Chronic corticosterone disrupts the circadian rhythm of CRH expression and m6A RNA methylation in the chicken hypothalamus

Background Corticotropin-releasing hormone (CRH), the major secretagogue of the hypothalamic-pituitary-adrenal (HPA) axis, is intricately intertwined with the clock genes to regulate the circadian rhythm of various body functions. N6-methyladenosine (m6A) RNA methylation is involved in the regulation of circadian rhythm, yet it remains unknown whether CRH expression and m6A modification oscillate with the clock genes in chicken hypothalamus and how the circadian rhythms change under chronic stress. Results Chronic exposure to corticosterone (CORT) eliminated the diurnal patterns of plasma CORT and melatonin levels in the chicken. The circadian rhythms of clock genes in hippocampus, hypothalamus and pituitary are all disturbed to different extent in CORT-treated chickens. The most striking changes occur in hypothalamus in which the diurnal fluctuation of CRH mRNA is flattened, together with mRNA of other feeding-related neuropeptides. Interestingly, hypothalamic m6A level oscillates in an opposite pattern to CRH mRNA, with lowest m6A level after midnight (ZT18) corresponding to the peak of CRH mRNA before dawn (ZT22). CORT diminished the circadian rhythm of m6A methylation with significantly increased level at night. Further site-specific m6A analysis on 3’UTR of CRH mRNA indicates that higher m6A on 3’UTR of CRH mRNA coincides with lower CRH mRNA at night (ZT18 and ZT22). Conclusions Our results indicate that chronic stress disrupts the circadian rhythms of CRH expression in hypothalamus, leading to dysfunction of HPA axis in the chicken. RNA m6A modification is involved in the regulation of circadian rhythms in chicken hypothalamus under both basal and chronic stress conditions. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s40104-022-00677-4.


Introduction
The hypothalamus plays an important role in the regulation of hypothalamic-pituitary-adrenal (HPA) axis [1], feeding behavior [2], and circadian rhythm [3]. Corticotropin-releasing hormone (CRH) released from hypothalamus stimulates pituitary ACTH secretion to modulate the activity of HPA axis [4]. Moreover, CRH is involved in the regulation of food intake [5] via interacting with appetite inhibiting proopiomelanocortin (POMC)/cocaine amphetamine-regulated transcript (CART) neurons and the appetite-inducing neuropeptide Y (NPY) and agouti-related protein (AgRP) neurons [6]. Both the HPA axis activity and the feeding behavior exhibit diurnal patterns, which indicates complex interactive networks with the master clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus [7]. SCN can projects to the pineal gland that secretes the hormone melatonin [8]. The core molecular clock consists of a transcriptional-translational autoregulatory "loop" with a positive arm and a negative arm [9]. The clock and bmal1 genes and their protein products comprise the positive arm, while the period (PER1, PER2, and PER3) and cryptochrome (CRY1, CRY2) genes and their protein products comprise the negative arm. An early research reported that the HPA system in the chicken displays a circadian rhythm [10]. Studies in mice indicate that CRH is intricately intertwined with the clock genes to regulate the circadian rhythm of various body functions [11]. However, the circadian rhythm of CRH expression in chicken hypothalamus has not been characterized.
CRH binds to CRH receptors type 1 (CRHR1) and type 2 (CRHR2) in the pituitary, causing the production and secretion of adrenocorticotropic hormone (ACTH) [12] to regulate the stress response of the body through corticosterone (CORT) synthesis and secretion from adrenal cortex [13,14]. CORT exerts a negative feedback regulation on CRH synthesis and secretion through its receptor, glucocorticoid receptor (GR), at different levels including hippocampus and hypothalamus [15]. Chronically elevated circulating CORT has detrimental physiological and cognitive effects [16], including HPA axis dysfunction and neuroinflammation [17], as well as depressive and anxiety-like behaviors in SD rats [18]. In addition, chronic stress causes irregular expression of circadian regulatory clock genes in mouse hippocampus [19], hypothalamus SCN [20] and pituitary [21]. However, it remains unknown how chronic CORT exposure affects the circadian rhythms of clock-related genes in the chicken brain, and how it is related to the circadian rhythm of CRH in hypothalamus. N 6 -methyladenosine (m 6 A) is the most prevalent modification in RNAs, which plays an important role in RNA splicing, degradation, and translation [22,23]. M 6 A level is finely balanced through interplay among m 6 A methyltransferases ("writers", such as METTL3, METTL14 and WTAP), demethylases ("erasers", such as fat mass and obesity-associated gene FTO and ALKBH5), and binding proteins ("readers", such as YTHDF1, YTHDF2 and YTHDF3) [24]. Chronic stress is reported to modulate m 6 A modification in the brain [25]. For instance, heat exposure for 6 h increases m 6 A RNA methylation levels in the hypothalamus of 3-day-old chickens [26]. Yet, chronic CORT treatment reduces the m 6 A methylation in chicken liver [27]. Moreover, m 6 A methylation has been reported to have circadian rhythm [28]. Clock gene CRY1/2 knockout mice show significantly lower m 6 A level and lost the circadian rhythm of m 6 A level in RNA [28]. However, studies in the chicken are scarce. Questions remain regarding whether m 6 A modification in chicken hypothalamus show a circadian rhythm, whether the m 6 A rhythmicity, if any, is interrupted by chronic CORT exposure, and whether m 6 A is involved in the regulation of CRH expression in chicken hypothalamus.
Therefore, the objectives of the present study were, firstly, to elaborate the effects of chronic CORT exposure on circadian rhythms of clock-related genes in different brain areas including hippocampus, hypothalamus and pituitary; secondly, to delineate the circadian rhythms of CRH mRNA expression and m 6 A methylation in chicken hypothalamus, and to reveal their responses to chronic CORT exposure; and thirdly, to investigate the possible link between m 6 A modification and CRH mRNA expression in chicken hypothalamus.

Ethics statement
The experimental protocol was approved by the Animal Ethics Committee of Nanjing Agricultural University. The project number is 31972638. The sampling procedures complied with the "Guidelines on Ethical Treatment of Experimental Animals" (2006) No.398 set by the Ministry of Science and Technology, China.

Animals and experimental design
Seventy 45-day-old male bantam chickens were purchased from Changzhou Lihua Livestock and Poultry Co., Ltd. After a three-day adaption, chickens were randomly divided into vehicle (CON) and corticosterone (CORT) group. Light regime was 12 light: 12 dark, with light on at 07:00 as zeitgeber time 0 (ZT0) and off at 19:00 as ZT12. Food and water were provided ad libitum. CORT (Sigma-Aldrich, St Louis, USA) was sonicated in saline with 0.1% Tween 80 and 0.2% DMSO until dissolved and protected from light. Chickens were injected (twice per day, 9:00-10:00 and 18:00-19:00) intraperitoneally with vehicle or CORT (4 mg/kg BW), according to previous publication [29], for 11 consecutive days. Daily food consumption and body weight were recorded every other day. By the end of the treatment, the chickens were sacrificed at the indicated time points (ZT2, ZT6, ZT10, ZT14, ZT18 and ZT22). Chickens were anesthetized with sodium pentobarbital and the brain was quickly separated from the skull. Hippocampus [30] and hypothalamus [31] were dissected as described in previous publications according to the chicken brain atlas [32]. Pituitary was removed as previously described [33]. Tissues collected were frozen immediately in liquid nitrogen and stored at − 80°C until use.

RNA isolation and real-time PCR
High quality total RNA was isolated from hippocampus, hypothalamus and pituitary using Trizol reagents (Invitrogen, Carlsbad, CA, USA). One microgram of RNA was reverse-transcribed according to the manufacturer's protocol (Vazyme Biotech, Nanjing, Jiangsu, China). Four microliter cDNA was diluted (1:25) and then used for real-time PCR in a QuantStudioTM 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Peptidylprolyl isomerase A (PPIA) was used as an internal control to normalize the technical variations. Data were analyzed using the method of 2 -ΔΔCT and presented relative to the CON group. All primers ( Table 1) were synthesized by Suzhou GENEWIZ Biological Technology Co., Ltd. (Suzhou, Jiangsu, China).

Analysis of mRNA m 6 A methylation by dot-blotting assay
Dot-blot analysis of mRNA m 6 A methylation was performed following a published procedure with minor modifications [34]. Briefly, total RNAs were isolated using the Trizol method and mRNAs were enriched by using GenElute™ mRNA Miniprep Kit (Sigma, Burlington, NJ, USA). The concentration and purity of mRNAs were measured by NanoDrop 2000. The mRNAs were denatured by heating at 95°C for 5 min, followed by chilling on ice immediately. Next, the mRNA (100 ng) was spotted directly onto the positively charged nylon membrane (GE Healthcare, Pittsburgh, PA, USA) and air dried for 5 min.
The membrane was then UV crosslinked in Ultraviolet Crosslinker, blocked with 5% of nonfat milk in TBST, and then incubated with anti-m 6 A antibody overnight at 4°C. HRP-conjugated anti-rabbit IgG secondary antibody was added to the membrane for 2 h at room temperature with gentle shaking and then developed with enhanced chemiluminescence. Methylene blue staining was used to verify that equal amount mRNA spotted on the membrane.

Single-base elongation and ligation-based qPCR amplification method (SELECT) assay
The SELECT assay for monitoring site-specific m 6 A levels in the 3′UTR of CRH mRNA was performed as described previously [35]. In brief, total RNA (2 μg) was mixed with 1 μL of 100 μmol/L dNTP (NEB, Ipswich, MA, USA), 2 μL of CutSmart buffer (NEB, Ipswich, MA, USA), and 2 μL each of 400 nmol/L up and down DNA probes ( Table 1). The total volume was adjusted to 17 μL with water. The DNA probes and RNA were annealed by incubating the mixture with a temperature gradient of 90°C for 1 min, 80°C for 1 min, 70°C for 1 min, 60°C for 1 min, 50°C for 1 min, and 40°C for 6 min. To the mixture was then added a 3 μL solution containing 0.01 U Bst 2.0 DNA polymerase, 0.5 U SplintR ligase, and 10 nmol ATP. After incubating at 40°C for 20 min and then at 80°C for 20 min, an aliquot (2 μL) of the reaction mixture was taken out for real-time qPCR analysis to quantify template abundance.

CORT ND ND
Values are means ± SEM. **P < 0.01, compared with CON group. ND represents not determined as there was no circadian rhythm

Statistical analysis
Data are presented as the mean ± standard error of the mean (SEM). The mRNA levels of clock-related genes and melatonin contents were analyzed using one-way analysis of variance (one-way ANOVA) with IBM SPSS Statistics 20 software (United States) to test the statistical significance of the differences among the six daily time points and confirm the daily variation (P ≤ 0.05), as the premise of cosinor analysis. To determine the circadian rhythmicity of each clockrelated gene profile, the mRNA levels of clock-related genes, as well as CORT and melatonin levels were analyzed separately using MATLAB 7.0 (MathWorks Inc., USA) based on unimodal cosinor regression [y = A + (B × cos (2π(x − C)/ 24))]. A, B and C represent the mesor, amplitude and acrophase, respectively. The results of regression analysis were considered significant at P ≤ 0.05, which was calculated using the number of samples, R 2 values and the number of predictors (mesor, amplitude and acrophase) from http:// www.danielsoper.com/statcalc3/calc.aspx?i1/415 [36]. Differences of the mesor, amplitude and acrophase between CON and CORT group were tested by one-way ANOVA followed by Fisher's least significant difference (LSD) post hoc test, considering P ≤ 0.05 to be significant.

Results
Effect of chronic CORT exposure on body weight, food intake, plasma CORT and melatonin concentration Chronic CORT exposure leads to growth retardation, with significantly lower body weight, as compared with their control counterparts, from the 5th day of CORT injection (D5) to D11 (Fig.1A). Interestingly, the feed intake was significantly increased on D3, D4 and D7, leading to significantly increased average daily feed intake (Fig.1B). Both CORT (Fig. 1C) and melatonin (Fig. 1D)  Values are means ± SEM. *P < 0.05, **P < 0.01, compared with CON group. ND represents not determined as there was no circadian rhythm levels in plasma exhibited diurnal pattern in CON group (P < 0.05, one-way ANOVA), which was eliminated in CORT group. The mesors of CORT level were significantly elevated (P < 0.01) by CORT injection, while the mesors of melatonin level did not change ( Table 2).

Effect of chronic CORT exposure on the circadian rhythm parameters of CRH in hypothalamus and CRH receptor genes in pituitary
In line with the abolished rhythmicity of clock genes in hypothalamus, the circadian pattern of CRH mRNA (Fig. 3A) in hypothalamus was significantly diminished in CORT group, so was the rhythmic expression of CRHR1 (Fig. 3B) and CRHR2 (Fig. 3C) mRNA in pituitary (P < 0.05, one-way ANOVA). Chronic CORT exposure significantly decreased the mesor (P < 0.05) and amplitude (P < 0.01) of CRH mRNA in hypothalamus, as well as CRHR1 and CRHR2 mRNA in pituitary (Table 6). In general, chronic CORT exposure significantly abolished (P < 0.05) the rise of CRH (Fig.  3A), CRHR1 (Fig. 3B) and CRHR2 (Fig. 3C) mRNA expression in the dark phase after midnight at ZT18 and ZT22.  Values are means ± SEM. *P < 0.05, compared with CON group. ND represents not determined as there was no circadian rhythm

Effect of chronic CORT exposure on the circadian rhythm parameters of feeding and inflammation-related genes in hypothalamus
In accordance with the alterations of CRH mRNA, the diurnal patterns of hypothalamic NPY (Fig. 4A), AGRP (Fig. 4B), POMC (Fig. 4C) and CART (Fig.  4D) RNA expression were also eliminated in CORT group (P < 0.05, one-way ANOVA). The expression pattern of "the hunger genes" NPY and AGRP were opposite to that of the "the satiety genes" POMC and CART, matching the diurnal pattern of feeding behavior in the chicken. Chronic CORT exposure significantly decreased (P < 0.01) the mesor and amplitude of all the 4 feeding regulatory genes in hypothalamus (Table 7). In addition, chronic CORT exposure significantly increased (P < 0.01) the hypothalamic expression of TNF-α, IL-1β and IL-6 mRNA, among which the circadian rhythms of TNFα and IL-6 mRNA was diminished (Additional file 1: Fig. S1).

Effect of chronic CORT exposure on the circadian rhythm parameters of m 6 A level and m 6 A related genes in hypothalamus
Interestingly, the global RNA m 6 A levels (Fig. 5A) exhibited diurnal pattern in CON group (P < 0.05, one-way ANOVA), higher m 6 A levels were detected in light phase. Chronic CORT exposure significantly disrupted this pattern with significantly decreased (P < 0.05) m 6 A levels in light phase at ZT6 and ZT10, but significantly increased (P < 0.05) m 6 A levels in dark phase at ZT14, ZT18 and ZT22. Meanwhile, chronic CORT exposure significantly (P < 0.01) decreased the amplitude of m 6 A levels and delayed the acrophase of m 6 A levels for 13.48 h (Table 8). Concurrently, chronic CORT exposure significantly increased (P < 0.05) the mesor of FTO (Fig. 5B) mRNA and decreased (P < 0.05) the mesor of YTHDF2 (Fig. 5F) and YTHDF3 (Fig. 5G) mRNA in hypothalamus ( Table 8).
Effect of chronic CORT exposure on the site-specific m 6 A levels in the 3'UTR of CRH mRNA in hypothalamus To explore the possible link between the site-specific m 6 A modification on CRH mRNA and CRH mRNA expression in hypothalamus, RNA samples from hypothalamus on ZT18 and ZT22 with significant changes in CRH mRNA were subjected to single-base elongation and ligation-based qPCR amplification method (SE-LECT) assay. Two specific m 6 A sites (Fig. 6A) were identified in the coding sequence (CDS) close to 3'UTR (X1) and 3'UTR (X2) of CRH mRNA, respectively, from published MeRIP-seq database [27]. N site located in the 5'UTR without consensus m 6 A motif was selected as a negative control. Chronic CORT exposure did not change the CT value on N site at either ZT 18 (Fig. 6B) or ZT 22 (Fig. 6E), compared with CON group. However, chronic CORT exposure significantly increased (P < 0.05) the CT value on both X1 (Fig. 6C, F) and X2 (Fig. 6D, G) at both time points (ZT18 and ZT22), which

CORT ND ND ND
Values are means ± SEM. *P < 0.05, **P < 0.01, compared with CON group. ND represents not determined as there was no circadian rhythm was in accordance with the significant decrease of CRH mRNA in hypothalamus at the same time points.

Discussion
In this study, we observed that chronic CORT exposure completely abolished the circadian rhythm of plasma melatonin levels in the chicken, indicating a disruption of the endogenous rhythmicity. The effects of CORT on plasma melatonin are biphasic, being stimulatory in the light phase when the melatonin levels are low, while inhibitory in the dark phase when the melatonin levels are high. The avian pineal gland receives circadian input through the release of norepinephrine during the day [37], and the dual effects of CORT on pineal melatonin synthesis are determined by the activation of different adrenoceptors (β or β + α1) during GR activation [38].
The circadian rhythms in birds are controlled by multiple circadian pacemakers in the central nervous system. Here we show, for the first time, the circadian expression of clock genes in chicken hippocampus, hypothalamus, and pituitary. All the 6 core clock genes show circadian rhythms in all the 3 brain areas, although the amplitude and the pattern of oscillation vary among genes and brain areas. It is noted that BMAL1 oscillates in an opposite pattern from PER2 and PER3, may be because they belong, respectively, to "negative arm" and "positive arm" of the circadian clock gene network [9]. Among 3 brain areas, hypothalamus shows more clear and significant rhythmicity and higher susceptibility to CORT treatment. This agrees with a previous publication that long-term administration of dexamethasone resulted in loss of the expression rhythms in Bmal1 and  Values are means ± SEM. *P < 0.05, **P < 0.01, compared with CON group. ND represents not determined as there was no circadian rhythm Clock genes in rat SCN [39]. The mechanisms by which chronic CORT alters the circadian gene expression in the chicken are largely unknown. It is likely that CORT directly regulates clock gene expression through GRmediated transcriptional regulation [40]. However, as melatonin was reported to play a key role in controlling circadian behavioral responses [41] and the loss of circadian rhythm of plasma melatonin corresponded to the diminished circadian pattern of clock genes in the hypothalamus of CORT-exposed chickens in this study. We speculate that chronic CORT may indirectly affects the expression rhythm of circadian clock gene through alterations in melatonin secretion. CRH is essential for stress adaptation by mediating HPA axis [1] and involved in the regulation of circadian rhythms [2]. Circadian variations of CRH neuron activity are driven by the SCN and likely mediate the characteristic circadian pattern of HPA axis activity [42]. Chronic unpredictable mild stress induces hyperactivity of HPA axis which is indicated by up-regulation of hypothalamic CRH mRNA expression in rats [43]. In contrast, chronic CORT exposure significantly decreased CRH expression in chicken hypothalamus during the dark phase with destroyed circadian rhythms. Many factors contribute to the disparity of the findings, including animal species (nocturnal rats vs. diurnal chickens), stress model, and the time points of the sampling. Accordingly, genes involved in feeding regulation, including satiety genes POMC and CART and hunger genes NPY and AgRP [44], show concerted circadian expression pattern, which is in agreement with a previous report that AgRP, NPY, POMC and CART genes are expressed in a circadian rhythm in the hypothalamus [45]. The same as CRH and its receptors, the circadian rhythm of these appetite-related genes is also destroyed in chickens subjected to chronic CORT exposure. These CORT-induced alterations in hypothalamic gene expression may associated, at least partly, with the disrupted feeding behavior in the chicken.
The m 6 A methylation plays important roles in the regulation of neurogenesis, circadian rhythm, cognitive function, and stress responses [46]. Here, we provide the  Values are means ± SEM. *P < 0.05, **P < 0.01, compared with CON group. ND represents not determined as there was no circadian rhythm first evidence that the global m 6 A level in chicken hypothalamus oscillates in a day, being higher in light phase and lower in dark phase. Interestingly, the circadian rhythm pattern of diurnal chickens is opposite to that reported in nocturnal animals. This makes sense as m 6 A is reported to participate in many stress responses [47], and higher m 6 A level corresponds to higher body activity. However, in this study, chronic CORT exposure disrupted the circadian rhythms of m 6 A methylation levels in hypothalamus. Based on the observation that significant decrease of CRH mRNA in the dark phase corresponds to the significant increase in m 6 A levels at the same time points, we speculate that m 6 A may be involved in the post-transcriptional regulation of CRH mRNA in chicken hypothalamus. Indeed, the two predicted m 6 A sites X1 and X2 were both hypermethylated at detected time points (ZT18 and ZT22). Therefore, it is likely that the decrease of CRH expression was due to m 6 A-mediated mRNA degradation [48]. Nevertheless, a functional verification study is required to elucidate the role of m 6 A on these sites in CRH gene regulation in chicken hypothalamus.

Conclusion
In conclusion, our study shows that chronic CORT exposure eliminated the diurnal patterns of plasma CORT and melatonin levels in the chicken. Hypothalamus is the most susceptible brain region to CORT treatment, as almost all the genes, including clock genes, CRH, and feeding-related genes, lost their circadian rhythmicity together with the global m 6 A level. Higher m 6 A on 3'UTR of CRH mRNA coincides with lower CRH mRNA at night, indicating a possible role of m 6 A in the post-transcriptional regulation of CRH expression in chicken hypothalamus. These findings provide evidence of CORTinduced disruption of central circadian rhythmicity in CRH expression that leads to dysfunction of HPA axis in the chicken, and also imply a role of RNA m 6 A modification in the regulation of circadian rhythms in the chicken.