Matthew Suderman

Disruption of histone methylation in developing sperm impairs offspring health transgenerationally

Generations affected by histone changes Parent and even grandparent environmental exposure can transmit adverse health effects to offspring. The mechanism of transmission is unclear, but some studies have implicated variations in DNA methylation. In a mouse model, Siklenka et al. found that alterations in histone methylation during sperm formation in one generation leads to reduced survival and developmental abnormalities in three subsequent generations (see the Perspective by McCarrey). Although changes in DNA methylation were not observed, altered sperm RNA content and abnormal gene expression in offspring were measured. Thus, chromatin may act as a mediator of molecular memory in transgenerational inheritance. Science, this issue p. 10.1126/science.aab2006; see also p. 634 Overexpression of a histone demethylase in the mouse germ line reveals a mode of transgenerational epigenetic inheritance. [Also see Perspective by McCarrey] INTRODUCTION Despite the father transmitting half of the heritable information to the embryo, the focus of preconception health has been the mother. Paternal effects have been linked to complex diseases such as cancer, diabetes, and obesity. These diseases are increasing in prevalence at rates that cannot be explained by genetics alone and highlight the potential for disease transmission via nongenetic inheritance, through epigenetic mechanisms. Epigenetic mechanisms include DNA methylation, posttranslational modifications of histones, and noncoding RNA. Studies in humans and animals suggest that epigenetic mechanisms may serve in the transmission of environmentally induced phenotypic traits from the father to the offspring. Such traits have been associated with altered gene expression and tissue function in first and second offspring generations, a phenomenon known as intergenerational or transgenerational inheritance, respectively. The mechanisms underlying such paternal epigenetic transmission are unclear. RATIONALE Sperm formation involves rapid cell division and distinctive transcription programs, resulting in a motile cell with highly condensed chromatin. Within the highly compacted sperm nucleus, few histones are retained in a manner that suggests an influential role in development. Despite being the major focus of studies in epigenetic inheritance, the role of DNA methylation in paternal epigenetic inheritance is unresolved, as only minor changes in DNA methylation in sperm at CpG-enriched regions have been associated with transmission of environmentally induced traits. Instead, there may be a combination of molecular mechanisms underlying paternal transgenerational epigenetic inheritance involving changes in histone states and/or RNA in sperm. The function of sperm histones and their modifications in embryonic development, offspring health, and epigenetic inheritance is unknown. By overexpressing the human KDM1A histone lysine 4 demethylase during mouse spermatogenesis, we generated a mouse model producing spermatozoa with reduced H3K4me2 within the CpG islands of genes implicated in development, and we studied the development and fitness of the offspring. RESULTS Male transgenic offspring were bred with C57BL/6 females, generating the experimental heterozygous transgenic (TG) and nontransgenic (nonTG) brothers. Each generation from TG and nonTG animals (F1 to F3 in our transgenerational studies) was bred with C57BL/6 females, and the offspring (pups from generations F1 to F4) were analyzed for intergenerational and transgenerational effects. We found that KDM1A overexpression in one generation severely impaired development and survivability of offspring. These defects lasted for two subsequent generations in the absence of KDM1A germline expression. We characterized histone and DNA methylation states in the sperm of TG and nonTG sires. Overexpression of KDM1A was associated with a specific loss of H3K4me2 at more than 2300 genes, including many developmental regulatory genes. Unlike in other examples of paternal transgenerational inheritance, we observed no changes in sperm DNA methylation associated with primarily CpG-enriched regions. Instead, we measured robust and analogous changes in sperm RNA content of TG and nonTG descendants, as well as in their offspring, at the two-cell stage. These changes in expression and the phenotypic abnormalities observed in offspring correlated with altered histone methylation levels at genes in sperm. This study demonstrates that KDM1A activity during sperm development has major developmental consequences for offspring and implicates histone methylation and sperm RNA as potential mediators of transgenerational inheritance. Our data emphasize the complexity of transgenerational epigenetic inheritance likely involving multiple molecular factors, including the establishment of chromatin states in spermatogenesis and sperm-borne RNA. CONCLUSION Correct histone methylation during spermatogenesis is critical for offspring development and survival over multiple generations. These findings demonstrate the potential of histone methylation as a molecular mechanism underlying paternal epigenetic inheritance. Its modification by environmental influences may alter embryo development and complex disease transmission across generations. An essential next step is to establish functional links between environmental exposures, the composition of the sperm epigenome, and consequent altered gene expression and metabolic processes in offspring. Considering the mounting evidence, it may soon be reasonable to suggest that future fathers protect their sperm epigenome. Disruption of histone methylation in developing sperm by exposure to the KDM1A transgene in one generation severely impaired development and survivability of offspring. These defects were transgenerational and occurred in nonTG descendants in the absence of KDM1A germline expression. Developmental defects in offspring and embryos were associated with altered RNA expression in sperm and embryos. A father’s lifetime experiences can be transmitted to his offspring to affect health and development. However, the mechanisms underlying paternal epigenetic transmission are unclear. Unlike in somatic cells, there are few nucleosomes in sperm, and their function in epigenetic inheritance is unknown. We generated transgenic mice in which overexpression of the histone H3 lysine 4 (H3K4) demethylase KDM1A (also known as LSD1) during spermatogenesis reduced H3K4 dimethylation in sperm. KDM1A overexpression in one generation severely impaired development and survivability of offspring. These defects persisted transgenerationally in the absence of KDM1A germline expression and were associated with altered RNA profiles in sperm and offspring. We show that epigenetic inheritance of aberrant development can be initiated by histone demethylase activity in developing sperm, without changes to DNA methylation at CpG-rich regions.

Definition of the Landscape of Promoter DNA Hypomethylation in Liver Cancer

We use hepatic cellular carcinoma (HCC), one of the most common human cancers, as a model to delineate the landscape of promoter hypomethylation in cancer. Using a combination of methylated DNA immunoprecipitation and hybridization with comprehensive promoter arrays, we have identified approximately 3,700 promoters that are hypomethylated in tumor samples. The hypomethylated promoters appeared in clusters across the genome suggesting that a high-level organization underlies the epigenomic changes in cancer. In normal liver, most hypomethylated promoters showed an intermediate level of methylation and expression, however, high-CpG dense promoters showed the most profound increase in gene expression. The demethylated genes are mainly involved in cell growth, cell adhesion and communication, signal transduction, mobility, and invasion; functions that are essential for cancer progression and metastasis. The DNA methylation inhibitor, 5-aza-2-deoxycytidine, activated several of the genes that are demethylated and induced in tumors, supporting a causal role for demethylation in activation of these genes. Previous studies suggested that MBD2 was involved in demethylation of specific human breast and prostate cancer genes. Whereas MBD2 depletion in normal liver cells had little or no effect, we found that its depletion in human HCC and adenocarcinoma cells resulted in suppression of cell growth, anchorage-independent growth and invasiveness as well as an increase in promoter methylation and silencing of several of the genes that are hypomethylated in tumors. Taken together, the findings define the potential functional role of hypomethylation in cancer.

Maternal pre-pregnancy BMI and gestational weight gain, offspring DNA methylation and later offspring adiposity: findings from the Avon Longitudinal Study of Parents and Children

Background: Evidence suggests that in utero exposure to undernutrition and overnutrition might affect adiposity in later life. Epigenetic modification is suggested as a plausible mediating mechanism. Methods: We used multivariable linear regression and a negative control design to examine offspring epigenome-wide DNA methylation in relation to maternal and offspring adiposity in 1018 participants. Results: Compared with neonatal offspring of normal weight mothers, 28 and 1621 CpG sites were differentially methylated in offspring of obese and underweight mothers, respectively [false discovert rate (FDR)-corrected P-valueu2009<u20090.05), with no overlap in the sites that maternal obesity and underweight relate to. A positive association, where higher methylation is associated with a body mass index (BMI) outside the normal range, was seen at 78.6% of the sites associated with obesity and 87.9% of the sites associated with underweight. Associations of maternal obesity with offspring methylation were stronger than associations of paternal obesity, supporting an intrauterine mechanism. There were no consistent associations of gestational weight gain with offspring DNA methylation. In general, sites that were hypermethylated in association with maternal obesity or hypomethylated in association with maternal underweight tended to be positively associated with offspring adiposity, and sites hypomethylated in association with maternal obesity or hypermethylated in association with maternal underweight tended to be inversely associated with offspring adiposity. Conclusions: Our data suggest that both maternal obesity and, to a larger degree, underweight affect the neonatal epigenome via an intrauterine mechanism, but weight gain during pregnancy has little effect. We found some evidence that associations of maternal underweight with lower offspring adiposity and maternal obesity with greater offspring adiposity may be mediated via increased DNA methylation.

Prenatal and early life influences on epigenetic age in children: a study of mother-offspring pairs from two cohort studies

DNA methylation-based biomarkers of aging are highly correlated with actual age. Departures of methylation-estimated age from actual age can be used to define epigenetic measures of child development or age acceleration (AA) in adults. Very little is known about genetic or environmental determinants of these epigenetic measures of aging. We obtained DNA methylation profiles using Infinium HumanMethylation450 BeadChips across five time-points in 1018 mother–child pairs from the Avon Longitudinal Study of Parents and Children. Using the Horvath age estimation method, we calculated epigenetic age for these samples. AA was defined as the residuals from regressing epigenetic age on actual age. AA was tested for associations with cross-sectional clinical variables in children. We identified associations between AA and sex, birth weight, birth by caesarean section and several maternal characteristics in pregnancy, namely smoking, weight, BMI, selenium and cholesterol level. Offspring of non-drinkers had higher AA on average but this difference appeared to resolve during childhood. The associations between sex, birth weight and AA found in ARIES were replicated in an independent cohort (GOYA). In children, epigenetic AA measures are associated with several clinically relevant variables, and early life exposures appear to be associated with changes in AA during adolescence. Further research into epigenetic aging, including the use of causal inference methods, is required to better our understanding of aging.

Longitudinal analysis of DNA methylation associated with birth weight and gestational age

Gestational age (GA) and birth weight have been implicated in the determination of long-term health. It has been hypothesized that changes in DNA methylation may mediate these long-term effects. We obtained DNA methylation profiles from cord blood and peripheral blood at ages 7 and 17 in the same children from the Avon Longitudinal Study of Parents and Children. Repeated-measures data were used to investigate changes in birth-related methylation during childhood and adolescence. Ten developmental phenotypes (e.g. height) were analysed to identify possible mediation of health effects by DNA methylation. In cord blood, methylation at 224 CpG sites was found to be associated with GA and 23 CpG sites with birth weight. Methylation changed in the majority of these sites over time, but neither birth characteristic was strongly associated with methylation at age 7 or 17 (using a conservative correction for multiple testing of P < 1.03 × 10–7), suggesting resolution of differential methylation by early childhood. Associations were observed between birth weight-associated CpG sites and phenotypic characteristics in childhood. One strong association involved birth weight, methylation of a CpG site proximal to the NFIX locus and bone mineral density at age 17. Analysis of serial methylation from birth to adolescence provided evidence for a lack of persistence of methylation differences beyond early childhood. Sites associated with birth weight were linked to developmental genes and have methylation levels which are associated with developmental phenotypes. Replication and interrogation of causal relationships are needed to substantiate whether methylation differences at birth influence the association between birth weight and development.

Overlapping signatures of chronic pain in the DNA methylation landscape of prefrontal cortex and peripheral T cells.

We tested the hypothesis that epigenetic mechanisms in the brain and the immune system are associated with chronic pain. Genome-wide DNA methylation assessed in 9 months post nerve-injury (SNI) and Sham rats, in the prefrontal cortex (PFC) as well as in T cells revealed a vast difference in the DNA methylation landscape in the brain between the groups and a remarkable overlap (72%) between differentially methylated probes in T cells and prefrontal cortex. DNA methylation states in the PFC showed robust correlation with pain score of animals in several genes involved in pain. Finally, only 11 differentially methylated probes in T cells were sufficient to distinguish SNI or Sham individual rats. This study supports the plausibility of DNA methylation involvement in chronic pain and demonstrates the potential feasibility of DNA methylation markers in T cells as noninvasive biomarkers of chronic pain susceptibility.

Human leukocyte telomere length is associated with DNA methylation levels in multiple subtelomeric and imprinted loci.

In humans, leukocyte telomere length (LTL) is positively correlated with lifespan, and shorter LTL is associated with increased risk of age-related disease. In this study we tested for association between telomere length and methylated cytosine levels. Measurements of mean telomere length and DNA methylation at >450,000 CpG sites were obtained for both blood (N = 24) and EBV-transformed cell-line (N = 36) DNA samples from men aged 44–45 years. We identified 65 gene promoters enriched for CpG sites at which methylation levels are associated with leukocyte telomere length, and 36 gene promoters enriched for CpG sites at which methylation levels are associated with telomere length in DNA from EBV-transformed cell-lines. We observed significant enrichment of positively associated methylated CpG sites in subtelomeric loci (within 4u2005Mb of the telomere) (P < 0.01), and also at loci in imprinted regions (P < 0.001). Our results pave the way for further investigations to help elucidate the relationships between telomere length, DNA methylation and gene expression in health and disease.

Stilbenoids remodel the DNA methylation patterns in breast cancer cells and inhibit oncogenic NOTCH signaling through epigenetic regulation of MAML2 transcriptional activity

Summary We established the role for epigenetics in regulation of NOTCH signaling in breast cancer. This may constitute a common mechanism of activation of oncogenic signals. Our study provides support for epigenetic-targeting strategies in anticancer approaches.

DNA methylation and substance-use risk: a prospective, genome-wide study spanning gestation to adolescence

Epigenetic processes have been implicated in addiction; yet, it remains unclear whether these represent a risk factor and/or a consequence of substance use. Here, we believe we conducted the first genome-wide, longitudinal study to investigate whether DNA methylation patterns in early life prospectively associate with substance use in adolescence. The sample comprised of 244 youth (51% female) from the Avon Longitudinal Study of Parents and Children (ALSPAC), with repeated assessments of DNA methylation (Illumina 450k array; cord blood at birth, whole blood at age 7) and substance use (tobacco, alcohol and cannabis use; age 14–18). We found that, at birth, epigenetic variation across a tightly interconnected genetic network (n=65 loci; q<0.05) associated with greater levels of substance use during adolescence, as well as an earlier age of onset amongst users. Associations were specific to the neonatal period and not observed at age 7. Key annotated genes included PACSIN1, NEUROD4 and NTRK2, implicated in neurodevelopmental processes. Several of the identified loci were associated with known methylation quantitative trait loci, and consequently likely to be under significant genetic control. Collectively, these 65 loci were also found to partially mediate the effect of prenatal maternal tobacco smoking on adolescent substance use. Together, findings lend novel insights into epigenetic correlates of substance use, highlight birth as a potentially sensitive window of biological vulnerability and provide preliminary evidence of an indirect epigenetic pathway linking prenatal tobacco exposure and adolescent substance use.

Maternal BMI at the start of pregnancy and offspring epigenome-wide DNA methylation: findings from the pregnancy and childhood epigenetics (PACE) consortium

&NA; Pre‐pregnancy maternal obesity is associated with adverse offspring outcomes at birth and later in life. Individual studies have shown that epigenetic modifications such as DNA methylation could contribute. Within the Pregnancy and Childhood Epigenetics (PACE) Consortium, we meta‐analysed the association between pre‐pregnancy maternal BMI and methylation at over 450,000 sites in newborn blood DNA, across 19 cohorts (9,340 mother‐newborn pairs). We attempted to infer causality by comparing the effects of maternal versus paternal BMI and incorporating genetic variation. In four additional cohorts (1,817 mother‐child pairs), we meta‐analysed the association between maternal BMI at the start of pregnancy and blood methylation in adolescents. In newborns, maternal BMI was associated with small (<0.2% per BMI unit (1 kg/m2), P < 1.06 × 10‐7) methylation variation at 9,044 sites throughout the genome. Adjustment for estimated cell proportions greatly attenuated the number of significant CpGs to 104, including 86 sites common to the unadjusted model. At 72/86 sites, the direction of the association was the same in newborns and adolescents, suggesting persistence of signals. However, we found evidence for acausal intrauterine effect of maternal BMI on newborn methylation at just 8/86 sites. In conclusion, this well‐powered analysis identified robust associations between maternal adiposity and variations in newborn blood DNA methylation, but these small effects may be better explained by genetic or lifestyle factors than a causal intrauterine mechanism. This highlights the need for large‐scale collaborative approaches and the application of causal inference techniques in epigenetic epidemiology.