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.

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.

SUMMARY 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 non-expressing 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.

Maternal-to-zygotic transition (MZT) is essential for the formation of a new individual, but is still poorly understood despite recent progress in analysis of gene expression and DNA methylation in early embryogenesis1–9. Dynamic histone modifications may have important roles in MZT10–13, but direct measurements of chromatin states have been hindered by technical difficulties in profiling histone modifications from small quantities of cells. Recent improvements allow for 500 cell-equivalents of chromatin per reaction, but require 10,000 cells for initial steps14 or require a highly specialized microfluidics device that is not readily available15. We developed a micro-scale chromatin immunoprecipitation and sequencing (μChlP-seq) method, which we used to profile genome-wide histone H3 lysine methylation (H3K4me3) and acetylation (H3K27ac) in mouse immature and metaphase II oocytes and in 2-cell and 8-cell embryos. Notably, we show that ~22% of the oocyte genome is associated with broad H3K4me3 domains that are anti-correlated with DNA methylation. The H3K4me3 signal becomes confined to transcriptional-start-site regions in 2-cell embryos, concomitant with the onset of major zygotic genome activation. Active removal of broad H3K4me3 domains by the lysine demethylases KDM5A and KDM5B is required for normal zygotic genome activation and is essential for early embryo development. Our results provide insight into the onset of the developmental program in mouse embryos and demonstrate a role for broad H3K4me3 domains in MZT.

Summary Differential methylation of the two parental genomes in placental mammals is essential for genomic imprinting and embryogenesis. To systematically study this epigenetic process, we have generated a base-resolution, allele specific DNA methylation (ASM) map in the mouse genome. We find parent-of-origin dependent (imprinted) ASM at 1,952 CG dinucleotides. These imprinted CGs form 55 discrete clusters including virtually all known germline differentially methylated regions (DMRs) and 24 previously unknown DMRs, with some occurring at microRNA genes. We also identify sequence dependent ASM at 131,765 CGs. Interestingly, methylation at these sites exhibits a strong dependence on the immediate adjacent bases, allowing us to define a conserved sequence preference for the mammalian DNA methylation machinery. Finally, we report a surprising presence of non-CG methylation in the adult mouse brain, with some showing evidence of imprinting. Our results provide a resource for understanding the mechanisms of imprinting and allele-specific gene expression in mammalian cells.

SR Ca2+ ATPase 2a (SERCA2a) is a critical ATPase responsible for Ca2+ re-uptake during excitation-contraction coupling. Impaired SR Ca2+ uptake resulting from decreased expression and reduced activity of SERCA2a is a hallmark of heart failure (HF)1. Accordingly, restoration of SERCA2a expression by gene transfer has proven to be effective in improving cardiac function in HF patients2 as well as in animal models3. The small ubiquitin-related modifier (SUMO) can be conjugated to lysine residues of target proteins4, which is involved in most cellular process5. Here, we show that SERCA2a is SUMOylated at lysine 480 and 585 and that this SUMOylation is essential for preserving SERCA2a ATPase activity and stability. The levels of SUMO1 and SUMOylation of SERCA2a itself were greatly reduced in failing hearts. SUMO1 restitution by adeno-associated virus-mediated gene delivery maintained protein abundance of SERCA2a and significantly improved cardiac function in HF mice. This effect was comparable to SERCA2a gene delivery. Moreover, SUMO1 overexpression in isolated cardiomyocytes augmented contractility and accelerated Ca2+ decay. Transgene-mediated SUMO1 overexpression rescued pressure overload-induced cardiac dysfunction concomitantly with increased SERCA2a function. By contrast, down-regulation of SUMO1 using shRNA accelerated pressure overload-induced deterioration of cardiac function and was accompanied by decreased SERCA2a function. However, knockdown of SERCA2a resulted in severe contractile dysfunction both in vitro and in vivo, which was not rescued by overexpression of SUMO1. Taken together, our data show that SUMOylation is a critical post-translational modification that regulates SERCA2a function and provides a platform for the design of novel therapeutic strategies for HF.

The Encyclopedia of DNA Elements (ENCODE) project has established a genomic resource for mammalian development, profiling a diverse panel of mouse tissues at 8 developmental stages from 10.5 days after conception until birth, including transcriptomes, methylomes and chromatin states. Here we systematically examined the state and accessibility of chromatin in the developing mouse fetus. In total we performed 1,128 chromatin immunoprecipitation with sequencing (ChIP–seq) assays for histone modifications and 132 assay for transposase-accessible chromatin using sequencing (ATAC–seq) assays for chromatin accessibility across 72 distinct tissue-stages. We used integrative analysis to develop a unified set of chromatin state annotations, infer the identities of dynamic enhancers and key transcriptional regulators, and characterize the relationship between chromatin state and accessibility during developmental gene regulation. We also leveraged these data to link enhancers to putative target genes and demonstrate tissue-specific enrichments of sequence variants associated with disease in humans. The mouse ENCODE data sets provide a compendium of resources for biomedical researchers and achieve, to our knowledge, the most comprehensive view of chromatin dynamics during mammalian fetal development to date.

Summary In mammals, cytosine methylation (5mC) is widely distributed throughout the genome, but is notably depleted from active promoters and enhancers. While the role of DNA methylation in promoter silencing has been well documented, the function of this epigenetic mark at enhancers remains unclear. Recent experiments have demonstrated that enhancers are enriched for 5-hydroxymethylcytosine (5hmC), an oxidization product of the Tet family of 5mC dioxygenases and an intermediate of DNA demethylation. These results support the involvement of Tet proteins in regulation of dynamic DNA methylation at enhancers. By mapping DNA methylation and hydroxymethylation at base resolution, we find that deletion of Tet2 causes extensive loss of 5hmC at enhancers, accompanied by enhancer hypermethylation, reduction of enhancer activity, and delayed gene induction in the early steps of differentiation. Our results reveal that DNA demethylation modulates enhancer activity, and its disruption influences the timing of transcriptome reprogramming during cellular differentiation.