Understanding Spatial Genome Organization: Methods and Insights

Abstract

The manner by which eukaryotic genomes are packaged into nuclei while maintaining crucial nuclear functions remains one of the fundamental mysteries in biology. Over the last ten years, we have witnessed rapid advances in both microscopic and nucleic acid-based approaches to map genome architecture, and the application of these approaches to the dissection of higher-order chromosomal structures has yielded much new information. It is becoming increasingly clear, for example, that interphase chromosomes form stable, multilevel hierarchical structures. Among them, self-associating domains like so-called topologically associating domains (TADs) appear to be building blocks for large-scale genomic organization. This review describes features of these broadly-defined hierarchical structures, insights into the mechanisms underlying their formation, our current understanding of how interactions in the nuclear space are linked to gene regulation, and important future directions for the field.

Keywords: 4D nucleome; Chromatin; Chromosome; Epigenomics; Hi-C.

Figure 1

High-throughput biochemical techniques for probing the nucleome High-throughput methods for probing the nucleome can broadly be grouped into three classes. (1) Methods detecting protein–DNA interactions include ChIP-seq, where antibodies specific to proteins of interest are used to co-precipitate crosslinked genomic DNA, and DNA adenine methyltransferase identification (DamID), in which a bacterial adenine methylase is used to methylate physically-proximal adenines. (2) Methods detecting RNA–DNA interactions include ChIRP, CHART, and RAP. Crosslinked chromatin is sheared and then hybridized to biotinylated anti-sense oligonucleotides specific to a transcript or transcripts of interest. In all of these methods, tagged or purified DNA is used to create a massively-parallel sequencing library. (3) The 3C family of methods are used to probe DNA–DNA interactions. While there are many different types of 3C assay, all 3C-based methods share the same core concept: chromatin interactions are measured by proximity ligation of fragmented and crosslinked chromatin. The key differences between these methods lie in how chromatin interactions are detected following proximity ligation. In ChIA-PET, crosslinked chromatin complexes are fragmented by sonication and chromatin interactions mediated by a protein of interest are enriched by ChIP before performing the proximity ligation. In 3C, 4C, and 5C, chromatin interactions of interest are enriched by PCR using locus-specific primers. In Hi-C and its variants, the valid chromatin interactions are enriched through a streptavidin-biotin-mediated pull-down. In targeted Hi-C methods, such as Capture-C, Capture Hi-C, and targeted DNase Hi-C, chromatin interactions of interest are enriched by applying hybrid capture technologies to 3C or Hi-C libraries.