The three dimensional organisation of the chromosomes within the nucleus is important in forming interactions between enhancers and silencers. Each chromosome resides within its own ‘territory’, giving it a specific area of the nucleus that it fills. Lamina associated domains (LADs) are regions of the chromosome bound to the nuclear lamina, and are transcriptionally silent. LADs help hold the chromosomes in the correct positions. DNA-DNA interactions can occur on very large linear distances, but due to the three dimensional organisation of the nucleus, distant regulatory regions can be in close proximity.
Distal enhancers can be located many thousands of bases away from the gene on which they have an impact.
These DNA-DNA interactions can be studied by chromosomal conformation capture. This is done by treating the DNA with formaldehyde to form crosslinks between the strands. In the case of 3C, this allows the use of primers of known sequences to determine whether there are interactions between two known regions. 3C requires the knowledge of which regions are being studied, as the primers must be specific. Other techniques are more flexible. Hi-C, the most recent development in chromosome conformation capture, labels the crosslinks with biotin. This can then be precipitated with an antibody, and the excess DNA degraded with a DNAse. Sequencing can then allow the mapping of crosslinked regions to a reference genome. 5C uses universal primers to amplify crosslinked regions, before using microarray or sequencing to identify the interacting regions.