ИСТИНА

Development of a panel of the so-called C-methods – experimental protocols that allow the study of the 3D organization of the eukaryotic genome – has permitted to make observations resulting in a new concept in molecular genetics. In became evident that the 3D genome organization constitutes a part of epigenetic mechanisms essential for maintaining the identity of differentiated cells. In this respect, the assembly of distant regulatory elements in common activating complexes – active chromatin hubs – appears to be of primary importance. Here we show that one of the principal assumptions behind the C-methods is not correct. All C-methods are based on the “proximity ligation” which is preferential cross-ligation of interacting DNA fragments that remain joined by protein bridges after solubilization from formaldehyde-fixed nuclei. We show that the proximity ligation in the 3C procedure really occurs within non-lysed nuclei inside a cage formed by cross-linked chromatin fibers. This finding allows a new interpretation of the results of 3C analysis. Our data suggest that regulatory elements participating in formation of an active chromatin hub do not necessarily form a common complex stabilized by protein bridges, but rather are recruited to the same nuclear compartment where they retain a certain degree of mobility. This model is further supported by demonstration that treatment of nuclei be agents inducing decompaction of chromatin performed before the fixation with formaldehyde results in a decrease of 3C signal.