Live imaging of chromatin distribution in muscle nuclei reveals novel principles of nuclear architecture and chromatin compartmentalization

Abstract

Packaging of the chromatin within the nucleus serves as an important factor in the regulation of transcriptional output. However, information on chromatin architecture on nuclear scale in fully differentiated cells, under physiological conditions and in live organisms, is largely unavailable. Here, we imaged nuclei and chromatin in muscle fibers of live, intact Drosophila larvae. In contrast to the common view that chromatin is distributed throughout the nuclear volume, we show that the entire chromatin, including active and repressed regions, forms a peripheral layer underneath the nuclear lamina, leaving a chromatin-devoid compartment at the nucleus center. Importantly, visualization of nuclear compartmentalization required imaging of un-fixed nuclei embedded within their intrinsic tissue environment, with preserved nuclear volume. Upon fixation of similar muscle nuclei, we observed an average of three-fold reduction in nuclear volume caused by dehydration and evidenced by nuclear flattening. In these conditions, the peripheral chromatin layer was not detected anymore, demonstrating the importance of preserving native biophysical tissue environment. We further show that nuclear compartmentalization is sensitive to the levels of lamin C, since over-expression of lamin C-GFP in muscle nuclei resulted in detachment of the peripheral chromatin layer from the lamina and its collapse into the nuclear center. Computer simulations of chromatin distribution recapitulated the peripheral chromatin organization observed experimentally, when binding of lamina associated domains (LADs) was incorporated with chromatin selfattractive interactions. Reducing the number of LADs led to collapse of the chromatin, similarly to our observations following lamin C over-expression. Taken together, our findings reveal a novel mode of mesoscale organization of chromatin within the nucleus in a live organism, in which the chromatin forms a peripheral layer separated from the nuclear interior.This architecture may be essential for robust transcriptional regulation in fully differentiated cells.

Mesoscale phase separation of chromatin in the nucleus

“…However, if chromatin were a self-avoiding polymer (with no self-attraction), its radius of gyration in aqueous solution would typically be larger than the diameter of the nucleus. Thus, under confinement, chromatin would fill the entire volume of nucleus, which is not consistent with the observation ( Amiad-Pavlov et al, 2020 ) of peripheral or central chromatin organization. In contrast, the study presented here illustrates in an intuitive manner, how chromatin-lamina interactions control peripheral and other organizational modes that show chromatin-aqueous phase separation.…”

“…In contrast, the study presented here illustrates in an intuitive manner, how chromatin-lamina interactions control peripheral and other organizational modes that show chromatin-aqueous phase separation. The experimental observations (see Appendix 1—figure 1 ) of peripheral organization demonstrate the condensation of chromatin in the outer part of the nucleus, indicating the presence of chromatin self-attraction ( Amiad-Pavlov et al, 2020 ). This motivates our model which focuses on attractive interactions within the chromatin so that it acts in the nucleus as a polymer in a relatively bad solvent.…”

“…More recently, we have presented (see Appendix 1—figure 1 ) intact-organism imaging of Drosophila larvae nuclei where the chromatin was labeled by H2B-RFP, and the nuclear envelope was labeled by Nesprin/Klar-GFP. These experiments reveal and quantify chromatin-aqueous phase separation and its control by changes in the nuclear volume and the interaction of chromatin with the lamina (part of the nuclear envelope) at the nuclear periphery ( Amiad-Pavlov et al, 2020 ). Here, we demonstrate theoretically that it is the competition of these two effects, together with the self-attraction of the chromatin to itself that determine whether the chromatin is conventionally or peripherally distributed within the nucleus; we also show that more complex organizational modes are also possible.…”

“…We therefore fixed the polymer monomer size, molecular weight, and persistence length, and instead varied the physical properties of the nucleus, including its volume and chromatin-lamina interactions. This was motivated by the intact-organism experiments ( Amiad-Pavlov et al, 2020 ). Comparing the live to fixed cells showed a reduction in the nuclear volume by a factor of about three and a transition from peripheral (live) to conventional (fixed) chromatin organization.…”

“…This depends on the availability of these proteins and the nature of their binding to the lamina. In the experiments ( Amiad-Pavlov et al, 2020 ) that induced lamin C overexpression, there were significant changes in the chromatin organization with much less bonding of the chromatin to the lamina. Lamin overexpression has been suggested to repress the bonding activity of LBR proteins that bind the LAD domains to lamin B ( Buxboim et al, 2017 ; Cho et al, 2017 ).…”