Elucidating the mechanism of MBD protein LLPS and its role in heterochromatin formation and transcriptional repression.

Abstract

Membraneless organelles (MLOs) self-assemble into condensed, biochemically distinct microenvironments through liquid-liquid phase separation (LLPS). Heterochromatin, most recently considered an MLO, assembles through weak, multivalent interactions with its associated proteins that contain intrinsically disordered regions. However, the details of the complex molecular interactions between heterochromatin-associated proteins and methylated DNA that drive LLPS have not been fully explored. It is crucial that we elucidate the molecular mechanisms involved in this process as it regulates vital nuclear processes such as transcriptional repression. Its 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 interpret methylated residues on heterochromatin's underlying DNA. We utilize biochemical and biophysical techniques to (1) determine the conditions and properties that promote MBD2 and MBD3 LLPS and elucidate the molecular mechanism(s) that underpin this process and (2) identify the role binding partners have on MBD2 and MBD3 LLPS. LLPS droplet formation is monitored using UV-Vis spectroscopy and differential interference contrast (DIC) and fluorescence microscopy. To better understand the molecular basis that drives LLPS, small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) is used to obtain structural and dynamic details of MBD2 and MBD3. Our results provide details into the mechanism(s) by which MBD2 and MBD3 undergo LLPS individually and how this process is enhanced by binding to each other and methylated DNA. Uncovering the driving forces that assemble MBD protein-based LLPS droplets will give us insight into the higher-order organization of heterochromatin and how it functions within this structure. Additionally, understanding how disease-related mutations lead to aberrant formation of condensates or the inability to form condensates will provide novel therapeutic targets.