A new type of molecular map reveals the 3-D architecture of the genome in individual cells1.
DNA folds and winds around proteins called histones to fit into the nucleus. The shape of this coiled complex, called chromatin, influences which genetic instructions are read and followed.
Scientists can work out the 3-D structure of the genome by breaking open cell nuclei, chemically freezing the folded chromatin in place and then sequencing stretches of DNA that intersect in the folded form. This technique, called Hi-C, reveals the approximate size and location of different DNA loops based on data from millions of cells. But it does not divulge variation in chromosome structure between cells.
The new approach, described 6 April in Nature, combines Hi-C with high-resolution images of chromosomes inside cells. The images provide a road map for analyzing Hi-C data from single cells.
To create the images, researchers engineered mouse embryonic stem cells to express two fluorescent tags. One of them labels a type of histone that acts as a spool for DNA. The other sticks to a histone found at the centromere, a region near the midpoint of a chromosome. Together, the tags outline the shape of each chromosome when viewed through a microscope.
The researchers used Hi-C to freeze the DNA folds inside eight of the cells. But instead of breaking open the nuclei to access the DNA, they created tiny holes in the nuclear membranes that allowed the Hi-C reagents inside. Confining the freezing step to the nucleus makes the reactions more efficient so that the researchers are able to analyze the small amount of chromatin in a single cell.
The researchers then used available computer software to match the Hi-C data from a single cell to the images of that cell’s chromosomes.
The new method revealed considerable variation in the fine structure of the genome from one cell to the next. It showed that small regions of chromosomes called topologically associating domains can end up in diverse locations. These chromosomal neighborhoods contain sequences that touch and regulate one another, rarely influencing distant DNA segments.
Scientists can use the method to determine how different patterns of DNA folding influence gene expression. Atypical folding patterns have been implicated in autism.