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Pathologic inflammation in response to injury, infection, or oxidative stress is a proposed mechanism relating cognitive decline to dementia. The kynurenine pathway and thioredoxin-interacting protein (TXNIP) activity regulate inflammation and neurotoxicity in Alzheimer disease (AD). We examined cognitive deficits, kynurenine pathway mediators, TXNIP, and oxidative damage in the cerebrum and spleen, including inflammatory cytokine production by stimulated splenocytes, from female triple transgenic (3xTg-AD) mice in early and late stages of disease progression, and characterized tissue-specific epigenetic regulation of Txnip gene expression. We show that cognitive deficits in 7-month–old 3xTg-AD mice are associated with a stable increase in cerebrum and spleen tryptophan metabolites, with a concomitant increase in amyloid β 40 (Aβ40)/Aβ42 and tau/hyperphosphorylated tau pathologies and a coordinated reduction in spleen proinflammatory cytokine production in 17-month–old mice. The enhanced cerebrum TXNIP expression is associated with increased histone acetylation, transcription factor [Aβ42 or CCCTC-binding factor (CTCF)] binding, and Txnip promoter hypomethylation, whereas the attenuated spleen TXNIP expression is associated with increased histone methylation, reduced CTCF binding, and Txnip promoter hypermethylation. These results suggest a causal relationship among epigenomic state, TXNIP expression, cerebral-spleen tryptophan metabolism, inflammatory cytokine production, and cognitive decline; and they provide a potential mechanism for Txnip gene regulation in normal and pathologic conditions, suggesting TXNIP levels may be a useful predictive or diagnostic biomarker for Aβ40/Aβ42 targeted AD therapies.

Exposing a male rat to an obesogenic high-fat diet (HFD) influences attractiveness to potential female mates, the subsequent interaction of female mates with infant offspring, and the development of stress-related behavioral and neural responses in offspring. To examine the stomach and fecal microbiome’s potential roles, fecal samples from 44 offspring and stomach samples from offspring and their fathers were collected and bacterial community composition was studied by 16 small subunit ribosomal RNA (16S rRNA) gene sequencing. Paternal diet (control, high-fat), maternal housing conditions (standard or semi-naturalistic housing), and maternal care (quality of nursing and other maternal behaviors) affected the within-subjects alpha-diversity of the offspring stomach and fecal microbiomes. We provide evidence from beta-diversity analyses that paternal diet and maternal behavior induced community-wide shifts to the adult offspring gut microbiome. Additionally, we show that paternal HFD significantly altered the adult offspring Firmicutes to Bacteroidetes ratio, an indicator of obesogenic potential in the gut microbiome. Additional machine-learning analyses indicated that microbial species driving these differences converged on Bifidobacterium pseudolongum. These results suggest that differences in early-life care induced by paternal diet and maternal care significantly influence the microbiota composition of offspring through the microbiota-gut-brain axis, having implications for adult stress reactivity.

The membrane-associated mucin (MAM) domain containing glycosylphosphatidylinositol anchor 2 protein single knock-out mice (MDGA2+/- ) are models of ASD. We examined the behavioral phenotypes of male and female MDGA2+/- and wildtype mice on C57BL6/NJ and C57BL6/N backgrounds at 2 months of age and measured MDGA2, neuroligin 1 and neuroligin 2 levels at 7 months. Mice on the C57BL6/NJ background performed better than those on the C57BL6/N background in visual ability and in learning and memory performance in the Morris water maze and differed in measures of motor behavior and anxiety. Mice with the MDGA2+/- genotype differed from WT mice in motor, social and repetitive behavior and anxiety, but most of these effects involved interactions between MDGA2+/- genotype and background strain. The background strain also influenced MDGA2 levels and NLGN2 association in MDGA2+/- mice. Our findings emphasize the importance of the background strain used in studies of genetically modified mice.

Cancer dissemination is initiated by the movement of cells into the vasculature which has been reported to be triggered by EMT (epithelial to mesenchymal transition). Cellular dissemination also requires proteases that remodel the extracellular matrix. The protease, plasmin is a prominent player in matrix remodeling and invasion. Despite the contribution of both EMT and the plasminogen activation (PA) system to cell dissemination, these processes have never been functionally linked. We reveal that canonical Smad-dependent TGFβ1 signaling and FOXC2-mediated PI3K signaling in cells undergoing EMT reciprocally modulate plasminogen activation partly by regulating the plasminogen receptor, S100A10 and the plasminogen activation inhibitor, PAI-1. Plasminogen activation and plasminogen-dependent invasion were more prominent in epithelial-like cells and were partly dictated by the expression of S100A10 and PAI-1.

The membrane-associated mucin (MAM) domain containing glycosylphosphatidylinositol anchor 2 protein single knock-out mice (MDGA2

Pancreatic cancer is arguably the deadliest cancer type. The efficacy of current therapies is often hindered by the inability to predict patient outcome. As such, the development of tools for early detection and risk prediction is key for improving outcome and quality of life. Here, we introduce the plasminogen receptor S100A10 as a novel predictive biomarker and a driver of pancreatic tumor growth and invasion. We demonstrated that S100A10 mRNA and protein are overexpressed in human pancreatic tumors compared to normal ducts and nonductal stroma. S100A10 mRNA and methylation status were predictive of overall survival and recurrence‐free survival across multiple patient cohorts. S100A10 expression was driven by promoter methylation and the oncogene KRAS. S100A10 knockdown reduced surface plasminogen activation, invasiveness, and in vivo growth of pancreatic cancer cell lines. These findings delineate the clinical and functional contribution of S100A10 as a biomarker in pancreatic cancer.

Cancer cells with a less differentiated stem-like phenotype are more resistant to therapeutic manipulations than their differentiated counterparts, and are considered as one of the main causes of cancer persistence and relapse. As such, induction of differentiation in cancer stem-like cells (CSLCs) has emerged as an alternative strategy to enhance the efficacy of anticancer therapies. CSLCs are metabolically distinct from differentiated cells, and any aberration from the intrinsic metabolic state can induce differentiation of CSLCs. Therefore, metabolism-related molecular targets, with a capacity to promote differentiation within CSLCs, are of therapeutic importance. Here, we demonstrate that phosphoglycerate dehydrogenase (PHGDH), an essential enzyme catalyzing the synthesis of amino acid serine, is important for maintaining the poorly differentiated, stem-like state of CSLCs. Our data shows that PHGDH deficiency impairs the tumorsphere formation capacity in embryonal carcinoma stem-like cells (ECSLCs), breast cancer stem-like cells (BCSLCs) and patient-derived brain tumor-initiating cells (BTICs), which is accompanied by the reduced expression of characteristic stemness-promoting factors, such as Oct4, Nanog, Sox-2, and Bmi-1. Mechanistically, PHGDH deficiency in ECSLCs promotes differentiation to various lineages via degradation of Oct4 and by increasing the stability of differentiation marker β3-tubulin. Furthermore, PHGDH inhibition promotes p-mTOR independent but Beclin-1-dependent autophagy, independent of apoptosis. When studied in combination, the inhibition of both PHGDH and p-mTOR in ECSLCs causes further augmentation of autophagy, and additionally promotes apoptosis, demonstrating the clinical applicability of PHGDH-based manipulations in cancer therapies. Recapitulating these in vitro findings in CSLC models, the intratumoral PHGDH expression in patient-derived tumors is positively correlated with the mRNA levels of stemness factors, especially Oct4, and cancer patients co-expressing high levels of PHGDH and Oct4 display significantly lower survival than those with low PHGDH/Oct4 co-expression. Altogether, this study identifies a clinically-relevant role for PHGDH in the regulation of stemness-differentiation axis within CSLCs.

Paternal preconception risk factors (e.g. stress, diet, drug use) correlate with metabolic dysfunction in offspring, which is often comorbid with depressive and anxiety-like phenotypes. Detection of these risk factors or deleterious phenotypes informs a female about prevailing ecological demands, in addition to potential adverse environment-induced phenotypes that may be disseminated to her offspring. We examined whether a F0 male rat's prior exposure to an obesogenic high-fat diet (HFD) influences a female's attraction towards a male, subsequent mother-infant interactions and the development of defensive (emotional) responses in the F1 offspring. Females displayed less interest in the HFD exposed F0 males relative to control diet-exposed F0 males. Dams that reared F1 offspring in larger, semi-naturalistic housing provided more licking and grooming and active arched-back-nursing behavior. However, some of these effects interacted with paternal experience. F0 HFD and maternal rearing environment revealed sex-dependent, between group differences in F1 offspring wean weight, juvenile social interactions and anxiety-like behavior in adolescence. Our results show for the first time in mammals that male exposure to HFD may contribute to stable behavioral variation among females in courtship, maternal care, even when the females are not directly exposed to a HFD, and anxiety-like behavior in F1 offspring. Furthermore, when offspring were exposed to a predatory threat, hypothalamic Crf gene regulation was influenced by early housing. These results, together with our previous findings, suggest that paternal experience and maternal rearing conditions can influence maternal behavior and development of defensive responses of offspring.

Avoiding detection and destruction by immune cells is key for tumor initiation and progression. The important role of cancer stem cells (CSCs) in tumor initiation has been well established, yet their ability to evade immune detection and targeting is only partly understood. To investigate the ability of breast CSCs to evade immune detection, we identified a highly tumorigenic population in a spontaneous murine mammary tumor based on increased aldehyde dehydrogenase activity. We performed tumor growth studies in immunocompetent and immunocompromised mice. In immunocompetent mice, growth of the spontaneous mammary tumor was restricted; however, the Aldefluor+ population was expanded, suggesting inherent resistance mechanisms. Gene expression analysis of the sorted tumor cells revealed that the Aldefluor+ tumor cells has decreased expression of transporter associated with antigen processing (TAP) genes and co-stimulatory molecule CD80, which would decrease susceptibility to T cells. Similarly, the Aldefluor+ population of patient tumors and 4T1 murine mammary cells had decreased expression of TAP and co-stimulatory molecule genes. In contrast, breast CSCs identified by CD44+CD24− do not have decreased expression of these genes, but do have increased expression of C-X-C chemokine receptor type 4. Decitabine treatment and bisulfite pyrosequencing suggests that DNA hypermethylation contributes to decreased TAP gene expression in Aldefluor+ CSCs. TAP1 knockdown resulted in increased tumor growth of 4T1 cells in immunocompetent mice. Together, this suggests immune evasion mechanisms in breast CSCs are marker specific and epigenetic silencing of TAP1 in Aldefluor+ breast CSCs contributes to their enhanced survival under immune pressure.

Dysregulation of DNA methylation is an established feature of breast cancers. DNA demethylating therapies like decitabine are proposed for the treatment of triple-negative breast cancers (TNBC) and indicators of response need to be identified. For this purpose, we characterized the effects of decitabine in a panel of 10 breast cancer cell lines and observed a range of sensitivity to decitabine that was not subtype specific. Knockdown of potential key effectors demonstrated the requirement of deoxycytidine kinase (DCK) for decitabine response in breast cancer cells. In treatment-naïve breast tumors, DCK was higher in TNBCs, and DCK levels were sustained or increased post chemotherapy treatment. This suggests that limited DCK levels will not be a barrier to response in patients with TNBC treated with decitabine as a second-line treatment or in a clinical trial. Methylome analysis revealed that genome-wide, region-specific, tumor suppressor gene–specific methylation, and decitabine-induced demethylation did not predict response to decitabine. Gene set enrichment analysis of transcriptome data demonstrated that decitabine induced genes within apoptosis, cell cycle, stress, and immune pathways. Induced genes included those characterized by the viral mimicry response; however, knockdown of key effectors of the pathway did not affect decitabine sensitivity suggesting that breast cancer growth suppression by decitabine is independent of viral mimicry. Finally, taxol-resistant breast cancer cells expressing high levels of multidrug resistance transporter ABCB1 remained sensitive to decitabine, suggesting that the drug could be used as second-line treatment for chemoresistant patients.

The fatal neurodegenerative disorder Niemann-Pick type C (NPC) is caused in most cases by mutations in NPC1, which encodes the late endosomal NPC1 protein. Loss of NPC1 disrupts cholesterol trafficking from late endosomes to the endoplasmic reticulum and plasma membrane, causing cholesterol accumulation in late endosomes/lysosomes. Neurons are particularly vulnerable to this cholesterol trafficking defect, but the pathogenic mechanisms through which NPC1 deficiency causes neuronal dysfunction remain largely unknown. Herein, we have investigated amino acid metabolism in cerebella of NPC1-deficient mice at different stages of NPC disease. Imbalances in amino acid metabolism were evident from increased branched chain amino acid and asparagine levels and altered expression of key enzymes of glutamine/glutamate metabolism in presymptomatic and early symptomatic NPC1-deficient cerebellum. Increased levels of several amino acid intermediates of one-carbon metabolism indicated disturbances in folate and methylation pathways. Alterations in DNA methylation were apparent in decreased expression of DNA methyltransferase 3a and methyl-5'-cytosine-phosphodiester-guanine-domain binding proteins, reduced 5-methylcytosine immunoreactivity in the molecular and Purkinje cell layers, demethylation of genome-wide repetitive LINE-1 elements, and hypermethylation in specific promoter regions of single-copy genes in NPC1-deficient cerebellum at early stages of the disease. Alterations in amino acid metabolism and epigenetic changes in the cerebellum at presymptomatic stages of NPC disease represent previously unrecognized mechanisms of NPC pathogenesis.

Acute promyelocytic leukemia (APL) is characterized by arrested differentiation of promyelocytes. Patients treated with all-trans retinoic acid (ATRA) alone experience relapse, while patients treated with ATRA and arsenic trioxide (ATO) are often relapse-free. This suggests sustained changes have been elicited by the combination therapy. To understand the lasting effects of the combination therapy, we compared the effects of ATRA and ATO on NB4 and ATRA-resistant NB4-MR2 APL cells during treatment versus post treatment termination. After treatment termination, NB4 cells treated with ATRA or ATO reverted to non-differentiated cells, while combination-treated cells remained terminally differentiated. This effect was diminished in NB4-MR2 cells. This suggests combination treatment induced more permanent changes. Combination treatment induced higher expression of target genes (e.g., transglutaminase 2 and retinoic acid receptor beta), which in NB4 cells was sustained post treatment termination. To determine whether sustained epigenetic changes were responsible, we quantified the enrichment of histone modifications by chromatin immunoprecipitation, and CpG methylation by bisulfite-pyrosequencing. While ATRA and combination treatment induced similar histone acetylation enrichment, combination treatment induced greater demethylation of target genes, which was sustained. Therefore, sustained demethylation of target genes by ATRA and ATO combination treatment is associated with lasting differentiation and gene expression changes.

Cancer dissemination is initiated by the movement of cells into the vasculature which has been reported to be triggered by EMT (epithelial to mesenchymal transition). Cellular dissemination also requires proteases that remodel the extracellular matrix. The protease, plasmin is a prominent player in matrix remodeling and invasion. Despite the contribution of both EMT and the plasminogen activation (PA) system to cell dissemination, these processes have never been functionally linked. We reveal that canonical Smad-dependent TGFβ1 signaling and FOXC2-mediated PI3K signaling in cells undergoing EMT reciprocally modulate plasminogen activation partly by regulating the plasminogen receptor, S100A10 and the plasminogen activation inhibitor, PAI-1. Plasminogen activation and plasminogen-dependent invasion were more prominent in epithelial-like cells and were partly dictated by the expression of S100A10 and PAI-1.

Cognition is comprised of the faculties: perception, creativity, intuition, and ratiocination. Optimal levels of cognition are needed for independent functioning and balanced living. With an aging population that continues to grow, dietary supplements that tilt the balance towards maintenance of cognition are being marketed for vulnerable populations facing these challenges. Randomized clinical trials provide the causal inference necessary to define the efficacy of emerging nutraceuticals. Cognition testing, in particular, requires a battery of tests that encompass all brain regions involved in cognition so as to provide endpoints necessary for product validation. The lack of well controlled studies for comparison analyses, limited sample sizes, ambiguous dosages, and poor cognitive measures result in data that cannot be compared across studies to determine the efficacy of supplements claiming to enhance cognition. Clinical trials for the nutraceutical industry should consider the multifaceted nature of supplements, where clinical endpoints must be comprehensive while remaining feasible. Combining endpoints of cognition with physiological biomarkers of immunity and metabolism to arrive at a global index for cognitive health may be necessary for claim substantiation in order to fully justify and scientifically validate improvements in cognitive health. The issues and needs of a global index will be discussed here.

Avoiding detection and destruction by immune cells is key for tumor initiation and progression. The important role of cancer stem cells (CSCs) in tumor initiation has been well established, yet their ability to evade immune detection and targeting is only partly understood. To investigate the ability of breast CSCs to evade immune detection, we identified a highly tumorigenic population in a spontaneous murine mammary tumor based on increased aldehyde dehydrogenase activity. We performed tumor growth studies in immunocompetent and immunocompromised mice. In immunocompetent mice, growth of the spontaneous mammary tumor was restricted; however, the Aldefluor

The influence of early life experience and degree of parental-infant attachment on emotional development in children and adolescents has been comprehensively studied. Structural and mechanistic insight into the biological foundation and maintenance of mammalian defensive systems (metabolic, immune, nervous and behavioral) is slowly advancing through the emerging field of developmental molecular (epi)genetics. Initial evidence revealed that differential nurture early in life generates stable differences in offspring hypothalamic-pituitary-adrenal (HPA) regulation, in part, through chromatin remodeling and changes in DNA methylation of specific genes expressed in the brain, revealing physical, biochemical and molecular paths for the epidemiological concept of gene-environment interactions. Herein, a primary molecular mechanism underpinning the early developmental programming and lifelong maintenance of defensive (emotional) responses in the offspring is the alteration of chromatin domains of specific genomic regions from a condensed state (heterochromatin) to a transcriptionally accessible state (euchromatin). Conversely, DNA methylation promotes the formation of heterochromatin, which is essential for gene silencing, genomic integrity and chromosome segregation. Therefore, inter-individual differences in chromatin modifications and DNA methylation marks hold great potential for assessing the impact of both early life experience and effectiveness of intervention programs—from guided psychosocial strategies focused on changing behavior to pharmacological treatments that target chromatin remodeling and DNA methylation enzymes to dietary approaches that alter cellular pools of metabolic intermediates and methyl donors to affect nutrient bioavailability and metabolism. In this review article, we discuss the potential molecular mechanism(s) of gene regulation associated with chromatin modeling and programming of endocrine (e.g., HPA and metabolic or cardiovascular) and behavioral (e.g., fearfulness, vigilance) responses to stress, including alterations in DNA methylation and the role of DNA repair machinery. From parental history (e.g., drugs, housing, illness, nutrition, socialization) to maternal-offspring exchanges of nutrition, microbiota, antibodies and stimulation, the nature of nurture provides not only mechanistic insight into how experiences propagate from external to internal variables, but also identifies a composite therapeutic target, chromatin modeling, for gestational/prenatal stress, adolescent anxiety/depression and adult-onset neuropsychiatric disease.

We have identified a novel, epigenetic-based strategy to identify breast cancer patients who will benefit from an existing anticancer agent, all-trans retinoic acid (atRA). Triple-negative breast cancer (TNBC) is among the most aggressive breast cancers and lacks targeted therapies. TNBCs are a heterogeneous group of breast cancers with a wide range of gene expression profiles and drug sensitivities. Profiling these differences will lead to the development of more effective treatment options for TNBC. We hypothesize that atRA can be an effective therapy for a subset of TNBC patients. To test our hypothesis, we profiled the response of 12 TNBC cell lines to atRA using in vivo tumor growth assays. We found that atRA treatment had a range of effects on the tumor growth of TNBC cell lines: significantly decreasing tumor growth in HCC70, SUM149, and HCC1937, while increasing the growth of MDA-MB-231, MDA-MB-436, and Du4475 tumors. Gene expression and methylation analysis of these 12 cell lines revealed subtype-specific expression of atRA-inducible genes due to silencing by DNA methylation, e.g., of the atRA-inducible tumor-suppressor gene RARRES1. RARRES1 is silenced by methylation in cell lines where atRA promotes tumor growth, but is hypomethylated and expressed in cells that can be effectively treated with atRA. Use of the distinct gene expression and methylation profiles allowed us to accurately predict the response of 4 patient-derived xenografts (PDXs) to RA treatment. Continued classification of TNBCs by gene expression and DNA methylation can predict the response of patient tumors to RA treatment, thus identifying a novel targeted therapy strategy for TNBCs. Citation Format: Krysta M. Coyle, Cheryl A. Dean, Dejan Vidovic, Ian Weaver, Carman Giacomantonio, Lucy Helyer, Paola Marcato. DNA methylation predicts response of triple-negative breast cancer to all-trans retinoic acid treatment [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A07.

Introduction Acute promyelocytic leukaemia (APL) results in the interrupted differentiation of granulocytes. Combination induction therapy using all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) is the most effective treatment of APL. DNA methylation and histone modifications are epigenetic mechanisms of gene regulation aberrant in APL. While ATRA treatment of APL NB4 cells restores granulocyte differentiation and induces significant genome-wide changes in histone modifications, it does not affect the aberrant DNA methylation. Similarly, ATO has comparable effects. Therefore, we hypothesise that the increased effectiveness of ATRA and ATO when combined is due to epigenetic changes at both the histone and DNA methylation level, resulting in profound changes in gene expression. Material and methods We identified the concentration of ATRA and ATO in combination that induced enhanced differentiation and cell death of NB4 cells in comparison to single agent treatment through fluorescence activated cells sorting (FACS) of CD11b and 7-AAD labelled cells at 72 hours and 96 hours post treatment termination. Quantitative PCR (qPCR) was performed to determine the expression levels of a panel of genes after 72 hours of treatment and 96 hours post treatment termination. Results and discussions ATRA and ATO in combination induced significant differentiation and cell death of NB4 cells after 72 hours. Interestingly, only the combination-treated cells maintained differentiation when treatment was terminated for 96 hours. Additionally,combination treatment significantly increased the expression of several genes important in APL compared to single agent treatment after 72 hours as well as 96 hours post treatment termination. Chromatin immunoprecipitation (ChIP) is currently being utilised to investigate the enrichment of chromatin with acetylated and methylated histones, and qPCR was performed to assess the enrichment of several genes of interest. Finally, bisulfite pyrosequencing is being performed to characterise the specific methylation pattern of these genes of interest. Conclusion ATRA and ATO in combination synergistically induces the differentiation of APL NB4 cells. Together, the ChIP and bisulfite pyrosequencing experiments will enable us to characterise the effects of combination treatment at the epigenetic level. Ultimately, understanding the epigenetic changes induced by ATO and ATRA synergy can help improve treatment in the clinic.

SummaryIncreasing evidence indicates that epigenetic changes regulate cell genesis. Here, we ask about neural precursors, focusing on CREB binding protein (CBP), a histone acetyltransferase that, when haploinsufficient, causes Rubinstein-Taybi syndrome (RTS), a genetic disorder with cognitive dysfunction. We show that neonatal cbp+/− mice are behaviorally impaired, displaying perturbed vocalization behavior. cbp haploinsufficiency or genetic knockdown with siRNAs inhibited differentiation of embryonic cortical precursors into all three neural lineages, coincident with decreased CBP binding and histone acetylation at promoters of neuronal and glial genes. Inhibition of histone deacetylation rescued these deficits. Moreover, CBP phosphorylation by atypical protein kinase C ζ was necessary for histone acetylation at neural gene promoters and appropriate differentiation. These data support a model in which environmental cues regulate CBP activity and histone acetylation to control neural precursor competency to differentiate, and indicate that cbp haploinsufficiency disrupts this mechanism, thereby likely causing cognitive dysfunction in RTS.

A major question in the biology of stress and environmental adaptation concerns the neurobiological basis of how neuroendocrine systems governing physiological regulatory mechanisms essential for life (metabolism, immune response, organ function) become harmful. The current view is that a switch from protection to damage occurs when vulnerable phenotypes are exposed to adverse environmental conditions. In accordance with this theory, sequelae of early life social and environmental stressors, such as childhood abuse, neglect, poverty, and poor nutrition, have been associated with the emergence of mental and physical illness (i.e., anxiety, mood disorders, poor impulse control, psychosis, and drug abuse) and an increased risk of common metabolic and cardiovascular diseases later in life. Evidence from animal and human studies investigating the associations between early life experiences (including parent-infant bonding), hypothalamus-pituitary-adrenal axis activity, brain development, and health outcome provide important clues into the neurobiological mechanisms that mediate the contribution of stressful experiences to personality development and the manifestation of illness. This review summarizes our current molecular understanding of how early environment influences brain development in a manner that persists through life and highlights recent evidence from rodent studies suggesting that maternal care in the first week of postnatal life establishes diverse and stable phenotypes in the offspring through epigenetic modification of genes expressed in the brain that shape neuroendocrine and behavioral stress responsivity throughout life.

Breast cancer subtyping, based on the expression of hormone receptors and other genes, can determine patient prognosis and potential options for targeted therapy. Among breast cancer subtypes, tumors of basal-like and claudin-low subtypes are typically associated with worse patient outcomes, are primarily classified as triple-negative breast cancers (TNBC), and cannot be treated with existing hormone-receptor-targeted therapies. Understanding the molecular basis of these subtypes will lead to the development of more effective treatment options for TNBC. In this study, we focus on retinoic acid receptor responder 1 (RARRES1) as a paradigm to determine if breast cancer subtype dictates protein function and gene expression regulation. Patient tumor dataset analysis and gene expression studies of a 26 cell-line panel, representing the five breast cancer subtypes, demonstrate that RARRES1 expression is greatest in basal-like TNBCs. Cell proliferation and tumor growth assays reveal that RARRES1 is a tumor suppressor in TNBC. Furthermore, gene expression studies, Illumina HumanMethylation450 arrays, and chromatin immunoprecipitation demonstrate that expression of RARRES1 is retained in basal-like breast cancers due to hypomethylation of the promoter. Additionally, expression of the cancer stem cell marker, aldehyde dehydrogenase 1A3, which provides the required ligand (retinoic acid) for RARRES1 transcription, is also specific to the basal-like subtype. We functionally demonstrate that the combination of promoter methylation and retinoic acid signaling dictates expression of tumor suppressor RARRES1 in a subtype-specific manner. These findings provide a precedent for a therapeutically-inducible tumor suppressor and suggest novel avenues of therapeutic intervention for patients with basal-like breast cancer.

The genome is programmed by the epigenome, which is comprised of chromatin and a covalent modification of DNA by methylation. Epigenetic patterns are sculpted during development to shape the diversity of gene expression programs in the different cell types of the organism. The epigenome of the developing fetus is especially sensitive to maternal nutrition, and exposure to environmental toxins as well as psychological stress. It is postulated here that not only chemicals but also exposure of the young pup to social behavior, such as maternal care, could affect the epigenome. Since epigenetic programming defines the state of expression of genes, epigenetic differences could have the same consequences as genetic polymorphisms. We will propose here a mechanism linking maternal behavior and epigenetic programming and we will discuss the prospect that similar epigenetic variations generated during early life play a role in generating inter-individual differences in human behavior. We speculate that exposures to different environmental toxins, which affect the epigenetic machinery might alter long-established epigenetic programs in the brain.

The nature of maternal care that an infant receives can effect the child's emotional and cognitive development, which is endured into adulthood. Similarly, maternal behavior in rodents is associated with long-term programming of individual differences in behavioral and hypothalamic-pituitary-adrenal responses to stress in the offspring. One critical question is how is such 'environmental programming' established and sustained in the offspring? This review discusses a novel mechanism to explain how maternal licking/grooming behavior in the rat can alter the hippocampal glucocorticoid receptor (GR) expression in the offspring, which concomitantly alters the HPA axis and the stress responsiveness of these animals. Both in vivo and in vitro studies show that maternal behavior increases GR expression in the offspring via increased hippocampal serotonergic tone accompanied by increased histone acetylase transferase activity, histone acetylation and DNA demethylation mediated by the transcription factor NGFI-A. In summary, this research demonstrates that an epigenetic state of a gene can be established through early-in-life experience, and is potentially reversible in adulthood. Accordingly, epigenetic modifications of specific genomic regions in response to variations in environmental conditions might serve as a major source of variation in biological and behavioral phenotypes.

Early-life experience has long-term consequences on behavior and stress responsivity of the adult. We previously proposed that early-life experience results in stable epigenetic programming of glucocorticoid receptor gene expression in the hippocampus. The aim of this study was to examine the global effect of early-life experience on the hippocampal transcriptome and the development of stress-mediated behaviors in the offspring and whether such effects were reversible in adulthood. Adult offspring were centrally infused with saline vehicle, the histone deacetylase inhibitor trichostatin A (TSA), or the essential amino acid l -methionine. The animals were assessed in an unfamiliar open-field arena, and the hippocampal transcriptome of each animal was evaluated by microarray analysis. Here we report that TSA and methionine treatment reversed the effect of maternal care on open-field behavior. We identified >900 genes stably regulated by maternal care. A fraction of these differences in gene expression is reversible by either the histone deacetylase inhibitor TSA or the methyl donor l -methionine. These results suggest that early-life experience has a stable and broad effect on the hippocampal transcriptome and anxiety-mediated behavior, which is potentially reversible in adulthood.

Stress responses in the adult rat are programmed early in life by maternal care and associated with epigenomic marking of the hippocampal exon 17glucocorticoid receptor (GR) promoter. To examine whether such epigenetic programming is reversible in adult life, we centrally infused the adult offspring with the essential amino acidl-methionine, a precursor toS-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 17promoter, 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.

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.

Naturally occurring variations in maternal care influence hippocampal development in the rat. In the present study we found that variations in maternal licking/grooming (LG) during the first week of life are associated with altered hippocampal expression of BAX (group-1 tumor necrosis factor family mediated cell death effector) in 90-day-old male offspring. BAX-like immunoreactivity on western blots is significantly increased in the adult offspring of low-level LG mothers. There is no effect of maternal care on levels of either B-cell lymphoma-2 (BCL-2) (group-II mitochondria mediated cell death suppressor) or BAD (group-III endoplasmic reticulum mediated cell death effector). The most striking biochemical event in apoptosis is DNA fragmentation. Terminal deoxynucleotidyl transerferase (Tdt)-mediated dUTP-biotin nick-end labeling (TUNEL) and 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI) staining showed that the number of TUNEL-positive cells in both the dentate gyrus and CA1 region of the hippocampus is significantly increased in the adult offspring of low-level LG mothers. In conclusion, we propose that hippocampal neurons in the offspring of low-level LG mothers may be more vulnerable to loss through apoptosis.

We have identified a novel strategy to identify breast cancer patients who will benefit from an existing anti-cancer agent, retinoic acid (RA). While RA has not yet achieved success in the treatment of breast cancers, we hypothesized that it can be an effective therapy for a subset of triple-negative breast cancer (TNBC) patients. TNBC is among the most aggressive breast cancers, and lacks targeted therapies. TNBCs can be further subtyped into basal-like and claudin-low, which differ in gene expression and drug sensitivities. Understanding the molecular basis of these subtypes will lead to the development of more effective treatment options for TNBC. To test this hypothesis, we performed tumor growth assays on TNBC cell lines and patient-derived xenografts (PDXs). We found that RA treatment decreased the tumor growth of four basal-like TNBC cell lines (MDA-MB-468, HCC70, SUM149, HCC1937). In contrast, RA increased the tumor growth of two claudin-low TNBC cell lines (MDA-MB-231, MDA-MB-436). Gene expression and methylation analysis of these affected cell lines revealed subtype-specific expression of RA-inducible genes due to silencing by DNA methylation, e.g. of the RA-inducible tumor-suppressor gene RARRES1. RARRES1 is silenced by methylation in claudin-low cell lines, but is hypomethylated and expressed in basal-like cells. Use of the subtype-specific expression and methylation profiles allowed us to accurately predict the response of 4 PDXs to RA treatment. Continued classification of TNBCs into these two subtypes will enable clinical use of RA, in part due to the subtype-specific hypomethylation of RA-inducible tumor suppressor genes including RARRES1. We have identified additional subtype-specific biomarkers which can predict the response of patient tumors to RA treatment, thus identifying a novel targeted therapy strategy for TNBCs. Citation Format: Krysta Mila Coyle, Cheryl A. Dean, Dejan Vidovic, Ian C. Weaver, Carman A. Giacomantonio, Paola Marcato. Retinoic acid: An effective therapy for basal-like breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3064. doi:10.1158/1538-7445.AM2017-3064

Adaptation to environmental changes is based on the perpetual generation of new phenotypes. Modern biology has focused on the role of epigenetic mechanisms in facilitating the adaptation of organisms to changing environments through alterations in gene expression. Inherited and/or acquired epigenetic factors are relatively stable and have regulatory roles in numerous genomic activities that translate into phenotypic outcomes. Evidence that dietary and pharmacological interventions have the potential to reverse environment-induced modification of epigenetic states (e.g., early life experience, nutrition, medication, infection) has provided an additional stimulus for understanding the biological basis of individual differences in cognitive abilities and disorders of the brain. It has been suggested that accurate quantification of the relative contribution of heritable genetic and epigenetic variation is essential for understanding phenotypic divergence and adaptation in changing environments, a process requiring stable modulation of gene expression. The main challenge for epigenetics in psychology and psychiatry is to determine how experiences and environmental cues, including the nature of our nurture, influence the expression of neuronal genes to produce long-term individual differences in behavior, cognition, personality, and mental health. To this end, focusing on DNA and histone modifications and their initiators, mediators and readers may provide new inroads for understanding the molecular basis of phenotypic plasticity and disorders of the brain. In this chapter, we review recent discoveries highlighting epigenetic aspects of normal brain development and mental illness, as well as discuss some future directions in the field of behavioral epigenetics.

SummaryMaltreatment in childhood affects mental health over the life course. New research shows that early life experiences alter the genome in a way that can be measured in peripheral blood samples decades later. These findings suggest a new strategy for exploring gene–environment interactions and open opportunities for translational epigenomic research.

Adaptation to environmental changes is based on the perpetual generation of new phenotypes. Modern biology has focused on the role of epigenetic mechanisms in facilitating the adaptation of organisms to changing environments through alterations in gene expression. Inherited and/or acquired epigenetic factors are relatively stable and have regulatory roles in numerous genomic activities that translate into phenotypic outcomes. Evidence that dietary and pharmacological interventions have the potential to reverse environment-induced modification of epigenetic states (e.g., early life experience, nutrition, medication, infection) has provided an additional stimulus for understanding the biological basis of individual differences in cognitive abilities and disorders of the brain. It has been suggested that accurate quantification of the relative contribution of heritable genetic and epigenetic variation is essential for understanding phenotypic divergence and adaptation in changing environments, a process requiring stable modulation of gene expression. The main challenge for epigenetics in psychology and psychiatry is to determine how experiences and environmental cues, including the nature of our nurture, influence the expression of neuronal genes to produce long-term individual differences in behavior, cognition, personality, and mental health. To this end, focusing on DNA and histone modifications and their initiators, mediators and readers may provide new inroads for understanding the molecular basis of phenotypic plasticity and disorders of the brain. In this chapter, we review recent discoveries highlighting epigenetic aspects of normal brain development and mental illness, as well as discuss some future directions in the field of behavioral epigenetics.

Adaptation to environmental changes is based on the perpetual generation of new phenotypes. Modern biology has focused on the role of epigenetic mechanisms in facilitating the adaptation of organisms to changing environments through alterations in gene expression. Inherited and/or acquired epigenetic factors are relatively stable and have regulatory roles in numerous genomic activities that translate into phenotypic outcomes. Evidence that dietary and pharmacological interventions have the potential to reverse environment-induced modification of epigenetic states (e.g., early life experience, nutrition, medication, infection etc.) has provided an additional stimulus for understanding the biological basis of individual differences in cognitive abilities and disorders of the brain. It has been suggested that the accurate quantification of the relative contribution of heritable genetic and epigenetic variation is essential for understanding phenotypic divergence and adaptation in changing environments, a process requiring stable modulation of gene expression. The main challenge for epigenetics in psychology and psychiatry is to determine how experiences and environmental cues influence the expression of neuronal genes to produce long-term individual differences in behavior, cognition, personality and mental health. To this end, focusing on DNA and histone modifications and their initiators, mediators and readers may provide new inroads for understanding the molecular basis phenotypic plasticity and disorders of the brain. In this review, we survey recent developments revealing epigenetic aspects of mental illness, as well as review some of the challenges of current approaches and some future directions in the field of behavioral epigenetics.

Detecting past experiences with predators of a potential mate informs a female about prevailing ecological threats, in addition to stress-induced phenotypes that may be disseminated to offspring. We examined whether prior exposure of a male rat to a predator (cat) odor influences the attraction of a female toward a male, subsequent mother–infant interactions and the development of defensive (emotional) responses in the offspring. Females displayed less interest in males that had experienced predator odor. Mothers that reared young in larger, seminaturalistic housing provided more licking and grooming and active arched back-nursing behavior toward their offspring compared with dams housed in standard housing, although some effects interacted with paternal experience. Paternal predation risk and maternal rearing environment revealed sex-dependent differences in offspring wean weight, juvenile social interactions, and anxiety-like behavior in adolescence. Additionally, paternal predator experience and maternal housing independently affected variations incrfgene promoter acetylation andcrfgene expression in response to an acute stressor in offspring. Our results show for the first time in mammals that variation among males in their predator encounters may contribute to stable behavioral variation among females in preference for mates and maternal care, even when the females are not directly exposed to predator threat. Furthermore, when offspring were exposed to the same threat experienced by the father, hypothalamiccrfgene regulation was influenced by paternal olfactory experience and early housing. These results, together with our previous findings, suggest that paternal stress exposure and maternal rearing conditions can influence maternal behavior and the development of defensive responses in offspring.

The retinoic acid (RA) signalling pathway plays an important role in breast cancer progression and has either a pro-tumorigenic or tumor-suppressive role depending upon the effector function of RA-inducible genes that are expressed or epigenetically silenced. To study this paradigm in breast cancer, we focused on a controversial RA-inducible gene, the retinoic acid receptor responder 1 (RARRES1) protein that is often hypermethylated in cancer and has been reported to have tumor-suppressive function in prostate and nasopharyngeal carcinomas. However, in a study focused on a rare subtype of breast cancer, inflammatory breast cancer, RARRES1 is pro-tumorigenic. This functional discrepancy requires further investigation to determine its role in breast cancer in general. First, analysis of patient data sets revealed that RARRES1 is predominantly expressed in triple-negative breast cancers (TNBCs). Knockdown of RARRES1 in claudin-low MDA-MB-231 and basal-like MDA-MB-468 and HCC1937 significantly increased tumor growth and cell proliferation, suggesting RARRES1 has a tumor suppressive function in (TNBC), regardless of position on the differentiation hierarchy. Expression analyses of 24 breast cancer cell lines (including 18 TNBC and 2 normal-like cell lines) revealed that RARRES1 is predominantly expressed in basal-like TNBC cells We found that RARRES1 expression is dependent on, and strongly correlates with, the cancer stem cell marker, and RA-producing, ALDH1A3 in fixed breast cancer patient samples. Immunohistochemistry of the same patient tumor samples revealed RARRES1 expression is localized to the endoplasmic reticulum. Importantly, however, the presence of ALDH1A3 or RA is not sufficient to induce RARRES1 expression. RARRES1 is hypermethylated in claudin-low breast cancer cell lines, and release of this silencing is required for full induction of RARRES1 expression. We have identified sites of regulation by methylation in RARRES1 using Illumina 450K methylation arrays and 5-methylcytosine ChIP. We conclude that RARRES1 is an ALDH1A3/RA-inducible tumor suppressor in TNBC with methylation and expression profiles distinct to the differentiation hierarchy observed in breast cancer. Citation Format: Krysta M. Coyle, Patrick Murphy, Dejan Vidovic, Cheryl A. Dean, Margaret L. Thomas, Derek Clements, Mohammad Sultan, Ahmad Vaghar-Kashani, Carman Giacomantonio, Lucy Helyer, Ian Weaver, Shashi Gujar, Patrick WK Lee, Paola Marcato. ALDH1A3-inducible RARRES1 is a tumor suppressor in triple-negative breast cancer and is methylated in claudin-low breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3661.

Developmental plasticity refers to the potential for intraindividual change. Traditionally, the relationship between the human genome and the environment has been presented under the framework of gene–environment interactions. However, adaptive phenotypic plasticity emerges from more than just genotype. In humans and nonhuman primates, the nature of mother–infant interactions early in life has a profound role in mediating variation in offspring phenotype, including emotional and cognitive development, which is endured through life. One critical question: How is this “environmental programming” established and maintained in the offspring? Evidence from rodent studies suggests that maternal care in the first week of postnatal life establishes diverse and stable phenotypes in the offspring through epigenetic modification of genes expressed in the brain, which shape neuroendocrine and behavioral stress responsivity throughout life. This research demonstrates that the epigenetic state of a gene can be established through early in life experience and is potentially reversible in adulthood. These findings may well form the molecular basis for understanding potential mechanisms of environmental and developmental determinates of individual differences in human stress reactivity and health outcomes. Henceforth, epigenetic modifications of specific genomic regions in response to variations in environmental conditions might serve as a major source of variation in biological and behavioral phenotypes.

Human epidemiological and animal studies show that many chronic adult conditions have their antecedents in compromised fetal and early postnatal development. Mother–infant interactions are the primary source of social stimulation and influence physiological and cellular defense mechanisms resulting in persistent changes in offspring phenotype. These effects appear to be mediated, in part, by neonatal programming of the hypothalamic–pituitary–adrenal (HPA) axis and glucocorticoid function in the neuroendocrine system. Rodent models provide evidence that epigenetic mechanisms are involved: natural variations in maternal care influence HPA stress reactivity in offspring via long-term changes in tissue-specific gene expression. Both in vivo and in vitro studies show that maternal licking and grooming increases glucocorticoid receptor expression in the offspring through increased hippocampal serotonergic tone accompanied by increased histone acetyltransferase activity, histone acetylation and DNA demethylation mediated by the transcription factor NGFI-A. These effects are reversed by early postnatal cross-fostering and by pharmacological manipulations in adulthood, including Trichostatin A (TSA) and l-methionine administration. Maternal care influences the maternal behavior of female offspring, an effect that appears to be related to epigenetic regulation of estrogen receptor-α expression in the medial preoptic area, providing a mechanism for trans-generational inheritance of maternal behavior from mother to offspring. Experience-induced changes in the epigenotype provide a potential mechanism to explain how physiological adaptations to changes in the early environment result in permanent programming and affect risk to disease later in life.

The early nurturing environment has persistent influences on developmental programming of inter-individual differences in metabolic and endocrine function that contribute to emotional and cognitive performance through life. These effects are mediated, in part, through neonatal programming of hypothalamic-pituitary-adrenal (HPA) axis function. Animal models support this hypothesis. For example, in the rat natural variations in maternal care influence HPA axis stress reactivity in the offspring via long-term changes in tissue-specific gene expression. Studies in vivo and in vitro show that maternal licking and grooming increases glucocorticoid receptor expression in the offspring via increased hippocampal serotonergic tone accompanied by increased histone acetylase transferase activity, histone acetylation and DNA demethylation mediated by the transcription factor nerve growth factor-inducible protein-A. These effects are reversed by early postnatal cross-fostering and by pharmacological manipulations, including trichostatin A (TSA) and l-methionine administration in adulthood. These studies demonstrate that an epigenetic state of a gene can be established through early in life experience, and is potentially reversible in adult life. Accordingly, epigenetic modifications in target gene promoters in response to environmental demand may ensure stable yet dynamic regulation that mediates persistent changes in biological and behavioral phenotype over the lifespan.

In evolutionary developmental biology, a common theme across all life forms is the ability to vary phenotype in response to environmental conditions, a process termed phenotypic plasticity. In humans and non-human primates, the nature of mother-infant interactions in early postnatal life has a profound role in mediating variation in offspring phenotype, including emotional and cognitive development, which is endured through life. Similarly, maternal behavior in rodents is associated with long-term programming of individual differences in behavioral and hypothalamic-pituitary-adrenal (HPA) responses to stress in the offspring. One critical question is how is such “environmental programming” established and sustained in the offspring?

The tumor-suppressive function of the retinoic acid (RA)-inducible gene, retinoic acid receptor responder 1 (RARRES1), has been reported in many cancer types; however, in inflammatory breast cancer, RARRES1 is pro-tumorigenic. Furthermore, high transcript levels of RARRES1 are associated with more aggressive triple-negative breast cancers (TNBCs) and poorer patient outcomes. This suggests that RARRES1 may also be oncogenic and requires investigation to determine its role in breast cancer. Expression analyses of 20 breast cancer cell lines (including 18 TNBC and two estrogen-receptor-positive cell lines) revealed that RARRES1 is predominantly expressed in basal-like breast cancer cells, but is often methylated and silenced in claudin-low breast cancer cells. We have identified possible sites of regulation by methylation in RARRES1 using Illumina 450K methylation arrays and 5-methylcytosine ChIP. Basal-like cells express higher levels of the cancer stem cell (CSC) marker ALDH1A3. Expression of RARRES1 is dependent on, and strongly correlates with ALDH1A3 expression in fixed breast cancer patient samples. Immunohistochemistry of the same patient tumor samples revealed RARRES1 expression is localized to the endoplasmic reticulum. Finally, knockdown of RARRES1 in claudin-low MDA-MB-231 and basal-like MDA-MB-468 and HCC1937 significantly increased cell proliferation and tumor growth, suggesting RARRES1 has a tumor suppressive function regardless of position on the differentiation hierarchy. We conclude that RARRES1 is a tumor suppressor in TNBC with methylation and expression profiles distinct to the differentiation hierarchy observed in breast cancer. Citation Format: Krysta Mila Coyle, Dejan Vidovic, Cheryl A. Dean, Margaret Lois Thomas, Mohammad Sultan, Derek Clements, Ahmad Vaghar-Kashani, Melissa Wallace, Ian Weaver, Carman A. Giacomantonio, Lucy Helyer, Paola Marcato. Expression of the tumor suppressor gene RARRES1 in the differentiation hierarchy of breast cancer is regulated by DNA methylation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A18.

Recent observations suggest that DNA methylation plays an important role in memory and long-term potentiation (LTP) in the hippocampus and is involved in programming the offspring epigenome in response to maternal care. Global DNA methylation is believed to be stable postnatally and to be similar across tissues in the adult mammal. It has also been a long held belief that DNA methyltransferases (DNMTs) play a very limited role in postmitotic tissues. Recent data suggests a more dynamic role for DNA methylation in the brain postnatally, therefore we examined the global state of methylation and the expression of the known DNMTs in the different regions of the hippocampus. We observed strikingly different levels of global methylation in the adult rat dentate gyrus (DG) and CA1 region in comparison with the CA2 and CA3 regions. mRNA levels of DNA methyltransferases exhibited similar regional specificity and were correlated with global DNA methylation levels. These regional differences in global methylation and expression of the DNA methylation machinery in the adult brain are consistent with the emerging hypothesis that DNA methylation may play a dynamic physiological role in the adult brain.

Background Epigenetic mechanisms may be involved in the reprogramming of gene expression in response to stressful stimuli. This investigation determined whether epigenetic phenomena might similarly be associated with suicide/depression. Methods The expression of DNA methyltransferase (DNMT) mRNA was assessed in several brain regions of individuals who had committed suicide and had been diagnosed with major depression relative to that of individuals who had died suddenly as a result of factors other than suicide. Results The DNMT gene transcripts' expression was altered in several brains regions of suicides, including frontopolar cortex, amygdala, and the paraventricular nucleus of the hypothalamus. Importantly, an increase of both mRNA and protein expression was found in the frontopolar cortex. In addition, although transcript abundance of various forms of DNMT was highly correlated in normal control subjects, this coordination of DNMT isoform expression was diminished in suicide brain. Further, within the frontopolar cortex, gene-specific aberrations in DNA methylation were apparent in the γ-aminobutyric acid (GABA) A receptor α1 subunit promoter region, the transcript of which is underexpressed in suicide/major depressive disorder (MDD) brains. Indeed, three cytosine/guanosine sites were hypermethylated relative to control subjects. Finally, we found that DNMT-3B mRNA abundance was inversely correlated to α1 mRNA abundance. Conclusions These data show that DNMT mRNA expression was altered in suicide brain, and this change in expression in the frontopolar cortex was associated with increased methylation of a gene whose mRNA expression has previously been shown to be reduced. These observations suggest that epigenetic mechanisms may be associated with altered gene expression in suicide/MDD.

DNA methylation has been viewed as a stable component of the epigenome, established during development and fixed thereafter. Here we have found that the DNA methylation pattern varies during a single cell cycle, with the global levels of DNA methylation decreased in G(1) and increase during S phase. There was little change in the DNA methylation levels in repetitive sequences throughout the cell cycle. However using a human CpG island microarray it was revealed that 174 CG-containing sequences were differentially methylated between G(1) and S. Seventy-five percent of all the variations in DNA methylation detected in unique sequences represented hypomethylation at G(0), with changes occurring in both CpG islands and non-CpG islands. This is the first demonstration of a dynamic DNA methylation pattern within a single cell cycle of a mature somatic cell. These data are important for our understanding of the stability of DNA methylation patterns in somatic cells.

A hallmark of vertebrate genes is that actively transcribed genes are hypomethylated in critical regulatory sequences. However, the mechanisms that link gene transcription and DNA hypomethylation are unclear. Using a trichostatin A (TSA)-induced replication-independent demethylation assay with HEK 293 cells, we show that RNA transcription is required for DNA demethylation. Histone acetylation precedes but is not sufficient to trigger DNA demethylation. Following histone acetylation, RNA polymerase II (RNAP II) interacts with the methylated promoter. Inhibition of RNAP II transcription with actinomycin D, alpha-amanitin, or CDK7-specific small interfering RNA inhibits DNA demethylation. H3 trimethyl lysine 4 methylation, a marker of actively transcribed genes, was associated with the cytomegalovirus promoter only after demethylation. TSA-induced demethylation of the endogenous cancer testis gene GAGE follows a similar sequence of events and is dependent on RNA transcription as well. These data suggest that DNA demethylation follows rather than precedes early transcription and point towards a novel function for DNA demethylation as a memory of actively transcribed genes.

DNA methyltransferase 1 (DNMT1) is an important component of the epigenetic machinery and is responsible for copying DNA methylation patterns during cell division. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming. Knockdown of DNMT1 leads to inhibition of DNA replication, but the mechanism has been unclear. Here we show that depletion of DNMT1 with either antisense or small interfering RNA (siRNA) specific to DNMT1 activates a cascade of genotoxic stress checkpoint proteins, resulting in phosphorylation of checkpoint kinases 1 and 2 (Chk1 and -2), gammaH2AX focus formation, and cell division control protein 25a (CDC25a) degradation, in an ataxia telangiectasia mutated-Rad3-related (ATR)-dependent manner. siRNA knockdown of ATR blocks the response to DNMT1 depletion; DNA synthesis continues in the absence of DNMT1, resulting in global hypomethylation. Similarly, the response to DNMT1 knockdown is significantly attenuated in human mutant ATR fibroblast cells from a Seckel syndrome patient. This response is sensitive to DNMT1 depletion, independent of the catalytic domain of DNMT1, as indicated by abolition of the response with ectopic expression of either DNMT1 or DNMT1 with the catalytic domain deleted. There is no response to short-term treatment with 5-aza-deoxycytidine (5-aza-CdR), which causes demethylation by trapping DNMT1 in 5-aza-CdR-containing DNA but does not cause disappearance of DNMT1 from the nucleus. Our data are consistent with the hypothesis that removal of DNMT1 from replication forks is the trigger for this response.

Increased levels of pup licking/grooming and arched-back nursing by rat mothers over the first week of life alter the epigenome at a glucocorticoid receptor gene promoter in the hippocampus of the offspring. Differences in the DNA methylation pattern between the offspring of High and Low licking/grooming--arched-back mothers emerge over the first week of life, are reversed with cross-fostering, persist into adulthood and are associated with altered histone acetylation and transcription factor (NGFI-A) binding to the glucocorticoid receptor promoter. Central infusion of the adult offspring with the histone deacetylase inhibitor trichostatin A removes the previously defined epigenomic group differences in histone acetylation, DNA methylation, NGFI-A binding, glucocorticoid receptor expression, and hypothalamic-pituitary-adrenal responses to stress, thus suggesting a causal relation between the epigenomic state, glucocorticoid receptor expression and the effects of maternal care on stress responses in the offspring. These findings demonstrate that an epigenomic state of a gene can be established through a behavioral mode of programming and that in spite of the inherent stability of this epigenomic mark, it is dynamic and potentially reversible.

This paper describes the results of a series of studies showing that variations in mother-pup interactions program the development of individual differences in behavioral and endocrine stress responses in the rat. These effects are associated with altered expression of genes in brain regions, such as the amygdala, hippocampus, and hypothalamus, that regulate the expression of stress responses. Studies from evolutionary biology suggest that such "maternal effects" are common and often associated with variations in the quality of the maternal environment. Together these findings suggest an epigenetic process whereby the experience of the mother alters the nature of the parent-offspring interactions and thus the phenotype of the offspring.

Environmental conditions in early life permanently alter the development of glucocorticoid receptor gene expression in the hippocampus and hypothalamic-pituitary-adrenal responses to acute or chronic stress. In part, these effects can involve an activation of ascending serotonergic pathways and subsequent changes in the expression of transcription factors that might drive glucocorticoid receptor expression in the hippocampus. This paper summarizes the evidence in favor of these pathways as well as recent studies describing regulatory targets within the chromatin structure of the promoter region of the rat hippocampal glucocorticoid receptor gene.