Comparative epigenomics by machine learning approach for neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is the second most common pediatric solid tumor. Because the number of genetic mutations found in tumors are small, even in some patients with unfavorable NB, epigenetic variation is expected to play an important role in NB progression. DNA methylation is a major epigenetic mechanism, and its relationship with NB prognosis has been a concern. One limitation with the analysis of variation in DNA methylation is the lack of a suitable analytical model. Therefore, in this study, we performed a random forest (RF) analysis of the DNA methylome data of NB from multiple databases. RESULTS: RF is a popular machine learning model owing to its simplicity, intuitiveness, and computational cost. RF analysis identified novel intermediate-risk patient groups with characteristic DNA methylation patterns within the low-risk group. Feature selection analysis based on probe annotation revealed that enhancer-annotated regions had strong predictive power, particularly for MYCN-amplified NBs. We developed a gene-based analytical model to identify candidate genes related to disease progression, such as PRDM8 and FAM13A-AS1. RF analysis revealed sufficient predictive power compared to other machine learning models. CONCLUSIONS: RF is a useful tool for DNA methylome analysis in cancer epigenetic studies, and has potential to identify a novel cancer-related genes.

pENCODE: a plant encyclopedia of DNA elements.

ENCODE projects exist for many eukaryotes, including humans, but as of yet no defined project exists for plants. A plant ENCODE would be invaluable to the research community and could be more readily produced than its metazoan equivalents by capitalizing on the preexisting infrastructure provided from similar projects. Collecting and normalizing plant epigenomic data for a range of species will facilitate hypothesis generation, cross-species comparisons, annotation of genomes, and an understanding of epigenomic functions throughout plant evolution. Here, we discuss the need for such a project, outline the challenges it faces, and suggest ways forward to build a plant ENCODE.

Genome-wide mapping of histone modifications during axenic growth in two species of Leptosphaeria maculans showing contrasting genomic organization.

Leptosphaeria maculans 'brassicae' (Lmb) and Leptosphaeria maculans 'lepidii' (Lml) are closely related phytopathogenic species that exhibit a large macrosynteny but contrasting genome structure. Lmb has more than 30% of repeats clustered in large repeat-rich regions, while the Lml genome has only a small amount of evenly distributed repeats. Repeat-rich regions of Lmb are enriched in effector genes, expressed during plant infection. The distinct genome structures of Lmb and Lml provide an excellent model for comparing the organization of pathogenicity genes in relation to the chromatin landscape in two closely related phytopathogenic fungi. Here, we performed chromatin immunoprecipitation (ChIP) during axenic culture, targeting histone modifications typical for heterochromatin or euchromatin, combined with transcriptomic analysis to analyze the influence of chromatin organization on gene expression. In both species, we found that facultative heterochromatin is enriched with genes lacking functional annotation, including numerous effector and species-specific genes. Notably, orthologous genes located in H3K27me3 domains are enriched with effector genes. Compared to other fungal species, including Lml, Lmb is distinct in having large H3K9me3 domains associated with repeat-rich regions that contain numerous species-specific effector genes. Discovery of these two distinctive heterochromatin landscapes now raises questions about their involvement in the regulation of pathogenicity, the dynamics of these domains during plant infection and the selective advantage to the fungus to host effector genes in H3K9me3 or H3K27me3 domains.

Epigenomics and genotype-phenotype association analyses reveal conserved genetic architecture of complex traits in cattle and human.

BACKGROUND: Lack of comprehensive functional annotations across a wide range of tissues and cell types severely hinders the biological interpretations of phenotypic variation, adaptive evolution, and domestication in livestock. Here we used a combination of comparative epigenomics, genome-wide association study (GWAS), and selection signature analysis, to shed light on potential adaptive evolution in cattle. RESULTS: We cross-mapped 8 histone marks of 1300 samples from human to cattle, covering 178 unique tissues/cell types. By uniformly analyzing 723 RNA-seq and 40 whole genome bisulfite sequencing (WGBS) datasets in cattle, we validated that cross-mapped histone marks captured tissue-specific expression and methylation, reflecting tissue-relevant biology. Through integrating cross-mapped tissue-specific histone marks with large-scale GWAS and selection signature results, we for the first time detected relevant tissues and cell types for 45 economically important traits and artificial selection in cattle. For instance, immune tissues are significantly associated with health and reproduction traits, multiple tissues for milk production and body conformation traits (reflecting their highly polygenic architecture), and thyroid for the different selection between beef and dairy cattle. Similarly, we detected relevant tissues for 58 complex traits and diseases in humans and observed that immune and fertility traits in humans significantly correlated with those in cattle in terms of relevant tissues, which facilitated the identification of causal genes for such traits. For instance, PIK3CG, a gene highly specifically expressed in mononuclear cells, was significantly associated with both age-at-menopause in human and daughter-still-birth in cattle. ICAM, a T cell-specific gene, was significantly associated with both allergic diseases in human and metritis in cattle. CONCLUSION: Collectively, our results highlighted that comparative epigenomics in conjunction with GWAS and selection signature analyses could provide biological insights into the phenotypic variation and adaptive evolution. Cattle may serve as a model for human complex traits, by providing additional information beyond laboratory model organisms, particularly when more novel phenotypes become available in the near future.

A comparative analysis of chromatin accessibility in cattle, pig, and mouse tissues.

BACKGROUND: Although considerable progress has been made towards annotating the noncoding portion of the human and mouse genomes, regulatory elements in other species, such as livestock, remain poorly characterized. This lack of functional annotation poses a substantial roadblock to agricultural research and diminishes the value of these species as model organisms. As active regulatory elements are typically characterized by chromatin accessibility, we implemented the Assay for Transposase Accessible Chromatin (ATAC-seq) to annotate and characterize regulatory elements in pigs and cattle, given a set of eight adult tissues. RESULTS: Overall, 306,304 and 273,594 active regulatory elements were identified in pig and cattle, respectively. 71,478 porcine and 47,454 bovine regulatory elements were highly tissue-specific and were correspondingly enriched for binding motifs of known tissue-specific transcription factors. However, in every tissue the most prevalent accessible motif corresponded to the insulator CTCF, suggesting pervasive involvement in 3-D chromatin organization. Taking advantage of a similar dataset in mouse, open chromatin in pig, cattle, and mice were compared, revealing that the conservation of regulatory elements, in terms of sequence identity and accessibility, was consistent with evolutionary distance; whereas pig and cattle shared about 20% of accessible sites, mice and ungulates only had about 10% of accessible sites in common. Furthermore, conservation of accessibility was more prevalent at promoters than at intergenic regions. CONCLUSIONS: The lack of conserved accessibility at distal elements is consistent with rapid evolution of enhancers, and further emphasizes the need to annotate regulatory elements in individual species, rather than inferring elements based on homology. This atlas of chromatin accessibility in cattle and pig constitutes a substantial step towards annotating livestock genomes and dissecting the regulatory link between genome and phenome.

ICGEC: a comparative method for measuring epigenetic conservation of genes via the integrated signal from multiple histone modifications between cell types.

BACKGROUND: Histone post-translational modifications play crucial roles in epigenetic regulation of gene expression and are known to be associated with the phenotypic differences of different cell types. Therefore, it is of fundamental importance to dissect the genes and pathways involved in such a phenotypic variation at the level of epigenetics. However, the existing comparative approaches are largely based on the differences, especially the absolute difference in the levels of individual histone modifications of genes under contrasting conditions. Thus, a method for measuring the overall change in the epigenetic circumstance of each gene underpinned by multiple types of histone modifications between cell types is lacking. RESULTS: To address this challenge, we developed ICGEC, a new method for estimating the degree of epigenetic conservation of genes between two cell lines. Different from existing comparative methods, ICGEC provides a reliable score for measuring the relative change in the epigenetic context of corresponding gene between two conditions and simultaneously produces a score for each histone mark. The application of ICGEC to the human embryonic stem cell line H1 and four H1-derived cell lines with available epigenomic data for the same 16 types of histone modifications indicated high robustness and reliability of ICGEC. Furthermore, the analysis of the epigenetically dynamic and conserved genes which were defined based on the ICGEC output results demonstrated that ICGEC can deepen our understanding of the biological processes of cell differentiation to overcome the limitations of traditional expression analysis. Specifically, the ICGEC-derived differentiation-direction-specific genes were shown to have putative functions that are well-matched with cell identity. Additionally, H3K79me1 and H3K27ac were found to be the main histone marks accounting for whether an epigenetically dynamic gene was differentially expressed between two cell lines. CONCLUSIONS: The use of ICGEC creates a convenient and robust way to measure the overall epigenetic conservation of individual genes and marks between two conditions. Thus, it provides a basis for exploring the epigenotype-phenotype relationship. ICGEC can be deemed a state-of-the-art method tailored for comparative epigenomic analysis of changes in cell dynamics.

Comparative epigenomics reveals evolution of duplicated genes in potato and tomato.

The evolution of duplicated genes after polyploidization has been the subject of many evolutionary biology studies. Potato (Solanum tuberosum) and tomato (Solanum lycopersicum) are the first two sequenced genomes of asterids, and share a common polyploidization event. However, the epigenetic role of DNA methylation on the evolution of duplicated genes derived from polyploidization is not fully understood. Here, we explore the role of the DNA methylation in the evolution of duplicated genes in potato and tomato. The overall levels of DNA methylation are different, although patterns of DNA methylation are similar in potato and tomato. Different types of duplicated genes can display different methylation patterns in potato and tomato. In addition, we found that differences in the methylation levels between duplicated genes were associated with gene expression divergence. In particular, for the majority of duplicated gene pairs, one copy is always hyper- or hypo-methylated compared with the other copy across different tomato fruit ripening stages, and these genes are enriched for specific function related to transcription factor (TF) activity. Furthermore, transcription of hundreds of duplicated TFs was shown to be regulated by DNA methylation during fruit ripening stages in tomato, some of which are well-known fruit ripening TFs. Taken together, our results support the notion that DNA methylation may facilitate divergent evolution of duplicated genes and play roles in important biological processes such as tomato fruit ripening.

Comparison of reprogramming factor targets reveals both species-specific and conserved mechanisms in early iPSC reprogramming.

BACKGROUND: Both human and mouse fibroblasts can be reprogrammed to pluripotency with Oct4, Sox2, Klf4, and c-Myc (OSKM) transcription factors. While both systems generate pluripotency, human reprogramming takes considerably longer than mouse. RESULTS: To assess additional similarities and differences, we sought to compare the binding of the reprogramming factors between the two systems. In human fibroblasts, the OSK factors initially target many more closed chromatin sites compared to mouse. Despite this difference, the intra- and intergenic distribution of target sites, target genes, primary binding motifs, and combinatorial binding patterns between the reprogramming factors are largely shared. However, while many OSKM binding events in early mouse cell reprogramming occur in syntenic regions, only a limited number is conserved in human. CONCLUSIONS: Our findings suggest similar general effects of OSKM binding across these two species, even though the detailed regulatory networks have diverged significantly.

Identifying co-opted transposable elements using comparative epigenomics.

The human genome gives rise to different epigenomic landscapes that define each cell type and can be deregulated in disease. Recent efforts by ENCODE, the NIH Roadmap and the International Human Epigenome Consortium (IHEC) have made significant advances towards assembling reference epigenomic maps of various tissues. Notably, these projects have found that approximately 80% of human DNA was biochemically active in at least one epigenomic assay while only approximately 10% of the sequence displayed signs of purifying selection. Given that transposable elements (TEs) make up at least 50% of the human genome and can be actively transcribed or act as regulatory elements either for their own purposes or be co-opted for the benefit of their host; we are interested in exploring their overall contribution to the "functional" genome. Traditional methods used to identify functional DNA have relied on comparative genomics, conservation analysis and low throughput validation assays. To discover co-opted TEs, and distinguish them from noisy genomic elements, we argue that comparative epigenomic methods will also be important.

Tissue-specific DNA methylation is conserved across human, mouse, and rat, and driven by primary sequence conservation.

BACKGROUND: Uncovering mechanisms of epigenome evolution is an essential step towards understanding the evolution of different cellular phenotypes. While studies have confirmed DNA methylation as a conserved epigenetic mechanism in mammalian development, little is known about the conservation of tissue-specific genome-wide DNA methylation patterns. RESULTS: Using a comparative epigenomics approach, we identified and compared the tissue-specific DNA methylation patterns of rat against those of mouse and human across three shared tissue types. We confirmed that tissue-specific differentially methylated regions are strongly associated with tissue-specific regulatory elements. Comparisons between species revealed that at a minimum 11-37% of tissue-specific DNA methylation patterns are conserved, a phenomenon that we define as epigenetic conservation. Conserved DNA methylation is accompanied by conservation of other epigenetic marks including histone modifications. Although a significant amount of locus-specific methylation is epigenetically conserved, the majority of tissue-specific DNA methylation is not conserved across the species and tissue types that we investigated. Examination of the genetic underpinning of epigenetic conservation suggests that primary sequence conservation is a driving force behind epigenetic conservation. In contrast, evolutionary dynamics of tissue-specific DNA methylation are best explained by the maintenance or turnover of binding sites for important transcription factors. CONCLUSIONS: Our study extends the limited literature of comparative epigenomics and suggests a new paradigm for epigenetic conservation without genetic conservation through analysis of transcription factor binding sites.

Comparative analysis of sperm DNA methylation supports evolutionary acquired epigenetic plasticity for organ speciation.

Aim: To perform a comparative epigenomic analysis of DNA methylation in spermatozoa from humans, mice, rats and mini-pigs. Materials & methods: Genome-wide DNA methylation analysis was used to compare the methylation profiles of orthologous CpG sites. Transcription profiles of early embryo development were analyzed to provide insight into the association between sperm methylation and gene expression programming. Results: We identified DNA methylation variation near genes related to the central nervous system and signal transduction. Gene expression dynamics at different time points of preimplantation stages were modestly associated with spermatozoal DNA methylation at the nearest promoters. Conclusion: Conserved genomic regions subject to epigenetic variation across different species were associated with specific organ functions, suggesting their potential contribution to organ speciation and long-term adaptation to the environment.

Stability of DNA methylation and chromatin accessibility in structurally diverse maize genomes.

Accessible chromatin and unmethylated DNA are associated with many genes and cis-regulatory elements. Attempts to understand natural variation for accessible chromatin regions (ACRs) and unmethylated regions (UMRs) often rely upon alignments to a single reference genome. This limits the ability to assess regions that are absent in the reference genome assembly and monitor how nearby structural variants influence variation in chromatin state. In this study, de novo genome assemblies for four maize inbreds (B73, Mo17, Oh43, and W22) are utilized to assess chromatin accessibility and DNA methylation patterns in a pan-genome context. A more complete set of UMRs and ACRs can be identified when chromatin data are aligned to the matched genome rather than a single reference genome. While there are UMRs and ACRs present within genomic regions that are not shared between genotypes, these features are 6- to 12-fold enriched within regions between genomes. Characterization of UMRs present within shared genomic regions reveals that most UMRs maintain the unmethylated state in other genotypes with only ∼5% being polymorphic between genotypes. However, the majority (71%) of UMRs that are shared between genotypes only exhibit partial overlaps suggesting that the boundaries between methylated and unmethylated DNA are dynamic. This instability is not solely due to sequence variation as these partially overlapping UMRs are frequently found within genomic regions that lack sequence variation. The ability to compare chromatin properties among individuals with structural variation enables pan-epigenome analyses to study the sources of variation for accessible chromatin and unmethylated DNA.

Comparative Analyses of Sperm DNA Methylomes Among Three Commercial Pig Breeds Reveal Vital Hypomethylated Regions Associated With Spermatogenesis and Embryonic Development.

Identifying epigenetic changes is essential for an in-depth understanding of phenotypic diversity and pigs as the human medical model for anatomizing complex diseases. Abnormal sperm DNA methylation can lead to male infertility, fetal development failure, and affect the phenotypic traits of offspring. However, the whole genome epigenome map in pig sperm is lacking to date. In this study, we profiled methylation levels of cytosine in three commercial pig breeds, Landrace, Duroc, and Large White using whole-genome bisulfite sequencing (WGBS). The results showed that the correlation of methylation levels between Landrace and Large White pigs was higher. We found that 1,040-1,666 breed-specific hypomethylated regions (HMRs) were associated with embryonic developmental and economically complex traits for each breed. By integrating reduced representation bisulfite sequencing (RRBS) public data of pig testis, 1743 conservated HMRs between sperm and testis were defined, which may play a role in spermatogenesis. In addition, we found that the DNA methylation patterns of human and pig sperm showed high similarity by integrating public data from WGBS and chromatin immunoprecipitation sequencing (ChIP-seq) in other mammals, such as human and mouse. We identified 2,733 conserved HMRs between human and pig involved in organ development and brain-related traits, such as NLGN1 (neuroligin 1) containing a conserved-HMR between human and pig. Our results revealed the similarities and diversity of sperm methylation patterns among three commercial pig breeds and between human and pig. These findings are beneficial for elucidating the mechanism of male fertility, and the changes in commercial traits that undergo strong selection.

Comparative Epigenomics Reveals Host Diversity of the Trichinella Epigenomes and Their Effects on Differential Parasitism.

Comparative epigenomics provides new insights on evolutionary biology in relation with complex interactions between species and their environments. In the present study, we focus on deciphering the conservation and divergence of DNA methylomes during Trichinella evolution. Whole-genome bisulfite sequencing and RNA-seq were performed on the two clades of Trichinella species, in addition to whole-genome sequencing. We demonstrate that methylation patterns of sing-copy orthologous genes (SCOs) of the 12 Trichinella species are host-related and can mirror known phylogenetic relationships. Among these SCOs, we identify a panel of genes exhibiting hyper-/hypo-methylated features in gene-bodies or respective promoters that play pivotal roles in transcriptome regulation. These hyper-/hypo-methylated SCOs are also of functional significance across developmental stages, as they are highly enriched species-specific and stage-specific expressed genes both in Ad and ML stages. We further identify a set of parasitism-related functional genes that exhibit host-related differential methylation and expression among those SCOs, including p53-like transcription factor and Cdc37 that are of functional significance for elucidating differential parasitology between the two clades of Trichinella. This comparative epigenome study can help to decipher the environmental effects on differential adaptation and parasitism of the genus Trichinella.

Predicted Archaic 3D Genome Organization Reveals Genes Related to Head and Spinal Cord Separating Modern from Archaic Humans.

High coverage sequences of archaic humans enabled the reconstruction of their DNA methylation patterns. This allowed comparing gene regulation between human groups, and linking such regulatory changes to phenotypic differences. In a previous work, a detailed comparison of DNA methylation in modern humans, archaic humans, and chimpanzees revealed 873 modern human-derived differentially methylated regions (DMRs). To understand the regulatory implications of these DMRs, we defined differentially methylated genes (DMGs) as genes that harbor DMRs in their promoter or gene body. While most of the modern human-derived DMRs could be linked to DMGs, many others remained unassigned. Here, we used information on 3D genome organization to link ~70 out of the remaining 288 unassigned DMRs to genes. Combined with the previously identified DMGs, we reinforce the enrichment of these genes with vocal and facial anatomy, and additionally find significant enrichment with the spinal column, chin, hair, and scalp. These results reveal the importance of 3D genomic organization in understanding gene regulation by DNA methylation.

Comparative Epigenomics Reveals that RNA Polymerase II Pausing and Chromatin Domain Organization Control Nematode piRNA Biogenesis.

Piwi-interacting RNAs (piRNAs) are important for genome regulation across metazoans, but their biogenesis evolves rapidly. In Caenorhabditis elegans, piRNA loci are clustered within two 3-Mb regions on chromosome IV. Each piRNA locus possesses an upstream motif that recruits RNA polymerase II to produce an ∼28 nt primary transcript. We used comparative epigenomics across nematodes to gain insight into the origin, evolution, and mechanism of nematode piRNA biogenesis. We show that the piRNA upstream motif is derived from core promoter elements controlling snRNA transcription. We describe two alternative modes of piRNA organization in nematodes: in C. elegans and closely related nematodes, piRNAs are clustered within repressive H3K27me3 chromatin, while in other species, typified by Pristionchus pacificus, piRNAs are found within introns of active genes. Additionally, we discover that piRNA production depends on sequence signals associated with RNA polymerase II pausing. We show that pausing signals synergize with chromatin to control piRNA transcription.

Comparative epigenomics in the Brassicaceae reveals two evolutionarily conserved modes of PRC2-mediated gene regulation.

BACKGROUND: Polycomb Repressive Complexes 2 (PRC2) are multi-protein chromatin modifiers that are evolutionarily conserved among eukaryotes and play key roles in the regulation of gene expression, notably through the trimethylation of lysine 27 of histone H3 (H3K27me3). Although PRC2-mediated gene regulation has been studied in many organisms, few studies have explored in depth the evolutionary conservation of PRC2 targets. RESULTS: Here, we compare the H3K27me3 epigenomic profiles for the two closely related species Arabidopsis thaliana and Arabidopsis lyrata and the more distant species Arabis alpina, three Brassicaceae that diverged from each other within the past 24 million years. Using a robust set of gene orthologs present in the three species, we identify two classes of evolutionarily conserved PRC2 targets, which are characterized by either developmentally plastic or developmentally constrained H3K27me3 marking across species. Constrained H3K27me3 marking is associated with higher conservation of promoter sequence information content and higher nucleosome occupancy compared to plastic H3K27me3 marking. Moreover, gene orthologs with constrained H3K27me3 marking exhibit a higher degree of tissue specificity and tend to be involved in developmental functions, whereas gene orthologs with plastic H3K27me3 marking preferentially encode proteins associated with metabolism and stress responses. In addition, gene orthologs with constrained H3K27me3 marking are the predominant contributors to higher-order chromosome organization. CONCLUSIONS: Our findings indicate that developmentally plastic and constrained H3K27me3 marking define two evolutionarily conserved modes of PRC2-mediated gene regulation that are associated with distinct selective pressures operating at multiple scales, from DNA sequence to gene function and chromosome architecture.

Comparative Epigenomic Profiling of the DNA Methylome in Mouse and Zebrafish Uncovers High Interspecies Divergence.

The DNA methylation landscape is dynamically patterned during development and distinct methylation patterns distinguish healthy from diseased cells. However, whether tissue-specific methylation patterns are conserved across species is not known. We used comparative methylome analysis of base-resolution DNA methylation profiles from the liver and brain of mouse and zebrafish generated by reduced representation bisulfite sequencing to identify the conserved and divergent aspects of the methylome in these commonly used vertebrate model organisms. On average, 24% of CpGs are methylated in mouse livers and the pattern of methylation was highly concordant among four male mice from two different strains. The same level of methylation (24.2%) was identified in mouse brain. In striking contrast, zebrafish had 63 and 70% of CpG methylation in the liver and brain, respectively. This is attributed, in part, to the higher percentage of the zebrafish genome occupied by transposable elements (52% vs. 45% in mice). Thus, the species identity was more significant in determining methylome patterning than was the similarity in organ function. Conserved features of the methylome across tissues and species was the exclusion of methylation from promoters and from CpG islands near transcription start sites, and the clustering of methylated CpGs in gene bodies and intragenic regions. These data suggest that DNA methylation reflects species-specific genome structure, and supports the notion that DNA methylation in non-promoter regions may contribute to genome evolution.

Toward understanding the evolution of vertebrate gene regulatory networks: comparative genomics and epigenomic approaches.

Vertebrates, as most animal phyla, originated >500 million years ago during the Cambrian explosion, and progressively radiated into the extant classes. Inferring the evolutionary history of the group requires understanding the architecture of the developmental programs that constrain the vertebrate anatomy. Here, I review recent comparative genomic and epigenomic studies, based on ChIP-seq and chromatin accessibility, which focus on the identification of functionally equivalent cis-regulatory modules among species. This pioneer work, primarily centered in the mammalian lineage, has set the groundwork for further studies in representative vertebrate and chordate species. Mapping of active regulatory regions across lineages will shed new light on the evolutionary forces stabilizing ancestral developmental programs, as well as allowing their variation to sustain morphological adaptations on the inherited vertebrate body plan.