A meta-analysis of pre-pregnancy maternal body mass index and placental DNA methylation identifies 27 CpG sites with implications for mother-child health.

Higher maternal pre-pregnancy body mass index (ppBMI) is associated with increased neonatal morbidity, as well as with pregnancy complications and metabolic outcomes in offspring later in life. The placenta is a key organ in fetal development and has been proposed to act as a mediator between the mother and different health outcomes in children. The overall aim of the present work is to investigate the association of ppBMI with epigenome-wide placental DNA methylation (DNAm) in 10 studies from the PACE consortium, amounting to 2631 mother-child pairs. We identify 27 CpG sites at which we observe placental DNAm variations of up to 2.0% per 10 ppBMI-unit. The CpGs that are differentially methylated in placenta do not overlap with CpGs identified in previous studies in cord blood DNAm related to ppBMI. Many of the identified CpGs are located in open sea regions, are often close to obesity-related genes such as GPX1 and LGR4 and altogether, are enriched in cancer and oxidative stress pathways. Our findings suggest that placental DNAm could be one of the mechanisms by which maternal obesity is associated with metabolic health outcomes in newborns and children, although further studies will be needed in order to corroborate these findings.

Wilson Disease: Intersecting DNA Methylation and Histone Acetylation Regulation of Gene Expression in a Mouse Model of Hepatic Copper Accumulation.

BACKGROUND & AIMS: The pathogenesis of Wilson disease (WD) involves hepatic and brain copper accumulation resulting from pathogenic variants affecting the ATP7B gene and downstream epigenetic and metabolic mechanisms. Prior methylome investigations in human WD liver and blood and in the Jackson Laboratory (Bar Harbor, ME) C3He-Atp7btx-j/J (tx-j) WD mouse model revealed an epigenetic signature of WD, including changes in histone deacetylase (HDAC) 5. We tested the hypothesis that histone acetylation is altered with respect to copper overload and aberrant DNA methylation in WD. METHODS: We investigated class IIa HDAC4 and HDAC5 and H3K9/H3K27 histone acetylation in tx-j mouse livers compared with C3HeB/FeJ (C3H) control in response to 3 treatments: 60% kcal fat diet, D-penicillamine (copper chelator), and choline (methyl group donor). Experiments with copper-loaded hepatoma G2 cells were conducted to validate in vivo studies. RESULTS: In 9-week tx-j mice, HDAC5 levels increased significantly after 8 days of a 60% kcal fat diet compared with chow. In 24-week tx-j mice, HDAC4/5 levels were reduced 5- to 10-fold compared with C3H, likely through mechanisms involving HDAC phosphorylation. HDAC4/5 levels were affected by disease progression and accompanied by increased acetylation. D-penicillamine and choline partially restored HDAC4/5 and H3K9ac/H3K27ac to C3H levels. Integrated RNA and chromatin immunoprecipitation sequencing analyses revealed genes regulating energy metabolism and cellular stress/development, which, in turn, were regulated by histone acetylation in tx-j mice compared with C3H mice, with Pparα and Pparγ among the most relevant targets. CONCLUSIONS: These results suggest dietary modulation of class IIa HDAC4/5, and subsequent H3K9/H3K27 acetylation/deacetylation can regulate gene expression in key metabolic pathways in the pathogenesis of WD.

Stable DNMT3L overexpression in SH-SY5Y neurons recreates a facet of the genome-wide Down syndrome DNA methylation signature.

BACKGROUND: Down syndrome (DS) is characterized by a genome-wide profile of differential DNA methylation that is skewed towards hypermethylation in most tissues, including brain, and includes pan-tissue differential methylation. The molecular mechanisms involve the overexpression of genes related to DNA methylation on chromosome 21. Here, we stably overexpressed the chromosome 21 gene DNA methyltransferase 3L (DNMT3L) in the human SH-SY5Y neuroblastoma cell line and assayed DNA methylation at over 26 million CpGs by whole genome bisulfite sequencing (WGBS) at three different developmental phases (undifferentiated, differentiating, and differentiated). RESULTS: DNMT3L overexpression resulted in global CpG and CpG island hypermethylation as well as thousands of differentially methylated regions (DMRs). The DNMT3L DMRs were skewed towards hypermethylation and mapped to genes involved in neurodevelopment, cellular signaling, and gene regulation. Consensus DNMT3L DMRs showed that cell lines clustered by genotype and then differentiation phase, demonstrating sets of common genes affected across neuronal differentiation. The hypermethylated DNMT3L DMRs from all pairwise comparisons were enriched for regions of bivalent chromatin marked by H3K4me3 as well as differentially methylated sites from previous DS studies of diverse tissues. In contrast, the hypomethylated DNMT3L DMRs from all pairwise comparisons displayed a tissue-specific profile enriched for regions of heterochromatin marked by H3K9me3 during embryonic development. CONCLUSIONS: Taken together, these results support a mechanism whereby regions of bivalent chromatin that lose H3K4me3 during neuronal differentiation are targeted by excess DNMT3L and become hypermethylated. Overall, these findings demonstrate that DNMT3L overexpression during neurodevelopment recreates a facet of the genome-wide DS DNA methylation signature by targeting known genes and gene clusters that display pan-tissue differential methylation in DS.

Low-pass whole genome bisulfite sequencing of neonatal dried blood spots identifies a role for RUNX1 in Down syndrome DNA methylation profiles.

Neonatal dried blood spots (NDBS) are a widely banked sample source that enables retrospective investigation into early life molecular events. Here, we performed low-pass whole genome bisulfite sequencing (WGBS) of 86 NDBS DNA to examine early life Down syndrome (DS) DNA methylation profiles. DS represents an example of genetics shaping epigenetics, as multiple array-based studies have demonstrated that trisomy 21 is characterized by genome-wide alterations to DNA methylation. By assaying over 24 million CpG sites, thousands of genome-wide significant (q < 0.05) differentially methylated regions (DMRs) that distinguished DS from typical development and idiopathic developmental delay were identified. Machine learning feature selection refined these DMRs to 22 loci. The DS DMRs mapped to genes involved in neurodevelopment, metabolism, and transcriptional regulation. Based on comparisons with previous DS methylation studies and reference epigenomes, the hypermethylated DS DMRs were significantly (q < 0.05) enriched across tissues while the hypomethylated DS DMRs were significantly (q < 0.05) enriched for blood-specific chromatin states. A ~28 kb block of hypermethylation was observed on chromosome 21 in the RUNX1 locus, which encodes a hematopoietic transcription factor whose binding motif was the most significantly enriched (q < 0.05) overall and specifically within the hypomethylated DMRs. Finally, we also identified DMRs that distinguished DS NDBS based on the presence or absence of congenital heart disease (CHD). Together, these results not only demonstrate the utility of low-pass WGBS on NDBS samples for epigenome-wide association studies, but also provide new insights into the early life mechanisms of epigenomic dysregulation resulting from trisomy 21.

mtDNA depletion-like syndrome in Wilson disease.

BACKGROUND AND AIMS: Wilson disease (WD) is caused by mutations in the copper transporter ATP7B, with its main pathology attributed to copper-mediated oxidative damage. The limited therapeutic effect of copper chelators and the early occurrence of mitochondrial deficits, however, undermine the prevalence of this mechanism. METHODS: We characterized mitochondrial DNA copy number and mutations as well as bioenergetic deficits in blood from patients with WD and in livers of tx-j mice, a mouse model of hepatic copper accumulation. In vitro experiments with hepatocytes treated with CuSO4 were conducted to validate in vivo studies. RESULTS: Here, for the first time, we characterized the bioenergetic deficits in WD as consistent with a mitochondrial DNA depletion-like syndrome. This is evidenced by enriched DNA synthesis/replication pathways in serum metabolomics and decreased mitochondrial DNA copy number in blood of WD patients as well as decreased mitochondrial DNA copy number, increased citrate synthase activity, and selective Complex IV deficit in livers of the tx-j mouse model of WD. Tx-j mice treated with the copper chelator penicillamine, methyl donor choline or both ameliorated mitochondrial DNA damage but further decreased mitochondrial DNA copy number. Experiments with copper-loaded HepG2 cells validated the concept of a direct copper-mitochondrial DNA interaction. CONCLUSIONS: This study underlines the relevance of targeting the copper-mitochondrial DNA pool in the treatment of WD separate from the established copper-induced oxidative stress-mediated damage.

Integrated analysis of a compendium of RNA-Seq datasets for splicing factors.

A vast amount of public RNA-sequencing datasets have been generated and used widely to study transcriptome mechanisms. These data offer precious opportunity for advancing biological research in transcriptome studies such as alternative splicing. We report the first large-scale integrated analysis of RNA-Seq data of splicing factors for systematically identifying key factors in diseases and biological processes. We analyzed 1,321 RNA-Seq libraries of various mouse tissues and cell lines, comprising more than 6.6 TB sequences from 75 independent studies that experimentally manipulated 56 splicing factors. Using these data, RNA splicing signatures and gene expression signatures were computed, and signature comparison analysis identified a list of key splicing factors in Rett syndrome and cold-induced thermogenesis. We show that cold-induced RNA-binding proteins rescue the neurite outgrowth defects in Rett syndrome using neuronal morphology analysis, and we also reveal that SRSF1 and PTBP1 are required for energy expenditure in adipocytes using metabolic flux analysis. Our study provides an integrated analysis for identifying key factors in diseases and biological processes and highlights the importance of public data resources for identifying hypotheses for experimental testing.

Genetic variants drive altered epigenetic regulation of endotoxin response in BTBR macrophages.

The BTBR T+Itpr3tf/J (BTBR) mouse has been used as a complex genetic model of Autism Spectrum Disorders (ASD). While the specific mechanisms underlying BTBR behavioral phenotypes are poorly understood, prior studies have implicated profound differences in innate immune system control of pro-inflammatory cytokines. Innate immune activation and elevated pro-inflammatory cytokines are also detected in blood of children with ASD. In this study, we examined how underlying BTBR genetic variants correspond to strain-specific changes in chromatin accessibility, resulting in a pro-inflammatory response specifically in BTBR bone marrow derived macrophages (BMDM). In response to repeated lipopolysaccharide (LPS) treatments, C57BL/6J (C57) BMDM exhibited intact endotoxin tolerance. In contrast, BTBR BMDM exhibited hyper-responsive expression of genes that were normally tolerized in C57. This failure in formation of endotoxin tolerance in BTBR was mirrored at the level of chromatin accessibility. Using ATAC-seq, we specifically identified promoter and enhancer regions with strain-specific differential chromatin accessibility both at baseline and in response to LPS. Regions with strain-specific differences in chromatin accessibility were significantly enriched for BTBR genetic variants, such that an average of 22% of the differential chromatin regions had at least one variant. Together, these results demonstrate that BTBR genetic variants contribute to altered chromatin responsiveness to endotoxin challenge resulting in hyper-responsive innate immunity in BTBR. These findings provide evidence for an interaction between complex genetic variants and differential epigenetic regulation of innate immune responses.

MeCP2 isoform e1 mutant mice recapitulate motor and metabolic phenotypes of Rett syndrome.

Mutations in the X-linked gene MECP2 cause the majority of Rett syndrome (RTT) cases. Two differentially spliced isoforms of exons 1 and 2 (MeCP2-e1 and MeCP2-e2) contribute to the diverse functions of MeCP2, but only mutations in exon 1, not exon 2, are observed in RTT. We previously described an isoform-specific MeCP2-e1-deficient male mouse model of a human RTT mutation that lacks MeCP2-e1 while preserving expression of MeCP2-e2. However, RTT patients are heterozygous females that exhibit delayed and progressive symptom onset beginning in late infancy, including neurologic as well as metabolic, immune, respiratory and gastrointestinal phenotypes. Consequently, we conducted a longitudinal assessment of symptom development in MeCP2-e1 mutant females and males. A delayed and progressive onset of motor impairments was observed in both female and male MeCP2-e1 mutant mice, including hind limb clasping and motor deficits in gait and balance. Because these motor impairments were significantly impacted by age-dependent increases in body weight, we also investigated metabolic phenotypes at an early stage of disease progression. Both male and female MeCP2-e1 mutants exhibited significantly increased body fat compared to sex-matched wild-type littermates prior to weight differences. Mecp2e1-/y males exhibited significant metabolic phenotypes of hypoactivity, decreased energy expenditure, increased respiratory exchange ratio, but decreased food intake compared to wild-type. Untargeted analysis of lipid metabolites demonstrated a distinguishable profile in MeCP2-e1 female mutant liver characterized by increased triglycerides. Together, these results demonstrate that MeCP2-e1 mutation in mice of both sexes recapitulates early and progressive metabolic and motor phenotypes of human RTT.

Epigenetic changes of the thioredoxin system in the tx-j mouse model and in patients with Wilson disease.

Wilson disease (WD) is caused by mutations in the copper transporter ATP7B, leading to copper accumulation in the liver and brain. Excess copper inhibits S-adenosyl-L-homocysteine hydrolase, leading to variable WD phenotypes from widespread alterations in DNA methylation and gene expression. Previously, we demonstrated that maternal choline supplementation in the Jackson toxic milk (tx-j) mouse model of WD corrected higher thioredoxin 1 (TNX1) transcript levels in fetal liver. Here, we investigated the effect of maternal choline supplementation on genome-wide DNA methylation patterns in tx-j fetal liver by whole-genome bisulfite sequencing (WGBS). Tx-j Atp7b genotype-dependent differences in DNA methylation were corrected by choline for genes including, but not exclusive to, oxidative stress pathways. To examine phenotypic effects of postnatal choline supplementation, tx-j mice were randomized to one of six treatment groups: with or without maternal and/or continued choline supplementation, and with or without copper chelation with penicillamine (PCA) treatment. Hepatic transcript levels of TXN1 and peroxiredoxin 1 (Prdx1) were significantly higher in mice receiving maternal and continued choline with or without PCA treatment compared to untreated mice. A WGBS comparison of human WD liver and tx-j mouse liver demonstrated a significant overlap of differentially methylated genes associated with ATP7B deficiency. Further, eight genes in the thioredoxin (TXN) pathway were differentially methylated in human WD liver samples. In summary, Atp7b deficiency and choline supplementation have a genome-wide impact, including on TXN system-related genes, in tx-j mice. These findings could explain the variability of WD phenotype and suggest new complementary treatment options for WD.

UBE3A-mediated regulation of imprinted genes and epigenome-wide marks in human neurons.

The dysregulation of genes in neurodevelopmental disorders that lead to social and cognitive phenotypes is a complex, multilayered process involving both genetics and epigenetics. Parent-of-origin effects of deletion and duplication of the 15q11-q13 locus leading to Angelman, Prader-Willi, and Dup15q syndromes are due to imprinted genes, including UBE3A, which is maternally expressed exclusively in neurons. UBE3A encodes a ubiquitin E3 ligase protein with multiple downstream targets, including RING1B, which in turn monoubiquitinates histone variant H2A.Z. To understand the impact of neuronal UBE3A levels on epigenome-wide marks of DNA methylation, histone variant H2A.Z positioning, active H3K4me3 promoter marks, and gene expression, we took a multi-layered genomics approach. We performed an siRNA knockdown of UBE3A in two human neuroblastoma cell lines, including parental SH-SY5Y and the SH(15M) model of Dup15q. Genes differentially methylated across cells with differing UBE3A levels were enriched for functions in gene regulation, DNA binding, and brain morphology. Importantly, we found that altering UBE3A levels had a profound epigenetic effect on the methylation levels of up to half of known imprinted genes. Genes with differential H2A.Z peaks in SH(15M) compared to SH-SY5Y were enriched for ubiquitin and protease functions and associated with autism, hypoactivity, and energy expenditure. Together, these results support a genome-wide epigenetic consequence of altered UBE3A levels in neurons and suggest that UBE3A regulates an imprinted gene network involving DNA methylation patterning and H2A.Z deposition.

Autism and Cancer Share Risk Genes, Pathways, and Drug Targets.

Autism is a neurodevelopmental disorder, diagnosed behaviorally by social and communication deficits, repetitive behaviors, and restricted interests. Recent genome-wide exome sequencing has revealed extensive overlap in risk genes for autism and for cancer. Understanding the genetic commonalities of autism(s) and cancer(s), with a focus on mechanistic pathways, could lead to repurposed therapeutics.

Self-reported pregnancy exposures and placental DNA methylation in the MARBLES prospective autism sibling study.

Human placenta is a fetal-derived tissue that offers a unique sample of epigenetic and environmental exposures present in utero. In the MARBLES prospective pregnancy study of high-risk younger siblings of children with autism spectrum disorder (ASD), pregnancy and environmental factors collected by maternal interviews were examined as predictors of placental DNA methylation, including partially methylated domains (PMDs), an embryonic feature of the placental methylome. DNA methylation data from MethylC-seq analysis of 47 placentas of children clinically diagnosed at 3 years with ASD or typical development using standardized assessments were examined in relation to: child's gestational age, birth-weight, and diagnosis; maternal pre-pregnancy body mass index, smoking, education, parity, height, prenatal vitamin and folate intake; home ownership; pesticides professionally applied to lawns or gardens or inside homes, pet flea/tick pouches, collars, or soaps/shampoos used in the 3 months prior to or during pregnancy. Sequencing run, order, and coverage, and child race and sex were considered as potential confounders. Akaike information criterion was used to select the most parsimonious among candidate models. Final prediction models used sandwich estimators to produce homoscadisticity-robust estimates of the 95% confidence interval (CI) and P-values controlled the false discovery rate at 5%. The strongest, most robust associations were between pesticides professionally applied outside the home and higher average methylation over PMDs [0.45 (95% CI 0.17, 0.72), P = 0.03] and a reduced proportion of the genome in PMDs [-0.42 (95% CI - 0.67 to -0.17), P = 0.03]. Pesticide exposures could alter placental DNA methylation more than other factors.

Chimeric MicroRNA-1291 Biosynthesized Efficiently in Escherichia coli Is Effective to Reduce Target Gene Expression in Human Carcinoma Cells and Improve Chemosensitivity.

In contrast to the growing interests in studying noncoding RNAs (ncRNAs) such as microRNA (miRNA or miR) pharmacoepigenetics, there is a lack of efficient means to cost effectively produce large quantities of natural miRNA agents. Our recent efforts led to a successful production of chimeric pre-miR-27b in bacteria using a transfer RNA (tRNA)-based recombinant RNA technology, but at very low expression levels. Herein, we present a high-yield expression of chimeric pre-miR-1291 in common Escherichia coli strains using the same tRNA scaffold. The tRNA fusion pre-miR-1291 (tRNA/mir-1291) was then purified to high homogeneity using affinity chromatography, whose primary sequence and post-transcriptional modifications were directly characterized by mass spectrometric analyses. Chimeric tRNA/mir-1291 was readily processed to mature miR-1291 in human carcinoma MCF-7 and PANC-1 cells. Consequently, recombinant tRNA/mir-1291 reduced the protein levels of miR-1291 target genes, including ABCC1, FOXA2, and MeCP2, as compared with cells transfected with the same doses of control methionyl-tRNA scaffold with a sephadex aptamer (tRNA/MSA). In addition, tRNA-carried pre-miR-1291 suppressed the growth of MCF-7 and PANC-1 cells in a dose-dependent manner, and significantly enhanced the sensitivity of ABCC1-overexpressing PANC-1 cells to doxorubicin. These results indicate that recombinant miR-1291 agent is effective in the modulation of target gene expression and chemosensitivity, which may provide insights into high-yield bioengineering of new ncRNA agents for pharmacoepigenetics research.

Characterization of timed changes in hepatic copper concentrations, methionine metabolism, gene expression, and global DNA methylation in the Jackson toxic milk mouse model of Wilson disease.

BACKGROUND: Wilson disease (WD) is characterized by hepatic copper accumulation with progressive liver damage to cirrhosis. This study aimed to characterize the toxic milk mouse from The Jackson Laboratory (Bar Harbor, ME, USA) (tx-j) mouse model of WD according to changes over time in hepatic copper concentrations, methionine metabolism, global DNA methylation, and gene expression from gestational day 17 (fetal) to adulthood (28 weeks). METHODS: Included liver histology and relevant biochemical analyses including hepatic copper quantification, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) liver levels, qPCR for transcript levels of genes relevant to methionine metabolism and liver damage, and DNA dot blot for global DNA methylation. RESULTS: Hepatic copper was lower in tx-j fetuses but higher in weanling (three weeks) and adult tx-j mice compared to controls. S-adenosylhomocysteinase transcript levels were significantly lower at all time points, except at three weeks, correlating negatively with copper levels and with consequent changes in the SAM:SAH methylation ratio and global DNA methylation. CONCLUSION: Compared to controls, methionine metabolism including S-adenosylhomocysteinase gene expression is persistently different in the tx-j mice with consequent alterations in global DNA methylation in more advanced stages of liver disease. The inhibitory effect of copper accumulation on S-adenosylhomocysteinase expression is associated with progressively abnormal methionine metabolism and decreased methylation capacity and DNA global methylation.

Methylation and gene expression responses to ethanol feeding and betaine supplementation in the cystathionine beta synthase-deficient mouse.

BACKGROUND: Alcoholic steatohepatitis (ASH) is caused in part by the effects of ethanol (EtOH) on hepatic methionine metabolism. METHODS: To investigate the phenotypic and epigenetic consequences of altered methionine metabolism in this disease, we studied the effects of 4-week intragastric EtOH feeding with and without the methyl donor betaine in cystathionine beta synthase (CßS) heterozygous C57BL/6J mice. RESULTS: The histopathology of early ASH was induced by EtOH feeding and prevented by betaine supplementation, while EtOH feeding reduced and betaine supplementation maintained the hepatic methylation ratio of the universal methyl donor S-adenosylmethionine (SAM) to the methyltransferase inhibitor S-adenosylhomocysteine (SAH). MethylC-seq genomic sequencing of heterozygous liver samples from each diet group found 2 to 4% reduced methylation in gene bodies, but not promoter regions of all autosomes of EtOH-fed mice, each of which were normalized in samples from mice fed the betaine-supplemented diet. The transcript levels of nitric oxide synthase (Nos2) and DNA methyltransferase 1 (Dnmt1) were increased, while those of peroxisome proliferator receptor-α (Pparα) were reduced in EtOH-fed mice, and each was normalized in mice fed the betaine-supplemented diet. DNA pyrosequencing of CßS heterozygous samples found reduced methylation in a gene body of Nos2 by EtOH feeding that was restored by betaine supplementation and was correlated inversely with its expression and positively with SAM/SAH ratios. CONCLUSIONS: The present study has demonstrated relationships among EtOH induction of ASH with aberrant methionine metabolism that was associated with gene body DNA hypomethylation in all autosomes and was prevented by betaine supplementation. The data imply that EtOH-induced changes in selected gene transcript levels and hypomethylation in gene bodies during the induction of ASH are a result of altered methionine metabolism that can be reversed through dietary supplementation of methyl donors.

Maternal choline modifies fetal liver copper, gene expression, DNA methylation, and neonatal growth in the tx-j mouse model of Wilson disease.

Maternal diet can affect fetal gene expression through epigenetic mechanisms. Wilson disease (WD), which is caused by autosomal recessive mutations in ATP7B encoding a biliary copper transporter, is characterized by excessive hepatic copper accumulation, but variability in disease severity. We tested the hypothesis that gestational supply of dietary methyl groups modifies fetal DNA methylation and expression of genes involved in methionine and lipid metabolism that are impaired prior to hepatic steatosis in the toxic milk (tx-j) mouse model of WD. Female C3H control and tx-j mice were fed control (choline 8 mmol/Kg of diet) or choline-supplemented (choline 36 mmol/Kg of diet) diets for 2 weeks throughout mating and pregnancy to gestation day 17. A second group of C3H females, half of which were used to cross foster tx-j pups, received the same diet treatments that extended during lactation to 21 d postpartum. Compared with C3H, fetal tx-j livers had significantly lower copper concentrations and significantly lower transcript levels of Cyclin D1 and genes related to methionine and lipid metabolism. Maternal choline supplementation prevented the transcriptional deficits in fetal tx-j liver for multiple genes related to cell growth and metabolism. Global DNA methylation was increased by 17% in tx-j fetal livers after maternal choline treatment (P<0.05). Maternal dietary choline rescued the lower body weight of 21 d tx-j mice. Our results suggest that WD pathogenesis is modified by maternal in utero factors, including dietary choline.

How has the study of the human placenta aided our understanding of partially methylated genes?

While the human genome sequence is relatively uniform between the cells of an individual, the DNA methylation of the genome (methylome) has unique features in different cells, tissues and stages of development. Recent genome-wide sequencing of the methylome has revealed large partially methylated domains (PMDs) in the human placenta. Unlike CpG islands and Polycomb-regulated regions, which can also have low levels of methylation, placental PMDs cover approximately 37% of the human genome and are associated with inaccessible chromatin and the repression of tissue-specific genes. Here, we summarize the interesting biological questions that have arisen as a result of finding PMDs in the human placenta, including how PMDs form, what they do, how they evolved and how they might be relevant to human disease.

R-loop formation at Snord116 mediates topotecan inhibition of Ube3a-antisense and allele-specific chromatin decondensation.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are oppositely imprinted autism-spectrum disorders with known genetic bases, but complex epigenetic mechanisms underlie their pathogenesis. The PWS/AS locus on 15q11-q13 is regulated by an imprinting control region that is maternally methylated and silenced. The PWS imprinting control region is the promoter for a one megabase paternal transcript encoding the ubiquitous protein-coding Snrpn gene and multiple neuron-specific noncoding RNAs, including the PWS-related Snord116 repetitive locus of small nucleolar RNAs and host genes, and the antisense transcript to AS-causing ubiquitin ligase encoding Ube3a (Ube3a-ATS). Neuron-specific transcriptional progression through Ube3a-ATS correlates with paternal Ube3a silencing and chromatin decondensation. Interestingly, topoisomerase inhibitors, including topotecan, were recently identified in an unbiased drug screen for compounds that could reverse the silent paternal allele of Ube3a in neurons, but the mechanism of topotecan action on the PWS/AS locus is unknown. Here, we demonstrate that topotecan treatment stabilizes the formation of RNA:DNA hybrids (R loops) at G-skewed repeat elements within paternal Snord116, corresponding to increased chromatin decondensation and inhibition of Ube3a-ATS expression. Neural precursor cells from paternal Snord116 deletion mice exhibit increased Ube3a-ATS levels in differentiated neurons and show a reduced effect of topotecan compared with wild-type neurons. These results demonstrate that the AS candidate drug topotecan acts predominantly through stabilizing R loops and chromatin decondensation at the paternally expressed PWS Snord116 locus. Our study holds promise for targeted therapies to the Snord116 locus for both AS and PWS.

The human placenta methylome.

Tissue-specific DNA methylation is found at promoters, enhancers, and CpG islands but also over larger genomic regions. In most human tissues, the vast majority of the genome is highly methylated (>70%). Recently, sequencing of bisulfite-treated DNA (MethylC-seq) has revealed large partially methylated domains (PMDs) in some human cell lines. PMDs cover up to 40% of the genome and are associated with gene repression and inactive chromatin marks. However, to date, only cultured cells and cancers have shown evidence for PMDs. Here, we performed MethylC-seq in full-term human placenta and demonstrate it is the first known normal tissue showing clear evidence of PMDs. We found that PMDs cover 37% of the placental genome, are stable throughout gestation and between individuals, and can be observed with lower sensitivity in Illumina 450K Infinium data. RNA-seq analysis confirmed that genes in PMDs are repressed in placenta. Using a hidden Markov model to map placental PMDs genome-wide and compare them to PMDs in other cell lines, we found that genes within placental PMDs have tissue-specific functions. For regulatory regions, methylation levels in promoter CpG islands are actually higher for genes within placental PMDs, despite the lower overall methylation of surrounding regions. Similar to PMDs, polycomb-regulated regions are hypomethylated but smaller and distinct from PMDs, with some being hypermethylated in placenta compared with other tissues. These results suggest that PMDs are a developmentally dynamic feature of the methylome that are relevant for understanding both normal development and cancer and may be of use as epigenetic biomarkers.

Wilson's disease: changes in methionine metabolism and inflammation affect global DNA methylation in early liver disease.

UNLABELLED: Hepatic methionine metabolism may play an essential role in regulating methylation status and liver injury in Wilson's disease (WD) through the inhibition of S-adenosylhomocysteine hydrolase (SAHH) by copper (Cu) and the consequent accumulation of S-adenosylhomocysteine (SAH). We studied the transcript levels of selected genes related to liver injury, levels of SAHH, SAH, DNA methyltransferases genes (Dnmt1, Dnmt3a, Dnmt3b), and global DNA methylation in the tx-j mouse (tx-j), an animal model of WD. Findings were compared to those in control C3H mice, and in response to Cu chelation by penicillamine (PCA) and dietary supplementation of the methyl donor betaine to modulate inflammatory and methylation status. Transcript levels of selected genes related to endoplasmic reticulum stress, lipid synthesis, and fatty acid oxidation were down-regulated at baseline in tx-j mice, further down-regulated in response to PCA, and showed little to no response to betaine. Hepatic Sahh transcript and protein levels were reduced in tx-j mice with consequent increase of SAH levels. Hepatic Cu accumulation was associated with inflammation, as indicated by histopathology and elevated serum alanine aminotransferase (ALT) and liver tumor necrosis factor alpha (Tnf-α) levels. Dnmt3b was down-regulated in tx-j mice together with global DNA hypomethylation. PCA treatment of tx-j mice reduced Tnf-α and ALT levels, betaine treatment increased S-adenosylmethionine and up-regulated Dnmt3b levels, and both treatments restored global DNA methylation levels. CONCLUSION: Reduced hepatic Sahh expression was associated with increased liver SAH levels in the tx-j model of WD, with consequent global DNA hypomethylation. Increased global DNA methylation was achieved by reducing inflammation by Cu chelation or by providing methyl groups. We propose that increased SAH levels and inflammation affect widespread epigenetic regulation of gene expression in WD.