Genome-wide high resolution parental-specific DNA and histone methylation maps uncover patterns of imprinting regulation in maize

Genetic imprinting is a specific epigenetic phenomenon in which a subset of genes is expressed depending on their parent-of-origin. Two types of chromatin modifications, DNA methylation and histone modification, are generally believed to be involved in the regulation of imprinting. However, the genome-wide correlation between allele-specific chromatin modifications and imprinted gene expression in maize remains elusive. Here we report genome-wide high resolution allele-specific maps of DNA methylation and histone H3 lysine 27 trimethylation (H3K27me3) in maize endosperm. For DNA methylation, thousands of parent-of-origin dependent differentially methylated regions (pDMRs) were identified. All pDMRs were uniformly paternally hypermethylated and maternally hypomethylated. We also identified 1131 allele-specific H3K27me3 peaks that are preferentially present in the maternal alleles. Maternally expressed imprinted genes (MEGs) and paternally expressed imprinted genes (PEGs) had different patterns of allele-specific DNA methylation and H3K27me3. Allele-specific expression of MEGs was not directly related to allele-specific H3K27me3, and only a subset of MEGs was associated with maternal-specific DNA demethylation, which was primarily located in the upstream and 5′ portion of gene body regions. In contrast, allele-specific expression of a majority of PEGs was related to maternal-specific H3K27me3, with a subgroup of PEGs also associated with maternal-specific DNA demethylation. Both pDMRs and maternal H3K27me3 peaks associated with PEGs are enriched in gene body regions. Our results indicate highly complex patterns of regulation on genetic imprinting in maize endosperm.Genetic imprinting is an epigenetic phenomenon, where genes are expressed in a parent-of-origin dependent manner in many plant species and mammals. Although first discovered in plants (Kermicle and Alleman 1990), research on genetic imprinting is much more advanced in mammals in terms of the number of imprinted genes identified and the understanding of their regulatory mechanisms (Koerner and Barlow 2010; Barlow 2011; Bartolomei and Ferguson-Smith 2011). Only a small number of imprinted genes were observed in plants for a long time since the phenomenon is highly specific to the triploid endosperm (Raissig et al. 2011). However, recent studies have indicated that genetic imprinting in plants is much more prevalent than previously thought, with hundreds of genes shown to be imprinted in several plant species (Gehring et al. 2011; Hsieh et al. 2011; Luo et al. 2011; Waters et al. 2011; Zhang et al. 2011). In contrast to mammals, where the regulation of genetic imprinting has been extensively studied (Koerner and Barlow 2010; Barlow 2011; Abramowitz and Bartolomei 2012), the understanding of regulation of parental imprinting in plants is highly limited.DNA methylation is one of the primary modifications reported to be associated with genetic imprinting. In Arabidopsis, DNA methylation around several maternally expressed imprinted protein-coding genes (MEG) including FWA, FIS2, and MPC was shown to be important for their maternally preferred expression, as all these genes exhibited biallelic expression in endosperm fertilized with met1 pollen (Kinoshita et al. 2004; Jullien et al. 2006; Tiwari et al. 2008). Two studies using RNA-seq in Arabidopsis also showed that a number of MEGs exhibited biallelic expression in paternal met1 endosperm, and the maternal alleles of dozens of paternally expressed imprinted genes (PEGs) were reactivated in maternal dme endosperm (Hsieh et al. 2011; Wolff et al. 2011). In maize, five confirmed endosperm MEGs (Fie1, Fie2, Mez1, Meg1, and Mee1) contain differentially methylated regions (DMRs) (Gutierrez-Marcos et al. 2004; Gutierrez-Marcos et al. 2006; Haun et al. 2007), and activation of the Fie1 maternal allele in the endosperm requires DNA demethylation of the maternal allele (Hermon et al. 2007).Another modification associated with genetic imprinting involves histone methylation. Polycomb repressive complex 2 (PRC2) is known to mediate the trimethylation of histone H3 lysine 27 (H3K27me3) (Schuettengruber and Cavalli 2009). Results on PHE1, the only well-studied PEG in plants, indicated that silencing of its maternal allele depends on a functional PRC2 complex in addition to DNA demethylation (Kohler et al. 2003, 2005; Makarevich et al. 2008). Recently, a number of MEGs and PEGs were shown to be biallelically expressed in maternal fie or fis2 endosperm (Hsieh et al. 2011; Wolff et al. 2011).Although the studies above suggest that DNA methylation and the PRC2 complex could be responsible for monoallelic expression of imprinted genes, there is not yet any general rule for the function of DNA and histone methylation on the regulation of genetic imprinting in plants. A high resolution genome-wide map of allele-specific DNA methylation and allele-specific histone modification will be crucial to gain better understanding of the regulation of genetic imprinting. Recently, several genome-wide studies have provided evidence that allele-specific patterns of DNA methylation or parent-of-origin dependent differentially methylated regions (pDMRs) are associated with some imprinted genes in mice and plants (Zhang et al. 2011; Ibarra et al. 2012; Xie et al. 2012; Rodrigues et al. 2013). Several studies in mammals also suggest a mutually exclusive relationship between allele-specific DNA methylation and histone modification or among different histone modifications (Xin et al. 2001; Fournier et al. 2002; Carr et al. 2007; Lindroth et al. 2008; Singh et al. 2010; Hon et al. 2012).Here we report a genome-wide analysis of allele-specific DMRs, H3K27me3, and imprinted gene expression in maize endosperm. Thousands of pDMRs and allele-specific H3K27me3 peaks were identified. Correlation of pDMRs, allele-specific H3K27me3 profile, and the expression of imprinted genes showed that MEGs and PEGs have different patterns of DNA methylation and H3K27me3. This study reveals complex patterns of genetic imprinting regulation in maize endosperm.     

Genome-wide nucleosome map and cytosine methylation levels of an ancient human genome

Epigenetic information is available from contemporary organisms, but is difficult to track back in evolutionary time. Here, we show that genome-wide epigenetic information can be gathered directly from next-generation sequence reads of DNA isolated from ancient remains. Using the genome sequence data generated from hair shafts of a 4000-yr-old Paleo-Eskimo belonging to the Saqqaq culture, we generate the first ancient nucleosome map coupled with a genome-wide survey of cytosine methylation levels. The validity of both nucleosome map and methylation levels were confirmed by the recovery of the expected signals at promoter regions, exon/intron boundaries, and CTCF sites. The top-scoring nucleosome calls revealed distinct DNA positioning biases, attesting to nucleotide-level accuracy. The ancient methylation levels exhibited high conservation over time, clustering closely with modern hair tissues. Using ancient methylation information, we estimated the age at death of the Saqqaq individual and illustrate how epigenetic information can be used to infer ancient gene expression. Similar epigenetic signatures were found in other fossil material, such as 110,000- to 130,000-yr-old bones, supporting the contention that ancient epigenomic information can be reconstructed from a deep past. Our findings lay the foundation for extracting epigenomic information from ancient samples, allowing shifts in epialleles to be tracked through evolutionary time, as well as providing an original window into modern epigenomics.Ancient DNA research started in the mid-1980s with the successful cloning and sequencing of a short mitochondrial DNA fragment from the quagga zebra, a species that became extinct in the early 20th century (Higuchi et al. 1984). Soon after, the invention of PCR unlocked access to this fragmented and degraded DNA material (Pääbo 1989), making it possible to amplify short gene markers of interest and compare their sequence to that from extant organisms. This illuminated a range of topics ranging from the reconstruction of the evolutionary origins of several now-extinct iconic mammals (Orlando et al. 2003; Krause et al. 2006), the evaluation of the possible role played by major past climatic changes in driving mega-fauna extinctions (Shapiro et al. 2004; Campos et al. 2010; Lorenzen et al. 2011), to the identification of the pathogens responsible for massive historical outbreaks (Taubenberger et al. 1997).However, before the advent of next-generation sequencing (NGS) platforms, the amount of ancient sequence information one had access to was limited to several tens of thousands of nucleotides at best (Noonan et al. 2005, 2006), and until very recently, sequencing whole ancient mitochondrial genomes was considered a major achievement (Cooper et al. 2001; Krause et al. 2006). Parallel sequencing of millions to billions of short DNA fragments has revolutionized ancient DNA research, and today a series of ancient genomes has been reconstructed from humans (Rasmussen et al. 2010, 2011; Keller et al. 2012; Raghavan et al. 2013), archaic hominins (Green et al. 2010; Reich et al. 2010; Meyer et al. 2012), the woolly mammoth (Miller et al. 2008), and several microbial pathogens (Bos et al. 2011; Martin et al. 2013; Schuenemann et al. 2013; Yoshida et al. 2013). Those mainly date back to recent historical periods or the Late Pleistocene, but most recently, the characterization of a 560,000- to 780,000-yr-old horse draft genome revealed that genomic information could be retrieved over much longer evolutionary time scales, probably up until the last million years (Orlando et al. 2013).Ancient genomes have provided important new insights into human evolution and dispersals (Rasmussen et al. 2010, 2011; Keller et al. 2012; Raghavan et al. 2013), revealing an admixture between contemporary human ancestors and archaic hominins (Green et al. 2010; Reich et al. 2010; Meyer et al. 2012) and multiple early human expansions into both Asia and North America (Rasmussen et al. 2010, 2011). The information gained from these samples has largely been limited to nucleotide polymorphisms. Unlike mutations, epigenetic modifications do not alter the underlying DNA sequence, but can be inherited across cell divisions and from parents to offspring and can control gene expression by reshaping cytosine methylation landscapes, nucleosome organization, and histone modification patterns. The range of biological processes that depend on some level of epigenetic regulation is diverse and includes imprinting (Bird 2002), transposition (Hollister and Gaut 2009), cell differentiation (Meissner et al. 2008), and cancer (Teschendorff et al. 2011). In this study, we use the Saqqaq genome that was retrieved from an ∼4000-yr-old tuft of hair of a Paleo-Eskimo from Greenland and sequenced to an average depth of 20× (Rasmussen et al. 2010). We demonstrate that NGS data can be used in the absence of bisulfite or further experimental treatment to directly infer genome-wide nucleosome organization and regional methylation levels, thereby providing the first survey of an ancient epigenome.     

A heterozygous IDH1R132H/WT mutation induces genome-wide alterations in DNA methylation

Monoallelic point mutations of the NADP⁺-dependent isocitrate dehydrogenases IDH1 and IDH2 occur frequently in gliomas, acute myeloid leukemias, and chondromas, and display robust association with specific DNA hypermethylation signatures. Here we show that heterozygous expression of the IDH1^R132H allele is sufficient to induce the genome-wide alterations in DNA methylation characteristic of these tumors. Using a gene-targeting approach, we knocked-in a single copy of the most frequently observed IDH1 mutation, R132H, into a human cancer cell line and profiled changes in DNA methylation at over 27,000 CpG dinucleotides relative to wild-type parental cells. We find that IDH1^R132H/WT mutation induces widespread alterations in DNA methylation, including hypermethylation of 2010 and hypomethylation of 842 CpG loci. We demonstrate that many of these alterations are consistent with those observed in IDH1-mutant and G-CIMP+ primary gliomas and can segregate IDH wild-type and mutated tumors as well as those exhibiting the G-CIMP phenotype in unsupervised analysis of two primary glioma cohorts. Further, we show that the direction of IDH1^R132H/WT-mediated DNA methylation change is largely dependent upon preexisting DNA methylation levels, resulting in depletion of moderately methylated loci. Additionally, whereas the levels of multiple histone H3 and H4 methylation modifications were globally increased, consistent with broad inhibition of histone demethylation, hypermethylation at H3K9 in particular accompanied locus-specific DNA hypermethylation at several genes down-regulated in IDH1^R132H/WT knock-in cells. These data provide insight on epigenetic alterations induced by IDH1 mutations and support a causal role for IDH1^R132H/WT mutants in driving epigenetic instability in human cancer cells.Mutations of the NADP⁺-dependent isocitrate dehydrogenase (IDH) genes IDH1 and IDH2 occur in >70% of Grade II–III gliomas and secondary glioblastomas (sGBM) (Balss et al. 2008; Parsons et al. 2008; Bleeker et al. 2009; Hartmann et al. 2009; Yan et al. 2009a,b; Gravendeel et al 2010), 15%–30% of acute myeloid leukemias (AMLs) (Mardis et al. 2009; Marcucci et al. 2010; Paschka et al. 2010; Wagner et al. 2010; Ward et al. 2010), 56% of chondrosarcomas (Amary et al. 2011), 87% of enchondromas, 70% of spindle cell hemangiomas (Pansuriya et al. 2011), 22%–28% of cholangiocarcinomas of intrahepatic origin (Borger et al. 2012; Kipp et al. 2012), and at lower frequencies in other malignancies, including colorectal cancer (Sjoblom et al. 2006), prostate carcinoma, and B-acute lymphoblastic leukemia (B-ALL) (Kang et al. 2009). IDH mutations occur early in tumor development and may either cause or predispose cells to become malignant (Ichimura et al. 2009; Watanabe et al. 2009). In human tumors, IDH mutations give rise to single amino acid substitutions at specific conserved residues, arginine 132 (R132) of IDH1 and arginine 140 (R140) or arginine 172 (R172) of IDH2. These IDH point mutations primarily occur as somatically acquired heterozygous events, with tumor cells showing one mutant allele and retention of the second wild-type allele, suggesting that the ratio of mutant to wild-type enzyme may be critical to its oncogenic activity. The IDH mutation leads to an attenuation of the normal catalytic activity, the oxidative decarboxylation of isocitrate to alpha-ketoglutarate (α-KG) (Yan et al. 2009b; Zhao et al. 2009), while concurrently imparting a gain of novel enzymatic function wherein α-KG is reduced, leading to the aberrant accumulation of the onco-metabolite D-2-hydroxyglutarate (D-2-HG) (Dang et al. 2009). Both reduction of cellular α-KG levels and accumulation of D-2-HG have the potential to contribute to altered cellular phenotypes through the inhibition of multiple Fe(II)/2-oxoglutarate-dependent dioxygenases (W Xu et al. 2011), a superfamily of enzymes involved in a wide range of biological functions, including DNA repair and chromatin modification, such as the AlkB family of oxidative demethylases, the Jumonji-C domain family of histone demethylases (JHDMs), and the TET family of methylcytosine hydroxylases (Loenarz and Schofield 2008; Tahiliani et al. 2009; Figueroa et al. 2010a; Chowdhury et al. 2011).Recent large-scale studies of DNA methylation distribution in primary tumors have identified biologically distinct subgroups of glioblastomas (GBMs) and AMLs associated with aberrant DNA methylation (Figueroa et al. 2010b; Noushmehr et al. 2010). A subset of primary GBMs exhibit the “CpG island methylator phenotype” (CIMP) and show concordant hypermethylation of a large number of CpG islands (Toyota and Issa 1999; Toyota et al. 1999; Noushmehr et al. 2010). Interestingly, in gliomas, the CIMP phenotype (termed G-CIMP) has a striking association with IDH1 mutation (Noushmehr et al. 2010; Christensen et al. 2011; Laffaire et al. 2011; Turcan et al. 2012). Similarly, IDH1 and IDH2 mutations robustly associate with specific global DNA hypermethylation phenotypes in AMLs (Figueroa et al. 2010a), enchondromas (Pansuriya et al. 2011), and low-grade gliomas (LGGs) (Turcan et al. 2012). The evidence linking IDH1 and IDH2 mutations with distinct DNA methylation phenotypes in primary human tumors raises the question of whether these mutations can drive oncogenesis through epigenetic reprogramming of cancer cells. Such epigenetic changes, including DNA hypermethylation and hypomethylation, can play fundamental roles in the initiation and progression of human cancer through regulation of gene expression (Jones and Baylin 2002, 2007). In addition to epigenetic classifications, gene expression-based molecular classification systems have been developed in GBM to distinguish clinically relevant molecular subclasses (Phillips et al. 2006; Li et al. 2009; Verhaak et al. 2010). GBMs with IDH1 mutations were shown to primarily exhibit a proneural gene expression signature (Verhaak et al. 2010), although specific gene-expression alterations resulting from mutant IDH1 proteins have not been elucidated.Despite the apparent correlation between IDH mutations and CpG island hypermethylation, the contribution of heterozygous expression of IDH1 point mutations to this phenotype has not been addressed. One limitation of the field has been the lack of model systems that recapitulate naturally occurring monoallelic point mutations observed in human tumors. Previous studies have relied on ectopic overexpression in human and mouse cell lines to study the effects of mutant IDH proteins (Dang et al. 2009; Yan et al. 2009b; Figueroa et al. 2010a; Ward et al. 2010; G Jin et al. 2011; Reitman et al. 2011; Lu et al. 2012; Turcan et al. 2012). To address this issue, and to faithfully recapitulate the naturally occurring genetic alterations, we utilized gene targeting to introduce heterozygous IDH1^R132H/WT substitutions in the human colorectal cancer cell line, HCT116. We determined the impact of this mutation on the genome-wide distribution of CpG methylation in the isogenic IDH1^R132H/WT cells and identified differentially methylated CpGs induced by the IDH1^R132H/WT mutation, which include both hyper- and hypomethylation events. Our data indicate that IDH1^R132H/WT mutations play a causal role in the widespread alteration of DNA and histone methylation observed in human cancers and can impact gene expression.     

H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type–independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors.Genomic DNA methylation is known to change during lifetime and aging (Jaenisch and Bird 2003). Some changes play important roles in development, but others occur stochastically without any apparent biological purpose (Fraga 2009; Feil and Fraga 2012). These molecular alterations, which are known as epigenetic drift, are currently being investigated as they have been proposed to account for many age-related diseases (Bjornsson et al. 2004; Heyn et al. 2013; Timp and Feinberg 2013). Various recent studies using 1.5K and 27K Illumina methylation arrays have identified a group of gene promoters in blood that becomes hypermethylated during aging (Christensen et al. 2009; Rakyan et al. 2010; Teschendorff et al. 2010; Bell et al. 2012; Fernandez et al. 2012). Interestingly, some of these studies have also shown that these DNA sequences are enriched in bivalent chromatin domains in embryonic stem cells (Rakyan et al. 2010; Fernandez et al. 2012; Heyn et al. 2012) and repressive histone marks such as H3K9me3 and H3K27me3 in differentiated cells (Rakyan et al. 2010), and that many of them are also frequently hypermethylated in cancer. However, drawing conclusions from some of these studies is limited by their low genome coverage (< 0.1%) and the location of the sequences analyzed (mainly at gene promoters). Further studies using Infinium HumanMethylation450 BeadChip and larger cohorts (Heyn et al. 2012; Hannum et al. 2013; Johansson et al. 2013) have, though, corroborated most of the previous observations with the 27K methylation arrays and have, in addition, identified new sets of genes that become hypermethylated and hypomethylated during aging in humans. Finally, a recent study that analyzed the genome-wide DNA methylation status of newborns, middle-aged individuals, and centenarians confirmed the results obtained with the methylation arrays and showed that aging is associated with overall hypomethylation, which primarily occurs at repetitive DNA sequences (Heyn et al. 2012). Most of the above studies were conducted with whole blood, and consequently, changes in cell heterogeneity during aging could have affected the results (Calvanese et al. 2012; Houseman et al. 2012). However, some genes presented consistent changes in different tissues, which indicates that in some cases, the changes truly are associated with aging (Rakyan et al. 2010; Horvath et al. 2012). Interestingly, Houseman et al. (2012) have recently reported an algorithm that, using the DNA methylation values of certain genes, estimates the relative proportion of the different blood cell types in a specific sample. This algorithm was successfully used by Liu et al. (2013) in a study to identify DNA methylation alterations associated with rheumatoid arthritis.In addition to the studies using blood, other works have identified specific DNA methylation signatures of aging in differentiated cell types, including the brain (Hernandez et al. 2011; Numata et al. 2012; Guintivano et al. 2013; Lister et al. 2013), muscle (Zykovich et al. 2014), and saliva (Bocklandt et al. 2011). Two studies have analyzed DNA methylation during aging in human adult stem cells: Bork et al. (2010) used 27K methylation arrays to analyze the DNA methylation status of mesenchymal stem cells (MSCs) obtained from young (21–50 yr) and old donors (53–85 yr) and found similar DNA methylation changes over time during prolonged in vitro culture and in vivo aging. Using the same methylation arrays, Bocker et al. (2011) observed a bimodal pattern of methylation changes in older hematopoietic progenitor cells, with hypomethylation of differentiation-associated genes, as well as de novo methylation events resembling epigenetic mutations. Recent studies in mice have revealed a number of genome-wide alterations in DNA methylation (Taiwo et al. 2013) that might play an important role in the functional decline of hematopoietic stem cells (HSCs) during aging (Beerman et al. 2013). To study the role of DNA methylation in adult stem cell aging further, the present study used the HumanMethylation450 BeadChip to characterize the genome-wide DNA methylation status of bone marrow MSCs obtained from individuals aged between 2 and 92 yr. We then systematically compared our results with previously published data to identify the chromatin signatures associated with DNA methylation changes in adult stem cells and to determine whether these changes were also present in other tissues. Finally, we analyzed monozygotic (MZ) twins of different ages to determine the effect of genetic factors on the DNA methylation changes during aging identified in our study.