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