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.

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.

Integrative analysis of 111 reference human epigenomes.

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.

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.

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.

Dynamic and rapid changes in the transcriptome and epigenome during germination and in developing rice (Oryza sativa) coleoptiles under anoxia and re-oxygenation.

Detailed molecular profiling of Oryza sativa (rice) was carried out to uncover the features that are essential for germination and early seedling growth under anoxic conditions. Temporal analysis of the transcriptome and methylome from germination to young seedlings under aerobic and anaerobic conditions revealed 82% similarity in the transcriptome and no differences in the epigenome up to 24 h. Following germination, significant changes in the transcriptome and DNA methylation were observed between 4-day aerobically and anaerobically grown coleoptiles. A link between the epigenomic state and cell division versus cell elongation is suggested, as no differences in DNA methylation were observed between 24-h aerobically and anaerobically germinating embryos, when there is little cell division. After that, epigenetic changes appear to correlate with differences between cell elongation (anaerobic conditions) versus cell division (aerobic conditions) in the coleoptiles. Re-oxygenation of 3-day anaerobically grown seedlings resulted in rapid transcriptomic changes in DNA methylation in these coleoptiles. Unlike the transcriptome, changes in DNA methylation upon re-oxygenation did not reflect those seen in aerobic coleoptiles, but instead, reverted to a pattern similar to dry seeds. Reversion to the 'dry seed' state of DNA methylation upon re-oxygenation may act to 'reset the clock' for the rapid molecular changes and cell division that result upon re-oxygenation.

Epigenomic programming contributes to the genomic drift evolution of the F-Box protein superfamily in Arabidopsis.

Comparisons within expanding sequence databases have revealed a dynamic interplay among genomic and epigenomic forces in driving plant evolution. Such forces are especially obvious within the F-Box (FBX) superfamily, one of the largest and most polymorphic gene families in land plants, where its frequent lineage-specific expansions and contractions provide an excellent model to assess how genetic variation impacted gene function before and after speciation. Previous phylogenetic comparisons based on orthology, diversity, and expression patterns identified three plant FBX groups--Common, Lineage-Specific, and Pseudo(genized)--whose emergences are consistent with genomic drift evolution. Here, we examined this variance within Arabidopsis thaliana by evaluating SNPs for all 877 FBX loci from 432 naturally occurring accessions and their relationships to variations in natural selection, expression, and DNA/histone methylation. In line with their phenotypic importance, Common FBX loci have low polymorphism but high deleterious mutation rates indicative of stringent functional constraints. In contrast, the Lineage-Specific and Pseudo groups are enriched in genes with basal expression and higher SNP density and more correlated with methylation marks (RNA-directed DNA methylation and histone H3K27 trimethylation) that promote transcriptional silencing. Taken together, we propose that reversible epigenomic modifications helped shape FBX gene evolution by transcriptionally suppressing the adverse effects of gene dosage imbalance and harmful FBX alleles that arise during genomic drift, while simultaneously allowing innovations to emerge through epigenomic reprogramming.

RF1 knockout allows ribosomal incorporation of unnatural amino acids at multiple sites.

Stop codons have been exploited for genetic incorporation of unnatural amino acids (Uaas) in live cells, but their low incorporation efficiency, which is possibly due to competition from release factors, limits the power and scope of this technology. Here we show that the reportedly essential release factor 1 (RF1) can be knocked out from Escherichia coli by 'fixing' release factor 2 (RF2). The resultant strain JX33 is stable and independent, and it allows UAG to be reassigned from a stop signal to an amino acid when a UAG-decoding tRNA-synthetase pair is introduced. Uaas were efficiently incorporated at multiple UAG sites in the same gene without translational termination in JX33. We also found that amino acid incorporation at endogenous UAG codons is dependent on RF1 and mRNA context, which explains why E. coli tolerates apparent global suppression of UAG. JX33 affords a unique autonomous host for synthesizing and evolving new protein functions by enabling Uaa incorporation at multiple sites.

Active DNA demethylation at enhancers during the vertebrate phylotypic period.

The vertebrate body plan and organs are shaped during a conserved embryonic phase called the phylotypic stage. However, the mechanisms that guide the epigenome through this transition and their evolutionary conservation remain elusive. Here we report widespread DNA demethylation of enhancers during the phylotypic period in zebrafish, Xenopus tropicalis and mouse. These enhancers are linked to developmental genes that display coordinated transcriptional and epigenomic changes in the diverse vertebrates during embryogenesis. Binding of Tet proteins to (hydroxy)methylated DNA and enrichment of 5-hydroxymethylcytosine in these regions implicated active DNA demethylation in this process. Furthermore, loss of function of Tet1, Tet2 and Tet3 in zebrafish reduced chromatin accessibility and increased methylation levels specifically at these enhancers, indicative of DNA methylation being an upstream regulator of phylotypic enhancer function. Overall, our study highlights a regulatory module associated with the most conserved phase of vertebrate embryogenesis and suggests an ancient developmental role for Tet dioxygenases.

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.

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.

Global epigenomic reconfiguration during mammalian brain development.

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.

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.

Surveillance of 3' Noncoding Transcripts Requires FIERY1 and XRN3 in Arabidopsis.

Eukaryotes possess several RNA surveillance mechanisms that prevent undesirable aberrant RNAs from accumulating. Arabidopsis XRN2, XRN3, and XRN4 are three orthologs of the yeast 5'-to-3' exoribonuclease, Rat1/Xrn2, that function in multiple RNA decay pathways. XRN activity is maintained by FIERY1 (FRY1), which converts the XRN inhibitor, adenosine 3', 5'-bisphosphate (PAP), into 5'AMP. To identify the roles of XRNs and FRY1 in suppression of non-coding RNAs, strand-specific genome-wide tiling arrays and deep strand-specific RNA-Seq analyses were carried out in fry1 and xrn single and double mutants. In fry1-6, about 2000 new transcripts were identified that extended the 3' end of specific mRNAs; many of these were also observed in genotypes that possess the xrn3-3 mutation, a partial loss-of-function allele. Mutations in XRN2 and XRN4 in combination with xrn3-3 revealed only a minor effect on 3' extensions, indicating that these genes may be partially redundant with XRN3. We also observed the accumulation of 3' remnants of many DCL1-processed microRNA (miRNA) precursors in fry1-6 and xrn3-3. These findings suggest that XRN3, in combination with FRY1, is required to prevent the accumulation of 3' extensions that arise from thousands of mRNA and miRNA precursor transcripts.

Transgenerational epigenetic instability is a source of novel methylation variants.

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.