Tet3 mediates stable glucocorticoid-induced alterations in DNA methylation and Dnmt3a/Dkk1 expression in neural progenitors

Developmental exposure to excess glucocorticoids (GCs) has harmful neurodevelopmental effects, which include persistent alterations in the differentiation potential of embryonic neural stem cells (NSCs). The mechanisms, however, are largely unknown. Here, we investigated the effects of dexamethasone (Dex, a synthetic GC analog) by MeDIP-like genome-wide analysis of differentially methylated DNA regions (DMRs) in NSCs isolated from embryonic rat cortices. We found that Dex-induced genome-wide DNA hypomethylation in the NSCs in vitro. Similarly, in utero exposure to Dex resulted in global DNA hypomethylation in the cerebral cortex of 3-day-old mouse pups. Dex-exposed NSCs displayed stable changes in the expression of the DNA methyltransferase Dnmt3a, and Dkk1, an essential factor for neuronal differentiation. These alterations were dependent on Tet3 upregulation. In conclusion, we propose that GCs elicit strong and persistent effects on DNA methylation in NSCs with Tet3 playing an essential role in the regulation of Dnmt3a and Dkk1. Noteworthy is the occurrence of similar changes in Dnmt3a and Dkk1 gene expression after exposure to excess GC in vivo.Glucocorticoid (GC) hormones are critical for the terminal maturation of organs, but fetal exposure to high levels of GCs have detrimental effects on the development of the nervous system, including impaired neurogenesis, alterations of the hypothalamic-pituitary-adrenal axis, and behavioral changes.^{1, 2, 3, 4, 5, 6, 7, 8} The fetus is protected from surges of GC by placental enzymes (namely 11bHSD2) that convert circulating GC into inactive, water soluble metabolites.⁹ Conditions that are associated with high fetal GC levels include severe maternal stress, placental failure, and exogenous administration of GC agonists in cases of high risk of premature delivery (reviewed in Harris and Seckl¹⁰). We have previously shown that neurons and neural stem cells (NSCs) of rats prenatally exposed to high levels of the synthetic GC dexamethasone (Dex) exhibit a long-lasting increased susceptibility to oxidative stress.^{11, 12} Dex treatment in vitro decreases NSC proliferation, neuronal differentiation, and modifies the expression of genes associated with cellular senescence and mitochondrial functions in a GC receptor (GR)-mediated manner.¹³ The phenotypical alterations are associated with a decrease in total DNA methylation and the expression of DNA methyltransferases (DNMTs), and notably these global changes persists in ''daughter'' NSCs never directly exposed to Dex, suggesting a bona fide epigenetic mechanism.¹³DNA methylation is catalyzed and maintained by DNMTs (Dnmt1, Dnmt3a, and Dnmt3b).¹⁴ Dnmt1 and Dnmt3a are required for proper proliferation as well as neuronal and glial differentiation of NSCs.^{15, 16, 17} Genetic deletion of Dnmt3a leads to premature glial differentiation,^{16, 18, 19} and conditional knockout mice exhibit decreased adult neurogenesis.^{17, 20, 21} The understanding of the dynamic regulation of DNA methylation has increased significantly with the discovery of the ten-eleven translocation (Tet) family of methylcytosine dioxygenases (Tet1, Tet2, and Tet3). Tets catalyze the oxidation of 5-methylcytosine (5-mC) and generate 5-mC derivatives, including 5-hydroxymethylcytosine (5-hmC). Recent reports have demonstrated that deficiency of Tet1 is associated with impaired embryonic and adult neurogenesis^{22, 23} whereas overexpression of Tet1 impairs memory formation in mice.²⁴ Tet3 is required for normal survival, proliferation, and differentiation of neural progenitor cells, but the mechanisms involved are not clarified.^{25, 26} Hence, a fine tuning of both Dnmts and Tets appears to be critical for the correct development and function of the brain.In this study, we aimed at elucidating the mechanisms underlying the programming effects of the GC agonist Dex on the epigenome in cortical NSCs. By analyzing genome-wide DNA methylation with a MeDIP-like approach, we found a dramatic decrease in DNA methylation and identified numerous differentially methylated regions (DMRs) in Dex-exposed proliferating NSCs. The genome-wide Dex-induced changes in methylation were associated with a downregulation of Dnmt3a and an upregulation of Tet3 in both parent (P) NSCs and daughter (D) cells, which were never directly exposed to Dex. Interestingly, we found a similar global DNA hypomethylation along with Dnmt3a downregulation and Tet1–3 upregulation in the cerebral cortex of pups exposed to Dex in utero. We have previously identified Dickkopf 1 (Dkk1) as a direct target of Dex acting via GR binding to the Dkk1 promoter.²⁷ Proteins of the Dkk family inhibit the canonical Wnt signaling and are essential for brain development.^{28, 29} We now show that the Dex-induced Dkk1 upregulation is heritable and dependent on Tet3 expression. Our results show that transient exposure to excess GC have dramatic and long-lasting effects on the epigenome of NSCs and specifically point to a critical Tet3-mediated dysregulation of Dnmt3a and Dkk1, both essential factors for proper forebrain development.     

Gestational dietary betaine supplementation suppresses hepatic expression of lipogenic genes in neonatal piglets through epigenetic and glucocorticoid receptor-dependent mechanisms

Methyl donors play critical roles in nutritional programming through epigenetic regulation of gene expression. Here we fed gestational sows with control or betaine-supplemented diets (3 g/kg) throughout the pregnancy to explore the effects of maternal methyl-donor nutrient on neonatal expression of hepatic lipogenic genes. Betaine-exposed piglets demonstrated significantly lower liver triglyceride content associated with down-regulated hepatic expression of lipogenic genes acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD) and sterol regulatory element-binding protein-1c. Moreover, s-adenosyl methionine to s-adenosyl homocysteine ratio was elevated in the liver of betaine-exposed piglets, which was accompanied by DNA hypermethylation on FAS and SCD gene promoters and more enriched repression histone mark H3K27me3 on SCD gene promoter. Furthermore, glucocorticoid receptor (GR) binding to SCD gene promoter was diminished along with reduced serum cortisol and liver GR protein content in betaine-exposed piglets. GR-mediated SCD gene regulation was confirmed in HepG2 cells in vitro. Dexamethasone (Dex) drastically increased the luciferase activity of porcine SCD promoter, while the deletion of GR response element on SCD promoter significantly attenuated Dex-mediated SCD transactivation. In addition, miR-let-7e, miR-1285 and miR-124a, which respectively target porcine SCD, ACC and GR, were significantly up-regulated in the liver of betaine-exposed piglets, being in accordance with decreased protein content of these three genes. Taken together, our results suggest that maternal dietary betaine supplementation during gestation attenuates hepatic lipogenesis in neonatal piglets via epigenetic and GR-mediated mechanisms.     

Timing of glucocorticoid administration determines severity of lipid metabolism and behavioral effects in rats

Glucocorticoids (GCs) are a group of steroid hormones secreted by the adrenal glands in circadian cycles, and the dysregulation of GC signaling has been suggested to cause metabolic syndrome. Even though prolonged GC exposure is associated with serious side effects such as metabolic syndrome and central nervous system disorders, the use of GCs in anti-inflammatory and immunosuppressive therapies has been continuously rising. Meanwhile, the exact mechanisms by which GCs can influence the lipid metabolism as well as behavior and how they are affected by time remain unknown. In this study, the effects of two different long-term GC dosing regimens on lipid metabolism and behavior were investigated. Male Wistar rats received daily administrations of the GC dexamethasone sodium phosphate (DEX, 0.5 mg/kg body weight) at either ZT0 (Dex0) or ZT12 (Dex12). After 6 weeks of treatment, DEX-treated rats, especially those treated at ZT0, had higher hepatic lipid accumulation and serum triglyceride levels and less locomotor activity than did control rats. In addition, serum levels of corticosterone, 5-hydroxy tryptamine and norepinephrine were decreased in the Dex0 group but not in the Dex12 group compared to the control group. Furthermore, quantitative real-time polymerase chain reaction analysis indicated that the chronic administration of GCs at ZT0 upregulated genes related to glycolysis and lipid synthesis and downregulated genes related to fatty acid β-oxidation in the liver more remarkably than administration at ZT12. Both DEX-treated groups displayed severely altered expression patterns of the core clock genes Bmal1 and Per2 in the liver and in fat. In addition, the expression of glutamate aspartate transporter, glial fibrillary acidic protein and glutamate transporter-1, astrocyte-related genes important for maintaining nervous system functions, was drastically decreased in the hippocampus of DEX-treated rats, especially when DEX was given at ZT0. In conclusion, our findings confirm that the severity of side effects, indicated by altered lipid metabolism and behavioral activity, depends on the timing of GC administration and is associated with the degree of glucocorticoid receptor dysfunction after dosing at disparate time points.     

Selective non-steroidal glucocorticoid receptor agonists attenuate inflammation but do not impair intestinal epithelial cell restitution in vitro

Introduction

Despite the excellent anti-inflammatory and immunosuppressive action of glucocorticoids (GCs), their use for the treatment of inflammatory bowel disease (IBD) still carries significant risks in terms of frequently occurring severe side effects, such as the impairment of intestinal tissue repair. The recently-introduced selective glucocorticoid receptor (GR) agonists (SEGRAs) offer anti-inflammatory action comparable to that of common GCs, but with a reduced side effect profile.

Methods

The in vitro effects of the non-steroidal SEGRAs Compound A (CpdA) and ZK216348, were investigated in intestinal epithelial cells and compared to those of Dexamethasone (Dex). GR translocation was shown by immunfluorescence and Western blot analysis. Trans-repressive effects were studied by means of NF-κB/p65 activity and IL-8 levels, trans-activation potency by reporter gene assay. Flow cytometry was used to assess apoptosis of cells exposed to SEGRAs. The effects on IEC-6 and HaCaT cell restitution were determined using an in vitro wound healing model, cell proliferation by BrdU assay. In addition, influences on the TGF-β- or EGF/ERK1/2/MAPK-pathway were evaluated by reporter gene assay, Western blot and qPCR analysis.

Results

Dex, CpdA and ZK216348 were found to be functional GR agonists. In terms of trans-repression, CpdA and ZK216348 effectively inhibited NF-κB activity and IL-8 secretion, but showed less trans-activation potency. Furthermore, unlike SEGRAs, Dex caused a dose-dependent inhibition of cell restitution with no effect on cell proliferation. These differences in epithelial restitution were TGF-β-independent but Dex inhibited the EGF/ERK1/2/MAPK-pathway important for intestinal epithelial wound healing by induction of MKP-1 and Annexin-1 which was not affected by CpdA or ZK216348.

Conclusion

Collectively, our results indicate that, while their anti-inflammatory activity is comparable to Dex, SEGRAs show fewer side effects with respect to wound healing. The fact that SEGRAs did not have a similar effect on cell restitution might be due to a different modulation of EGF/ERK1/2 MAPK signalling.     

Histone code of genes induced by co-treatment with a glucocorticoid hormone agonist and a p44/42 MAPK inhibitor in human small intestinal Caco-2 cells

Background

Inactivation of glucocorticoid hormones and p44/42 mitogen-activated protein kinase (MAPK) is thought to be important in small intestinal maturation and expression of genes related to intestinal differentiation and functions.

Methods

We investigated target genes induced by co-treatment for 48 h with a glucocorticoid hormone agonist, dexamethasone (Dex), and a p44/42 MAPK inhibitor, PD98059 (PD), in a small intestine-like cell line (Caco-2) using microarray analysis. We also investigated whether expression changes of the target genes induced by the co-treatment are associated with histone modifications around these genes.

Results

Co-treatment of Caco-2 cells with Dex and PD enhanced several genes related to intestinal differentiation and functions such as SCNN1A, FXYD3, LCT and LOX. Induction of the SCNN1A gene was associated with increased presence of acetylated histone H3 and H4 and di-methylated histone H3 at lysine (K) 4 around the transcribed region of the gene, and induction of the FXYD3 gene was associated with increased presence of acetylated histones H3 and H4 from the promoter/enhancer to the transcribed region of the gene. Induction of LCT and LOX genes was associated with increased presence of acetylated histone H4 on the promoter/enhancer region of the genes.

Conclusions

Histone acetylation and/or histone H3 K4 methylation around the promoter/enhancer or/and transcribed regions of target genes are associated with induction of the genes by co-treatment with Dex and PD in Caco-2 cells.

General significance

The histone code is specific to each gene with respect to induction by glucocorticoid hormone and inhibition of p44/42 MAPK in Caco-2 cells.     

Gestational diabetes mellitus epigenetically affects genes predominantly involved in metabolic diseases

Offspring exposed to gestational diabetes mellitus (GDM) have an increased risk for chronic diseases, and one promising mechanism for fetal metabolic programming is epigenetics. Therefore, we postulated that GDM exposure impacts the offspring’s methylome and used an epigenomic approach to explore this hypothesis. Placenta and cord blood samples were obtained from 44 newborns, including 30 exposed to GDM. Women were recruited at first trimester of pregnancy and followed until delivery. GDM was assessed after a 75-g oral glucose tolerance test at 24–28 weeks of pregnancy. DNA methylation was measured at > 485,000 CpG sites (Infinium HumanMethylation450 BeadChips). Ingenuity Pathway Analysis was conducted to identify metabolic pathways epigenetically affected by GDM. Our results showed that 3,271 and 3,758 genes in placenta and cord blood, respectively, were potentially differentially methylated between samples exposed or not to GDM (p-values down to 1 × 10⁻⁰⁶; none reached the genome-wide significance levels), with more than 25% (n = 1,029) being common to both tissues. Mean DNA methylation differences between groups were 5.7 ± 3.2% and 3.4 ± 1.9% for placenta and cord blood, respectively. These genes were likely involved in the metabolic diseases pathway (up to 115 genes (11%), p-values for pathways = 1.9 × 10⁻¹³ < p < 4.0 × 10⁻⁰³; including diabetes mellitus p = 4.3 × 10⁻¹¹). Among the differentially methylated genes, 326 in placenta and 117 in cord blood were also associated with newborn weight. Our results therefore suggest that GDM has epigenetic effects on genes preferentially involved in the metabolic diseases pathway, with consequences on fetal growth and development, and provide supportive evidence that DNA methylation is involved in fetal metabolic programming.     

Analysis of Epigenetic Factors in Mouse Embryonic Neural Stem Cells Exposed to Hyperglycemia

Background

Maternal diabetes alters gene expression leading to neural tube defects (NTDs) in the developing brain. The mechanistic pathways that deregulate the gene expression remain unknown. It is hypothesized that exposure of neural stem cells (NSCs) to high glucose/hyperglycemia results in activation of epigenetic mechanisms which alter gene expression and cell fate during brain development.

Methods and Findings

NSCs were isolated from normal pregnancy and streptozotocin induced-diabetic pregnancy and cultured in physiological glucose. In order to examine hyperglycemia induced epigenetic changes in NSCs, chromatin reorganization, global histone status at lysine 9 residue of histone H3 (acetylation and trimethylation) and global DNA methylation were examined and found to be altered by hyperglycemia. In NSCs, hyperglycemia increased the expression of Dcx (Doublecortin) and Pafah1b1 (Platelet activating factor acetyl hydrolase, isoform 1b, subunit 1) proteins concomitant with decreased expression of four microRNAs (mmu-miR-200a, mmu-miR-200b, mmu-miR-466a-3p and mmu-miR-466 d-3p) predicted to target these genes. Knockdown of specific microRNAs in NSCs resulted in increased expression of Dcx and Pafah1b1 proteins confirming target prediction and altered NSC fate by increasing the expression of neuronal and glial lineage markers.

Conclusion/Interpretation

This study revealed that hyperglycemia alters the epigenetic mechanisms in NSCs, resulting in altered expression of some development control genes which may form the basis for the NTDs. Since epigenetic changes are reversible, they may be valuable therapeutic targets in order to improve fetal outcomes in diabetic pregnancy.     

Dosing schedule-dependent attenuation of dexamethasone-induced muscle atrophy in mice

Many inflammatory and autoimmune diseases are treated using synthetic glucocorticoids. However, excessive glucocorticoid can often cause unpredictable effects including muscle atrophy. Endogenous glucocorticoid levels robustly fluctuate in a circadian manner and peak just before the onset of the active phase in both humans and nocturnal rodents. The present study determines whether muscle atrophy induced by exogenous glucocorticoid can be avoided by optimizing dosing times. We administered single daily doses of the glucocorticoid analog dexamethasone (Dex) to mice for 10 days at the times of day corresponding to peak (early night) or trough (early morning) endogenous glucocorticoid levels. Administration at the acrophase of endogenous glucocorticoids significantly attenuated Dex-induced wasting of the gastrocnemius (Ga) and tibialis anterior (TA) muscles that comprise mostly fast-twitch muscle fibers. Real-time RT-PCR revealed that the Dex-induced mRNA expression of genes encoding the atrophy-related ubiquitin ligases Muscle Atrophy F-box (Fbxo32, also known as MAFbx/Atrogin-1) and Muscle RING finger 1 (Trim63, also known as MuRF1) in the Ga and TA muscles was significantly attenuated by Dex when administered during the early night. Dex negligibly affected the weight of the soleus (So) muscle that mostly comprises slow-twitch muscle fibers, but significantly and similarly decreased the weight of the spleen at both dosing times. These results suggest that glucocorticoid-induced muscle atrophy can be attenuated by optimizing the dosing schedule.     

Childhood adversity and epigenetic modulation of the leukocyte glucocorticoid receptor: preliminary findings in healthy adults

Background

A history of early adverse experiences is an important risk factor for adult psychopathology. Changes in stress sensitivity and functioning of the hypothalamic-pituitary-adrenal (HPA) axis may underlie the association between stress and risk for psychiatric disorders. Preclinical work in rodents has linked low levels of maternal care to increased methylation of the promoter region of the glucocorticoid receptor (GR) gene, as well as to exaggerated hormonal and behavioral responses to stress. Recent studies have begun to examine whether early-life stress leads to epigenetic modifications of the GR gene in humans.

Methods

We examined the degree of methylation of a region of the promoter of the human GR gene (NR3C1) in leukocyte DNA from 99 healthy adults. Participants reported on their childhood experiences of parental behavior, parental death or desertion, and childhood maltreatment. On a separate day, participants completed the dexamethasone/corticotropin-releasing hormone (Dex/CRH) test, a standardized neuroendocrine challenge test.

Results

Disruption or lack of adequate nurturing, as measured by parental loss, childhood maltreatment, and parental care, was associated with increased NR3C1 promoter methylation (p<.05). In addition, NR3C1 promoter methylation was linked to attenuated cortisol responses to the Dex/CRH test (p<.05).

Conclusions

These findings suggest that childhood maltreatment or adversity may lead to epigenetic modifications of the human GR gene. Alterations in methylation of this gene could underlie the associations between childhood adversity, alterations in stress reactivity, and risk for psychopathology.     

Role of nephrin phosphorylation inducted by dexamethasone and angiotensin II in podocytes

The phosphorylation of nephrin plays an important role in maintaining the normal structure and function in podocytes. Dexamethasone (Dex) is usually used to treat glomerular diseases with proteinuria. In this study, we observated the effect of Dex and angiotensin II (AngII) on the change of nephrin phosphorylation in cultured podocytes. In vitro, cultured podocytes were exposed to AngII (10^?6 mol/L) pretreated with or without Dex (100 nM) for different time periods. Nck or Fyn were silenced by small interfering RNA (siRNA), nephrin and its phosphorylation expression were analyzed by Western blotting. In vitro, the phosphorylation of nephrin was significantly reduced after AngII stimulation (P < 0.05). Dex significantly resisted podocyte injury inducted by AngII via increasing the phosphorylation of nephrin (P < 0.05), siRNA silencing Nck can partially inhibited nephrin phosphorylation, siRNA silencing Fyn can completely inhibited nephrin phosphorylation. Phosphorylation of nephrin is important for the survival status of podocytes. Glucocorticoid treatment for human glomerulonephritis may exert its function by regulating Nck and Fyn complex to promote phosphorylation of nephrin. These results elucidate a novel mechanism of glucocorticoid treatment for glomerulonephritis.     

Dexamethasone inhibits apoptosis in C6 glioma cells through increased expression of Bcl-XL

The glucocorticoid dexamethasone (Dex) has been reported to modulate a number of signaling pathways and physiological processes, including apoptosis. This study was carried out to investigate the cytoprotective mechanism of Dex in C6 glioma cells. Pre-treatment of cells with Dex inhibited apoptosis induced by staurosporine, etoposide and thapsigargin. Apoptosis inhibition correlated with blockade of mitochondrial cytochrome c release, abolition of caspase-3 activity along with inhibition of caspase-9 and PARP cleavage. Dex-mediated cytoprotection coincided with the induction of the anti-apoptotic protein, Bcl-X_L. The specific glucocorticoid receptor antagonist, RU486, reversed the anti-apoptotic effect of Dex and prevented Bcl-X_L induction. Here, we show for the first time that knockdown of Bcl-X_L expression with siRNA reversed the protective effects of the glucocorticoid in glioma cells. We conclude that Dex-mediated inhibition of apoptosis in C6 glioma cells is through induction of Bcl-X_L.     

Epigenetic regulation of ganglioside expression in neural stem cells and neuronal cells

The structural diversity and localization of cell surface glycosphingolipids (GSLs), including gangliosides, in glycolipid-enriched microdomains (GEMs, also known as lipid rafts) render them ideally suited to play important roles in mediating intercellular recognition, interactions, adhesion, receptor function, and signaling. Gangliosides, sialic acid-containing GSLs, are most abundant in the nerve tissues. The quantity and expression pattern of gangliosides in brain change drastically throughout development and these changes are mainly regulated through stage-specific expression of glycosyltransferase genes. We previously demonstrated for the first time that efficient histone acetylation of the glycosyltransferase genes in mouse brain contributes to the developmental alteration of ganglioside expression. We further demonstrated that acetylation of histones H3 and H4 on the N-acetylgalactosaminyltransferase I (GalNAcT, GA2/GM2/GD2/GT2-synthase; B4galnt1) gene promoter resulted in recruitment of trans-activation factors. In addition, we showed that epigenetic activation of the GalNAcT gene was detected and accompanied by an apparent induction of neuronal differentiation of neural stem cells (NSCs) responding to an exogenous supplement of ganglioside GM1. Most recently, we found that nuclear GM1 binds with acetylated histones on the promoters of the GalNAcT as well as on the NeuroD1 genes in differentiated neurons. Here, we will introduce epigenetic regulation of ganglioside synthase genes in neural development and neuronal differentiation of NSCs.     

Loss of TBL1XR1 Disrupts Glucocorticoid Receptor Recruitment to Chromatin and Results in Glucocorticoid Resistance in a B-Lymphoblastic Leukemia Model

Although great advances have been made in the treatment of pediatric acute lymphoblastic leukemia, up to one of five patients will relapse, and their prognosis thereafter is dismal. We have previously identified recurrent deletions in TBL1XR1, which encodes for an F-box like protein responsible for regulating the nuclear hormone repressor complex stability. Here we model TBL1XR1 deletions in B-precursor ALL cell lines and show that TBL1XR1 knockdown results in reduced glucocorticoid receptor recruitment to glucocorticoid responsive genes and ultimately decreased glucocorticoid signaling caused by increased levels of nuclear hormone repressor 1 and HDAC3. Reduction in glucocorticoid signaling in TBL1XR1-depleted lines resulted in resistance to glucocorticoid agonists, but not to other chemotherapeutic agents. Importantly, we show that treatment with the HDAC inhibitor SAHA restores sensitivity to prednisolone in TBL1XR1-depleted cells. Altogether, our data indicate that loss of TBL1XR1 is a novel driver of glucocorticoid resistance in ALL and that epigenetic therapy may have future application in restoring drug sensitivity at relapse.