Uncovering a new phase: the interactions that mediate MBD2 and MBD3 LLPS

Abstract

Chromatin structure and organization controls DNA's accessibility to regulatory factors and influences gene regulation. Heterochromatin, or condensed chromatin containing mostly silenced genes, self-assembles through weak, multivalent interactions with its associated proteins that contain intrinsically disordered regions (IDRs) and undergoes liquid-liquid phase separation (LLPS). However, the details of the intricate molecular interactions that drive heterochromatin LLPS are not fully understood. It is crucial that we uncover the molecular mechanisms involved as it regulates vital nuclear functions, and dysregulation is implicated in neurological disorders and cancer. Here, we focus on two members of the methyl-CpG-binding domain (MBD) family of proteins, MBD2 and MBD3, that recognize and interpret methylated residues on heterochromatin's underlying DNA. We use an integrated approach to explore the driving forces that allow them to undergo LLPS and how known interactors influence this process. Using computational approaches that assess amino acid sequence features, we found that MBD2 and MBD3 are highly disordered proteins predicted to undergo LLPS. Although they are highly similar in sequence, they have distinct clustering patterns of certain residue types that suggest the molecular basis of how they phase separate differs between them. We have tested these predictions in vitro and in cellulo and have demonstrated their ability to phase separate individually, together and with methylated DNA using UV-Vis spectroscopy and microscopy. Through truncations of MBD2 and MBD3, we have found that their ability to undergo LLPS is dictated by a balance between hydrophobic interactions, likely arising from their associative domains, and electrostatic interactions, arising from their highly charged termini, occurring within or between the proteins and DNA. Finally, using scattering techniques such as small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS), we have demonstrated that MBD2 and MBD3 are self-interacting proteins that form large assemblies. We propose that MBD2 and MBD3, through their ability to self-interact via hydrophobic and electrostatic forces, undergo LLPS and foster a biochemically unique environment to sequester binding partners and perform their functions as transcriptional repressors and heterochromatin organizers. Uncovering the driving forces that assemble MBD protein-based droplets will give us insight into the higher-order, LLPS-mediated organization of heterochromatin and how it functions within this structure. Additionally, understanding how disease-related aberrations influence biomolecular condensate dynamics will provide novel therapeutic targets.