ИСТИНА

Background: The DNA transcription regulation in chromatin, which is required for controlled genes expression, occurs with the participation of various regulatory factors, in particular, the FACT chaperone. It has been previously shown that yeast FACT (yFACT) is able to ATP-independently and reversibly unfold nucleosomes in the presence of Nhp6 protein, reorganizing nucleosomes in nearly linear structures. However, the detailed mechanism of this process remains unclear. Methods: Here we studied the artificially prepared subnucleosomes (SNs) missing one H2A/H2B dimer and reorganized by yFACT in presence of Nhp6. Images were taken using JEOL 2100 TEM. Micrographs were captured with 2.31 Å pixel size. EM images pre-processing and single particles analysis were performed in CryoSPARC. Total number of single particles used for the final analysis was ~490 000. Results: Based on 2D-data analysis we found structural intermediates, emphasizing the gradual unfolding of SNs by FACT, which were similar to intact nucleosomes (Ns) reorganized by yFACT/Nhp6 (Sivkina et al, 2022). The distribution of 2D averages and their characteristic views are presented on Figure 1(A, B). Comparison of the linear dimensions demonstrated similar “beads-on-a-thread” architecture of unfolded SNs and Ns intermediates. Structural analysis of yFACT (Figure 2a,b) and previously described ”joint”-like role of dimerization domains of Spt16/Pob3 propose the potential localization of yFACT domains mapped on Figure 2c. Conclusions: Based on our EM data we propose that the mechanism of subnucleosome reorganization follows the same steps as in intact nucleosomes. This work was supported by the Russian State Foundation (Grant 19-74-30003). Electron microscopy was performed on Unique scientific installation ‘3D-EMC’