The spatial organization of the genome is intimately linked to its biological function, yet our understanding of higher order genomic structure is coarse, fragmented and incomplete. In the nucleus of eukaryotic cells, interphase chromosomes occupy distinct chromosome territories (CT), and numerous models have been proposed for how chromosomes fold within CTs1. These models, however, provide only few mechanistic details about the relationship between higher order chromatin structure and genome function. Recent advances in genomic technologies have led to rapid revolutions in the study of 3D genome organization. In particular, Hi-C has been introduced as a method for identifying higher order chromatin interactions genome wide2. In the present study, we investigated the 3D organization of the human and mouse genomes in embryonic stem cells and terminally differentiated cell types at unprecedented resolution. We identify large, megabase-sized local chromatin interaction domains, which we term “topological domains”, as a pervasive structural feature of the genome organization. These domains correlate with regions of the genome that constrain the spread of heterochromatin. The domains are stable across different cell types and highly conserved across species, suggesting that topological domains are an inherent property of mammalian genomes. Lastly, we find that the boundaries of topological domains are enriched for the insulator binding protein CTCF, housekeeping genes, tRNAs, and SINE retrotransposons, suggesting that these factors may play a role in establishing the topological domain structure of the genome.
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 The study of 5-hydroxylmethylcytosines (5hmC) has been hampered by the lack of a method to map it at single-base resolution on a genome-wide scale. Affinity purification-based methods cannot precisely locate 5hmC nor accurately determine its relative abundance at each modified site. We here present a genome-wide approach, Tet-assisted Bisulfite Sequencing (TAB-Seq), for mapping 5hmC at base resolution and quantifying the relative abundance of 5hmC as well as 5mC when combined with traditional bisulfite sequencing. Application of this method to embryonic stem cells not only confirms widespread distribution of 5hmC in the mammalian genome, but also reveals sequence bias and strand asymmetry at 5hmC sites. We observe high levels of 5hmC and reciprocally low levels of 5mC near but not on transcription factor binding sites. Additionally, the relative abundance of 5hmC varies significantly among distinct functional sequence elements, suggesting different mechanisms for 5hmC deposition and maintenance.
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.
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.
Nuclear architecture defects have been shown to correlate with the manifestation of a number of human diseases as well as aging1-4. It is then plausible that diseases whose manifestations correlate with aging might be connected to the appearance of nuclear aberrations over time. We decided to evaluate nuclear organization in the context of aging-associated disorders by focusing on a Leucine Rich Repeat Kinase 2 (LRRK2) dominant mutation (G2019S) shown to associate with familial and sporadic Parkinson’s Disease (PD), as well as impairment of adult neurogenesis in mice5. Here, we report on the generation of PD patient-derived induced pluripotent stem cells (iPSCs) and the implications of LRRK2(G2019S) in human neural stem cell (NSC) populations. Mutant NSCs showed increased susceptibility to proteasomal stress as well as passage-dependent deficiencies in clonal expansion and neuronal differentiation. Disease phenotypes were rescued by targeted correction of the LRRK2(G2019S) mutation with its wild-type counterpart in PD-iPSCs and recapitulated upon targeted knock-in of LRRK2(G2019S) in human embryonic stem cells (hESCs). Analysis of human brain tissue showed nuclear envelope impairment in clinically diagnosed Parkinson’s patients. Altogether, our results identify the nucleus as a previously unknown cellular organelle in Parkinson’s pathology and may help open new avenues for PD diagnoses as well as potential development of therapeutics targeting this fundamental cell structure.
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