Epigenetic Traits Research Articles

Abstract P1-04-01: Epigenome-wide association study for breast cancer risk using whole genome and target captured bisulphite sequencing: A pooled case-control study nested in the breakthrough generations study

Abstract Background: The field of epigenetic epidemiology has rapidly advanced and recent work has discovered epigenetic markers of breast cancer risk in white blood cell (WBC) DNA. Using Epigenome-Wide Association Studies (EWAS) on the Illumina 450k methylation array, we and others have shown epigenome-wide hypomethylation (-0.2%, p<2.2x10-16) in incident breast cancer cases compared with controls in several prospective cohorts. We have proposed a mechanism that involves cancer risk exposures, lifetime and environmental events, that alter the epigenome and stably modifies an individual's cancer risk. However, more work is needed to establish the clinical utility of this observation, the underlying causes of this variation and to determine whether the 1.7% of CpG sites targeted by the 450k array are representative of the remaining 98% of the epigenome that has not yet been interrogated. Our overall aim is to identify epigenetic traits within the epigenome that are associated with the risk of developing breast cancer. Methods: We conducted an EWAS using whole genome bisulphite sequencing (WGBS) of WBC DNA from incident breast cancer cases (n=548) compared to matched controls (n=548) from a prospective cohort (Breakthrough Generations Study) using a DNA pooling approach. Eight DNA pools were prepared in sequencing libraries and sequenced on the Hiseq2500 at PE100bp reads, resulting in ~10-fold coverage per CpG, per library, across ~20 million mappable CpG sites. Each pooled sample was also analysed in triplicate on the Illumina 450k methylation array for validation. We used Agilent target capture bisulphite sequencing (TCBS) for technical validation in a subset of breast cancer cases (n=48) and matched controls (n=48), individually barcoded and sequenced on the MiSeq at PE150bp, aiming for >1000 fold coverage of 425 kb targeted sequence. Results: Interrogation of specific genomic regions showed that gene-body methylation averages tended to be hypomethylated in cases, while CpG island averages identified both hypo- and hypermethylation. We have validated the same direction of change in 40/51 CpG islands that were covered by the Illumina 450K methylation array and have developed a target capture panel for validation of 960 gene body regions and 224 CpG island regions that were identified as significantly different between cases and controls (average -11%, FDR<5%). Analysis of TCBS data is ongoing. Conclusions: Results indicate that epigenome-wide hypomethylation and methylation in specific sites, particularly gene bodies, measured in pre-diagnostic blood samples may be predictive of breast cancer risk, and may thus be useful as a risk biomarker. Citation Format: Flanagan JM, Curry E, Stirling L, Flower K, Orr N, Tomczyk K, Coulson P, Jones M, Ashworth A, Swerdlow A, Brown R, Garcia-Closas M. Epigenome-wide association study for breast cancer risk using whole genome and target captured bisulphite sequencing: A pooled case-control study nested in the breakthrough generations study [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-04-01.

Genetic and Epigenetic Intra-tumour Heterogeneity in Colorectal Cancer

IntroductionColorectal cancer (CRC) is a highly heterogeneous disease, with pathologically similar cancers having completely different responses to treatment and patient survival. Intra-tumour heterogeneity (defined as distinct morphological and phenotypic differences) has recently been demonstrated to be an important factor in the development and behaviour of cancer cells and can be used to determine response to anticancer therapy.MethodPatients with resected CRC had DNA extracted from eight defined tumour areas which were analysed for two genetic mutations (BRAF and KRAS) and one epigenetic trait (CpG island methylator phenotype/CIMP). Normal adjacent tissue was studied as control.ResultsTwelve patients with CRC were included. Intra-tumoural heterogeneity for KRAS mutation was seen in 2 patients (17%). There was no statistical evidence of CIMP status heterogeneity (p = 0.85), but 6 of the 12 patients (50%) demonstrated at least one heterogeneous area within the tumour.DiscussionIntra-tumoural heterogeneity for both genetic and epigenetic factors in CRC is more prevalent than previously thought in Stage II and Stage III CRC. This study provides new insight into epigenetic heterogeneity of CRC and supports the development of a more targeted biopsy strategy to support expansion of personalised treatment.

Transgenerational transmission of an anticholinergic endophenotype with memory dysfunction

Impaired cholinergic neurotransmission associated with cognitive dysfunction occurs in various mental disorders of different etiologies including Alzheimer's disease and postalcoholic dementia and others. To address the question whether there exists a common endophenotype with a defined genetic and/or epigenetic signature causing mental dysfunction in these disorders, we investigated 2 generations of offspring born to alcohol-treated mothers. Here, we show that memory impairment and reduced synthesis of acetylcholine occurs in both F1 (exposed to ethanol in utero) and F2 generation (never been exposed to ethanol). Effects in the F2 generation are most likely consequences of transgenerationally transmitted epigenetic modifications in stem cells induced by alcohol. This clearly documents the role of ancestral history of drug abuse on the brain development of subsequent generations. The results further suggest an epigenetic trait for an anticholinergic endophenotype associated with cognitive dysfunction which might be relevant to our understanding of mental impairment in neurodegenerative disorders such as Alzheimer's disease and related disorders.

Stable Epigenetic Variants Selected from an Induced Hypomethylated Fragaria vesca Population.

Epigenetic inheritance was transmitted through selection over five generations of extreme early, but not late flowering time phenotypic lines in Fragaria vesca. Epigenetic variation was initially artificially induced using the DNA demethylation reagent 5-azacytidine (5-azaC). It is the first report to explore epigenetic variant selection and phenotypic trait inheritance in strawberry. Transmission frequency of these traits was determined across generations. The early flowering (EF4) and late stolon (LS) phenotypic traits were successfully transmitted across five and three generations through meiosis, respectively. Stable mitotic transmission of the early flowering phenotype was also demonstrated using clonal daughters derived from the 4th Generation (S4) mother plant. In order to further explore the DNA methylation patterns underlying the early flowering trait, the standard MSAP method using isoschizomers Hpa II/Msp I, and newly modified MSAP method using isoschizomers Tfi I/Pfe I which detected DNA methylation at CG, CHG, CHH sites were used in two early flowering lines, EF lines 1 (P2) and EF lines 2 (P3), and control lines (P1). A significant reduction in the number of fully-methylated bands was detected in P2 and P3 when compared to P1 using the novel MSAP method. In the standard MSAP, the symmetric CG and CHG methylation was maintained over generations in the early flowering lines based on the clustering in P2 and P3, the novel MSAP approach revealed the asymmetric CHH methylation pattern was not maintained over generations. This study provides evidence of stable selection of phenotypic traits, particularly early flowering through both meiosis and mitosis, which is meaningful to both breeding programs and commercial horticulture. The maintenance in CG and CHG methylation over generations suggests the early flowering phenotype might be related to DNA methylation alterations at the CG or CHG sites. Finally, this work provides a new approach for studying the role of epigenetics on complex quantitative trait improvement in strawberry, as well as providing a tool to expand phenotypic diversity and expedite potential new horticulture cultivar releases through either seed or vegetative propagation.

Oct4 Methylation-Mediated Silencing As an Epigenetic Barrier Preventing Müller Glia Dedifferentiation in a Murine Model of Retinal Injury.

Müller glia (MG) is the most abundant glial type in the vertebrate retina. Among its many functions, it is capable of responding to injury by dedifferentiating, proliferating, and differentiating into every cell types lost to damage. This regenerative ability is notoriously absent in mammals. We have previously reported that cultured mammalian MG undergoes a partial dedifferentiation, but fails to fully acquire a progenitor phenotype and differentiate into neurons. This might be explained by a mnemonic mechanism comprised by epigenetic traits, such as DNA methylation. To achieve a better understanding of this epigenetic memory, we studied the expression of pluripotency-associated genes, such as Oct4, Nanog, and Lin28, which have been reported as necessary for regeneration in fish, at early times after NMDA-induced retinal injury in a mouse experimental model. We found that although Oct4 is expressed rapidly after damage (4 hpi), it is silenced at 24 hpi. This correlates with a significant decrease in the DNA methyltransferase Dnmt3b expression, which returns to basal levels at 24 hpi. By MS-PCR, we observed a decrease in Oct4 methylation levels at 4 and 12 hpi, before returning to a fully methylated state at 24 hpi. To demonstrate that these changes are restricted to MG, we separated these cells using a GLAST antibody coupled with magnetic beads. Finally, intravitreous administration of the DNA-methyltransferase inhibitor SGI-1027 induced Oct4 expression at 24 hpi in MG. Our results suggest that mammalian MG injury-induced dedifferentiation could be restricted by DNA methylation, which rapidly silences Oct4 expression, preventing multipotency acquisition.

Identification of activated enhancers and linked transcription factors in breast, prostate, and kidney tumors by tracing enhancer networks using epigenetic traits.

BackgroundAlthough technological advances now allow increased tumor profiling, a detailed understanding of the mechanisms leading to the development of different cancers remains elusive. Our approach toward understanding the molecular events that lead to cancer is to characterize changes in transcriptional regulatory networks between normal and tumor tissue. Because enhancer activity is thought to be critical in regulating cell fate decisions, we have focused our studies on distal regulatory elements and transcription factors that bind to these elements.ResultsUsing DNA methylation data, we identified more than 25,000 enhancers that are differentially activated in breast, prostate, and kidney tumor tissues, as compared to normal tissues. We then developed an analytical approach called Tracing Enhancer Networks using Epigenetic Traits that correlates DNA methylation levels at enhancers with gene expression to identify more than 800,000 genome-wide links from enhancers to genes and from genes to enhancers. We found more than 1200 transcription factors to be involved in these tumor-specific enhancer networks. We further characterized several transcription factors linked to a large number of enhancers in each tumor type, including GATA3 in non-basal breast tumors, HOXC6 and DLX1 in prostate tumors, and ZNF395 in kidney tumors. We showed that HOXC6 and DLX1 are associated with different clusters of prostate tumor-specific enhancers and confer distinct transcriptomic changes upon knockdown in C42B prostate cancer cells. We also discovered de novo motifs enriched in enhancers linked to ZNF395 in kidney tumors.ConclusionsOur studies characterized tumor-specific enhancers and revealed key transcription factors involved in enhancer networks for specific tumor types and subgroups. Our findings, which include a large set of identified enhancers and transcription factors linked to those enhancers in breast, prostate, and kidney cancers, will facilitate understanding of enhancer networks and mechanisms leading to the development of these cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0102-4) contains supplementary material, which is available to authorized users.

VII. Moving toward integrative feminist evolutionary behavioral sciences

There is a long history of mostly antagonist interactions between feminist and evolutionary scholarship in the behavioral sciences. However, recent theoretical and empirical advances have highlighted that “nature” and “nurture” are not mutually exclusive, or even divorceable, levels of explanation. New developments in evolutionary theory, articulated under the name of “the extended synthesis,” show significant promise for integrating feminist and evolutionary approaches to the study of human behavior. The extended synthesis provides feminist inquiry and research an expanded ground on which to enter the conversation, such as with the inclusion of “fast” evolution – the idea that genetic and epigenetic (environmentally influenced gene expression) traits can change within a generation – which demonstrates that there has in fact been evolutionary time to (adaptively) alter behavior and physiology. This integration has the potential to lead to new research paradigms that bridge the old divide between “nature” and “nurture”, resulting in transdisciplinary frameworks that show the interaction effects between each. Feminism and feminist psychology will be more complete descriptors and analyzers of human behavior in this model, and crucial interaction effects historically ignored by both the sciences and humanities may now receive due consideration.

Abstract LB-301: Paternal overweight leads to reprogramming of daughters’ breast cancer risk in a mouse model

Abstract Background: Obesity and overweight are known to induce epigenetic changes. Acquired epigenetic traits have been shown to be transmitted from parents to offspring via the germ-line (eggs and sperm). Maternal weight and nutrition in pregnancy lead to reprogramming of the mammary gland tissue and breast cancer risk in the offspring, yet no studies have investigated the impact of fathers’ body weight on offspring's risk of this disease. Here we investigated whether paternal overweight can epigenetically reprogram father's germ-line and increase daughters’ likelihood of developing breast cancer, using a mouse model. Methods: Male mice were fed control or obesity-inducing diets from weaning until sexual maturity; at this point, males were housed together with female mice reared on control diet. Once a vaginal plug was detected, males were euthanized for sperm collection. Pregnant dams were fed the control diet throughout pregnancy and after giving birth. Pups were fed the control diet throughout the experiment. A subset of female offspring was euthanized for mammary tissue harvesting. Another female offspring sub-set was treated with 9,12-dimethylbenz[a]anthracene (DMBA) to induce mammary tumors. Results: We found that overweight in male mice alters their sperm epigenome, including the miRNA expression profile and DNA methylation patterns. We also observed that, compared to control offspring, the female offspring of overweight fathers had higher birth weight (p = 0.013).This increase in body weight persisted through early adulthood but disappeared as animals aged (p = 0.6). The mammary tissue of overweight fathers’ offspring had higher numbers of undifferentiated structures (terminal end buds; TEBs; p = 0.017) and ductal elongation (p = 0.004) compared to control daughters. In addition, their mammary miRNA expression profile was altered with some of the same changes observed in fathers. These changes in normal mammary tissue were associated with higher rates of DMBA-induced mammary tumors (p = 0.02) in daughters of overweight fathers. Down-stream analyses showed that the hypoxia signaling pathway which is regulated by miR-874, one of the miRNAs down-regulated in their mammary tissue, was active in normal mammary glands and tumors. Conclusions: Using a mouse model, our study provides evidence that paternal overweight, around the time of conception, reprograms daughters’ mammary gland development, epigenetic profile and increase their mammary cancer risk. Studies are underway to determine whether the observed epigenetic changes are functionally responsible for increased breast cancer risk observed in the offspring of overweight males. If confirmed in humans, our findings could have important implications since it supports the notion that the familial or heritable aspect of breast cancer can be due not only to genetic factors but could also result from ancestral exposure and possibly inherited through modifiers of gene expression such as epigenetic mechanisms. Citation Format: Camile Castilho Fontelles, Elissa Carney, Johan Clarke, Nguyen Nguyen, Chao Yin, Lu Jin, Leena Hilakivi-Clarke, Thomas Prates Ong, Zuolin Cheng, Yi Fu, Yue Wang, Sonia M. De Assis. Paternal overweight leads to reprogramming of daughters’ breast cancer risk in a mouse model. [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 LB-301.

Obesity: epigenetic aspects.

Epigenetics, defined as inheritable and reversible phenomena that affect gene expression without altering the underlying base pair sequence has been shown to play an important role in the etiopathogenesis of obesity. Obesity is associated with extensive gene expression changes in tissues throughout the body. Epigenetics is emerging as perhaps the most important mechanism through which the lifestyle-choices we make can directly influence the genome. Considerable epidemiological, experimental and clinical data have been amassed showing that the risk of developing disease in later life is dependent on early life conditions, mainly operating within the normative range of developmental exposures. In addition to the 'maternal' interactions, there has been increasing interest in the epigenetic mechanisms through which 'paternal' influences on offspring development can be achieved. Nutrition, among many other environmental factors, is a key player that can induce epigenetic changes not only in the directly exposed organisms but also in subsequent generations through the transgenerational inheritance of epigenetic traits. Overall, significant progress has been made in the field of epigenetics and obesity and the first potential epigenetic markers for obesity that could be detected at birth have been identified. Fortunately, epigenetic phenomena are dynamic and rather quickly reversible with intensive lifestyle changes. This is a very promising and sustainable resolution to the obesity pandemic.

The Effects of Epigenetics on Stress Response

Despite the vast amount of resources at the disposal of humanity today, the intricacies of human biology are often a mystery. The chemical and biological products of the human genome have been well studied and documented, but many of the chemical and neurological pathways are missing quite a few details. The human stress response is one of the most primal and valuable functions of this code that developed as a self- preservation mechanism (Hans, 1975) to naturally increase the odds of procreation. However, this function is prone to overload, particularly in individuals with certain epigenetic traits instilled by early life events, or even events taking place before their life began. Left unchecked, this overclocked stress response can lead to irate outward behavior with no known cause, and even worse, no known treatments. These irate behaviors can be seen on the experimental level; mice who are not adequately groomed by their mothers expressed an increase glucorticoid receptor (GR) response than mice with adequate grooming (Radtke et al., 2011). In human studies, these GR reactions are responsible for a myriad of mental disorders including suicidal tendencies, psychopathy, and increased aggression. Gene therapy is possible for these epigenetic factors, opening up new possibilities for treatment of mental disorders.

Colorectal cancer carcinogenesis: a review of mechanisms

Colorectal cancer (CRC) is the second most common cancer in women and the third most common in men globally. CRC arises from one or a combination of chromosomal instability, CpG island methylator phenotype, and microsatellite instability. Genetic instability is usually caused by aneuploidy and loss of heterozygosity. Mutations in the tumor suppressor or cell cycle genes may also lead to cellular transformation. Similarly, epigenetic and/or genetic alterations resulting in impaired cellular pathways, such as DNA repair mechanism, may lead to microsatellite instability and mutator phenotype. Non-coding RNAs, more importantly microRNAs and long non-coding RNAs have also been implicated at various CRC stages. Understanding the specific mechanisms of tumorigenesis and the underlying genetic and epigenetic traits is critical in comprehending the disease phenotype. This paper reviews these mechanisms along with the roles of various non-coding RNAs in CRCs.

254 SYSTEMS BIOLOGY OF SPERM AND BULL FERTILITY

Bull fertility, the ability of the spermatozoon to fertilize and activate the egg as well as support early embryonic development, is crucial for efficient production of cattle. With the major advances in fundamental science of reproduction, such as genomics, and in biotechnology, such as assisted reproductive technologies, bull influence on herd production has been accentuated. In addition to providing half of the genome for the zygote, sperm also contribute proteins, transcripts, and epigenetic traits that play important roles in sperm physiology, fertilization, and early embryonic development. Recently, using sperm from Holstein bulls with different in vivo fertility data, we have demonstrated that sperm Protamine 1 protein and a panel of microRNA are associated with bull fertility. In addition, molecular and cellular biology experiments on the analyses of sperm nuclear proteins showed that bull sperm have significant levels of histones that are also associated with bull fertility. This presentation will convey the results of most relevant research on discovery of sperm biomolecular markers associated with semen quality and bull fertility. Because of the available field data in bull fertility and the physiological similarities between bovine and other mammals, research results stemming from the systems biology of bull fertility are applicable to other species as well.

Insights into the Social Structure of the PPNB Site of Kfar HaHoresh, Israel, Based on Dental Remains.

One of the central questions of the transition from mobile hunter-gatherers to sedentary farming communities concerns the establishment of new social structures and group identities. Along with other important factors, such as territory, ideology or economy, biological relationships might have played a decisive role in defining social groups. We therefore systematically analyzed teeth and jaw remains from nine sites in the Near East dating from the Natufian to the Late PPNB as primary proxy data for the reconstruction of familial relationships. This paper presents the results of morphological analyses on the teeth of the individuals from Kfar HaHoresh, one of the investigated Pre-Pottery Neolithic B sites. Kfar HaHoresh is located in the Nazareth hills of Galilee (32°42'20'' N 35°16'28'' E), Israel. Different statistical methods were applied to our data of epigenetic traits with the aim of determining biological relationships within the community, whereby the data of the eight other sites were used as cross-references. Our comparison of the traits of all individuals from Kfar HaHoresh indicates a rather heterogeneous community, but clearly shows one cluster belonging to a quite homogenous group, suggesting close biological relations between females and sub-adults. Interestingly, none of the male individuals belongs to this cluster, although their number outweighs that of the female individuals. This might suggest matrilocal residence patterns. However, due to the incomplete preservation of the teeth along with several other uncertainties, our conclusion must be seen as preliminary. A cross-examination of the results on skeletons excavated after our investigation should also be taken into consideration.

Epigenetics: The karma of oil palms.

Despite their clonal origin, some oil palm trees develop fruits that give almost no oil. It emerges that the number of methyl groups attached to a DNA region called Karma determine which plants are defective. See Letter p.533 A flower and fruit abnormality known as 'mantled' can develop in some agricultural oil palm cultivars derived from tissue culture and the resulting mantled palms can become unproductive. Mantling is widely regarded as an epigenetic trait, but has not been fully explained. Meilina Ong-Abdullah et al. have undertaken a genome-wide, unbiased, DNA methylation analysis to look for loci epigenetically associated with the mantled phenotype. They find that hypomethylation of a single Karma family retrotransposon embedded in the intron of a homeotic gene is common to all mantled clones and associated with aberrant splicing and termination of the gene transcript. Loss of methylation — dubbed the Bad Karma epiallele — predicts a loss of oil palm yield and this property should enable screening for higher-performing clones at the plantlet stage.

Epigenetic inheritance and the missing heritability.

Genome-wide association studies of complex physiological traits and diseases consistently found that associated genetic factors, such as allelic polymorphisms or DNA mutations, only explained a minority of the expected heritable fraction. This discrepancy is known as “missing heritability”, and its underlying factors and molecular mechanisms are not established. Epigenetic programs may account for a significant fraction of the “missing heritability.” Epigenetic modifications, such as DNA methylation and chromatin assembly states, reflect the high plasticity of the genome and contribute to stably alter gene expression without modifying genomic DNA sequences. Consistent components of complex traits, such as those linked to human stature/height, fertility, and food metabolism or to hereditary defects, have been shown to respond to environmental or nutritional condition and to be epigenetically inherited. The knowledge acquired from epigenetic genome reprogramming during development, stem cell differentiation/de-differentiation, and model organisms is today shedding light on the mechanisms of (a) mitotic inheritance of epigenetic traits from cell to cell, (b) meiotic epigenetic inheritance from generation to generation, and (c) true transgenerational inheritance. Such mechanisms have been shown to include incomplete erasure of DNA methylation, parental effects, transmission of distinct RNA types (mRNA, non-coding RNA, miRNA, siRNA, piRNA), and persistence of subsets of histone marks.

The IGF1 P2 promoter is an epigenetic QTL for circulating IGF1 and human growth.

BackgroundEven if genetics play an important role, individual variation in stature remains unexplained at the molecular level. Indeed, genome-wide association study (GWAS) have revealed hundreds of variants that contribute to the variability of height but could explain only a limited part of it, and no single variant accounts for more than 0.3% of height variance. At the interface of genetics and environment, epigenetics contributes to phenotypic diversity. Quantifying the impact of epigenetic variation on quantitative traits, an emerging challenge in humans, has not been attempted for height. Since insulin-like growth factor 1 (IGF1) controls postnatal growth, we tested whether the CG methylation of the two promoters (P1 and P2) of the IGF1 gene is a potential epigenetic contributor to the individual variation in circulating IGF1 and stature in growing children.ResultsChild height was closely correlated with serum IGF1. The methylation of a cluster of six CGs located within the proximal part of the IGF1 P2 promoter showed a strong negative association with serum IGF1 and growth. The highest association was for CG-137 methylation, which contributed 13% to the variance of height and 10% to serum IGF1. CG methylation (studied in children undergoing surgery) was approximately 50% lower in liver and growth plates, indicating that the IGF1 promoters are tissue-differentially methylated regions (t-DMR). CG methylation was inversely correlated with the transcriptional activity of the P2 promoter in mononuclear blood cells and in transfection experiments, suggesting that the observed association of methylation with the studied traits reflects true biological causality.ConclusionsOur observations introduce epigenetics among the individual determinants of child growth and serum IGF1. The P2 promoter of the IGF1 gene is the first epigenetic quantitative trait locus (QTLepi) reported in humans. The CG methylation of the P2 promoter takes place among the multifactorial factors explaining the variation in human stature.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0062-8) contains supplementary material, which is available to authorized users.

Epigenetic mechanisms in neurological and neurodegenerative diseases.

The role of epigenetic mechanisms in the function and homeostasis of the central nervous system (CNS) and its regulation in diseases is one of the most interesting processes of contemporary neuroscience. In the last decade, a growing body of literature suggests that long-term changes in gene transcription associated with CNS’s regulation and neurological disorders are mediated via modulation of chromatin structure. “Epigenetics”, introduced for the first time by Waddington in the early 1940s, has been traditionally referred to a variety of mechanisms that allow heritable changes in gene expression even in the absence of DNA mutation. However, new definitions acknowledge that many of these mechanisms used to perpetuate epigenetic traits in dividing cells are used by neurons to control a variety of functions dependent on gene expression. Indeed, in the recent years these mechanisms have shown their importance in the maintenance of a healthy CNS. Moreover, environmental inputs that have shown effects in CNS diseases, such as nutrition, that can modulate the concentration of a variety of metabolites such as acetyl-coenzyme A (acetyl-coA), nicotinamide adenine dinucleotide (NAD+) and beta hydroxybutyrate (β-HB), regulates some of these epigenetic modifications, linking in a precise way environment with gene expression. This manuscript will portray what is currently understood about the role of epigenetic mechanisms in the function and homeostasis of the CNS and their participation in a variety of neurological disorders. We will discuss how the machinery that controls these modifications plays an important role in processes involved in neurological disorders such as neurogenesis and cell growth. Moreover, we will discuss how environmental inputs modulate these modifications producing metabolic and physiological alterations that could exert beneficial effects on neurological diseases. Finally, we will highlight possible future directions in the field of epigenetics and neurological disorders.

Genome-wide analysis of DNA methylation before-and after exercise in the thoroughbred horse with MeDIP-Seq.

Athletic performance is an important criteria used for the selection of superior horses. However, little is known about exercise-related epigenetic processes in the horse. DNA methylation is a key mechanism for regulating gene expression in response to environmental changes. We carried out comparative genomic analysis of genome-wide DNA methylation profiles in the blood samples of two different thoroughbred horses before and after exercise by methylated-DNA immunoprecipitation sequencing (MeDIP-Seq). Differentially methylated regions (DMRs) in the pre-and post-exercise blood samples of superior and inferior horses were identified. Exercise altered the methylation patterns. After 30 min of exercise, 596 genes were hypomethylated and 715 genes were hypermethylated in the superior horse, whereas in the inferior horse, 868 genes were hypomethylated and 794 genes were hypermethylated. These genes were analyzed based on gene ontology (GO) annotations and the exercise-related pathway patterns in the two horses were compared. After exercise, gene regions related to cell division and adhesion were hypermethylated in the superior horse, whereas regions related to cell signaling and transport were hypermethylated in the inferior horse. Analysis of the distribution of methylated CpG islands confirmed the hypomethylation in the gene-body methylation regions after exercise. The methylation patterns of transposable elements also changed after exercise. Long interspersed nuclear elements (LINEs) showed abundance of DMRs. Collectively, our results serve as a basis to study exercise-based reprogramming of epigenetic traits.

Effect of plant growth regulators on calyx abscission, fruit quality, and auxin-repressed protein (ARP) gene expression in fruitlets of ‘Dangshansuli’ pear (Pyrus bretschneideri Rehd.)

SummarySepals of a pome fruit flower are collectively called the calyx. Calyxes may be shed to produce calyx-shed fruit (CSF), or remain to produce calyx-persistent fruit (CPF). To investigate the relationship between calyx shedding and phytohormone concentrations in fruitlets and fruit quality in ‘Dangshansuli’ pear (Pyrus bretschneideri Rehd.), endogenous levels of gibberellins, indole-3-acetic acid, abscisic acid, and zeatin riboside were measured in CSF and CPF at different stages of development. Manual excision of the calyx at an early stage of fruitlet development was also performed. The auxin-repressed protein gene (ARP) was cloned, and relative levels of ARP gene expression were determined in fruitlets on trees treated by spraying separately with water (control) or each of the plant growth regulators (PGRs) gibberellic acid (GA3), paclobutrazol (Pac), or 1-naphthaleneacetic acid (NAA) using real-time quantitative reverse transcription-PCR. The results indicated that manual calyx excision at an early stage of fruit development altered only the shape and appearance, but not the inner quality of the fruit. Higher endogenous levels of all gibberellins in fruitlets at 10 - 20 d after full bloom (DAFB) played an important role in calyx persistency on fruitlets. The ARP gene was cloned from fruitlets of ‘Dangshansuli’ pear and named PbARP. The full-length cDNA was 707 bp with a 5'-untranslated region (UTR) of 40 bp, a 3'-UTR of 316 bp, and a coding region of 351 bp, encoding a deduced polypeptide containing 116 amino acids.GA3 [100 mg l-1 of 90% (w/w)] inhibited expression of the PbARP gene and increased calyx persistency, whereas 100 mg l-1 of 15% (w/w) Pac enhanced PbARP gene expression and accelerated calyx shedding. Higher levels of PbARP gene expression in fruitlets of ‘Dangshansuli’ pear were associated with calyx shedding.The percentage of CSF increased by > 20% following treatment with 100 mg l-1 of 15% (w/w) Pac, 100 ml l-1 of an emulsifiable concentrate of 40% (w/v) flusilazole (Fls), a mixture of 100 mg l-1 of 15% (w/w) Pac plus 50 mg l-1 of 99% (w/w) benzylaminopurine (BA), or a mixture of 100 ml l-1 of 40% (w/v) Fls plus 50 mg l-1 of 99% (w/w) BA, with no significant difference in fruit set percentage or fruit quality. Persistence or shedding of the calyx in pear fruit is therefore an epigenetic trait that can be regulated by the application of PGRs.

Epigenetic regulation of p14ARF and p16INK4A expression in cutaneous and uveal melanoma

Inactivation of p14ARF and p16INK4A by epigenetic changes in cutaneous and uveal melanoma has been here investigated. Compared with melanocytes, p14ARF mRNA reduction and p16INK4A inactivation were frequently noticed. No association between p14ARF promoter methylation and mRNA levels was found, whereas aberrant p16INK4A methylation was associated with gene silencing (p<0.001). Comparative analysis within melanomas of different Breslow's thicknesses showed that drastic reductions in p14ARF and p16INK4A expression appeared at the level of thin/intermediate and intermediate/thick transitions. The effects of 5-aza-2′-deoxycytidine (5-aza-dC) and suberanilohydroxamic acid (SAHA) on in vivo binding of DNA methyltransferases (DNMTs) and acetyl histone H3/H4 to p14ARF and p16INK4A promoters were tested together with the impact of ectopic expression of p14ARF and p16INK4A on cell proliferation, migration, and invasion. SAHA treatment induced H3 and H4 hyperacetylation at the p14ARF promoter followed by increased p14ARF expression, whereas exposure to 5-aza-dC decreased the recruitment of DNMT1 and DNMT3b at the p16INK4A promoter and reactivated p16INK4A. Studies on promoter-associated di-methyl histone H3 (Lys4) levels ruled out an involvement of this epigenetic trait on p14ARF and p16INK4A expression. The enforced expression of p14ARF or p16INK4A and, even more so, their co-expression, significantly reduced cell proliferation, migration and invasion. Our data pinpoint: i) a frequent impairment of p14ARF and p16INK4A gene expression by epigenetic modifications in melanoma; ii) histone hypoacetylation as the dominant mechanism of p14ARF silencing; and iii) 5′ CpG promoter methylation as the major mechanism of p16INK4A gene inactivation. Collectively, our data suggest that selected epi-drugs may be useful in melanoma treatment.