Increased Arterial Responsiveness to Angiotensin II in Mice Conceived by Assisted Reproductive Technologies.

Since the first report in 1978, the number of individuals conceived by Assisted Reproductive Technologies (ART) has grown incessantly. In parallel, with the recent emergence of possible underlying mechanisms of ART-induced epigenetic changes in the renin-angiotensin system, the cardiovascular repercussions of ART in mice and human offspring (including arterial hypertension, vascular dysfunction, and cardiac remodeling) have become increasingly recognized. Here, we hypothesized that ART may increase arterial responsiveness to angiotensin II (ANG II) by epigenetically modifying the expression of its receptors. To test this hypothesis, we assessed the vasoconstrictor responsiveness to ANG II in isolated aortas from ART and control mice. We also examined ANG II receptor (ATR) type 1 and 2 expression and the promoter methylation of the At1aR, At1bR and At2R genes. We found that the vasoconstrictor response to ANG II was markedly increased in ART mice compared to controls. This exaggerated vasoconstrictor responsiveness in ART mice correlated with a significant increase in the ANG II receptor (ATR) type 1 to ATR type 2 protein expression ratio in the aorta; this was mainly driven by an increase in AT1R expression, and by hypomethylation of two CpG sites located in the At1bR gene promoter leading to increased transcription of the gene. We conclude that in mice, ART increase the vasoconstrictor response to ANG II in the aorta by epigenetically causing an imbalance between the expression of vasoconstrictor (AT1R) and vasodilator (AT2R) ANG II receptors. Unbalanced expression of AT1R and AT2R receptors seems to be a novel mechanism contributing to ART-induced arterial hypertension in mice.

The L1-dependant and Pol III transcribed Alu retrotransposon, from its discovery to innate immunity.

The 300 bp dimeric repeats digestible by AluI were discovered in 1979. Since then, Alu were involved in the most fundamental epigenetic mechanisms, namely reprogramming, pluripotency, imprinting and mosaicism. These Alu encode a family of retrotransposons transcribed by the RNA Pol III machinery, notably when the cytosines that constitute their sequences are de-methylated. Then, Alu hijack the functions of ORF2 encoded by another transposons named L1 during reverse transcription and integration into new sites. That mechanism functions as a complex genetic parasite able to copy-paste Alu sequences. Doing that, Alu have modified even the size of the human genome, as well as of other primate genomes, during 65 million years of co-evolution. Actually, one germline retro-transposition still occurs each 20 births. Thus, Alu continue to modify our human genome nowadays and were implicated in de novo mutation causing diseases including deletions, duplications and rearrangements. Most recently, retrotransposons were found to trigger neuronal diversity by inducing mosaicism in the brain. Finally, boosted during viral infections, Alu clearly interact with the innate immune system. The purpose of that review is to give a condensed overview of all these major findings that concern the fascinating physiology of Alu from their discovery up to the current knowledge.

Intergenerational Transmission of DNA Methylation Signatures Associated with Early Life Stress.

Early life stress in humans (i.e. maltreatment, violence exposure, loss of a loved one) and in rodents (i.e. disrupted attachment or nesting, electric shock, restraint, predator odor) occurs during critical steps of neural circuit formation. ELS in humans is associated with increased risk for developmental psychopathology, including anxious and depressive phenotypes. The biological mechanisms underlying these potentially persistent maladaptive changes involve long-term epigenetic modifications, which have been suggested to be potentially transmissible to subsequent generations. DNA methylation is an epigenetic mechanism that modifies gene expression patterns in response to environmental challenges and influences mutation rates. It remains to be seen whether a functionally relevant fraction of DNA methylation marks can escape genome-wide erasures that occur in primordial germ cells and after fertilization within the zygote. Early life-stress-triggered changes in epigenetic mediated transmission of acquired behavioral traits among humans have been assessed mainly by targeting genes involved in the hypothalamic-pituitary-adrenal (HPA) axis, such as NR3C1 and FKBP5. Recently, researchers examining epigenetic transmission have begun to apply genome-wide approaches. In humans, reduced representation bisulfite sequencing (RRBS) was performed on peripheral samples that were obtained from individuals who were prenatally exposed to the "Dutch Hunger Winter", resulting in two Differentially Methylated Regions (DMRs) in INSR and CPTIA genes that were functionally, biologically and technically validated, and significantly associated with birth weights and LDL cholesterol levels in offspring. In rodents, non-genomic intergenerational transmission of anxiety which was associated with differentially methylated enhancers that were putatively involved in lipid signaling and synaptic/neurotransmission in hippocampal granule cells, was discovered also using RRBS. Finally, transgenerational transmission of altered behaviors was associated with sperm-derived microRNAs produced by ELS male mice. The field of epigenetic transmission is just beginning to enter the epigenomic era by using genome-wide analyses. Such approaches remain of strong interest to human studies, first in order to help to assess the relevance of the previous targeted studies, and second to discover new important epigenetic modifications of potential clinical importance. New discoveries may help to assess how transmittable the negative impact of stress may be to offspring. The latter may open doors for future treatments and resilience-promoting interventions, as well as new approaches to treat the effects of childhood trauma before the onset of psychiatric disorder.

METHYLATION OF SEROTONIN RECEPTOR 3A IN ADHD, BORDERLINE PERSONALITY, AND BIPOLAR DISORDERS: LINK WITH SEVERITY OF THE DISORDERS AND CHILDHOOD MALTREATMENT.

BACKGROUND: Serotonin 3A receptor (5-HT3A R) is associated at the genetic and epigenetic levels with a variety of psychiatric disorders and interacts with early-life stress such as childhood maltreatment. We studied the impact of childhood maltreatment on the methylation status of the 5-HT3A R and its association with clinical severity outcomes in relation with a functional genetic polymorphism. METHODS: Clinical severity indexes of 346 bipolar, borderline personality, and adult attention deficit hyperactivity disorders patients were tested for association with the DNA methylation status of eight 5-HT3A R gene CpGs. Relationship between the functional variant rs1062613 (C > T) and methylation status on severity of the disorders were also assessed. RESULTS: Childhood maltreatment was associated with higher severity of the disease (higher number of mood episodes, history of suicide attempts, hospitalization, and younger age at onset) across disorders and within each individual disorder. This effect was mediated by two 5-HT3A R CpGs. Compared to T allele carriers, CC carriers had higher methylation status at one CpG located 1 bp upstream of this variant. CONCLUSIONS: This study shows that epigenetic modification of the 5-HT3A R is involved in the mechanism underlying the relationship between maltreatment in childhood and the severity of several psychiatric disorders in adulthood.

Epigenomic Changes after Acupuncture Treatment in Patients Suffering from Burnout.

INTRODUCTION: The effects of acupuncture treatment in patients suffering from burnout may imply an epigenetic control mediated by DNA methylation changes. In this observational study, a genome-wide characterization of epigenetic changes in blood DNA, before and after acupuncture treatment, was performed in a cohort of 11 patients suffering from burnout. METHODS: Burnout was assessed using the Maslach Burnout Inventory (MBI) and DNA was extracted from blood samples and analyzed by Illumina EPIC BeadChip. RESULTS: Before acupuncture, all patients suffered of emotional exhaustion (EE) (MBI-EE score, 44 ± 6), 81% suffered of depersonalization (DP) (MBI-DP score, 16 ± 6), and 72% of low feelings of personal accomplishment (PA) (MBI-PA score, 29 ± 9). After acupuncture, all MBI dimensions improved significantly (EE, 16 ± 11 [p = 1.5 × 10-4]; DP, 4 ± 5 [p = 5.3 × 10-4]; and PA, 40 ± 6 [p = 4.1 × 10-3]). For each patient, both methylomes obtained before and after acupuncture co-clustered in the multidimensional scaling plot, indicating a high level of similarity. Genes corresponding to the 10 most differentially methylated CpGs showed enrichment in the brain dopaminergic signaling, steroid synthesis and in the insulin sensitivity pathways. CONCLUSION: Acupuncture treatment was found to be highly effective on all burnout dimensions and the epigenetic targets identified were involved in some major disturbances of this syndrome.

Genome-Wide Epigenomic Analyses in Patients With Nociceptive and Neuropathic Chronic Pain Subtypes Reveals Alterations in Methylation of Genes Involved in the Neuro-Musculoskeletal System.

Nociceptive pain involves the activation of nociceptors without damage to the nervous system, whereas neuropathic pain is related to an alteration in the central or peripheral nervous system. Chronic pain itself and the transition from acute to chronic pain may be epigenetically controlled. In this cross-sectional study, a genome-wide DNA methylation analysis was performed using the blood DNA reduced representation bisulfite sequencing (RRBS) technique. Three prospective cohorts including 20 healthy controls (CTL), 18 patients with chronic nociceptive pain (NOCI), and 19 patients with chronic neuropathic pain (NEURO) were compared at both the single CpG and differentially methylated region (DMR) levels. Genes with DMRs were seen in the NOCI and NEURO groups belonged to the neuro-musculoskeletal system and differed between NOCI and NEURO patients. Our results demonstrate that the epigenetic disturbances accompanying nociceptive pain are very different from those accompanying neuropathic pain. In the former, among others, the epigenetic disturbance observed would affect the function of the opioid analgesic system, whereas in the latter it would affect that of the GABAergic reward system. This study presents biological findings that help to characterize NOCI- and NEURO-affected pathways and opens the possibility of developing epigenetic diagnostic assays. PERSPECTIVE: Our results help to explain the various biological pathways modifications underlying the different clinical manifestations of nociceptive and neuropathic pains. Furthermore, the new targets identified in our study might help to discover more specific treatments for nociceptive or neuropathic pains.

Altered DNA methylation in estrogen-responsive repetitive sequences of spermatozoa of infertile men with shortened anogenital distance.

BACKGROUND: It has been suggested that antenatal exposure to environmental endocrine disruptors is responsible for adverse trends in male reproductive health, including male infertility, impaired semen quality, cryptorchidism and testicular cancer, a condition known as testicular dysgenesis syndrome. Anogenital distance (AGD) is an anthropomorphic measure of antenatal exposure to endocrine disruptors, with higher exposure levels leading to shortened AGD. We hypothesized that exposure to endocrine disruptors could lead to changes in DNA methylation during early embryonic development, which could then persist in the sperm of infertile men with shortened AGD. RESULTS: Using fluorescence activated cell sorting based on staining with either YO-PRO-1 (YOPRO) or chromomycin-3 (CMA3), we isolated four sperm fractions from eleven infertile men with short AGD and ten healthy semen donors. We examined DNA methylation in these sorted spermatozoa using reduced representation bisulfite sequencing. We found that fractions of spermatozoa from infertile men stained with CMA3 or YOPRO were more likely to contain transposable elements harboring an estrogen receptor response element (ERE). Abnormal sperm (as judged by high CMA3 or YOPRO staining) from infertile men shows substantial hypomethylation in estrogenic Alu sequences. Conversely, normal sperm fractions (as judged by low CMA3 or YO-PRO-1 staining) of either healthy donors or infertile patients were more likely to contain hypermethylated Alu sequences with ERE. CONCLUSIONS: Shortened AGD, as related to previous exposure to endocrine disruptors, and male infertility are accompanied by increased presence of hormonal response elements in the differentially methylated regulatory sequences of the genome of sperm fractions characterized by chromatin decondensation and apoptosis.

The relationship of maternal and child methylation of the glucocorticoid receptor NR3C1 during early childhood and subsequent child psychopathology at school-age in the context of maternal interpersonal violence-related post-traumatic stress disorder.

Introduction: Interpersonal violent (IPV) experiences when they begin in childhood and continue in various forms during adulthood often lead to chronic post-traumatic stress disorder (PTSD) that is associated in multiple studies with hypocortisolism and lower percentage of methylation of the promoter region of the gene coding for the glucocorticoid receptor (NR3C1). This prospective, longitudinal study examined the relationship of NR3C1 methylation among mothers with IPV-related PTSD and their toddlers and then looked at the relationship of maternal NR3C1 methylation and child psychopathology at school age. Methods: Forty-eight mothers were evaluated for life-events history and post-traumatic stress disorder via structured clinical interview when their children were ages 12-42 months (mean age 26.7 months, SD 8.8). Their children's psychopathology in terms of internalizing symptoms and externalizing behaviors was evaluated using the Child Behavior Checklist at ages 5-9 years (mean age 7 years, SD 1.1). Percentage of methylation for the NR3C1 gene promoter region was assessed from DNA extracted from maternal and child saliva using bisulfite pyrosequencing. Data analysis involved parametric and non-parametric correlations and multiple linear and logistic regression modeling. Results: Logistic regression models using child NR3C1 methylation as the dependent variable and maternal NR3C1 methylation and PTSD group status as predictors, as well as the interaction indicated that all three of these significantly predicted child NR3C1 methylation. These findings remained significant when controlling for child age, sex and maternal child abuse history. Overall, maternal NR3C1 methylation when children were toddlers was negatively and significantly associated with child externalizing behavior severity at school age. Discussion: We found that correlations between mothers and their children of NR3C1 methylation levels overall and at all individual CpG sites of interest were significant only in the IPV-PTSD group. The latter findings support that NR3C1 methylation in mothers positively and statistically significantly correlates with NR3C1 methylation in their children only in presence of IPV-PTSD in the mothers. This maternal epigenetic signature with respect to this glucocorticoid receptor is significantly associated with child behavior that may well pose a risk for intergenerational transmission of violence and related psychopathology.

Daptomycin resistance mechanisms in clinically derived Staphylococcus aureus strains assessed by a combined transcriptomics and proteomics approach.

OBJECTIVES: The development of daptomycin resistance in Staphylococcus aureus is associated with clinical treatment failures. The mechanism(s) of such resistance have not been clearly defined. METHODS: We studied an isogenic daptomycin-susceptible (DAP(S)) and daptomycin-resistant (DAP(R)) S. aureus strain pair (616; 701) from a patient with relapsing endocarditis during daptomycin treatment, using comparative transcriptomic and proteomic techniques. RESULTS: Minor differences in the genome content were found between strains by DNA hybridization. Transcriptomic analyses identified a number of genes differentially expressed in important functional categories: cell division; metabolism of bacterial envelopes; and global regulation. Of note, the DAP(R) isolate exhibited reduced expression of the major cell wall autolysis gene coincident with the up-regulation of genes involved in cell wall teichoic acid production. Using quantitative (q)RT-PCR on the gene cadre putatively involved in cationic peptide resistance, we formulated a putative regulatory network compatible with microarray data sets, mainly implicating bacterial envelopes. Of interest, qRT-PCR of this same gene cadre from two distinct isogenic DAP(S)/DAP(R) clinical strain pairs revealed evidence of other strain-dependent networks operative in the DAP(R) phenotype. Comparative proteomics of 616 versus 701 revealed a differential abundance of proteins in various functional categories, including cell wall-associated targets and biofilm formation proteins. Phenotypically, strains 616 and 701 showed major differences in their ability to develop bacterial biofilms in the presence of the antibacterial lipid, oleic acid. CONCLUSIONS: Compatible with previous in vitro observations, in vivo-acquired DAP(R) in S. aureus is a complex, multistep phenomenon involving: (i) strain-dependent phenotypes; (ii) transcriptome adaptation; and (iii) modification of the lipid and protein contents of cellular envelopes.

Altered BDNF Methylation in Patients with Chronic Musculoskeletal Pain and High Biopsychosocial Complexity.

PURPOSE: The INTERMED instrument, which was developed to measure patient's biopsychosocial (BPS) complexity, represents a powerful diagnostic and therapeutic tool. Epigenetic changes are the interface between signals from the environment and genetic modifications, affecting gene expression, in particular, by DNA methylation of CpG dinucleotides in promotor regions of the corresponding genes. The brain-derived neurotrophic factor (BDNF) gene plays a crucial role in the central sensitization (CS) of pain. In this study, we hypothesized that chronic pain modifies the methylation levels of the BDNF gene in a manner that is interconnected with the BPS status. PATIENTS AND METHODS: Fifty-eight chronic musculoskeletal pain patients (CMSP) were enrolled in the study. DNA was extracted from blood samples, the methylation levels of 13 CpG sites in the BDNF promoter were measured by pyrosequencing, and association studies with various patient parameters and the INTERMED scores were performed. RESULTS: Interestingly, a negative correlation (-0.40) was found between the total INTERMED scores and the average CpG methylation values of the BDNF gene, but no correlation was observed with the severity of pain, degree of anxiety, depression, or kinesiophobia and catastrophism. Moreover, the association was independent of age, sex and level of comorbidities. CONCLUSION: This result shows that CMSP, in association with its biopsychosocial context, epigenetically decreases the degree of methylation of the BDNF promoter and should therefore increase the level of BDNF transcription. It also suggests a role of the INTERMED tool to detect a relationship between the BPS complexity and the epigenetic control of a target gene. The possible upregulation of BDNF expression might be, at least in part, the signal for chronic pain-induced central sensitization (CS). This could partly explain why patients with a higher level of complexity feel more pain than those with lower complexity.

CodY in Staphylococcus aureus: a regulatory link between metabolism and virulence gene expression.

The repressor CodY is reported to inhibit metabolic genes mainly involved in nitrogen metabolism. We analyzed codY mutants from three unrelated Staphylococcus aureus strains (Newman, UAMS-1, and RN1HG). The mutants grew more slowly than their parent strains in a chemically defined medium. However, only codY mutants were able to grow in medium lacking threonine. An excess of isoleucine resulted in growth inhibition in the wild type but not in the codY mutants, indicating that isoleucine plays a role in CodY-dependent repression. Prototypic CodY-repressed genes including the virulence regulator agr are repressed after up-shift with isoleucine. The CodY-dependent repression of agr is consistent with the concomitant influence of CodY on typical agr-regulated genes such as cap, spa, fnbA, and coa. However, some of these virulence genes (e.g., cap, fnbA, and spa) were also regulated by CodY in an agr-negative background. Microarray analysis revealed that the large majority of CodY-repressed genes were involved in amino acid metabolism; CodY-activated genes were mainly involved in nucleotide metabolism or virulence. In summary, CodY in S. aureus not only acts as a repressor for genes involved in nitrogen metabolism but also contributes to virulence gene regulation by supporting as well as substituting for agr function.

Genetic resistance to DEHP-induced transgenerational endocrine disruption.

Di(2-ethylhexyl)phthalate (DEHP) interferes with sex hormones signaling pathways (SHP). C57BL/6J mice prenatally exposed to 300 mg/kg/day DEHP develop a testicular dysgenesis syndrome (TDS) at adulthood, but similarly-exposed FVB/N mice are not affected. Here we aim to understand the reasons behind this drastic difference that should depend on the genome of the strain. In both backgrounds, pregnant female mice received per os either DEHP or corn oil vehicle and the male filiations were examined. Computer-assisted sperm analysis showed a DEHP-induced decreased sperm count and velocities in C57BL/6J. Sperm RNA sequencing experiments resulted in the identification of the 62 most differentially expressed RNAs. These RNAs, mainly regulated by hormones, produced strain-specific transcriptional responses to prenatal exposure to DEHP; a pool of RNAs was increased in FVB, another pool of RNAs was decreased in C57BL/6J. In FVB/N, analysis of non-synonymous single nucleotide polymorphisms (SNP) impacting SHP identified rs387782768 and rs29315913 respectively associated with absence of the Forkhead Box A3 (Foxa3) RNA and increased expression of estrogen receptor 1 variant 4 (NM_001302533) RNA. Analysis of the role of SNPs modifying SHP binding sites in function of strain-specific responses to DEHP revealed a DEHP-resistance allele in FVB/N containing an additional FOXA1-3 binding site at rs30973633 and four DEHP-induced beta-defensins (Defb42, Defb30, Defb47 and Defb48). A DEHP-susceptibility allele in C57BL/6J contained five SNPs (rs28279710, rs32977910, rs46648903, rs46677594 and rs48287999) affecting SHP and six genes (Svs2, Svs3b, Svs4, Svs3a, Svs6 and Svs5) epigenetically silenced by DEHP. Finally, targeted experiments confirmed increased methylation in the Svs3ab promoter with decreased SEMG2 persisting across generations, providing a molecular explanation for the transgenerational sperm velocity decrease found in C57BL/6J after DEHP exposure. We conclude that the existence of SNP-dependent mechanisms in FVB/N inbred mice may confer resistance to transgenerational endocrine disruption.

Impact of oleic acid (cis-9-octadecenoic acid) on bacterial viability and biofilm production in Staphylococcus aureus.

Staphylococcus aureus is responsible for a broad variety of chronic infections. Most S. aureus clinical isolates show the capacity to adhere to abiotic surfaces and to develop biofilms. Because S. aureus growing in a biofilm is highly refractory to treatment, inhibition of biofilm formation represents a major therapeutic objective. We evaluated the effects of oleic acid on primary adhesion and biofilm production in eight genotypically different S. aureus strains as well as in the biofilm-negative Staphylococcus carnosus strain TM300. Oleic acid inhibited primary adhesion but increased biofilm production in every S. aureus strain tested. Staphylococcus aureus strain UAMS-1 was then selected as a model organism for studying the mechanisms triggered by oleic acid on the formation of a biofilm in vitro. Oleic acid inhibited the primary adhesion of UAMS-1 dose dependently with an IC(50) around 0.016%. The adherent bacterial population decreased proportionally with increasing concentrations of oleic acid whereas an opposite effect was observed on the planktonic population. Overall, the total bacterial counts remained stable. Macroscopic detachments and clumps were visible from the adherent bacterial population. In the presence of oleic acid, the expression of sigB, a gene potentially involved in bacterial survival through an effect on fatty acid composition, was not induced. Our results suggest a natural protective effect of oleic acid against primary adhesion.

Recipient rs1045642 Polymorphism Is Associated With Office Blood Pressure at 1-Year Post Kidney Transplantation: A Single Center Pharmacogenetic Cohort Pilot Study.

Background: Corticosteroids are associated with reduced bone mineral density (BMD), as well as water and salt retention, leading to hypertension. They are substrates for P-glycoprotein, a protein coded by the highly polymorphic ABCB1 gene. We hypothesized that one ABCB1 polymorphism, rs1045642, is associated with blood pressure and BMD parameters at 1-year post kidney transplantation (KT). Methods: Rs1045642 was genotyped using pyrosequencing in 40 KT recipients. Both dominant (CC vs. CT + TT) and codominant (CC vs. CT vs. TT) genetic models (analysis of variance from linear regressions) were adjusted for confounding variables (age, sex, type of nephropathy, glomerular filtration rate, and corticosteroid use at 1 year). Results: Rs1045642 genotypes were significantly associated with systolic (SBP) and diastolic (DBP) blood pressure 1-year post-transplantation, independent of the genetic model used (adjusted codominant model: SBP p-value = 0.015, DBP p-value = 0.038; adjusted dominant model: SBP p-value = 0.003, DBP p-value = 0.011). A non-statistically significant trend was observed for an association between rs1045642 and BMD change at 1-year post-KT. Conclusions: Rs1045642 is significantly associated with higher BP 1 year after KT. Further investigations are necessary to confirm the role of rs1045642 in corticosteroid-related adverse effects.

Testicular Dysgenesis Syndrome and Long-Lasting Epigenetic Silencing of Mouse Sperm Genes Involved in the Reproductive System after Prenatal Exposure to DEHP.

The endocrine disruptor bis(2-ethylhexyl) phthalate (DEHP) has been shown to exert adverse effects on the male animal reproductive system. However, its mode of action is unclear and a systematic analysis of its molecular targets is needed. In the present study, we investigated the effects of prenatal exposure to 300 mg/kg/day DEHP during a critical period for gonads differentiation to testes on male mice offspring reproductive parameters, including the genome-wide RNA expression and associated promoter methylation status in the sperm of the first filial generation. It was observed that adult male offspring displayed symptoms similar to the human testicular dysgenesis syndrome. A combination of sperm transcriptome and methylome data analysis allowed to detect a long-lasting DEHP-induced and robust promoter methylation-associated silencing of almost the entire cluster of the seminal vesicle secretory proteins and antigen genes, which are known to play a fundamental role in sperm physiology. It also resulted in the detection of a DEHP-induced promoter demethylation associated with an up-regulation of three genes apparently not relevant for sperm physiology and partially related to the immune system. As previously reported, DEHP induced an increase in mir-615 microRNA expression and a genome-wide decrease in microRNA promoter methylation. A functional analysis revealed DEHP-induced enrichments in down-regulated gene transcripts coding for peroxisome proliferator-activated receptors and tumor necrosis factor signaling pathways, and in up-regulated gene transcripts coding for calcium binding and numerous myosin proteins. All these enriched pathways and networks have been described to be associated in some way with the reproductive system. This study identifies a large new array of genes dysregulated by DEHP that may play a role in the complex system controlling the development of the male reproductive system.

The association of serotonin receptor 3A methylation with maternal violence exposure, neural activity, and child aggression.

BACKGROUND: Methylation of the serotonin 3A receptor gene (HTR3A) has been linked to child maltreatment and adult psychopathology. The present study examined whether HTR3A methylation might be associated with mothers' lifetime exposure to interpersonal violence (IPV), IPV-related psychopathology, child disturbance of attachment, and maternal neural activity. METHODS: Number of maternal lifetime IPV exposures and measures of maternal psychopathology including posttraumatic stress disorder (PTSD), major depression and aggressive behavior (AgB), and a measure of child attachment disturbance known as "secure base distortion" (SBD) were assessed in a sample of 35 mothers and children aged 12-42 months. Brain fMRI activation was assessed in mothers using 30-s silent film excerpts depicting menacing adult male-female interactions versus prosocial and neutral interactions. Group and continuous analyses were performed to test for associations between clinical and fMRI variables with DNA methylation. RESULTS: Maternal IPV exposure-frequency was associated with maternal PTSD; and maternal IPV-PTSD was in turn associated with child SBD. Methylation status of several CpG sites in the HTR3A gene was associated with maternal IPV and IPV-PTSD severity, AgB and child SBD, in particular, self-endangering behavior. Methylation status at a specific CpG site (CpG2_III) was associated with decreased medial prefrontal cortical (mPFC) activity in response to film-stimuli of adult male-female interactions evocative of violence as compared to prosocial and neutral interactions. CONCLUSIONS: Methylation status of the HTR3A gene in mothers is linked to maternal IPV-related psychopathology, trauma-induced brain activation patterns, and child attachment disturbance in the form of SBD during a sensitive period in the development of self-regulation.

Prenatal Exposure to DEHP Affects Spermatogenesis and Sperm DNA Methylation in a Strain-Dependent Manner.

Di-(2-ethylhexyl)phtalate (DEHP) is a plasticizer with endocrine disrupting properties found ubiquitously in the environment and altering reproduction in rodents. Here we investigated the impact of prenatal exposure to DEHP on spermatogenesis and DNA sperm methylation in two distinct, selected, and sequenced mice strains. FVB/N and C57BL/6J mice were orally exposed to 300 mg/kg/day of DEHP from gestation day 9 to 19. Prenatal DEHP exposure significantly decreased spermatogenesis in C57BL/6J (fold-change = 0.6, p-value = 8.7*10-4), but not in FVB/N (fold-change = 1, p-value = 0.9). The number of differentially methylated regions (DMRs) by DEHP-exposure across the entire genome showed increased hyper- and decreased hypo-methylation in C57BL/6J compared to FVB/N. At the promoter level, three important subsets of genes were massively affected. Promoters of vomeronasal and olfactory receptors coding genes globally followed the same trend, more pronounced in the C57BL/6J strain, of being hyper-methylated in DEHP related conditions. In contrast, a large set of micro-RNAs were hypo-methylated, with a trend more pronounced in the FVB/N strain. We additionally analyze both the presence of functional genetic variations within genes that were associated with the detected DMRs and that could be involved in spermatogenesis, and DMRs related with the DEHP exposure that affected both strains in an opposite manner. The major finding in this study indicates that prenatal exposure to DEHP can decrease spermatogenesis in a strain-dependent manner and affects sperm DNA methylation in promoters of large sets of genes putatively involved in both sperm chemotaxis and post-transcriptional regulatory mechanisms.

BDNF promoter I methylation correlates between post-mortem human peripheral and brain tissues.

Several psychiatric disorders have been associated with CpG methylation changes in CG rich promoters of the brain-derived neurotrophic factor (BDNF) mainly by extracting DNA from peripheral blood cells. Whether changes in peripheral DNA methylation can be used as a proxy for brain-specific alterations remains an open question. In this study we aimed to compare DNA methylation levels in BDNF promoter regions in human blood cells, muscle and brain regions using bisulfite-pyrosequencing. We found a significant correlation between the levels of BDNF promoter I methylation measured in quadriceps and vPFC tissues extracted from the same individuals (n = 98, Pearson, r = 0.48, p = 4.5 × 10(-7)). In the hippocampus, BDNF promoter I and IV methylation levels were strongly correlated (Pearson, n = 37, r = 0.74, p = 1.4 × 10(-7)). We found evidence for sex-dependent effect on BDNF promoter methylation levels in the various tissues and blood samples. Taken together, these data indicate a strong intra-individual correlation between peripheral and brain tissue. They also suggest that sex determines methylation patterns in BDNF promoter region across different types of tissue, including muscle, brain, and blood.

BDNF Methylation and Maternal Brain Activity in a Violence-Related Sample.

It is known that increased circulating glucocorticoids in the wake of excessive, chronic, repetitive stress increases anxiety and impairs Brain-Derived Neurotrophic Factor (BDNF) signaling. Recent studies of BDNF gene methylation in relation to maternal care have linked high BDNF methylation levels in the blood of adults to lower quality of received maternal care measured via self-report. Yet the specific mechanisms by which these phenomena occur remain to be established. The present study examines the link between methylation of the BDNF gene promoter region and patterns of neural activity that are associated with maternal response to stressful versus non-stressful child stimuli within a sample that includes mothers with interpersonal violence-related PTSD (IPV-PTSD). 46 mothers underwent fMRI. The contrast of neural activity when watching children-including their own-was then correlated to BDNF methylation. Consistent with the existing literature, the present study found that maternal BDNF methylation was associated with higher levels of maternal anxiety and greater childhood exposure to domestic violence. fMRI results showed a positive correlation of BDNF methylation with maternal brain activity in the anterior cingulate (ACC), and ventromedial prefrontal cortex (vmPFC), regions generally credited with a regulatory function toward brain areas that are generating emotions. Furthermore we found a negative correlation of BDNF methylation with the activity of the right hippocampus. Since our stimuli focus on stressful parenting conditions, these data suggest that the correlation between vmPFC/ACC activity and BDNF methylation may be linked to mothers who are at a disadvantage with respect to emotion regulation when facing stressful parenting situations. Overall, this study provides evidence that epigenetic signatures of stress-related genes can be linked to functional brain regions regulating parenting stress, thus advancing our understanding of mothers at risk for stress-related psychopathology.

Methylation of NR3C1 is related to maternal PTSD, parenting stress and maternal medial prefrontal cortical activity in response to child separation among mothers with histories of violence exposure.

Prior research has shown that mothers with Interpersonal violence-related posttraumatic stress disorder (IPV-PTSD) report greater difficulty in parenting their toddlers. Relative to their frequent early exposure to violence and maltreatment, these mothers display dysregulation of their hypothalamic pituitary adrenal axis (HPA-axis), characterized by hypocortisolism. Considering methylation of the promoter region of the glucocorticoid receptor gene NR3C1 as a marker for HPA-axis functioning, with less methylation likely being associated with less circulating cortisol, the present study tested the hypothesis that the degree of methylation of this gene would be negatively correlated with maternal IPV-PTSD severity and parenting stress, and positively correlated with medial prefrontal cortical (mPFC) activity in response to video-stimuli of stressful versus non-stressful mother-child interactions. Following a mental health assessment, 45 mothers and their children (ages 12-42 months) participated in a behavioral protocol involving free-play and laboratory stressors such as mother-child separation. Maternal DNA was extracted from saliva. Interactive behavior was rated on the CARE-Index. During subsequent fMRI scanning, mothers were shown films of free-play and separation drawn from this protocol. Maternal PTSD severity and parenting stress were negatively correlated with the mean percentage of methylation of NR3C1. Maternal mPFC activity in response to video-stimuli of mother-child separation versus play correlated positively to NR3C1 methylation, and negatively to maternal IPV-PTSD and parenting stress. Among interactive behavior variables, child cooperativeness in play was positively correlated with NR3C1 methylation. Thus, the present study is the first published report to our knowledge, suggesting convergence of behavioral, epigenetic, and neuroimaging data that form a psychobiological signature of parenting-risk in the context of early life stress and PTSD.