Thursday, April 25, 2024
4/25/2024
Project Summary 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 (IDRs). However, the details of the complex molecular interactions that drive the LLPS of functional heterochromatin, though, have not been fully explored. It is crucial that we elucidate the molecular mechanisms involved in this process as it regulates vital nuclear processes, and its dysregulation is implicated in neurological disorders and cancer. Here, we will 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 will explore the conditions and properties that allow them to undergo LLPS and how known interactors influence this process. We can then begin to understand how methylated DNA and its MBD reader proteins regulate heterochromatin formation and its functions. The goals of Aim 1 are to determine the conditions and properties that promote MBD2 and MBD3 LLPS and elucidate the molecular mechanism(s) that underpin this process. We can then relate these findings to their function in chromatin compaction and transcriptional repression. The goals of Aim 2 are to identify the role binding partners and methylated DNA have on MBD2 and MBD3 LLPS. We can then begin to piece together how interactions between critical heterochromatin-associated proteins and DNA drive LLPS and influences heterochromatin assembly and function. To address these aims, we will identify individual regions in MBD2 and MBD3 that drive LLPS by inducing LLPS in vitro under various solution conditions using full-length, truncated, and disease-relevant mutant constructs. Additionally, we will determine the effect of DNA methylation on MBD2 and MBD3, individually and in complex. LLPS droplet formation will be monitored using light sc attering techniques and differential interference contrast (DIC) and fluorescence microscopy. To better understand the molecular basis that drives LLPS, we will obtain structural and dynamic details of MBD2 and MBD3 at atomic resolution by nuclear magnetic resonance (NMR) spectroscopy. The results will 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 to methylated DNA. Uncovering the driving forces that assemble MBD protein-based LLPS 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 mutations lead to aberrant formation of condensates will provide novel therapeutic targets.
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