The current understanding of chromosome folding largely relies on methods based on chromosome conformation capture techniques (3C), such as Hi-C and 4C. In these methods, chromosomal interactions are detected as ligation products after chromatin crosslinking with formaldehyde, which ‘glues’ proteins and DNA together. However, these procedures have been criticized as sources of potential experimental bias. This raises the question of whether structures such as TADs and chromatin loops, which are intensively studied because of the fundamental implications for a range of research areas from epigenetic regulation to genetic disorders, really exist in living cells. To address this question we developed a technique called DamC, which combines DNA-methylation based detection of chromosomal interactions with next-generation sequencing. Using DamC we could confirm the existence of key structural features of chromosomes, notably TADs and chromatin loops. This not only validated 3C as an experimental method, but also provides a definitive proof of the fundamentals of chromosomal folding. These results have been published in Nature Structural and Molecular Biology (DOI: 10.1038/s41594-019-0231-0).
We have also made considerable progress in understanding the mechanisms that allow chromosomal interactions and TADs in particular to mediate functional communication between enhancers and promoters. To this aim, we have established a new method to quantitatively measure transcription from a promoter as a function of its distance from a cognate enhancer located at hundreds of different genomic positions within a TAD. Using this method in mouse embryonic stem cells, we have discovered that there is a quantitative, nonlinear relationship between transcription levels at the promoter and its contact probabilities with an enhancer. This explains why enhancers are able to communicate with promoters across large genomic distances within TADs, but are insulated by TAD boundaries. These results have been recently published in Nature (DOI: 10.1038/s41586-022-04570-y).
Finally, we measured the dynamics of chromosome loops in living cells using high-resolution live-cell microscopy of bacterial operators arrays inserted at targeted locations inside a TAD. Our results showed that cohesin and CTCF control the timing and duration of chromosomal interactions, and provided measurements of the duration of CTCF loops. This resulted in a publication in Nature Genetics (DOI: 10.1038/s41586-022-04570-y).
In addition we have collaborated with other laboratories to 1) better characterize the role of chromosome structure at the X inactivation center in mouse embryonic stem cells (van Bemmel et al., Nature Genetics 2019, DOI: 10.1038/ s41588-019-0412-0); and 2) determine the architecture of the regulatory network underlying X chromosome inactivation (Mutzel et al., Nature Structural and Molecular Biology 2019, DOI: 10.1038/s41594-019-0214-1). I have also coordinated and am the corresponding author on two reviews for Molecular Cell (McCord, Kaplan and Giorgetti 2019, DOI: 10.1016/j.molcel.2019.12.021) and Current Opinion in Genetics and Development (Mach and Giorgetti 2022, DOI: 10.1016/j.gde.2023.10205).
