DNA methylation is an important epigenetic mechanism for regulating the activity of the genome. Inter-individual differences in the epigenome, including the DNA methylome, are thought to account for the missing variance in disease susceptibility that has not been identified in Genome-Wide Association Studies (GWAS). Large-scale profiling of DNA methylation in population cohorts at the sample size of thousands to tens of thousands is necessary to characterize the epigenetic component of diseases susceptibility. Although whole genome bisulfite sequencing has been demonstrated in mammalian-size genomes, it is still too costly for a large sample size. In addition, only a very small fraction of CpG sites in the human genome is variable and carries information related to the epigenetic state of the cells, whereas the majority of CpG sites are static. For large-scale methylation sequencing projects, ideally the sequencing cost should be spent only on the informative sites in the genome.
We have previously developed a targeted bisulfite sequencing method based on bisulfite padlock probes (BSPP), which can quantify the absolute CpG methylation levels on an arbitrary set of genomic regions. In this talk I will present a second-generation BSPP method, which has a highly optimized protocol for production-scale methylation sequencing at a batch size of 96 samples. We also designed and optimized a set of ∼300,000 padlock probes targeting a set of carefully selected genomic regions (DMRs, enhancers, insulators, promoters, DNase I hypersensitive sites) throughout the entire human genome. A computational pipeline for probe design and efficient processing of methylation sequencing data was also developed. This method allows us to obtain highly accurate measurements of CpG and non-CpG methylation on >500,000 highly informative sites at the cost of <$250 per sample. Preliminary results on clinical samples processed with our second-generation BSPP method will be discussed.
The Epigenomics database at the National Center for Biotechnology Information (NCBI) is a new resource that has been created to serve as a comprehensive public resource for whole-genome epigenetic data sets (www.ncbi.nlm.nih.gov/epigenomics). Epigenetics is the study of stable and heritable changes in gene expression that occur independently of the primary DNA sequence. Epigenetic mechanisms include post-translational modifications of histones, DNA methylation, chromatin conformation and non-coding RNAs. It has been observed that misregulation of epigenetic processes has been associated with human disease. We have constructed the new resource by selecting the subset of epigenetics-specific data from general-purpose archives, such as the Gene Expression Omnibus, and Sequence Read Archives, and then subjecting them to further review, annotation and reorganization. Raw data is processed and mapped to genomic coordinates to generate ‘tracks’ that are a visual representation of the data. These data tracks can be viewed using popular genome browsers or downloaded for local analysis. The Epigenomics resource also provides the user with a unique interface that allows for intuitive browsing and searching of data sets based on biological attributes. Currently, there are 69 studies, 337 samples and over 1100 data tracks from five well-studied species that are viewable and downloadable in Epigenomics.
Epigenetics is focused on understanding the control of gene expression beyond what is encoded in the sequence of DNA. Central to growing interest in the field is the hope that more can be learned about the epigenetic regulatory mechanisms underlying processes of human development and disease. Researchers have begun to examine epigenetic alterations – such as changes in promoter DNA methylation, genomic imprinting, and expression of miRNA – to learn more about epigenetic regulation in the placenta, an organ whose proper development and function are crucial to the health growth and survival of the developing fetus. A number of studies are now making important links between alterations to appropriate epigenetic regulation in the placenta and diseases of gestation and early life. In addition, these studies are adding important insight into our understanding of trophoblast biology and differentiation as well as placental immunology. Examining epigenetic alterations in the placenta will prove especially important in the search for biomarkers of exposure, pathology, and disease risk and can provide critical insights into the biology of development and pathogenesis of disease. Thus, epigenetic alterations may aid in disease diagnosis and prognosis as well as in targeting new treatment and prevention strategies.
DNA methylation; environmental exposure; miRNA; imprinting
Epigenetics pertains to heritable alterations in gene expression that do not involve modification of the underlying genomic DNA sequence. Historically, the study of epigenetic mechanisms has focused on DNA methylation and histone modifications, but the concept of epigenetics has been more recently extended to include microRNAs as well. Epigenetic patterning is modified by environmental exposures and may be a mechanistic link between environmental risk factors and the development of disease. Epigenetic dysregulation has been associated with a variety of human diseases, including cancer, neurological disorders, and autoimmune diseases. In this review, we consider the role of epigenetics in common ocular diseases, with a particular focus on DNA methylation and microRNAs. DNA methylation is a critical regulator of gene expression in the eye and is necessary for the proper development and postmitotic survival of retinal neurons. Aberrant methylation patterns have been associated with age-related macular degeneration, susceptibility to oxidative stress, cataract, pterygium, and retinoblastoma. Changes in histone modifications have also been observed in experimental models of diabetic retinopathy and glaucoma. The expression levels of specific microRNAs have also been found to be altered in the context of ocular inflammation, retinal degeneration, pathological angiogenesis, diabetic retinopathy, and ocular neoplasms. Although the complete spectrum of epigenetic modifications remains to be more fully explored, it is clear that epigenetic dysregulation is an important contributor to common ocular diseases and may be a relevant therapeutic target.
Age-related macular generation; Cataract; Diabetic retinopathy; Epigenetics; DNA methylation; microRNA.
Epigenetics refers to changes in phenotype and gene expression that occur without alterations in DNA sequence. Epigenetic modifications of the genome can be acquired de novo and are potentially heritable. This review focuses on the emerging recognition of a role for epigenetics in the development of pulmonary arterial hypertension (PAH). Lessons learned from the epigenetics in cancer and neurodevelopmental diseases, such as Prader-Willi syndrome, can be applied to PAH. These syndromes suggest that there is substantial genetic and epigenetic cross-talk such that a single phenotype can result from a genetic cause, an epigenetic cause, or a combined abnormality. There are three major mechanisms of epigenetic regulation, including methylation of CpG islands, mediated by DNA methyltransferases, modification of histone proteins, and microRNAs. There is substantial interaction between these epigenetic mechanisms. Recently, it was discovered that there may be an epigenetic component to PAH. In PAH there is downregulation of superoxide dismutase 2 (SOD2) and normoxic activation of hypoxia inducible factor (HIF-1α). This decrease in SOD2 results from methylation of CpG islands in SOD2 by lung DNA methyltransferases. The partial silencing of SOD2 alters redox signaling, activates HIF-1α) and leads to excessive cell proliferation. The same hyperproliferative epigenetic abnormality occurs in cancer. These epigenetic abnormalities can be therapeutically reversed. Epigenetic mechanisms may mediate gene-environment interactions in PAH and explain the great variability in susceptibility to stimuli such as anorexigens, virus, and shunts. Epigenetics may be relevant to the female predisposition to PAH and the incomplete penetrance of BMPR2 mutations in familial PAH.
CpG islands; DNA methyl transferases; histone acetylation; small inhibitor RNA; superoxide dismutase 2
With the availability of complete genome sequences for a growing number of organisms, high-throughput methods for gene annotation and analysis of genome dynamics are needed. The application of whole-genome tiling microarrays for studies of global gene expression is providing a more unbiased view of the transcriptional activity within genomes. For example, this approach has led to the identification and isolation of many novel non-protein-coding RNAs (ncRNAs), which have been suggested to comprise a major component of the transcriptome that have novel functions involved in epigenetic regulation of the genome. Additionally, tiling arrays have been recently applied to the study of histone modifications and methylation of cytosine bases (DNA methylation). Surprisingly, recent studies combining the analysis of gene expression (transcriptome) and DNA methylation (methylome) using whole-genome tiling arrays revealed that DNA methylation regulates the expression levels of many ncRNAs. Further capture and integration of additional types of genome-wide data sets will help to illuminate additional hidden features of the dynamic genomic landscape that are regulated by both genetic and epigenetic pathways in plants.
The regulation of gene expression plays a pivotal role in complex phenotypes, and epigenetic mechanisms such as DNA methylation are essential to this process. The availability of next-generation sequencing technologies allows us to study epigenetic variation at an unprecedented level of resolution. Even so, our understanding of the underlying sources of epigenetic variability remains limited. Twin studies have played an essential role in estimating phenotypic heritability, and these now offer an opportunity to study epigenetic variation as a dynamic quantitative trait. High monozygotic twin discordance rates for common diseases suggest that unexplained environmental or epigenetic factors could be involved. Recent genome-wide epigenetic studies in disease-discordant monozygotic twins emphasize the power of this design to successfully identify epigenetic changes associated with complex traits. We describe how large-scale epigenetic studies of twins can improve our understanding of how genetic, environmental and stochastic factors impact upon epigenetics, and how such studies can provide a comprehensive understanding of how epigenetic variation affects complex traits.
During mammalian evolution, complex systems of epigenetic gene regulation have been established: Epigenetic mechanisms control tissue-specific gene expression, X chromosome inactivation in females and genomic imprinting. Studying DNA sequence conservation in imprinted genes, it becomes evident that evolution of gene function and evolution of epigenetic gene regulation are tightly connected. Furthermore, comparative studies allow the identification of DNA sequence features that distinguish imprinted genes from biallelically expressed genes. Among these features are CpG islands, tandem repeats and retrotransposed elements that are known to play major roles in epigenetic gene regulation. Currently, more and more genetic and epigenetic data sets become available. In future, such data sets will provide the basis for more complex investigations on epigenetic variation in human populations. Therein, an exciting topic will be the genetic and epigenetic variability of imprinted genes and its input on human disease.
Genomic imprinting; Mammalian evolution; Repetitive elements; DNA methylation; CpG island
Many cellular constituents in the human brain permanently exit from the cell cycle during pre- or early postnatal development, but little is known about epigenetic regulation of neuronal and glial epigenomes during maturation and aging, including changes in mood and psychosis spectrum disorders and other cognitive or emotional disease. Here, we summarize the current knowledge base as it pertains to genome organization in the human brain, including the regulation of DNA cytosine methylation and hydroxymethylation, and a subset of (altogether >100) residue-specific histone modifications associated with gene expression, and silencing and various other functional chromatin states. We propose that high-resolution mapping of epigenetic markings in postmortem brain tissue or neural cultures derived from induced pluripotent cells (iPS), in conjunction with transcriptome profiling and whole-genome sequencing, will increasingly be used to define the molecular pathology of specific cases diagnosed with depression, schizophrenia, autism, or other major psychiatric disease. We predict that these highly integrative explorations of genome organization and function will provide an important alternative to conventional approaches in human brain studies, which mainly are aimed at uncovering group effects by diagnosis but generally face limitations because of cohort size.
DNA methylation; DNA hydroxymethylation; post-translational histone modification; chromosome conformation; postmortem brain; psychiatric disease; DNA methylation; DNA hydroxymethylation; post-translational histone modification; chromosome conformation; postmortem brain; psychiatric disease
Methylation of CpG dinucleotides is a fundamental mechanism of epigenetic regulation in eukaryotic genomes. Development of methods for rapid genome wide methylation profiling will greatly facilitate both hypothesis and discovery driven research in the field of epigenetics. In this regard, a single molecule approach to methylation profiling offers several unique advantages that include elimination of chemical DNA modification steps and PCR amplification.
A single molecule approach is presented for the discernment of methylation profiles, based on optical mapping. We report results from a series of pilot studies demonstrating the capabilities of optical mapping as a platform for methylation profiling of whole genomes. Optical mapping was used to discern the methylation profile from both an engineered and wild type Escherichia coli. Furthermore, the methylation status of selected loci within the genome of human embryonic stem cells was profiled using optical mapping.
The optical mapping platform effectively detects DNA methylation patterns. Due to single molecule detection, optical mapping offers significant advantages over other technologies. This advantage stems from obviation of DNA modification steps, such as bisulfite treatment, and the ability of the platform to assay repeat dense regions within mammalian genomes inaccessible to techniques using array-hybridization technologies.
Epigenetic changes refer to heritable changes that may modulate gene expression without affecting DNA sequence. DNA methylation is one such heritable epigenetic change, which is causally associated with the transcription regulation of many genes in the mammalian genome. Altered DNA methylation has been implicated in a wide variety of human diseases including cancer. Understanding the regulation of DNA methylation is likely to improve the ability to diagnose and treat these diseases. With the advent of high-throughput RNA interference (RNAi) screens, answering epigenetic questions on a genomic scale is now possible. Two recent genome-wide RNAi screens have addressed the regulation of DNA methylation in cancer, leading to the identification of the regulators of epigenetic silencing by oncogenic RAS and how epigenetic silencing of the tumor suppressor RASSF1A is maintained. These RNAi screens have much wider applications, since similar screens can now be adapted to identify the mechanism of silencing of any human disease-associated gene that is epigenetically regulated. In this review, we discuss two recent genome-wide RNAi screens for epigenetic regulators and explore potential applications in understanding DNA methylation and gene expression regulation in mammalian cells. We also discuss some of the key unanswered questions in the field of DNA methylation and suggest genome-wide RNAi screens designed to answer them.
DNA methylation; Epigenetics; Transcription; RNA interference; Imprinting
The transcriptome of a cell is represented by a myriad of different RNA molecules with and without protein-coding capacities. In recent years, advances in sequencing technologies have allowed researchers to more fully appreciate the complexity of whole transcriptomes, showing that the vast majority of the genome is transcribed, producing a diverse population of non-protein coding RNAs (ncRNAs). Thus, the biological significance of non-coding RNAs (ncRNAs) have been largely underestimated. Amongst these multiple classes of ncRNAs, the long non-coding RNAs (lncRNAs) are apparently the most numerous and functionally diverse. A small but growing number of lncRNAs have been experimentally studied, and a view is emerging that these are key regulators of epigenetic gene regulation in mammalian cells. LncRNAs have already been implicated in human diseases such as cancer and neurodegeneration, highlighting the importance of this emergent field. In this article, we review the catalogs of annotated lncRNAs and the latest advances in our understanding of lncRNAs.
non-coding RNAs; regulation; long non-coding RNA; epigenetics
Genome-wide association studies have thus far failed to explain the observed heritability of complex human diseases. This is referred to as the “missing heritability” problem. However, these analyses have usually neglected to consider a role for epigenetic variation, which has been associated with many human diseases. We extend models of epigenetic inheritance to investigate whether environment-sensitive epigenetic modifications of DNA might explain observed patterns of familial aggregation. We find that variation in epigenetic state and environmental state can result in highly heritable phenotypes through a combination of epigenetic and environmental inheritance. These two inheritance processes together can produce familial covariances significantly higher than those predicted by models of purely epigenetic inheritance and similar to those expected from genetic effects. The results suggest that epigenetic variation, inherited both directly and through shared environmental effects, may make a key contribution to the missing heritability.
A number of studies have shown that transcriptome analysis in terms of chromosomal location can reveal regions of non-random transcriptional activity within the genome. Genomic clusters of differentially expressed genes can identify genomic patterns of structural organization, underlying copy number variations or long-range epigenetic regulation such as X-chromosome inactivation. Here we apply an integrative bioinformatics analysis to a collection of 315 freely available mouse pluripotent stem cell samples to discover transcriptional clusters in the genome. We show that over half of the analysed samples (56.83%) carry whole or partial-chromosome spanning clusters which recur in genomic regions previously implicated in chromosomal imbalances. Strikingly, we found that the presence of such large-clusters is linked to the differential expression of a limited number of genes, common to all samples carrying clusters irrespectively of the chromosome where the cluster is found. We have used these genes to train and test classification models that can predict samples that carry large-scale clusters on any chromosome with over 90% accuracy. Our findings suggest that there is a common downstream activation in these cells that affects a limited number of nodes. We propose that this effect is linked to selective advantage and identify potential driver genes.
There is increasing evidence that interindividual epigenetic variation is an etiological factor in common human diseases. Such epigenetic variation could be genetic or non-genetic in origin, and epigenome-wide association studies (EWASs) are underway for a wide variety of diseases/phenotypes. However, performing an EWAS is associated with a range of issues not typically encountered in genome-wide association studies (GWASs), such as the tissue to be analyzed. In many EWASs, it is not possible to analyze the target tissue in large numbers of live humans, and consequently surrogate tissues are employed, most commonly blood. But there is as yet no evidence demonstrating that blood is more informative than buccal cells, the other easily accessible tissue. To assess the potential of buccal cells for use in EWASs, we performed a comprehensive analysis of a buccal cell methylome using whole-genome bisulfite sequencing. Strikingly, a buccal vs. blood comparison reveals > 6X as many hypomethylated regions in buccal. These tissue-specific differentially methylated regions (tDMRs) are strongly enriched for DNaseI hotspots. Almost 75% of these tDMRs are not captured by commonly used DNA methylome profiling platforms such as Reduced Representational Bisulfite Sequencing and the Illumina Infinium HumanMethylation450 BeadChip, and they also display distinct genomic properties. Buccal hypo-tDMRs show a statistically significant enrichment near SNPs associated to disease identified through GWASs. Finally, we find that, compared with blood, buccal hypo-tDMRs show significantly greater overlap with hypomethylated regions in other tissues. We propose that for non-blood based diseases/phenotypes, buccal will be a more informative tissue for EWASs.
BS-seq; buccal; complex disease; epigenome wide association study; human
Neuropsychiatric disorders affect a large segment of the human population and result in large costs to society. The majority of such disorders have unknown underlying causes. Recent evidence suggests an important role for epigenetic regulation in the emergence of neuropsychiatric disease. Epigenetics may provide a link between genetic and environmental factors and behavior. Epigenetic signaling involves changes on the structure of chromatin; such changes are often triggered and maintained by the post-translational modification of chromatin proteins and/or DNA. Recent proteomic technologies have enabled the study of epigenetic mechanisms in a high-throughput manner. This review will provide an overview of the major epigenetic pathways and modern techniques for their study, before focusing on experimental evidence supporting a strong role for epigenetics in selected psychiatric disorders such as depression, schizophrenia and drug addiction. These results highlight a great need for the inclusion of the proteomic characterization of epigenetic mechanisms in the study of gene/disease associations in psychiatric disorders.
Epigenetics; chromatin; histone modification; DNA methylation; neuropsychiatric disorders
Epigenetics is defined as the study of all inheritable and potentially reversible changes in genome function that do not alter the nucleotide sequence within the DNA. Epigenetic mechanisms such as DNA methylation, histone modification, nucleosome positioning, and microRNAs (miRNAs) are essential to carry out key functions in the regulation of gene expression. Therefore, the epigenetic mechanisms are a window to understanding the possible mechanisms involved in the pathogenesis of complex diseases such as autoimmune diseases. It is noteworthy that autoimmune diseases do not have the same epidemiology, pathology, or symptoms but do have a common origin that can be explained by the sharing of immunogenetic mechanisms. Currently, epigenetic research is looking for disruption in one or more epigenetic mechanisms to provide new insights into autoimmune diseases. The identification of cell-specific targets of epigenetic deregulation will serve us as clinical markers for diagnosis, disease progression, and therapy approaches.
Epigenetic regulation of gene expression, through covalent modification of histones, is a key process controlling growth and development. Accordingly, the transcription factors regulating these processes are important targets of genetic diseases. However, surprisingly little is known about the relationship between aberrant epigenetic states, the cellular process affected, and their phenotypic consequences. By chromosomal breakpoint mapping in a patient with a Noonan syndrome–like phenotype that encompassed short stature, blepharoptosis, and attention deficit hyperactivity disorder, we identified haploinsufficiency of the histone acetyltransferase gene MYST histone acetyltransferase (monocytic leukemia) 4 (MYST4), as the underlying cause of the phenotype. Using acetylation, whole genome expression, and ChIP studies in cells from the patient, cell lines in which MYST4 expression was knocked down using siRNA, and the Myst4 querkopf mouse, we found that H3 acetylation is important for neural, craniofacial, and skeletal morphogenesis, mainly through its ability to specifically regulating the MAPK signaling pathway. This finding further elucidates the complex role of histone modifications in mammalian development and adds what we believe to be a new mechanism to the pathogenic phenotypes resulting from misregulation of the RAS signaling pathway.
A growing amount of evidence points towards alterations in epigenetic machinery as a leading cause in disease initiation and progression. Like genetic alterations, misregulation of the epigenetic regulators can lead to abnormal gene expression. However, unlike genetic events, the epigenetic machinery may be targeted pharmacologically, potentially resulting in the reversal of a particular epigenetic state. The success of DNA methyltransferase and histone deacetylase inhibitors represents a proof of concept for the use of therapies intended to target the epigenome in the treatment of hematological malignancies. Nevertheless, the molecular mechanisms underlying the efficacy of these agents have not been completely elucidated. Recently, a large number of studies sequencing cancer cell genomes identified recurring mutations of epigenetic regulators, providing new insights into the molecular underpinnings of cancer. Consequently, the efforts to identify specific epigenetic inhibitors have been expanded in order to target particular subsets of patients. This review will summarize the progress made using the currently available epigenetic therapies and discuss some of the more recently identified targets whose inhibition may present potential avenues for the treatment of hematologic malignancies.
epigenetics; methylation; MLL; EZH2
In vivo positioning and covalent modifications of nucleosomes play an important role in epigenetic regulation, but genome-wide studies of positioned nucleosomes and their modifications in human still remain limited.
This paper describes a novel computational framework to efficiently identify positioned nucleosomes and their histone modification profiles from nucleosome-resolution histone modification ChIP-Seq data. We applied the algorithm to histone methylation ChIP-Seq data in human CD4+ T cells and identified over 438,000 positioned nucleosomes, which appear predominantly at functionally important regions such as genes, promoters, DNase I hypersensitive regions, and transcription factor binding sites. Our analysis shows the identified nucleosomes play a key role in epigenetic gene regulation within those functionally important regions via their positioning and histone modifications.
Our method provides an effective framework for studying nucleosome positioning and epigenetic marks in mammalian genomes. The algorithm is open source and available at .
The identification of all epigenetic modifications implicated in gene expression is the next step for a better understanding of human biology in both normal and pathological states. This field is referred to as epigenomics, and it is defined as epigenetic changes (ie, DNA methylation, histone modifications and regulation by noncoding RNAs such as microRNAs) on a genomic scale rather than a single gene. Epigenetics modulate the structure of the chromatin, thereby affecting the transcription of genes in the genome. Different studies have already identified changes in epigenetic modifications in a few genes in specific pathways in cancers. Based on these epigenetic changes, drugs against different types of tumors were developed, which mainly target epimutations in the genome. Examples include DNA methylation inhibitors, histone modification inhibitors, and small molecules that target chromatin-remodeling proteins. However, these drugs are not specific, and side effects are a major problem; therefore, new DNA sequencing technologies combined with epigenomic tools have the potential to identify novel biomarkers and better molecular targets to treat cancers. The purpose of this review is to discuss current and emerging epigenomic tools and to address how these new technologies may impact the future of cancer management.
genomics; epigenomics; epigenetics; DNA methylation; histone modifications; new technologies; cancer management
Increasing evidence suggests that epigenetic regulation is key for the maintenance of the stem cell state. Chromatin is the physiological form of eukaryotic genomes and the substrate for epigenetic marking, including DNA methylation, post-translational modifications of histones, and the exchange of core histones with histone variants. The chromatin template undergoes significant reorganization during embryonic stem cell (ESC) differentiation and somatic cell reprogramming (SCR). Intriguingly, remodeling of the epigenome appears to be a crucial barrier that must be surmounted for efficient SCR. This area of research has gained significant attention due to the importance of ESCs in modeling and treating human disease. Here we review the epigenetic mechanisms that are critical for maintenance of the ESC state, ESC differentiation and SCR. We focus our attention on murine and human ESCs and induced pluripotent stem cells (iPSCs), and highlight pharmacological approaches used to study or manipulate cell fate where relevant.
The DNA of most vertebrates is depleted in CpG dinucleotides, the target for DNA methylation. The remaining CpGs tend to cluster in regions referred to as CpG islands (CGI). CGI have been useful as marking functionally relevant epigenetic loci for genome studies. For example, CGI are enriched in the promoters of vertebrate genes and thought to play an important role in regulation. Currently, CGI are defined algorithmically as an observed-to-expected ratio (O/E) of CpG greater than 0.6, G+C content greater than 0.5, and usually but not necessarily greater than a certain length. Here we find that the current definition leaves out important CpG clusters associated with epigenetic marks, relevant to development and disease, and does not apply at all to nonvertabrate genomes. We propose an alternative Hidden Markov model-based approach that solves these problems. We fit our model to genomes from 30 species, and the results support a new epigenomic view toward the development of DNA methylation in species diversity and evolution. The O/E of CpG in islands and nonislands segregated closely phylogenetically and showed substantial loss in both groups in animals of greater complexity, while maintaining a nearly constant difference in CpG O/E between islands and nonisland compartments. Lists of CGI for some species are available at http://www.rafalab.org.
The immunodeficiency, centromere instability and facial anomalies (ICF) syndrome is associated to mutations of the DNA methyl-transferase DNMT3B, resulting in a reduction of enzyme activity. Aberrant expression of immune system genes and hypomethylation of pericentromeric regions accompanied by chromosomal instability were determined as alterations driving the disease phenotype. However, so far only technologies capable to analyze single loci were applied to determine epigenetic alterations in ICF patients. In the current study, we performed whole-genome bisulphite sequencing to assess alteration in DNA methylation at base pair resolution. Genome-wide we detected a decrease of methylation level of 42%, with the most profound changes occurring in inactive heterochromatic regions, satellite repeats and transposons. Interestingly, transcriptional active loci and ribosomal RNA repeats escaped global hypomethylation. Despite a genome-wide loss of DNA methylation the epigenetic landscape and crucial regulatory structures were conserved. Remarkably, we revealed a mislocated activity of mutant DNMT3B to H3K4me1 loci resulting in hypermethylation of active promoters. Functionally, we could associate alterations in promoter methylation with the ICF syndrome immunodeficient phenotype by detecting changes in genes related to the B-cell receptor mediated maturation pathway.
CpG island; DNA methylation; DNA methyltransferase; DNMT3B; X chromosome; histone; immunodeficiency; transposon; whole genome bisulfite sequencing
The spatial conformation of a genome plays an important role in the long-range regulation of genome-wide gene expression and methylation, but has not been extensively studied due to lack of genome conformation data. The recently developed chromosome conformation capturing techniques such as the Hi-C method empowered by next generation sequencing can generate unbiased, large-scale, high-resolution chromosomal interaction (contact) data, providing an unprecedented opportunity to investigate the spatial structure of a genome and its applications in gene regulation, genomics, epigenetics, and cell biology. In this work, we conducted a comprehensive, large-scale computational analysis of this new stream of genome conformation data generated for three different human leukemia cells or cell lines by the Hi-C technique. We developed and applied a set of bioinformatics methods to reliably generate spatial chromosomal contacts from high-throughput sequencing data and to effectively use them to study the properties of the genome structures in one-dimension (1D) and two-dimension (2D). Our analysis demonstrates that Hi-C data can be effectively applied to study tissue-specific genome conformation, chromosome-chromosome interaction, chromosomal translocations, and spatial gene-gene interaction and regulation in a three-dimensional genome of primary tumor cells. Particularly, for the first time, we constructed genome-scale spatial gene-gene interaction network, transcription factor binding site (TFBS) – TFBS interaction network, and TFBS-gene interaction network from chromosomal contact information. Remarkably, all these networks possess the properties of scale-free modular networks.