BPS2026 – Ion-specific modulation of MBD3 phase separation via its acidic disordered region

Methyl-CpG-binding domain (MBD) family proteins regulate chromatin remodeling and gene expression by binding methylated DNA and modulating chromatin condensation level. Among them, MBD3 is a core component of the Nucleosome Remodeling and Deacetylase (NuRD) complex. Structurally, MBD3 is a modular protein, consists of a folded MBD domain and a C terminal intrinsically disordered region (C-IDR). Previous studies have investigated the role of intrinsically disordered regions (IDRs) in chromatin-associated proteins, which play a significant role in the phase-separation behavior of these proteins, facilitating the compartmentalization of nuclear processes, but their mechanistic roles remain poorly understood. We investigated the phase-separation behavior of the MBD3 C-IDR, which contains a highly acidic, DE-rich segment, and compared it with the GR-rich region of the closely related MBD2 protein. Using UV-Vis spectroscopy, differential interference contrast (DIC) microscopy, and dynamic light scattering (DLS), we demonstrate that Mg 2+ induces robust LLPS of the MBD3 C-IDR in a concentration- and temperature-dependent manner. In contrast, Ca 2+ promotes weaker or morphologically distinct condensates at comparable ionic strengths. Deletion of the DE-rich region abolishes LLPS behavior, highlighting its essential role in condensate formation. To our knowledge, this is the first demonstration that a DE-rich acidic tail within an MBD protein can drive metal ion-dependent phase separation. Our findings suggest that divalent cation binding to acidic IDRs represents a novel and emerging mechanism by which chromatin-associated proteins may self-assemble. This work provides new insight into how MBD3 may dynamically modulate chromatin structure in response to physiological fluctuations in metal ion concentrations, with broader implications for gene regulation and epigenetic plasticity.