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Single-Cell Hi-C Approach Models Chromosomes, Finding Variability

NEW YORK (GenomeWeb News) – By zooming in on the chromosomes of single cells, researchers led by Peter Fraser from the Babraham Institute in the UK found that chromosome structure varies between cells, as they reported in Nature today.

To get a better glimpse of chromosome structure, the researchers adapted the Hi-C approach, which enables the detection of long-range interactions among chromosomes, for single cells. Conventional Hi-C method, the researchers noted, gives an average conformation, while examining cells individually might enable researchers to investigate cell-to-cell variation. From this, the researchers noted that chromosome structure was more complex and variable than that of the traditional X-shape in which they are often depicted.

"The image of a chromosome, an X-shaped blob of DNA, is familiar to many but this microscopic portrait of a chromosome actually shows a structure that occurs only transiently in cells — at a point when they are just about to divide," Fraser noted in a statement. "The vast majority of cells in an organism have finished dividing and their chromosomes don't look anything like the X-shape. Chromosomes in these cells exist in a very different form and so far it has been impossible to create accurate pictures of their structure."

Chromosome structure and DNA folding are linked to both gene regulation and expression, with possible ramifications for aging, disease, and health.

The researchers' single-cell Hi-C approach begins with chromatin cross-linking, restriction enzyme digestion, biotin fill-in, and ligation in nuclei. By contrast, in conventional Hi-C, the ligation and dilution steps are performed after nuclear lysis.

They then selected nuclei under the microscope and, keeping them in separate tubes, reversed the cross-links and attached the Hi-C ligation junctions to beads before digesting the products to fragment DNA for amplification and pair-end sequencing.

Using a population of male mouse spleenic T-helper cells, the researchers generated both single-cell and conventional Hi-C libraries. They noted that single-cell contact maps "despite their inherent sparseness … clearly reflect hallmarks of chromosomal organization."

Further, by comparing the ensemble, conventional Hi-C maps with pooled single-cell maps, Fraser and his colleagues found that the pooled version contains the most features of the ensemble maps, which they said confirmed the validity of this their approach.

Diving deeper into the single-cell contact maps from the T-helper cells, the researchers uncovered some 1,400 domains, finding that such domains remain intact at the single-cell level, though they noted that interdomain contacts vary between individual cells. This, they said, suggests "large-scale differences in higher-order chromosome folding that are obscured in ensemble maps, averaged over millions of such structures."

Through modeling the conformations of the X chromosome, Fraser and his team found four regions with marked large-scale conformation differences between cells, underscoring that interdomain contacts are variable. However, they noted that despite such variability, the X chromosomes still shared some properties. For example, the researchers found that active domains tend to be located at the edge of chromosome territories.

"These unique images not only show us the structure of the chromosome, but also the path of the DNA in it, allowing us to map specific genes and other important features," Fraser added. "Using these 3D models, we have begun to unravel the basic principles of chromosome structure and its role in how our genome functions."