outcome of gene expression. Researchers believe that a variety of epigenetic mechanisms can be involved simultaneously in the regulation of gene expression. Taken together, our data suggest that CUMS induces a long-lasting increase in CRHR1 expression in the hypothalamus, which may correlates with a significant decrease of repressive chromatin state produced by the reduced H3k9 trimethylation levels at promoter of the Crhr1 gene. Meanwhile, we think that epigenetic modifications of the CRHR1 gene may be one reason for the CRHR1 expression in the CUMS model, and there may be any other regulatory pathways. More work need be further studied. Acknowledgments
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This study was supported by grants from the National Natural Science Foundation of China (30971040, 30900459, 81271496), the National Key Technology R&D Program during the 11th Five-Year of China (2007BAI17B05), the Nature Science Foundation of Hubei Province (2005ABA105) and the Youth Talent Foundation of Hubei Province Hygiene Department (QJX2008-23). We sincerely thank Dir. Wumin of the Life Sciences School of Wuhan University for technical advice. The funding sources played no role in the analysis and interpretation of data, in the writing of the report, or in the decision to submit the paper for publication.
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