Mixing chromatin fibers with different nucleosome repeat lengths changes dynamics of chromatin phase separation

The eukaryotic genome is organized into chromatin at multiple lengths and timescales. Liquid-liquid phase separation (LLPS) has recently emerged as a mechanism for the dynamic compartmentalization of chromatin. However, it remains unclear how cells can locally alter phase separation behavior to condense, decondense, and segregate specific regions of their genome. Selective interactions between chromatin fibers with different nucleosome repeat lengths (NRLs), as well as their incorporation into existing condensates composed of different NRL chromatin fibers, may provide a pathway for such processes. Using fluorescence microscopy, we investigated how these mechanisms influence the formation, coalescence, and maturation of chromatin condensates. Our results show distinct NRL-dependent mixing behaviors of chromatin before and after condensate formation. 167 and 197 NRL fibers, known to fold into compact fibers by strong nucleosome stacking interactions, formed amorphous condensates. In contrast, 172 and 202 NRL fibers, which only allow for weak stacking, formed spherical condensates. When NRLs were mixed, amorphous condensates exhibited localized clustering of identical NRLs. In spherical condensates, however, both NRLs were homogeneously distributed, with a varying NRL ratio per condensate. In addition, incorporation of 167 NRLs into pre-existing 172 NRL condensates resulted in a multiphase structure where 167 NRL fibers formed an outer layer. These findings present an intrinsic link between DNA sequence, nucleosome positioning, local chromatin configuration and multi-scale phase separation behavior. More broadly, they contribute to a deeper understanding of the dynamic methods of genome organization employed by eukaryotic organisms.