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Background: The aim of the present study was to investigate the association of fMRI blood oxygen-level dependent (BOLD) reactivity with the level of epigenetic methylation of SLC6A4 in blood DNA from a sample of healthy participants and patients with major depressive disorder (MDD). Methods: We investigated patients with MDD and healthy controls using fMRI and an emotional attention-shifting task. We assessed site-specific DNA methylation of a previously characterized SLC6A4 region in peripheral blood DNA using pyrosequencing. Results: Our study involved 25 patients with MDD and 35 healthy controls. Activation in the anterior insula elicited by negative emotional content was significantly positively associated with the degree of SLC6A4 methylation. Significantly negative associations were observed between activation in the posterior insula and the degree of SLC6A4 methylation when judging the geometry of pictures after seeing negative in contrast to positive emotional stimuli. Healthy controls with a high degree of SLC6A4 methylation depicted significantly more activity elicited by positive stimuli in limbic regions and more activity elicited by negative stimuli in limbic as well as cognitive control regions than those with a low degree of SLC6A4 methylation. Limitations: It is impossible to measure methylation directly in the brain and thus we assessed peripheral methylation of SLC6A4. Since the association was cross-sectional, no conclusion about cause and effect can be drawn. Conclusion: Our study provides further support to the hypothesis that particular DNA methylation states that are associated with brain function during emotion processing are detectable in the periphery., Introduction Childhood adversity, such as childhood maltreatment, plays an important role in a number of multifactorial mental disorders. Recent studies reveal that certain aspects of stress-related mental disorders result from [...]

Serotonin plays an important role in the etiology of depression. Serotonin is also crucial for brain development. For instance, animal studies have demonstrated that early disruptions in the serotonin system affect brain development and emotion regulation in later life. A plausible explanation is that environmental stressors reprogram the serotonin system through epigenetic processes by altering serotonin system gene expression. This in turn may affect brain development, including the hippocampus, a region with dense serotonergic innervations and important in stress-regulation. The aim of this study was to test whether greater DNA methylation in specific CpG sites at the serotonin transporter promoter in peripheral cells is associated with childhood trauma, depression, and smaller hippocampal volume. We were particularly interested in those CpG sites whose state of methylation in peripheral cells had previously been associated with in vivo measures of brain serotonin synthesis. Thirty-three adults with Major Depressive Disorder (MDD) (23 females) and 36 matched healthy controls (21 females) were included in the study. Depressive symptoms, childhood trauma, and high-resolution structural MRI for hippocampal volume were assessed. Site-specific serotonin transporter methylation was assessed using pyrosequencing. Childhood trauma, being male, and smaller hippocampal volume were independently associated with greater peripheral serotonin transporter methylation. Greater serotonin transporter methylation in the depressed group was observed only in SSRI-treated patients. These results suggest that serotonin transporter methylation may be involved in physiological gene-environment interaction in the development of stress-related brain alterations. The results provide some indications that site-specific serotonin transporter methylation may be a biomarker for serotonin-associated stress-related psychopathology. [ABSTRACT FROM AUTHOR]

Maternal care influences hypothalamic-pituitary-adrenal (HPA) function in the rat through epigenetic programming of glucocorticoid receptor expression. In humans, childhood abuse alters HPA stress responses and increases the risk of suicide. We examined epigenetic differences in a neuron-specific glucocorticoid receptor (NR3C1) promoter between postmortem hippocampus obtained from suicide victims with a history of childhood abuse and those from either suicide victims with no childhood abuse or controls. We found decreased levels of glucocorticoid receptor mRNA, as well as mRNA transcripts bearing the glucocorticoid receptor 1F splice variant and increased cytosine methylation of an NR3C1 promoter. Patch-methylated NR3C1 promoter constructs that mimicked the methylation state in samples from abused suicide victims showed decreased NGFI-A transcription factor binding and NGFI-A–inducible gene transcription. These findings translate previous results from rat to humans and suggest a common effect of parental care on the epigenetic regulation of hippocampal glucocorticoid receptor expression. [ABSTRACT FROM AUTHOR]

Maternal care alters epigenetic programming of glucocorticoid receptor (GR) gene expression in the hippocampus, and increased postnatal maternal licking/grooming (LG) behavior enhances nerve growth factor-inducible protein A (NGFI-A) transcription factor binding to the exon 17GR promoter within the hippocampus of the offspring. We tested the hypothesis that NGFI-A binding to the exon 17GR promoter sequence marks this sequence for histone acetylation and DNA demethylation and that such epigenetic alterations subsequently influence NGFI-A binding and GR transcription. We report that (1) NGFI-A binding to its consensus sequence is inhibited by DNA methylation, (2) NGFI-A induces the activity of exon 17GR promoter in a transient reporter assay, (3) DNA methylation inhibits exon 17GR promoter activity, and (4) whereas NGFI-A interaction with the methylated exon 17GR promoter is reduced, NGFI-A overexpression induces histone acetylation, DNA demethylation, and activation of the exon 17GR promoter in transient transfection assays. Site-directed mutagenesis assays demonstrate that NGFI-A binding to the exon 17GR promoter is required for such epigenetic reprogramming. In vivo, enhanced maternal LG is associated with increased NGFI-A binding to the exon 17GR promoter in the hippocampus of pups, and NGFI-A-bound exon 17GR promoter is unmethylated compared with unbound exon 17GRpromoter. Knockdown experiments of NGFI-A in hippocampal primary cell culture show that NGFI-A is required for serotonin-induced DNA demethylation and increased exon 17GR promoter expression. The data are consistent with the hypothesis that NGFI-A participates in epigenetic programming of GR expression. [ABSTRACT FROM AUTHOR]

Stress responses in the adult rat are programmed early in life by maternal care and associated with epigenomic marking of the hippocampal exon 17 glucocorticoid receptor (GR) promoter. To examine whether such epigenetic programming is reversible in adult life, we centrally infused the adult offspring with the essential amino acid L-methionine, a precursor to S-adenosyl-methionine that serves as the donor of methyl groups for DNA methylation. Here we report that methionine infusion reverses the effect of maternal behavior on DNA methylation, nerve growth factor-inducible protein-A binding to the exon 17 promoter, GR expression, and hypothalamic-pituitary- adrenal and behavioral responses to stress, suggesting a causal relationship among epigenomic state, GR expression, and stress responses in the adult offspring. These results demonstrate that, despite the inherent stability of the epigenomic marks established early in life through behavioral programming, they are potentially reversible in the adult brain. [ABSTRACT FROM AUTHOR]

Here we report that increased pup licking and grooming (LG) and arched-back nursing (ABN) by rat mothers altered the offspring epigenome at a glucocorticoid receptor (GR) gene promoter in the hippocampus. Offspring of mothers that showed high levels of LG and ABN were found to have differences in DNA methylation, as compared to offspring of 'low-LG-ABN' mothers. These differences emerged over the first week of life, were reversed with cross-fostering, persisted into adulthood and were associated with altered histone acetylation and transcription factor (NGFI-A) binding to the GR promoter. Central infusion of a histone deacetylase inhibitor removed the group differences in histone acetylation, DNA methylation, NGFI-A binding, GR expression and hypothalamic-pituitary-adrenal (HPA) responses to stress, suggesting a causal relation among epigenomic state, GR expression and the maternal effect on stress responses in the offspring. Thus we show that an epigenomic state of a gene can be established through behavioral programming, and it is potentially reversible. [ABSTRACT FROM AUTHOR]

Variations in maternal care in the rat influence the epigenetic state and transcriptional activity of glucocorticoid receptor (GR) gene in the hippocampus. The mechanisms underlying this maternal effect remained to be defined, including the nature of the relevant maternally regulated intracellular signalling pathways. We show here that increased maternal licking/grooming (LG), which stably enhances hippocampal GR expression, paradoxically increases hippocampal expression of the methyl-CpG binding domain protein-2 (MBD2) and MBD2 binding to the exon 17 GR promoter. Knockdown experiments of MBD2 in hippocampal primary cell culture show that MBD2 is required for activation of exon 17 GR promoter. Ectopic co-expression of nerve growth factor-inducible protein A (NGFI-A) with MBD2 in HEK 293 cells with site-directed mutagenesis of the NGFI-A response element within the methylated exon 17 GR promoter supports the hypothesis that MBD2 collaborates with NGFI-A in binding and activation of this promoter. These data suggest a possible mechanism linking signalling pathways, which are activated by behavioural stimuli and activation of target genes., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

Variations in maternal behavior are associated with differences in estrogen receptor (ER)-alpha expression in the medial preoptic area (MPOA) and are transmitted across generations such that, as adults, the female offspring of mothers that exhibit increased pup licking/grooming (LG) over the first week postpartum (i.e. high LG mothers) show increased ERalpha expression in the MPOA and are themselves high LG mothers. In the present studies, cross-fostering confirmed an association between maternal care and ERalpha expression in the MPOA; the biological offspring of low LG mothers fostered at birth to high LG dams show increased ERalpha expression in the MPOA. Cross-fostering the biological offspring of high LG mothers to low LG dams produces the opposite effect. We examined whether the maternal programing of ERalpha expression is associated with differences in methylation of the relevant ERalpha promoter. Levels of cytosine methylation across the ERalpha1b promoter were significantly elevated in the adult offspring of low, compared with high, LG mothers. Differentially methylated regions included a signal transducer and activator of transcription (Stat)5 binding site and the results of chromatin immunoprecipitation assays revealed decreased Stat5b binding to the ERalpha1b promoter in the adult offspring of low, compared with high, LG mothers. Finally, we found increased Stat5b levels in the MPOA of neonates reared by high, compared with low, LG mothers. These findings suggest that maternal care is associated with cytosine methylation of the ERalpha1b promoter, providing a potential mechanism for the programming of individual differences in ERalpha expression and maternal behavior in the female offspring.

To isolate Sinorhizobium meliloti mutants deficient in malate dehydrogenase (MDH) activity, random transposon Tn5tac1 insertion mutants were screened for conditional lethal phenotypes on complex medium. Tn5tac1 has an outward-oriented isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter (Ptac). The insertion in strain Rm30049 was mapped to the mdh gene, which was found to lie directly upstream of the genes encoding succinyl-CoA synthetase (sucCD) and 2-oxoglutarate dehydrogenase (sucAB and lpdA). Rm30049 required IPTG for wild-type growth in complex media, and had a complex growth phenotype in minimal media with different carbon sources. The mdh:: Tn5tacl insertion eliminated MDH activity under all growth conditions, and activities of succinyl-CoA synthetase, 2-oxoglutarate dehydrogenase, and succinate dehydrogenase were affected by the addition of IPTG. Reverse-transcriptase polymerase chain reaction (RT-PCR) studies confirmed that expression from Ptac was induced by IPTG and leaky in its absence. Alfalfa plants inoculated with Rm30049 were chlorotic and stunted, with small white root nodules, and had shoot dry weight and percent-N content values similar to those of uninoculated plants. Cosmid clone pDS15 restored MDH activity to Rm30049, complemented both the mutant growth and symbiotic phenotypes, and was found to carry six complete (sdhB, mdh, sucCDAB) and two partial (IpdA, sdhA) tricarboxylic acid cycle genes.