Uncovering the Molecular Interactions Underlying MBD2 and MBD3 Phase Separation

Chromatin organization
controls DNA’s accessibility
to regulatory
factors to influence gene expression. Heterochromatin, or transcriptionally
silent chromatin enriched in methylated DNA and methylated histone
tails, self-assembles through multivalent interactions with its associated
proteins into a condensed, but dynamic state. Liquid–liquid
phase separation (LLPS) of key heterochromatin regulators, such as
heterochromatin protein 1 (HP1), plays an essential role in heterochromatin
assembly and function. Methyl-CpG-binding protein 2 (MeCP2), the most
studied member of the methyl-CpG-binding domain (MBD) family of proteins,
has been recently shown to undergo LLPS in the absence and presence
of methylated DNA. These studies provide a new mechanistic framework
for understanding the role of methylated DNA and its readers in heterochromatin
formation. However, the details of the molecular interactions by which
other MBD family members undergo LLPS to mediate genome organization
and transcriptional regulation are not fully understood. Here, we
focus on two MBD proteins, MBD2 and MBD3, that have distinct but interdependent
roles in gene regulation. Using an integrated computational and experimental
approach, we uncover the homotypic and heterotypic interactions governing
MBD2 and MBD3 phase separation and DNA’s influence on this
process. We show that despite sharing the highest sequence identity
and structural homology among all the MBD protein family members,
MBD2 and MBD3 exhibit differing residue patterns resulting in distinct
phase separation mechanisms. Understanding the molecular underpinnings
of MBD protein condensation offers insights into the higher-order,
LLPS-mediated organization of heterochromatin.