A decade of 3C technologies: insights into nuclear organization

Abstract

Over the past 10 years, the development of chromosome conformation capture (3C) technology and the subsequent genomic variants thereof have enabled the analysis of nuclear organization at an unprecedented resolution and throughput. The technology relies on the original and, in hindsight, remarkably simple idea that digestion and religation of fixed chromatin in cells, followed by the quantification of ligation junctions, allows for the determination of DNA contact frequencies and insight into chromosome topology. Here we evaluate and compare the current 3C-based methods (including 4C [chromosome conformation capture-on-chip], 5C [chromosome conformation capture carbon copy], HiC, and ChIA-PET), summarize their contribution to our current understanding of genome structure, and discuss how shape influences genome function.For more than a century, researchers have been making inquiries into the organization of the nucleus, the largest and most easily discernable organelle in the eukaryotic cell. Early in the 20th Century, Cajal (1903) identified subnuclear structures that were later named Cajal bodies. Twenty-five years later, Heitz (1928) observed differentially staining chromatin in interphase nuclei of mosses and described it as heterochromatin and euchromatin. Interest in nuclear structure was further fueled by the realization that the nucleus contains genetic material in the form of DNA fibers. In humans, when unwound, the DNA measures ;2 m, ;200,000 times the diameter of an average mammalian cell nucleus. Packing DNA inside the nucleus therefore imposes tremendous organizational challenges. While already conceptually interesting, the shape of the genome becomes even more fascinating when one realizes that it also relates to genome functioning. Although we are still far from understanding this exact relationship, breakthrough technologies are now available for the systematic and detailed analysis of nuclear organization.Traditionally, nuclear organization is studied by microscopy, and thus it is appropriate to start by highlighting some important observations made under the microscope.However, the emphasis of this review is on novel genomics strategies that are based on chromosome conformation capture (3C) technology. Ten years ago, Dekker et al. (2002) developed 3C technology, a biochemical strategy to analyze contact frequencies between selected genomic sites in cell populations. Since then, various 3C-derived genomics methods have been developed. In comparison with microscopy, 3C-based methods enable more systematic DNA topology studies at a higher resolution: These technologies can put observations made on single genes in selected cells in the context of genomic behavior in cell populations. The generated DNA contact maps start teaching us the rules that dictate genome structure and functioning inside the cell. As we explain here, these rules are probabilistic, not deterministic, implying that they cannot predict the shape and functioning of the genome in individual cells. To investigate this, microscopy i...

Promoter architectures and developmental gene regulation

“…It appears that the enhancer loops form prior to gene activation and stably associate with paused RNAPII at promoters, keeping this loop topology ready for rapid activation of transcription by recruitment of additional factors [116]. The formation of chromatin loops brings the enhancer and its target promoter into close physical proximity in the nucleus and this feature is utilised by chromatin conformation capture experimental approaches [117] to detect long-range interactions genomewide [118] and to identify target promoters of specific regulatory elements. However, the knowledge about the specificity of promoter-enhancer interactions is still very limited.…”

Topologically-associating domains: gene warehouses adapted to serve transcriptional regulation

“…7 Using the Hi-C approach, Lieberman-Aiden and colleagues have demonstrated that in mammals, transcriptionally active and inactive regions of the genome are spatially segregated within the 3D nuclear space, forming A and B compartments, respectively. 8 Both compartments appear to be partitioned into megabase-and sub-megabase-scale topologically-associating (self-interacting) domains (TADs).…”

Single-cell Hi-C for genome-wide detection of chromatin interactions that occur simultaneously in a single cell

“…This organization is an emerging area of importance, as the function and activity of loci are strongly correlated with their 3D conformations 1,2 . To explore such chromosome interactions at a genome-wide level, chromosome conformation capture (3C)-based high-throughput methods, in particular Hi-C, have been extensively used [3][4][5] . Hi-C measures pairwise contact frequencies between all loci in the genome using populations of millions of cells.…”

“…Hi-C measures pairwise contact frequencies between all loci in the genome using populations of millions of cells. These data have led to advances in the understanding of higher-order organization of the genome, such as chromatin loop formation and the discovery of topologically associating domains [6][7][8] , and they have been used to build 3D models of chromatin 3,9,10 . However, it is important to note that Hi-C data represent an ensemble average of millions of cells, and decades of microscopy studies have shown that nuclear organization is not uniform, even among cells of a homogeneous population 11 .…”