ИСТИНА

Chomatin compaction level dynamically changes during the cell cycle and reaches the maximum density in mitotic chromosomes. Additional complexity in analysis of chromatin topology is variability in functional activity of chromatin domains which is manifested in formation of structurally distinct transcriptionally active euchromatin and inactive silenced heterochromatin. Molecular genomic techniques allow to get information how chromatin folds and interacts inside the nuclear space: to recognize interections between chromatin and nuclear proteins (DamID) or define contacts between different genomic loci (chromosome conformation capture techniques, 3C). Thereby structural chromatin compartments (LADs, TADs, TADs boundaries and inter-TADs) were observed and important links between topological state of chromatin domains and their functional state (active genes cooperation, reposition during differentiation/reprogramming) have been discovered. This genome-wide approaches provide details about nuclear architecture at the molecular level but still the question remains whether these "statistical" approaches accurately reflect chtomatin topology of individual domains at the single cell level. In this work we used various labeling techniques and different microscopic approachs to trace the structural state of individual chromatin domains. Combination of two diverse methods, 3C and TEM, demonstrated substantial limitations in 3C-technology (Gavrilov,2013). This observation have led us to combine superresolution optical microscopy and immunoEM microscopy, using non-destructive labeling of replication domains as a paradigm of TADs with EdU or expression of GFP-tagged PCNA: the former approach allowed us to define the condtions for best preservation of chromatin domains, the latter giving optimal spatial resolution while showing rich structural context of the nucleus. Detailed analysis allowed to recognize universal structural motifs within both euchromatic early replicating domains and in late-replicating heterochromatin. This motif is represented by higher order chromatin fibers ~200 nm thick in which replicating domains form compact segments structurally stable throughout the cell cycle including mitosis. In heterochromatic regions these fibers seems to coil tightly, thus the replication domains appear clustered. The structural persistence of replication domains at the ultrastructural level permited us to assume the hypothesis of preservation of TADs in mitosis, in contrast to the hypothesis of Naumova et al (2013) drawn from the 3C analysis of hypercondensed mitotic chromosomes stating the global rearrangement of TADs during the mitotic compaction. These contradctions call for futher efforts in bridging 3C approaches with microscopic analysis of individual TADs to elucidate structural basis of topologically associated domain defined by molecular approaches.