Joseph R. Ecker

Human DNA methylomes at base resolution show widespread epigenomic differences

DNA cytosine methylation is a central epigenetic modification that has essential roles in cellular processes including genome regulation, development and disease. Here we present the first genome-wide, single-base-resolution maps of methylated cytosines in a mammalian genome, from both human embryonic stem cells and fetal fibroblasts, along with comparative analysis of messenger RNA and small RNA components of the transcriptome, several histone modifications, and sites of DNA–protein interaction for several key regulatory factors. Widespread differences were identified in the composition and patterning of cytosine methylation between the two genomes. Nearly one-quarter of all methylation identified in embryonic stem cells was in a non-CG context, suggesting that embryonic stem cells may use different methylation mechanisms to affect gene regulation. Methylation in non-CG contexts showed enrichment in gene bodies and depletion in protein binding sites and enhancers. Non-CG methylation disappeared upon induced differentiation of the embryonic stem cells, and was restored in induced pluripotent stem cells. We identified hundreds of differentially methylated regions proximal to genes involved in pluripotency and differentiation, and widespread reduced methylation levels in fibroblasts associated with lower transcriptional activity. These reference epigenomes provide a foundation for future studies exploring this key epigenetic modification in human disease and development.

Highly Integrated Single-Base Resolution Maps of the Epigenome in Arabidopsis

Deciphering the multiple layers of epigenetic regulation that control transcription is critical to understanding how plants develop and respond to their environment. Using sequencing-by-synthesis technology we directly sequenced the cytosine methylome (methylC-seq), transcriptome (mRNA-seq), and small RNA transcriptome (smRNA-seq) to generate highly integrated epigenome maps for wild-type Arabidopsis thaliana and mutants defective in DNA methyltransferase or demethylase activity. At single-base resolution we discovered extensive, previously undetected DNA methylation, identified the context and level of methylation at each site, and observed local sequence effects upon methylation state. Deep sequencing of smRNAs revealed a direct relationship between the location of smRNAs and DNA methylation, perturbation of smRNA biogenesis upon loss of CpG DNA methylation, and a tendency for smRNAs to direct strand-specific DNA methylation in regions of RNA-DNA homology. Finally, strand-specific mRNA-seq revealed altered transcript abundance of hundreds of genes, transposons, and unannotated intergenic transcripts upon modification of the DNA methylation state.

HIV-1 Integration in the Human Genome Favors Active Genes and Local Hotspots

A defining feature of HIV replication is integration of the proviral cDNA into human DNA. The selection of chromosomal targets for integration is crucial for efficient viral replication, but the mechanism is poorly understood. Here we describe mapping of 524 sites of HIV cDNA integration on the human genome sequence. Genes were found to be strongly favored as integration acceptor sites. Global analysis of cellular transcription indicated that active genes were preferential integration targets, particularly genes that were activated in cells after infection by HIV-1. Regional hotspots for integration were also found, including a 2.4 kb region containing 1% of sites. These data document unexpectedly strong biases in integration site selection and suggest how selective targeting promotes aggressive HIV replication.

Genome-wide High-Resolution Mapping and Functional Analysis of DNA Methylation in Arabidopsis

Cytosine methylation is important for transposon silencing and epigenetic regulation of endogenous genes, although the extent to which this DNA modification functions to regulate the genome is still unknown. Here we report the first comprehensive DNA methylation map of an entire genome, at 35 base pair resolution, using the flowering plant Arabidopsis thaliana as a model. We find that pericentromeric heterochromatin, repetitive sequences, and regions producing small interfering RNAs are heavily methylated. Unexpectedly, over one-third of expressed genes contain methylation within transcribed regions, whereas only approximately 5% of genes show methylation within promoter regions. Interestingly, genes methylated in transcribed regions are highly expressed and constitutively active, whereas promoter-methylated genes show a greater degree of tissue-specific expression. Whole-genome tiling-array transcriptional profiling of DNA methyltransferase null mutants identified hundreds of genes and intergenic noncoding RNAs with altered expression levels, many of which may be epigenetically controlled by DNA methylation.

Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) offer immense potential for regenerative medicine and studies of disease and development. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem (ES) cells. However, it remains unknown how complete the reestablishment of ES-cell-like DNA methylation patterns is throughout the genome. Here we report the first whole-genome profiles of DNA methylation at single-base resolution in five human iPSC lines, along with methylomes of ES cells, somatic cells, and differentiated iPSCs and ES cells. iPSCs show significant reprogramming variability, including somatic memory and aberrant reprogramming of DNA methylation. iPSCs share megabase-scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming of non-CG methylation, and differences in CG methylation and histone modifications. Lastly, differentiation of iPSCs into trophoblast cells revealed that errors in reprogramming CG methylation are transmitted at a high frequency, providing an iPSC reprogramming signature that is maintained after differentiation.

Ethylene Biosynthesis and Signaling Networks

Despite its simple two-carbon structure, the olefin ethylene is a potent modulator of plant growth and development ([Ecker, 1995][1]). The plant hormone ethylene is involved in many aspects of the plant life cycle, including seed germination, root hair development, root nodulation, flower senescence

Genome-wide association study of 107 phenotypes in Arabidopsis thaliana inbred lines

Although pioneered by human geneticists as a potential solution to the challenging problem of finding the genetic basis of common human diseases, genome-wide association (GWA) studies have, owing to advances in genotyping and sequencing technology, become an obvious general approach for studying the genetics of natural variation and traits of agricultural importance. They are particularly useful when inbred lines are available, because once these lines have been genotyped they can be phenotyped multiple times, making it possible (as well as extremely cost effective) to study many different traits in many different environments, while replicating the phenotypic measurements to reduce environmental noise. Here we demonstrate the power of this approach by carrying out a GWA study of 107 phenotypes in Arabidopsis thaliana, a widely distributed, predominantly self-fertilizing model plant known to harbour considerable genetic variation for many adaptively important traits. Our results are dramatically different from those of human GWA studies, in that we identify many common alleles of major effect, but they are also, in many cases, harder to interpret because confounding by complex genetics and population structure make it difficult to distinguish true associations from false. However, a-priori candidates are significantly over-represented among these associations as well, making many of them excellent candidates for follow-up experiments. Our study demonstrates the feasibility of GWA studies in A. thaliana and suggests that the approach will be appropriate for many other organisms.

The NIH Roadmap Epigenomics Mapping Consortium

The NIH Roadmap Epigenomics Mapping Consortium aims to produce a public resource of epigenomic maps for stem cells and primary ex vivo tissues selected to represent the normal counterparts of tissues and organ systems frequently involved in human disease.

Global Epigenomic Reconfiguration During Mammalian Brain Development

Introduction Several lines of evidence point to a key role for dynamic epigenetic changes during brain development, maturation, and learning. DNA methylation (mC) is a stable covalent modification that persists in post-mitotic cells throughout their lifetime, defining their cellular identity. However, the methylation status at each of the ~1 billion cytosines in the genome is potentially an information-rich and flexible substrate for epigenetic modification that can be altered by cellular activity. Indeed, changes in DNA methylation have been implicated in learning and memory, as well as in age-related cognitive decline. However, little is known about the cell type–specific patterning of DNA methylation and its dynamics during mammalian brain development. The DNA methylation landscape of human and mouse neurons is dynamically reconfigured through development. Base-resolution analysis allowed identification of methylation in the CG and CH context (H = A, C, or T). Unlike other differentiated cell types, neurons accumulate substantial mCH during the early years of life, coinciding with the period of synaptogenesis and brain maturation. Methods We performed genome-wide single-base resolution profiling of the composition, patterning, cell specificity, and dynamics of DNA methylation in the frontal cortex of humans and mice throughout their lifespan (MethylC-Seq). Furthermore, we generated base-resolution maps of 5-hydroxymethylcytosine (hmC) in mammalian brains by TAB-Seq at key developmental stages, accompanied by RNA-Seq transcriptional profiling. Results Extensive methylome reconfiguration occurs during development from fetal to young adult. In this period, coincident with synaptogenesis, highly conserved non-CG methylation (mCH) accumulates in neurons, but not glia, to become the dominant form of methylation in the human neuronal genome. We uncovered surprisingly complex features of brain cell DNA methylation at multiple scales, first by identifying intragenic methylation patterns in neurons and glia that distinguish genes with cell type–specific activity. Second, we report a novel mCH signature that identifies genes escaping X-chromosome inactivation in neurons. Third, we find >100,000 developmentally dynamic and cell type–specific differentially CG-methylated regions that are enriched at putative regulatory regions of the genome. Finally, whole-genome detection of 5-hydroxymethylcytosine (hmC) at single-base resolution revealed that this mark is present in fetal brain cells at locations that lose CG methylation and become activated during development. CG-demethylation at these hmC-poised loci depends on Tet2 activity. Discussion Whole-genome single-base resolution methylcytosine and hydroxymethylcytosine maps revealed profound changes during frontal cortex development in humans and mice. These results extend our knowledge of the unique role of DNA methylation in brain development and function, and offer a new framework for testing the role of the epigenome in healthy function and in pathological disruptions of neural circuits. Overall, brain cell DNA methylation has unique features that are precisely conserved, yet dynamic and cell-type specific. Epigenetic Brainscape Epigenetic modifications and their potential changes during development are of high interest, but few studies have characterized such differences. Lister et al. (1237905, published online 4 July; see the Perspective by Gabel and Greenberg) report whole-genome base-resolution analysis of DNA cytosine modifications and transcriptome analysis in the frontal cortex of human and mouse brains at multiple developmental stages. The high-resolution mapping of DNA cytosine methylation (5mC) and one of its oxidation derivatives (5hmC) at key developmental stages provides a comprehensive resource covering the temporal dynamics of these epigenetic modifications in neurons compared to glia. The data suggest that methylation marks are dynamic during brain development in both humans and mice. A genome-wide map shows that DNA methylation in neurons and glial cells changes during development in humans and mice. [Also see Perspective by Gabel and Greenberg] DNA methylation is implicated in mammalian brain development and plasticity underlying learning and memory. We report the genome-wide composition, patterning, cell specificity, and dynamics of DNA methylation at single-base resolution in human and mouse frontal cortex throughout their lifespan. Widespread methylome reconfiguration occurs during fetal to young adult development, coincident with synaptogenesis. During this period, highly conserved non-CG methylation (mCH) accumulates in neurons, but not glia, to become the dominant form of methylation in the human neuronal genome. Moreover, we found an mCH signature that identifies genes escaping X-chromosome inactivation. Last, whole-genome single-base resolution 5-hydroxymethylcytosine (hmC) maps revealed that hmC marks fetal brain cell genomes at putative regulatory regions that are CG-demethylated and activated in the adult brain and that CG demethylation at these hmC-poised loci depends on Tet2 activity.

Retroviral DNA Integration: ASLV, HIV, and MLV Show Distinct Target Site Preferences

The completion of the human genome sequence has made possible genome-wide studies of retroviral DNA integration. Here we report an analysis of 3,127 integration site sequences from human cells. We compared retroviral vectors derived from human immunodeficiency virus (HIV), avian sarcoma-leukosis virus (ASLV), and murine leukemia virus (MLV). Effects of gene activity on integration targeting were assessed by transcriptional profiling of infected cells. Integration by HIV vectors, analyzed in two primary cell types and several cell lines, strongly favored active genes. An analysis of the effects of tissue-specific transcription showed that it resulted in tissue-specific integration targeting by HIV, though the effect was quantitatively modest. Chromosomal regions rich in expressed genes were favored for HIV integration, but these regions were found to be interleaved with unfavorable regions at CpG islands. MLV vectors showed a strong bias in favor of integration near transcription start sites, as reported previously. ASLV vectors showed only a weak preference for active genes and no preference for transcription start regions. Thus, each of the three retroviruses studied showed unique integration site preferences, suggesting that virus-specific binding of integration complexes to chromatin features likely guides site selection.