June 10, 2019

New method reveals principles of chromatin folding in vivo

Characterizing chromosome structure is fundamental to a better understanding of gene expression. Current experimental methods helped to build mechanistic models of chromosome folding, however they could not be formally validated so far by independent techniques. This is what the Giorgetti group just did – thanks to a new method they developed to measure chromosome structure quantitatively in living cells.

The linear molecules of DNA that constitute a eukaryotic genome have to be carefully organized within the nucleus to be able to correctly direct gene expression. Research has revealed a hierarchical organization into chromosomal territories, compartments, domains, and eventually chromatin loops that serve to bring transcriptional enhancers in proximity of their target promoters. In particular, TADs (Topologically Associating Domains), which were discovered in 2012, are considered a fundamental functional unit in chromosome folding.

The current understanding of chromosome folding largely relies on methods based on chromosome conformation capture techniques (3C). These methods quantify the number of interactions between genomic loci that are nearby in the three-dimensional space; chromosomal interactions are detected as ligation products after chromatin crosslinking with formaldehyde, which ‘glues’ proteins and DNA together in an irreversible manner. However, crosslinking and ligation have been criticized as sources of potential experimental bias. Notably, one cannot be sure that contacts between two loci detected by 3C methods really correspond to their actual physical proximity in living cells, since formaldehyde could also ‘glue’ together DNA sequences that are separated by large physical distances by crosslinking intervening proteins. This raises the question of whether structures such as TADs and chromatin loops detected by 3C methods actually exist in living cells.

The group of Luca Giorgetti was committed to find a method to study chromosome interactions that does not require crosslinking nor ligation. They developed a technique they called DamC, which combines DNA-methylation based detection of chromosomal interactions with next-generation sequencing and biophysical modelling of methylation kinetics. "An essential aspect of our method is that DNA methylation data can be interpreted in a very quantitative manner using physical modeling," says Giorgetti. "This in turn ensures that chromosomal interactions detected using DamC really reflect direct physical proximity between DNA sequences."

With their new method performed in mouse embryonic stem cells, the researchers could confirm the existence of key structural features of chromosomes, notably TADs and chromatin loops. "Up to now, important dogmas in nuclear organization, such as the existence of TADs, relied on methods which had never been verified in an independent manner," says Giorgetti. "Our new in vivo method validates the findings obtained using 3C methods; this not only validates 3C as an experimental method, but also provides a definitive proof of the fundamentals of chromosomal folding."