Genomics tools for unraveling chromosome architecture

Abstract

The spatial organization of chromosomes inside the cell nucleus is still poorly understood. This organization is guided by intra- and interchromosomal contacts and by interactions of specific chromosomal loci with relatively fixed nuclear 'landmarks' such as the nuclear envelope and the nucleolus. Researchers have begun to use new molecular genome-wide mapping techniques to uncover both types of molecular interactions, providing insights into the fundamental principles of interphase chromosome folding.

Figure 1

Cartoon of nucleus depicting the spatial interactions that contribute to the overall architecture of interphase chromosomes. Table on the right summarizes the techniques that are currently used to map the respective interactions genome-wide.

Figure 2

Mapping of interactions of the genome with nuclear landmarks, here illustrated for the NL. See text for explanation. Adenine-methylated DNA is specifically amplified using a PCR-based protocol that employs restriction endonucleases that selectively digest DNA depending on the adenine-methylation state, as described elsewhere,.

Figure 3

Principles of the major 3C-based technologies. All protocols start with treatment of cells with formaldehyde (not shown), resulting into crosslinking of DNA segments that are in close proximity of one another. After digestion with one or more restriction enzymes linked restriction fragments are intramolecularly ligated. In the case of Hi-C the ends of the restriction fragments are first filled in with biotinylated dNTPs prior to ligation to facilitate purification of ligation junctions using streptavidin-coated beads. Either single or multiple ligation events are detected directly (3C, 4C, 5C and Hi-C), or first immunoprecipitation is used to enrich for DNA that is associated with a protein of interest (ChIP-loop, Chia-PET). See table 1 for an overview of the different detection strategies and their scope.

Figure 4

Speculative cartoon model of chromatin organization. LADs may consist of relatively condensed chromatin (thick lines) and aggregate at the NL. Other repressed regions may interact with each other in the nuclear interior, as do active regions. Complexes formed by components of the transcription machinery (“transcription factories”) and CTCF may tether active regions together. Parts of only two chromosomes are depicted, each drawn in a different color for clarity. Most interactions occur within chromosomes, and relatively few between chromosomes.