Summary DNA cytosine methylation is a central epigenetic modification that plays 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 mRNA 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 they may utilize 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.

The human body is composed of diverse cell types with distinct functions. While it is known that lineage specification depends on cell specific gene expression, which in turn is driven by promoters, enhancers, insulators and other cis-regulatory DNA sequences for each gene1–3, the relative roles of these regulatory elements in this process is not clear. We have previously developed a chromatin immunoprecipitation-based microarray method (ChIP-chip) to locate promoters, enhancers, and insulators in the human genome4–6. Here, we use the same approach to identify these elements in multiple cell types and investigated their roles in cell type-specific gene expression. We observed that chromatin state at promoters and CTCF-binding at insulators are largely invariant across diverse cell types. By contrast, enhancers are marked with highly cell type-specific histone modification patterns, strongly correlate to cell type-specific gene expression programs on a global scale, and are functionally active in a cell type-specific manner. Our results defined over 55,000 potential transcriptional enhancers in the human genome, significantly expanding the current catalog of human enhancers and highlighting the role of these elements in cell type-specific gene expression.

The laboratory mouse is the most widely used mammalian model organism in biomedical research. The 2.6 × 109 bases of the mouse genome possess a high degree of conservation with the human genome1, so a thorough annotation of the mouse genome will be of significant value to understanding the function of the human genome. So far, most of the functional sequences in the mouse genome have yet to be found, and the cis-regulatory sequences in particular are still poorly annotated. Comparative genomics has been a powerful tool for the discovery of these sequences2, but on its own it cannot resolve their temporal and spatial functions. Recently, ChIP-Seq has been developed to identify cis-regulatory elements in the genomes of several organisms including humans, Drosophila melanogaster and Caenorhabditis elegans3–5. Here we apply the same experimental approach to a diverse set of 19 tissues and cell types in the mouse to produce a map of nearly 300,000 murine cis-regulatory sequences. The annotated sequences add up to 11% of the mouse genome, and include more than 70% of conserved non-coding sequences. We define tissue-specific enhancers and identify potential transcription factors regulating gene expression in each tissue or cell type. Finally, we show that much of the mouse genome is organized in to domains of coordinately regulated enhancers and promoters. Our results provide a resource for the annotation of functional elements in the mammalian genome and for the study of mechanisms regulating tissue-specific gene expression.

Higher order chromatin structure is emerging as an important regulator of gene expression. Although dynamic chromatin structures have been identified in the genome, the full scope of chromatin dynamics during mammalian development and lineage specification remains obscure. By mapping genome-wide chromatin interactions in human embryonic stem cells (hESC) and four hESC-derived lineages, we uncover extensive chromatin reorganization during lineage specification. We observe that while topological domain boundaries remain intact during differentiation, interactions both within and between domains change dramatically, altering 36% of active and inactive chromosomal “compartments” throughout the genome. By integrating chromatin interaction maps with haplotype-resolved epigenome and transcriptome datasets, we find widespread allelic bias in gene expression correlated with allele-biased chromatin states of linked promoters and distal enhancers. Our results therefore provide a global view of chromatin dynamics and a resource for studying long-range control of gene expression in distinct human cell lineages.

Summary Using an integrated approach, we here report the identification of 67 novel histone marks, a discovery that increases the current number of known histone marks by about 70%. We verified one of the newly-identified marks, lysine crotonylation (Kcr), as a novel, evolutionarily-conserved histone post-translational modification. The unique structure and genomic localization of Kcr suggest that it is mechanistically and functionally different from lysine acetylation (Kac), a previously-described post-translational modification. Specifically, in both human somatic and mouse male germ cell genomes, histone Kcr marks either active promoters or potential enhancers. In male germinal cells immediately following meiosis, Kcr is enriched on sex chromosomes and specifically marks testis-specific genes, including a significant proportion of X-linked genes that escape sex chromosome inactivation in haploid cells. These results therefore dramatically extend the repertoire of histone PTM sites and designate Kcr as a specific mark of active sex chromosome-linked genes in post-meiotic male germ cells.

SummaryAs the premier model organism in biomedical research, the laboratory mouse shares the majority of protein-coding genes with humans, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications, and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of other sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.

to mass spectrometry related experiments and analysis; R.H., Z.Y. and B.R. performed the library construction and next-generation sequencing for ChIP-seq and RNA-seq; M.H. and Y.G.Z. synthesized L-lactyl-CoA. H.H. and D.Z. analyzed ChIP-seq and RNA-seq data. G.Z. provided all primary BMDM cell cultures. D.M.C. carried out the bacterial infection experiments, C.C. carried out TAM experiments. Author Information. Y.Z. is a founder, board member, advisor to, and inventor on patents licensed to PTM Bio Inc. L.B. is co-founder and CSO of rMark Bio Inc., and founder and CEO of Onchilles Pharma Inc. Readers are welcome to comment on the online version of the paper. Data availability. The ChIP-seq and RNA-seq data have been made available at the Gene Expression Omnibus (GEO) repository under the accession number GSE115354. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 31 partner repository with the dataset identifier PXD014870. All other data are available from the authors upon reasonable request.

A large number of cis-regulatory sequences have been annotated in the human genome1,2, but defining their target genes remains a challenge3. One strategy is to identify the long-range looping interactions at these elements with the use of chromosome conformation capture (3C) based techniques4. However, previous studies lack either the resolution or coverage to permit a whole-genome, unbiased view of chromatin interactions. Here, we report a comprehensive chromatin interaction map generated in human fibroblasts using a genome-wide 3C analysis method (Hi-C)5. We determined over one million long-range chromatin interactions at 5–10kb resolution, and uncovered general principles of chromatin organization at different types of genomic features. We also characterized the dynamics of promoter-enhancer contacts upon TNF-α signaling in these cells. Unexpectedly, we found that TNF-α responsive enhancers are already in contact with their target promoters prior to signaling. Such pre-existing chromatin looping, which also exists in other cell types with different extra-cellular signaling, is a strong predictor of gene induction. Our observations suggest that the three-dimensional chromatin landscape, once established in a particular cell type, is rather stable and could influence the selection or activation of target genes by a ubiquitous transcription activator in a cell-specific manner.