Matthew D. Schultz

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.

Transgenerational Epigenetic Instability Is a Source of Novel Methylation Variants

Spontaneous methylation rates that may affect phenotype in the plant Arabidopsis are higher than the mutation rate. Epigenetic information, which may affect an organism’s phenotype, can be stored and stably inherited in the form of cytosine DNA methylation. Changes in DNA methylation can produce meiotically stable epialleles that affect transcription and morphology, but the rates of spontaneous gain or loss of DNA methylation are unknown. We examined spontaneously occurring variation in DNA methylation in Arabidopsis thaliana plants propagated by single-seed descent for 30 generations. We identified 114,287 CG single methylation polymorphisms and 2485 CG differentially methylated regions (DMRs), both of which show patterns of divergence compared with the ancestral state. Thus, transgenerational epigenetic variation in DNA methylation may generate new allelic states that alter transcription, providing a mechanism for phenotypic diversity in the absence of genetic mutation.

Epigenomic Analysis of Multilineage Differentiation of Human Embryonic Stem Cells

Epigenetic mechanisms have been proposed to play crucial roles in mammalian development, but their precise functions are only partially understood. To investigate epigenetic regulation of embryonic development, we differentiated human embryonic stem cells into mesendoderm, neural progenitor cells, trophoblast-like cells, and mesenchymal stem cells and systematically characterized DNA methylation, chromatin modifications, and the transcriptome in each lineage. We found that promoters that are active in early developmental stages tend to be CG rich and mainly engage H3K27me3 upon silencing in nonexpressing lineages. By contrast, promoters for genes expressed preferentially at later stages are often CG poor and primarily employ DNA methylation upon repression. Interestingly, the early developmental regulatory genes are often located in large genomic domains that are generally devoid of DNA methylation in most lineages, which we termed DNA methylation valleys (DMVs). Our results suggest that distinct epigenetic mechanisms regulate early and late stages of ES cell differentiation.

Patterns of population epigenomic diversity.

Natural epigenetic variation provides a source for the generation of phenotypic diversity, but to understand its contribution to such diversity, its interaction with genetic variation requires further investigation. Here we report population-wide DNA sequencing of genomes, transcriptomes and methylomes of wild Arabidopsis thaliana accessions. Single cytosine methylation polymorphisms are not linked to genotype. However, the rate of linkage disequilibrium decay amongst differentially methylated regions targeted by RNA-directed DNA methylation is similar to the rate for single nucleotide polymorphisms. Association analyses of these RNA-directed DNA methylation regions with genetic variants identified thousands of methylation quantitative trait loci, which revealed the population estimate of genetically dependent methylation variation. Analysis of invariably methylated transposons and genes across this population indicates that loci targeted by RNA-directed DNA methylation are epigenetically activated in pollen and seeds, which facilitates proper development of these structures.

Human body epigenome maps reveal noncanonical DNA methylation variation

Understanding the diversity of human tissues is fundamental to disease and requires linking genetic information, which is identical in most of an individual’s cells, with epigenetic mechanisms that could have tissue-specific roles. Surveys of DNA methylation in human tissues have established a complex landscape including both tissue-specific and invariant methylation patterns. Here we report high coverage methylomes that catalogue cytosine methylation in all contexts for the major human organ systems, integrated with matched transcriptomes and genomic sequence. By combining these diverse data types with each individuals’ phased genome, we identified widespread tissue-specific differential CG methylation (mCG), partially methylated domains, allele-specific methylation and transcription, and the unexpected presence of non-CG methylation (mCH) in almost all human tissues. mCH correlated with tissue-specific functions, and using this mark, we made novel predictions of genes that escape X-chromosome inactivation in specific tissues. Overall, DNA methylation in several genomic contexts varies substantially among human tissues.

Abnormalities in human pluripotent cells due to reprogramming mechanisms

Human pluripotent stem cells hold potential for regenerative medicine, but available cell types have significant limitations. Although embryonic stem cells (ES cells) from in vitro fertilized embryos (IVF ES cells) represent the ‘gold standard’, they are allogeneic to patients. Autologous induced pluripotent stem cells (iPS cells) are prone to epigenetic and transcriptional aberrations. To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method, genetically matched sets of human IVF ES cells, iPS cells and nuclear transfer ES cells (NT ES cells) derived by somatic cell nuclear transfer (SCNT) were subjected to genome-wide analyses. Both NT ES cells and iPS cells derived from the same somatic cells contained comparable numbers of de novo copy number variations. In contrast, DNA methylation and transcriptome profiles of NT ES cells corresponded closely to those of IVF ES cells, whereas iPS cells differed and retained residual DNA methylation patterns typical of parental somatic cells. Thus, human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal for cell replacement therapies.

Stress induced gene expression drives transient DNA methylation changes at adjacent repetitive elements

Cytosine DNA methylation (mC) is a genome modification that can regulate the expression of coding and non-coding genetic elements. However, little is known about the involvement of mC in response to environmental cues. Using whole genome bisulfite sequencing to assess the spatio-temporal dynamics of mC in rice grown under phosphate starvation and recovery conditions, we identified widespread phosphate starvation-induced changes in mC, preferentially localized in transposable elements (TEs) close to highly induced genes. These changes in mC occurred after changes in nearby gene transcription, were mostly DCL3a-independent, and could partially be propagated through mitosis, however no evidence of meiotic transmission was observed. Similar analyses performed in Arabidopsis revealed a very limited effect of phosphate starvation on mC, suggesting a species-specific mechanism. Overall, this suggests that TEs in proximity to environmentally induced genes are silenced via hypermethylation, and establishes the temporal hierarchy of transcriptional and epigenomic changes in response to stress. DOI: http://dx.doi.org/10.7554/eLife.09343.001

‘Leveling’ the playing field for analyses of single-base resolution DNA methylomes

Over the past few years the cost of DNA sequencing has plummeted while the numbers and lengths of sequencing reads have increased. This sequencing revolution has led to widespread adoption of methods to investigate genome-wide patterns of DNA methylation, collectively referred to as whole-genome bisulfite sequencing (WGBS). Single-base resolution DNA methylomes are now routinely being decoded by combining high-throughput sequencing with sodium bisulfite conversion, the gold-standard method for the detection of cytosine DNA methylation [1,2]. With increasing acquisition and analysis of DNA methylomes there is a growing need to reach a consensus on the definition(s) of the amount of methylation at a specific cytosine or region. ‘Methylation level’ is often poorly defined, and can vary significantly depending on the experimenter and the queries being addressed. Therefore, we propose a set of guidelines to be considered when analyzing ‘methylation levels’ from WGBS data.

The Developmental Potential of iPSCs Is Greatly Influenced by Reprogramming Factor Selection

Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliably high-quality iPSCs will be needed for future therapeutic applications. Here, we show that one major determinant of iPSC quality is the combination of reprogramming factors used. Based on tetraploid complementation, we found that ectopic expression of Sall4, Nanog, Esrrb, and Lin28 (SNEL) in mouse embryonic fibroblasts (MEFs) generated high-quality iPSCs more efficiently than other combinations of factors including OSKM. Although differentially methylated regions, transcript number of master regulators, establishment of specific superenhancers, and global aneuploidy were comparable between high- and low-quality lines, aberrant gene expression, trisomy of chromosome 8, and abnormal H2A.X deposition were distinguishing features that could potentially also be applicable to human.

Release Factor One Is Nonessential in Escherichia coli

Recoding a stop codon to an amino acid may afford orthogonal genetic systems for biosynthesizing new protein and organism properties. Although reassignment of stop codons has been found in extant organisms, a model organism is lacking to investigate the reassignment process and to direct code evolution. Complete reassignment of a stop codon is precluded by release factors (RFs), which recognize stop codons to terminate translation. Here we discovered that RF1 could be unconditionally knocked out from various Escherichia coli stains, demonstrating that the reportedly essential RF1 is generally dispensable for the E. coli species. The apparent essentiality of RF1 was found to be caused by the inefficiency of a mutant RF2 in terminating all UAA stop codons; a wild type RF2 was sufficient for RF1 knockout. The RF1-knockout strains were autonomous and unambiguously reassigned UAG to encode natural or unnatural amino acids (Uaas) at multiple sites, affording a previously unavailable model for studying code evolution and a unique host for exploiting Uaas to evolve new biological functions.