The 4 D nucleome project

t he human genome contains over 20,000 genes and a larger number of regulatory elements. Large-scale studies over the last decade have catalogued these components of our genome and the cell types in which they are active. The ENCODE, Roadmap Epigenome, International Human Epigenome Consortium, EpiGeneSys (http://www. epigenesys.eu/en/) and FANTOM projects1–4 have annotated thousands of genes and millions of candidate regulatory elements. However, our understanding of the mechanisms by which these elements exert regulatory effects on specific target genes across distances of kilobases, and in some cases megabases, remains incomplete. The spatial folding of chromosomes and their organization in the nucleus have profound effects on gene expression. For example, spatial proximity is necessary for enhancers to modulate transcription of target genes (for example, refs 5–7), and clustering of chromatin near the nuclear lamina is correlated with gene silencing and replication timing8,9. In addition, genome-wide association studies have identified large numbers of disease-associated loci, and the majority of these loci are located in distal, potentially regulatory, noncoding regions (for example, ref. 10). In cancer cells, genomic rearrangements frequently occur and these are at least in part guided by the three-dimensional organization of the nucleus11,12. These data emphasize the importance of distal elements for gene regulation and suggest an exciting opportunity to uncover the fundamental mechanisms of disease through the mapping of long-range chromatin interactions and three-dimensional genome organization. Therefore, to determine how the genome operates, we need to understand not only the linear encoding of information along chromosomes, but also its three-dimensional organization and its dynamics across time, that is, the ‘4D nucleome’. Concomitantly, we must pursue deeper knowledge of the biophysical and molecular factors that determine genome organization, and how this organization contributes to gene regulation and other nuclear activities. Here we outline the goals and strategies of the 4D Nucleome (4DN) Network. This Network builds on other consortia and efforts focusing on (epi-)genome analysis outlined above and adds spatial and temporal dimensions to explore how the genome is organized inside cells and how this relates to genome function. The nucleus is not a homogeneous organelle, but consists of distinct nuclear structures and non-chromatin bodies as well as defined chromosomal regions, such as centromeres, telomeres and insulator bodies, that have been shown to cluster with each other and other genomic regions to define distinct nuclear compartments13,14. Examples of nuclear structures include the nuclear lamina and nuclear pores. Examples of nuclear compartments include the heterochromatic compartment, while examples of nuclear bodies include nucleoli, nuclear speckles, paraspeckles, and Cajal and PML (promyelocytic leukaemia) bodies. Chromosome conformation capture (3C) approaches15,16 have yielded additional insights by characterizing genome-wide chromatin folding at kilobase resolution6,17,18. These studies have shown that the genome is compartmentalized in active and inactive spatial compartments at the scale of the nucleus, and that within each compartment, folding of chromatin fibres brings together loci and regulatory elements that are otherwise separated by large genomic distances. CTCF, the cohesin complex and other DNA-binding proteins, as well as RNAs, have roles in organizing chromatin domains and longrange interactions between DNA loci18–24. These studies indicate that the genome is intricately organized within the nucleus and that this organization has a critical role in gene regulation and activity. During the past decade marked innovation in chromosome and nuclear structure analysis has occurred. Genomic approaches for mapping chromatin interactions, such as 3C, 4C (circular 3C, or 3C-onchip), 5C (3C-carbon copy), Hi-C, and chromatin-interaction analysis by paired-end-tag sequencing (ChIA-PET)16, are yielding genome-wide chromatin interaction maps at unprecedented resolution. Live-cell and super-resolution microscopic approaches, combined with application of new ways (for example, CRISPR–Cas9-based systems) to visualize loci and sub-nuclear structures are beginning to provide detailed views of the organization and dynamics of chromatin inside (living) cells25–31. There has also been pronounced progress in analysing chromosome structural data, producing structural models for chromosome folding32,33. However, despite this progress, a comprehensive understanding of the 4D nucleome is still lacking. This is partly due to the fact that different experimental cell systems and approaches are used that together with the absence of shared The 4D Nucleome Network aims to develop and apply approaches to map the structure and dynamics of the human and mouse genomes in space and time with the goal of gaining deeper mechanistic insights into how the nucleus is organized and functions. The project will develop and benchmark experimental and computational approaches for measuring genome conformation and nuclear organization, and investigate how these contribute to gene regulation and other genome functions. Validated experimental technologies will be combined with biophysical approaches to generate quantitative models of spatial genome organization in different biological states, both in cell populations and in single cells.