Liquid-Liquid Phase Separation of Sp100-HMG: Driving Biogenesis and Functional Diversity of PML Nuclear Bodies

Abstract

Promyelocytic leukemia nuclear bodies (PML NBs) are membraneless organelles (0.1-1 m) integral to fundamental cellular processes, yet the molecular logic governing their hierarchical organization is unclear. Contrary to the classical "PML-sole-scaffold" model, we identify Sp100-HMG as the unique isoform that autonomously nucleates into liquid-like condensates. Our findings establish an "inside-out" assembly paradigm, where Sp100-HMG initiates a liquid core that recruits PML and other client proteins (DAXX, ATRX) through three distinct cooperative processes: I. Multimerization domain- and intrinsically disordered region (IDR)-mediated LLPS of Sp100-HMG, nucleating the core; II. C-terminal-dependent protein-protein interactions enriching client components; and III. SUMOylation-directed PML recruitment, facilitating the formation of a stabilizing peripheral shell. This assembly paradigm extends beyond Sp100-HMG, as evidenced by ZBTB16--a PML NB-associated oncoprotein implicated in acute promyelocytic leukemia -- adopting an analogous mechanism to organize PML-positive condensates. Using HEp-2 cells as a main model, we show this hierarchical assembly is critical for orchestrating transcriptional programs and cell-cycle dynamics. Together, our study defines a new biogenesis mode for PML-NBs, where a master liquid nucleator coordinates shell integrity and plasticity for nuclear homeostasis. Significance StatementPML nuclear bodies (PML NBs) serve as essential organizers of the cell nucleus, yet the assembly principles underlying their biogenesis and linking it to functional heterogeneity, spatial positioning, and compositional diversity remain elusive. This study overturns the long-standing "PML-centric" view by establishing an LLPS-driven, hierarchical "inside-out" assembly paradigm. We identify Sp100-HMG as a master liquid nucleator that initiates a core to organize the outer PML shell. This inner-core LLPS-driven assembly logic is shared by other PML NB-associated factors, such as the oncoprotein ZBTB16, providing a unified biophysical framework to explain the structural integrity and plasticity of nuclear condensates. Functionally, this organizational process is critical for shaping the compositional diversity and spatial specificity of PML bodies, thereby modulating transcriptional programs and cell-cycle dynamics in a context-dependent manner. These findings offer broad insights into how organized protein condensation sustains nuclear homeostasis and its dysfunction in diseases such as leukemia.