Tatiana Kisliouk

Dynamic changes in DNA methylation during thermal control establishment affect CREB binding to the brain-derived neurotrophic factor promoter

Thermal control establishment develops during a critical period by alterations in cellular properties in the frontal hypothalamus. These alterations may be modulated by the epigenetic code that determines the repertoire of transcribed proteins. Here we demonstrate transient changes in the expression of brain‐derived neurotrophic factor (Bdnf) during both thermal conditioning and re‐exposure of conditioned chicks to heat stress, relative to their age‐matched naive counterparts. These changes coincide with changes in CpG methylation pattern in the avian Bdnf promoter region. Reduction in methylation during heat conditioning was observed at a cAMP response element‐binding (CREB) site which coincided with both elevation in phospho‐CREB levels and its binding to the Bdnf promoter. At the same time, an increase in methylation was observed at two other CpG sites, accompanied by elevation of the DNA methyltransferase 3a (DNMT3a) expression. DNMT3a was also found to bind to the two elevated methyl CpG sites, but not to the CREB binding site. These data suggest that complex and dynamic changes in DNA methylation are involved in the regulation of Bdnf expression during thermotolerance acquisition.

Overweight and CpG methylation of the Pomc promoter in offspring of high-fat-diet-fed dams are not “reprogrammed” by regular chow diet in rats

This study aimed to determine whether epigenetic malprogramming induced by high‐fat diet (HFD) has an obesogenic effect on nonmated and mated female rats and their offspring. Further, it aimed to reprogram offsprings epigenetic malprogramming and phenotype by providing normal diet after weaning. Body weight (BW) was measured, and plasma and hypothalamic arcuate nuclei were collected for analysis of hormones, mRNA, and DNA CpG methylation of the promoter of Pomc, a key factor in control of food intake. In nonmated females, HFD decreased Pomc/leptin ratio by ~38%. This finding was associated with Pomc promoter hypermethylation. While heavier during pregnancy, during lactation HFD dams showed sharper BW decrease (2.5‐fold) and loss of Pomc promoter hypermethylation. Moreover, their weight loss was correlated with demethylation (r=‐0.707) and with gadd45b mRNA expression levels (r=0.905). Even though offspring of HFD dams ate standard chow from weaning, they displayed increased BW, Pomc promoter hypermethylation, and vulnerability to HFD challenge (3‐fold kilocalorie intake increase). These findings demonstrate a long‐term effect of maternal HFD on CpG methylation of the Pomc promoter in the offspring, which was not reprogrammed by standard chow from weaning. Further, the results suggest a possible mechanism of demethylation of the Pomc promoter following pregnancy and lactation.—Marco, A., Kisliouk, T., Tabachnik, T., Meiri, N., Weller, A. Overweight and CpG methylation of the Pomc promoter in offspring of high‐fat‐diet‐fed dams are not “reprogrammed” by regular chow diet in rats. FASEB J. 28, 4148‐4157 (2014). www.fasebj.org

High fat diet induces hypermethylation of the hypothalamic Pomc promoter and obesity in post-weaning rats

Impaired response of the brain to the leptin signal leads to a persisting dysregulation of food intake and energy balance. High plasma leptin or insulin should activate proopiomelanocortin (POMC), the precursor of the anorexigenic neuropeptide α-melanocyte-stimulating hormone (α-MSH) in the hypothalamic arcuate nucleus (ARC). Nevertheless, in obesity, this signal transduction pathway might be impaired. In this study we investigated whether chronic high fat (HF) diet consumption from post-weaning to adulthood increases CpG methylation of the Pomc promoter. The hypothesis that this would disrupt the essential binding of the transcription factor Sp1 to the Pomc promoter was tested. Male rats were raised from postnatal day 21 till 90 on either HF or standard diet. As a result HF fed rats were significantly heavier, with high leptin and insulin levels in their plasma but almost no changes in ARC mRNA expression levels of Pomc. The Pomc promoter area in the HF-treated rats was found to be hypermethylated. Furthermore, there was a direct correlation in individual rats between CpG methylation at specific sites that affect Sp1 binding and plasma leptin levels and/or body weight. Although, as expected the HF diet resulted in up-regulation of Sp1, the binding of Sp1 to the hypermethylated Pomc promoter was significantly reduced. Therefore, we suggest that hypermethylation on the promoter region of the Pomc gene can emerge at post-lactation periods and interfere with transcription factor binding, thus blocking the effects of high leptin levels, leading to obesity.

MiR-138 inhibits EZH2 methyltransferase expression and methylation of histone H3 at lysine 27, and affects thermotolerance acquisition.

Thermotolerance acquisition involves neuronal network remodeling and, hence, alteration in the repertoire of expressed proteins. We have previously demonstrated the role of histone H3 methylation at lysineu200327 (H3K27) by EZH2 methyltransferase in the regulation of gene expression during the critical period for the establishment of thermal control in chicks. Here we describe another level of biological regulation, demonstrating the inhibitory role of microRNAs (miRNAs) in the regulation of EZH2 expression in thermoregulatory system development and functioning. During heat conditioning in the critical period for the establishment of thermal control, a decrease in expression of the EZH2‐targeting miR‐138 occurred simultaneously with an increase in EZH2 levels in the preoptic anterior hypothalamus. Intracranial injection of miR‐138 during the critical period led to a transient reduction in EZH2 levels, which was accompanied by a decrease in H3K27 methylation. Injection of miR‐138 followed by heat conditioning also abolished EZH2 induction during heat conditioning. Moreover, this miR‐138‐induced inhibition of EZH2 during the critical period resulted in a long‐term effect on EZH2 expression. A week after the treatment, the EZH2 protein levels in conditioned and in nonconditioned chicks were different from those in their saline‐injected counterparts and the directions of change were opposite to each other. Finally, miR‐138 injection during the critical period disrupted the establishment of thermoregulation, manifested as a defective body temperature response to heat. These data demonstrate a role for miRNAs in regulating the expression of histone‐modifying enzymes, and thus emphasize the multilevel regulation mechanism which includes both epigenetic and miRNA regulatory mechanisms in neuronal network organization during the critical period of sensory development.

A critical role for dynamic changes in histone H3 methylation at the Bdnf promoter during postnatal thermotolerance acquisition.

As with other sensory mechanisms, determination of the thermal‐control set point is refined during a critical period of development by alterations in cellular properties in the frontal hypothalamus. These alterations in hypothalamic plasticity are achieved by renewal of the protein repertoire via activation or silencing of gene transcription, both of which are regulated by histone modifications. This study demonstrates induction of global histone H3 lysine 27 (H3K27) dimethylation, with no changes in its trimethylation levels, in the frontal hypothalamus, as well as at the promoter of the brain‐derived neurotrophic factor (BDNF) gene during thermal‐control establishment. Furthermore, antisense ‘knockdown’ of the H3K27‐specific methyltransferase, enhancer of zeste 2, which was induced in correlation with the dimethylation of H3K27, inhibited Bdnf mRNA expression and disrupted the establishment of thermoregulation. This phenotypic effect was partially rescued by intracranial injection of BDNF. The presented findings highlight the specific epigenetic role of chromatin modifications in thermal‐control establishment.

Epigenetic control of translation regulation: alterations in histone H3 lysine 9 post-translation modifications are correlated with the expression of the translation initiation factor 2B (Eif2b5) during thermal control establishment.

Thermal control set point is regulated by thermosensitive neurons of the preoptic anterior hypothalamus (PO/AH) and completes its development during postnatal critical sensory period. External stimuli, like increase in environmental temperature, influence the neuronal protein repertoire and, ultimately, cell properties via activation or silencing of gene transcription, both of which are regulated by the “histone code.” Here, we demonstrated an increase in global histone H3 lysine 9 (H3K9) acetylation as well as H3K9 dimethylation in chick PO/AH during heat conditioning at the critical period of sensory development. In contrast to the global profile of H3K9 modifications, acetylation and dimethylation patterns of H3K9 at the promoter of the catalytic subunit of eukaryotic translation initiation factor 2B (Eif2b5) were opposite to each other. During heat conditioning, there was an increase in H3K9 acetylation at the Eif2b5 promoter, simultaneously with decrease in H3K9 dimethylation. These alterations coincided with Eif2b5 mRNA induction. Moreover, exposure to excessive heat during the critical period resulted in long‐term effect on both H3K9 tagging at the Eif2b5 promoter and Eif2b5 mRNA expression. These data suggest a role for dynamic H3K9 post‐translational modifications in global translation regulation during the thermal control establishment.

DNA CpG Methylation (5-Methylcytosine) and Its Derivative (5-Hydroxymethylcytosine) Alter Histone Posttranslational Modifications at the Pomc Promoter, Affecting the Impact of Perinatal Diet on Leanness and Obesity of the Offspring

A maternal high-fat diet (HFD) alters the offsprings feeding regulation, leading to obesity. This phenomenon is partially mediated by aberrant expression of the hypothalamic anorexigenic neuropeptide proopiomelanocortin (POMC). Nevertheless, although some individual offspring suffer from morbid obesity, others escape the malprogramming. It is suggested that this difference is due to epigenetic programming. In this study, we report that in lean offspring of non-HFD–fed dams, essential promoter regions for Pomc expression were enriched with 5-hydroxymethylcytosine (5hmC) together with a reduction in the level of 5-methylcytosine (5mC). Moreover, 5hmC was negatively correlated whereas 5mC was positively correlated with body weight in offspring from both HFD- and control-fed dams. We further found that Pomc expression in obese offspring is determined by a two-step epigenetic inhibitory mechanism in which CpG methylation is linked with histone posttranslational modifications. An increase in CpG methylation at the Poxmc promoter enables binding of methyl-binding domain 1 (MBD1) to 5mC, but not to its derivative 5hmC. MBD1 then interacts with SET domain bifurcated 1 methyltransferase to promote bimethylation on the histone 3 lysine 9 residue, reducing Pomc mRNA expression. These results suggest an epigenetic regulatory mechanism that affects obesity-prone or resilient traits.

The balance between stress resilience and vulnerability is regulated by corticotropin-releasing hormone during the critical postnatal period for sensory development

Determining whether a stressful event will lead to stress‐resilience or vulnerability depends probably on an adjustable stress response set point, which is most likely effective during postnatal sensory development and involves the regulation of corticotrophin‐releasing hormone (CRH) expression. During the critical period of thermal‐control establishment in 3‐day‐old chicks, heat stress was found to render resilient or sensitized response, depending on the ambient temperature. These two different responses were correlated with the amount of activation of the hypothalamic–pituitary–adrenal (HPA) axis. The expression of CRH mRNA in the hypothalamic paraventricular nucleus was augmented during heat challenge a week after heat conditioning in chicks which were trained to be vulnerable to heat, while it declined in chicks that were trained to be resilient. To study the role of CRH in HPA‐axis plasticity, CRH or Crh‐antisense were intracranially injected into the third ventricle. CRH caused an elevation of both body temperature and plasma corticosterone level, while Crh‐antisense caused an opposite response. Moreover, these effects had long term implications by reversing a week later, heat resilience into vulnerability and vice versa. Chicks that had been injected with CRH followed by exposure to mild heat stress, normally inducing resilience, demonstrated, a week later, an elevation in body temperature, and Crh mRNA level similar to heat vulnerability, while Crh‐antisense injected chicks, which were exposed to harsh temperature, responded in heat resilience. These results demonstrate a potential role for CRH in determining the stress resilience/vulnerability balance.

Methyl CpG Level at Distal Part of Heat‐Shock Protein Promoter HSP70 Exhibits Epigenetic Memory for Heat Stress by Modulating Recruitment of POU2F1‐Associated Nucleosome Remodeling Deacetylase (NuRD) Complex

Depending on its stringency, exposure to heat in early life leads to either resilience or vulnerability to heat stress later in life. We hypothesized that epigenetic alterations in genes belonging to the cell proteostasis pathways are attributed to long‐term responses to heat stress. Epigenetic regulation of the mRNA expression of the molecular chaperone heat‐shock protein (HSP) 70 (HSPA2) was evaluated in the chick hypothalamus during the critical period of thermal‐control establishment on day 3 post‐hatch and during heat challenge on day 10. Both the level and duration of HSP70 expression during heat challenge a week after heat conditioning were more pronounced in chicks conditioned under harsh versus mild temperature. Analyzing different segments of the promoter in vitro indicated that methylation of a distal part altered its transcriptional activity. In parallel, DNA‐methylation level of this segment in vivo was higher in harsh‐ compared to mild‐heat‐conditioned chicks. Hypermethylation of the HSP70 promoter in high‐temperature‐conditioned chicks was accompanied by a reduction in both POU Class 2 Homeobox 1 (POU2F1) binding and recruitment of the nucleosome remodeling deacetylase (NuRD) chromatin‐remodeling complex. As a result, histone H3 acetylation levels at the HSP70 promoter were higher in harsh‐temperature‐conditioned chicks than in their mild‐heat‐conditioned counterparts. These results suggest that methylation level of a distal part of the HSP70 promoter and POU2F1 recruitment may reflect heat‐stress‐related epigenetic memory and may be useful in differentiating between individuals that are resilient or vulnerable to stress.

Thyroid Hormone-Dependent Epigenetic Regulation of Melanocortin 4 Receptor Levels in Female Offspring of Obese Rats

Maternal obesity is a risk factor for offspring obesity. The melanocortin 4 receptor (Mc4r) is one of the mediators of food intake and energy balance. The present study examined the epigenetic mechanisms underlying altered Mc4r levels in the hypothalamic paraventricular nucleus in the offspring of high-fat diet (HFD)-induced obese dams and sought to elucidate the role of thyroid hormones in epigenetic regulation and tagging of their nucleosome at the Mc4r promoter. Female Wistar rats were fed an HFD or standard chow from weaning through gestation and lactation. Epigenetic alterations were analyzed in the offspring on postnatal day 21 at the Mc4r promoter using chromatin immunoprecipitation and bisulfite sequencing. To study the role of triiodothyronine (T3) in Mc4r downregulation, dams received methimazole (MMI), an inhibitor of thyroid hormone production. Offspring of HFD-fed dams had a greater body weight, elevated plasma T3 concentrations, and lower Mc4r messenger RNA levels than controls. At the Mc4r promoter, offspring of HFD-fed mothers demonstrated increased histone 3 lysine 27 acetylation (H3K27ac) with a greater association to thyroid hormone receptor-β (TRβ), an inhibitor of Mc4r transcription. Moreover, TRβ coimmunoprecipitated with H3K27ac, supporting their presence in the same complex. Maternal MMI administration prevented the HFD reduction in Mc4r levels, the increase in TRβ, and the increase in the TRβ-H3K27ac association, providing further support for the role of T3 in downregulating Mc4r levels. These findings demonstrate that a perinatal HFD environment affects Mc4r regulation through a T3 metabolic pathway involving histone acetylation of its promoter.