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Background Programmed epigenetic modifications occurring at early postnatal brain developmental stages may have a long-lasting impact on brain function and complex behavior throughout life. Notably, it is now emerging that several genes that undergo perinatal changes in DNA methylation are associated with neuropsychiatric disorders. In this context, we envisaged that epigenetic modifications during the perinatal period may potentially drive essential changes in the genes regulating brain levels of critical neuromodulators such as d-serine and d-aspartate. Dysfunction of this fine regulation may contribute to the genesis of schizophrenia or other mental disorders, in which altered levels of d-amino acids are found. We recently demonstrated that Ddo, the d-aspartate degradation gene, is actively demethylated to ultimately reduce d-aspartate levels. However, the role of epigenetics as a mechanism driving the regulation of appropriate d-ser levels during brain development has been poorly investigated to date. Methods We performed comprehensive ultradeep DNA methylation and hydroxymethylation profiling along with mRNA expression and HPLC-based d-amino acids level analyses of genes controlling the mammalian brain levels of d-serine and d-aspartate. DNA methylation changes occurring in specific cerebellar cell types were also investigated. We conducted high coverage targeted bisulfite sequencing by next-generation sequencing and single-molecule bioinformatic analysis. Results We report consistent spatiotemporal modifications occurring at the Dao gene during neonatal development in a specific brain region (the cerebellum) and within specific cell types (astrocytes) for the first time. Dynamic demethylation at two specific CpG sites located just downstream of the transcription start site was sufficient to strongly activate the Dao gene, ultimately promoting the complete physiological degradation of cerebellar d-serine a few days after mouse birth. High amount of 5′-hydroxymethylcytosine, exclusively detected at relevant CpG sites, strongly evoked the occurrence of an active demethylation process. Conclusion The present investigation demonstrates that robust and selective demethylation of two CpG sites is associated with postnatal activation of the Dao gene and consequent removal of d-serine within the mouse cerebellum. A single-molecule methylation approach applied at the Dao locus promises to identify different cell-type compositions and functions in different brain areas and developmental stages.

Epigenetic mechanisms can mediate gene-environment interactions relevant for complex disorders. The BDNF gene is crucial for development and brain plasticity, is sensitive to environmental stressors, such as hypoxia, and harbors the functional SNP rs6265 (Val(66)Met), which creates or abolishes a CpG dinucleotide for DNA methylation. We found that methylation at the BDNF rs6265 Val allele in peripheral blood of healthy subjects is associated with hypoxia-related early life events (hOCs) and intermediate phenotypes for schizophrenia in a distinctive manner, depending on rs6265 genotype: in ValVal individuals increased methylation is associated with exposure to hOCs and impaired working memory (WM) accuracy, while the opposite is true for ValMet subjects. Also, rs6265 methylation and hOCs interact in modulating WM-related prefrontal activity, another intermediate phenotype for schizophrenia, with an analogous opposite direction in the 2 genotypes. Consistently, rs6265 methylation has a different association with schizophrenia risk in ValVals and ValMets. The relationships of methylation with BDNF levels and of genotype with BHLHB2 binding likely contribute to these opposite effects of methylation. We conclude that BDNF rs6265 methylation interacts with genotype to bridge early environmental exposures to adult phenotypes, relevant for schizophrenia. The study of epigenetic changes in regions containing genetic variation relevant for human diseases may have beneficial implications for the understanding of how genes are actually translated into phenotypes.

The spatio-temporal regulation of genes involved in the synthesis and degradation of D-serine and D-aspartate such as serine racemase (SR), D-amino acid oxidase (DAO), G72 and D-aspartate oxidase (DDO), play pivotal roles in determining the correct levels of these D-amino acids in the human brain. Here we provide a comprehensive analysis of mRNA expression and DNA methylation status of these genes in post-mortem samples from hippocampus, dorsolateral prefrontal cortex, and cerebellum from patients with schizophrenia and non-psychiatric controls. DNA methylation analysis was performed at an ultradeep level, measuring individual epialleles frequency by single molecule approach. Differential CpG methylation and expression was detected across different brain regions, although no significant correlations were found with diagnosis. G72 showed the highest CpG and non-CpG methylation degree, which may explain the repression of G72 transcription in the brain regions considered here. Conversely, in line with the sustained SR mRNA expression in the analyzed areas, very low methylation levels were detected at this gene’s regulatory regions. Furthermore, for DAO and DDO, our single-molecule methylation approach demonstrated that analysis of epiallele distribution was able to detect differences in DNA methylation representing area-specific methylation signatures, which are likely not detectable with targeted or genome-wide classic methylation analyses.

In this study we assessed the BDNF promoter IV methylation state of a large genomic region surrounding promoter IV and evaluated BDNF transcript IV expression from prefrontal cortex and striatum of 15 schizophrenic and 15 control subjects. In prefrontal cortex, a single CpG site at -93, appeared to be undermethylated in patients׳group. BDNF mRNA levels in frontal cortex and striatum were variable among individuals but did not associate with disease.

Although it is well known that RET gene is strongly activated by retinoic acid (RA) in neuroblastoma cells, the mechanisms underlying such activation are still poorly understood. Here we show that a complex series of molecular events, that include modifications of both chromatin and DNA methylation state, accompany RA-mediated RET activation. Our results indicate that the primary epigenetic determinants of RA-induced RET activation differ between enhancer and promoter regions. At promoter region, the main mark of RET activation was the increase of H3K4me3 levels while no significant changes of the methylation state of H3K27 and H3K9 were observed. At RET enhancer region a bipartite chromatin domain was detected in unstimulated cells and a prompt demethylation of H3K27me3 marked RET gene activation upon RA exposure. Moreover, ChIP experiments demonstrated that EZH2 and MeCP2 repressor complexes were associated to the heavily methylated enhancer region in the absence of RA while both complexes were displaced during RA stimulation. Finally, our data show that a demethylation of a specific CpG site at the enhancer region could favor the displacement of MeCP2 from the heavily methylated RET enhancer region providing a novel potential mechanism for transcriptional regulation of methylated RA-regulated loci.

We performed ultra-deep methylation analysis at single molecule level of the promoter region of developmentally regulated D-Aspartate oxidase (Ddo), as a model gene, during brain development and embryonic stem cell neural differentiation. Single molecule methylation analysis enabled us to establish the effective epiallele composition within mixed or pure brain cell populations. In this framework, an epiallele is defined as a specific combination of methylated CpG within Ddo locus and can represent the epigenetic haplotype revealing a cell-to-cell methylation heterogeneity. Using this approach, we found a high degree of polymorphism of methylated alleles (epipolymorphism) evolving in a remarkably conserved fashion during brain development. The different sets of epialleles mark stage, brain areas, and cell type and unravel the possible role of specific CpGs in favoring or inhibiting local methylation. Undifferentiated embryonic stem cells showed non-organized distribution of epialleles that apparently originated by stochastic methylation events on individual CpGs. Upon neural differentiation, despite detecting no changes in average methylation, we observed that the epiallele distribution was profoundly different, gradually shifting toward organized patterns specific to the glial or neuronal cell types. Our findings provide a deep view of gene methylation heterogeneity in brain cell populations promising to furnish innovative ways to unravel mechanisms underlying methylation patterns generation and alteration in brain diseases. We performed ultra-deep methylation analysis at single molecule level of the promoter region of developmentally regulated D-Aspartate oxidase (Ddo), as a model gene, during brain development and embryonic stem cell neural differentiation. Single molecule methylation analysis enabled us to establish the effective epiallele composition within mixed or pure brain cell populations. In this framework, an epiallele is defined as a specific combination of methylated CpG within Ddo locus and can represent the epigenetic haplotype revealing a cell-to-cell methylation heterogeneity. Using this approach, we found a high degree of polymorphism of methylated alleles (epipolymorphism) evolving in a remarkably conserved fashion during brain development. The different sets of epialleles mark stage, brain areas, and cell type and unravel the possible role of specific CpGs in favoring or inhibiting local methylation. Undifferentiated embryonic stem cells showed non-organized distribution of epialleles that apparently originated by stochastic methylation events on individual CpGs. Upon neural differentiation, despite detecting no changes in average methylation, we observed that the epiallele distribution was profoundly different, gradually shifting toward organized patterns specific to the glial or neuronal cell types. Our findings provide a deep view of gene methylation heterogeneity in brain cell populations promising to furnish innovative ways to unravel mechanisms underlying methylation patterns generation and alteration in brain diseases.

In order to supplement the cytopathological assessment of thyroid tumors, there is a need for new markers to correctly diagnose malignant thyroid lesions and avoid unnecessary and potentially harmful therapies for patients. The immunohistochemical expression of galectin-3 is currently considered to be the most accurate stand-alone marker for thyroid cancer diagnosis. The aim of this study was to establish whether the methylation state of the galectin-3 gene is a candidate molecular marker for thyroid malignancy. Thyroid specimens from 50 patients were analyzed, including 5 normal thyroid, 3 goiters, 39 papillary and 3 anaplastic thyroid carcinoma cases. High-resolution methylation analyses was performed to investigate the methylation state of a large genomic region (from −89 to +408) encompassing the galectin-3 transcriptional start site. Within this region, 5 CpG sites (nucleotide positions +134, +137, +142, +147 and +156) were observed to be differentially methylated among the samples and were further analyzed by the quantitative pyrosequencing technique. The hypomethylation of the +134, +137, +142, +147 and +156 CpG sites was observed to be markedly associated with cancer. Although the methylation degree of each single site was highly variable in non-neoplastic tissues, the average methylation state of the 5 CpG sites clearly distinguished cancer from the nonneoplastic thyroid tissues.

One of the most fascinating aspects of the field of epigenetics is the emerging ability of environmental factors to trigger epigenetic changes in eukaryotic cells, thus contributing to transient or stable, and potentially heritable, changes in gene expression program in the absence of alteration in DNA sequence. Epigenetic response may result in cell adaptation to environmental stimuli or, in some instances, may contribute to generation or progression of different kind of diseases. A paradigmatic case of disease that is accompanied by multiple epigenetic alterations is gastric cancer, among other relevant examples. In turn, Helicobacter pylori (Hp) infection has been associated as a leading cause of gastric cancer. One possible hypothesis is that Hp-gastric cell interaction initiates an epigenetic reprogramming of host cell genome that may favor tumorigenesis. Accordingly, an abundance of experimental evidence indicates that several epigenetic alterations underlie the gastric cancerogenesis process and that these alterations represent one of the major hallmarks of gastric cancer. However, several critical questions remain unanswered: Does Hp directly provoke epigenetic alterations? Which mechanisms underlie these phenomena? Based on currently available data, it is often arduous to discriminate between the epigenetic modifications directly triggered by Hp-gastric cell interaction and those alterations that are mediated by inflammation process or by many other molecular and genetic events occurring during the gastric cancer progression. We will review our present knowledge of epigenetic modifications and alterations proven to occur in host cells as a direct consequence of Hp infection.

Background Alterations of DNA methylation and expression of suicide-related genes occurring in specific brain's areas have been associated to suicidal behavior. In the BDNF pathway, TrkB gene in frontal cortex and hippocampus, and BDNF gene in Wernicke area have been found hypermethylated and down-regulated in suicide subjects as compared to controls. In this work we investigated whether epigenetic modifications of TrkB gene occur in Wernicke area of 18 suicide subjects as compared to 18 controls. Methods MassArray analysis was performed to determine the methylation degree of TrkB promoter in post-mortem samples. TrkB full length and TrkB-T1 mRNA levels were assessed by quantitative RT-PCR. Geometric averaging of four internal control genes was calculated for normalization of results. Results We found that TrkB and TrkB-T1 expression and promoter methylation in Wernicke area did not correlate with suicidal behavior whereas, in the same samples, the BDNF promoter IV was significantly hypermethylated in suicide with respect of controls. Limitation Data from a single brain's area in this study's sample. Conclusions Our data show that no correlation exists between TrkB gene methylation and suicide in Wernicke area, confirming that expression and methylation state of suicide-related genes, even belonging to the same pathway, may be specific for brain area.

COX-2 expression is altered in gastrointestinal diseases. Helicobacter pylori (Hp) infection may have a critical role in COX-2 disregulation. We undertook this study to investigate possible chromatin and DNA methylation changes occurring early during COX-2 gene activation as a direct consequence of Hp-gastric cells interaction. We show that Hp infection is followed by different expression, chromatin and DNA methylation changes including: (i) biphasic activation of COX-2 gene; (ii) rapid remodulation of HDACs expression and activity, increased acetylation and release of HDAC from COX-2 promoter; (iii) transient gradual increase of H3 acetylation and H3K4 dimethylation and decrease of H3K9 dimethylation; (iv) late and long-lasting increase of H3K27 trimethylation; (v) rapid cyclical DNA methylation/demethylation events at 8 specific CpG sites (-176, -136, +25, +36, +57, +82, +198, +231) surrounding the COX-2 gene transcriptional start site. Our data indicate that specific chromatin and DNA methylation changes occur at COX-2 gene in the first phases of Hp exposure in cultured gastric cells as a primary response to host-parasite interaction.

CONTEXT: Brain-derived neurotrophic factor (BDNF) plays a pivotal role in the pathophysiology of suicidal behavior and BDNF levels are decreased in the brain and plasma of suicide subjects. So far, the mechanisms leading to downregulation of BDNF expression are poorly understood. OBJECTIVES: To test the hypothesis that alterations of DNA methylation could be involved in the dysregulation of BDNF gene expression in the brain of suicide subjects. DESIGN: Three independent quantitative methylation techniques were performed on postmortem samples of brain tissue. BDNF messenger RNA levels were determined by quantitative real-time polymerase chain reaction. SETTING: Academic medical center. Patients or Other PARTICIPANTS: Forty-four suicide completers and 33 nonsuicide control subjects of white ethnicity. MAIN OUTCOME MEASURES: The DNA methylation degree at BDNF promoter IV and the genome-wide DNA methylation levels in the brain's Wernicke area. RESULTS: Postmortem brain samples from suicide subjects showed a statistically significant increase of DNA methylation at specific CpG sites in BDNF promoter/exon IV compared with nonsuicide control subjects (P < .001). Most of the CpG sites lying in the -300/+500 region, on both strands, had low or no methylation, with the exception of a few sites located near the transcriptional start site that had differential methylation, while genome-wide methylation levels were comparable among the subjects. The mean methylation degree at the 4 CpG sites analyzed by pyrosequencing was always less than 12.9% in the 33 nonsuicide control subjects, while in 13 of 44 suicide victims (30%), the mean methylation degree ranged between 13.1% and 34.2%. Higher methylation degree corresponded to lower BDNF messenger RNA levels. CONCLUSIONS: BDNF promoter/exon IV is frequently hypermethylated in the Wernicke area of the postmortem brain of suicide subjects irrespective of genome-wide methylation levels, indicating that a gene-specific increase in DNA methylation could cause or contribute to the downregulation of BDNF expression in suicide subjects. The reported data reveal a novel link between epigenetic alteration in the brain and suicidal behavior.

Background The release of LPS by bacteria stimulates both immune and specific epithelial cell types to release inflammatory mediators. It is known that LPS induces the release of IL-8 by intestinal mucosal cells. Because it is now emerging that bacteria may induce alteration of epigenetic patterns in host cells, we have investigated whether LPS-induced IL-8 activation in human intestinal epithelial cells involves changes of histone modifications and/or DNA methylation at IL-8 gene regulatory region. Results RT-PCR analysis showed that IL-8 mRNA levels rapidly increase after exposure of HT-29 cells to LPS. DNA demethylating agents had no effects on IL-8 expression, suggesting that DNA methylation was not involved in IL-8 gene regulation. Consistently we found that 5 CpG sites located around IL-8 transcription start site (-83, -7, +73, +119, +191) were unmethylated on both lower and upper strand either in LPS treated or in untreated HT-29 cells, as well as in normal intestinal mucosa. Conversely, pretreatment of HT-29 cells with deacetylase inhibitors strengthened the LPS-mediated IL-8 activation. Inhibitors of histone deacetylases could induce IL-8 mRNA expression also in the absence of LPS, suggesting that chromatin modifications could be involved in IL-8 gene regulation. Chromatin immunoprecipitation analyses showed that, concurrently with IL-8 activation, transient specific changes in H3 acetylation and H3K4, H3K9 and H3K27 methylation occurred at IL-8 gene promoter during LPS stimulation. Changes of H3-acetyl, H3K4me2 and H3K9me2 levels occurred early, transiently and corresponded to transcriptional activity, while changes of H3K27me3 levels at IL-8 gene occurred later and were long lasting. Conclusion The results showed that specific chromatin modifications occurring at IL-8 gene, including histone H3 acetylation and methylation, mark LPS-mediated IL-8 activation in intestinal epithelial cells while it is unlikely that DNA methylation of IL-8 promoter is directly involved in IL-8 gene regulation in these cells.

The most frequent mutation of Friedreich ataxia (FRDA) is the abnormal expansion of a GAA repeat located within the first intron of FXN gene. It is known that the length of GAA is directly correlated with disease severity. The effect of mutation is a severe reduction of mRNA. Recently, a link among aberrant CpG methylation, chromatin organisation and GAA repeat was proposed. METHODS: In this study, using pyrosequencing technology, we have performed a quantitative analysis of the methylation status of five CpG sites located within the region upstream of GAA repeat, in 67 FRDA patients. RESULTS: We confirm previous observation about differences in the methylation degree between FRDA individuals and controls. We showed a direct correlation between CpG methylation and triplet expansion size. Significant differences were found for each CpG tested (ANOVA p

Re-induction of fetal genes and/or re-expression of postnatal genes represent hallmarks of pathological cardiac remodeling, and are considered important in the progression of the normal heart towards heart failure (HF). Whether epigenetic modifications are involved in these processes is currently under investigation. Here we hypothesized that histone chromatin modifications may underlie changes in the gene expression program during pressure overload-induced HF. We evaluated chromatin marks at the promoter regions of the sarcoplasmic reticulum Ca(2+)ATPase (SERCA-2A) and β-myosin-heavy chain (β-MHC) genes (Atp2a2 and Myh7, respectively) in murine hearts after one or eight weeks of pressure overload induced by transverse aortic constriction (TAC). As expected, all TAC hearts displayed a significant reduction in SERCA-2A and a significant induction of β-MHC mRNA levels. Interestingly, opposite histone H3 modifications were identified in the promoter regions of these genes after TAC, including H3 dimethylation (me2) at lysine (K) 4 (H3K4me2) and K9 (H3K9me2), H3 trimethylation (me3) at K27 (H3K27me3) and dimethylation (me2) at K36 (H3K36me2). Consistently, a significant reduction of lysine-specific demethylase KDM2A could be found after eight weeks of TAC at the Atp2a2 promoter. Moreover, opposite changes in the recruitment of DNA methylation machinery components (DNA methyltransferases DNMT1 and DNMT3b, and methyl CpG binding protein 2 MeCp2) were found at the Atp2a2 or Myh7 promoters after TAC. Taken together, these results suggest that epigenetic modifications may underlie gene expression reprogramming in the adult murine heart under conditions of pressure overload, and might be involved in the progression of the normal heart towards HF.

Bacterial lipopolysaccharide (LPS) induces release of inflammatory mediators both in immune and epithelial cells. We investigated whether changes of epigenetic marks, including selected histone modification and DNA methylation, may drive or accompany the activation of COX-2 gene in HT-29 human intestinal epithelial cells upon exposure to LPS. Here we describe cyclical histone acetylation (H3), methylation (H3K4, H3K9, H3K27) and DNA methylation changes occurring at COX-2 gene promoter overtime after LPS stimulation. Histone K27 methylation changes are carried out by the H3 demethylase JMJD3 and are essential for COX-2 induction by LPS. The changes of the histone code are associated with cyclical methylation signatures at the promoter and gene body of COX-2 gene.

Increasing evidence points to a role for dysfunctional glutamate N-methyl-D-aspartate receptor (NMDAR) neurotransmission in schizophrenia. D-aspartate is an atypical amino acid that activates NMDARs through binding to the glutamate site on GluN2 subunits. D-aspartate is present in high amounts in the embryonic brain of mammals and rapidly decreases after birth, due to the activity of the enzyme D-aspartate oxidase (DDO). The agonistic activity exerted by D-aspartate on NMDARs and its neurodevelopmental occurrence make this D-amino acid a potential mediator for some of the NMDAR-related alterations observed in schizophrenia. Consistently, substantial reductions of D-aspartate and NMDA were recently observed in the postmortem prefrontal cortex of schizophrenic patients. Here we show that DDO mRNA expression is increased in prefrontal samples of schizophrenic patients, thus suggesting a plausible molecular event responsible for the D-aspartate imbalance previously described. To investigate whether altered D-aspartate levels can modulate schizophrenia-relevant circuits and behaviors, we also measured the psychotomimetic effects produced by the NMDAR antagonist, phencyclidine, in Ddo knockout mice (Ddo(-)(/-)), an animal model characterized by tonically increased D-aspartate levels since perinatal life. We show that Ddo(-/-) mice display a significant reduction in motor hyperactivity and prepulse inhibition deficit induced by phencyclidine, compared with controls. Furthermore, we reveal that increased levels of D-aspartate in Ddo(-/-) animals can significantly inhibit functional circuits activated by phencyclidine, and affect the development of cortico-hippocampal connectivity networks potentially involved in schizophrenia. Collectively, the present results suggest that altered D-aspartate levels can influence neurodevelopmental brain processes relevant to schizophrenia.