Native and senescent 3D organization of the human genome
Mammalian interphase chromosomes fold into a multitude of loops to fit into the confines of cell nuclei, and looping is tightly linked to their regulated function. The introduction of chromosome conformation capture (3C) revolutionized the study of nuclear organization. However, 3C-based methods rely on chemical crosslinking to stabilize spatial interactions. This step remains a “black box” for experimentalists as regards the biases it may introduce, and discrepancies between microscopy and 3C studies in model loci are now being reported. To address these concerns, we developed “i3C”, a novel technology that captures spatial chromatin interactions without a need for crosslinking. i3C relies on the use of close-to-physiological salt conditions to preserve nuclear structure, and exploits native forces that stabilize chromatin folding. The protocol is simple, it requires <1.5 days to conclude, and is sensitive enough to be performed on low cell counts. We apply i3C to different types of living cells, using various loci and treatments, and combine it with an orthogonal “TALEN-iD” approach that also foregoes crosslinking. Taken together, our data highlight the importance of the spatial restrictions imposed by topological domain formation, while i3C complements the existing toolkit for investigating 3D genome architecture by allowing the interrogation of native chromosome folding.
If you would like to attend this seminar, please use the "Contact us" link below to express interest and arrange site access.
Dr Sven Sewitz
The Brian Heap Seminar Room