From Atomic Interactions to Cellular Dynamics: Multiscale Insights into MORC2 Function

MORC2, an oncoprotein possessing a GHKL-type ATPase domain, is associated with H3K9me3-mediated epigenetic silencing and DNA damage response, implicated in various cancers and neurological disorders, including autosomal dominant early-onset Charcot-Marie-Tooth disease. Its complex biological functions require phosphorylation by the P21-activated kinase 1 (PAK1) pathway. Despite being phosphorylated and linked to gastric cancer progression, the precise mechanism underlying MORC2's phosphorylation-dependent chromatin remodeling activity remains poorly understood. Here, we purified full-length MORC2 and investigated its phosphorylation activity with various DNA substrates. Cryo-electron microscopy and quantitative crosslinking mass spectrometry reveals symmetric dimer formation upon phosphorylation and large rearrangements through the protein length. Hydrogen-deuterium mass spectrometry, surface plasmon resonance and DNA binding assays showed MORC2 possess multiple DNA biding sites with different binding affinities. Fluctuation Fluorescence Spectroscopy confirms MORC2 homodimerization in live cells. Chromatin immunoprecipitation coupled to sequencing, ATAC seq and Fluorescence Lifetime Imaging coupled with FRET demonstrate MORC2's preference for open DNA binding over the repressed chromatin marker H3K9me3. In vitro single-molecule analysis of MORC2 and its variants elucidates that MORC2 imparts epigenetic silencing by DNA compaction driven by ATP hydrolysis. This activity is precisely regulated by extensive phosphorylation at its C-terminus. Our findings provide direct evidence of how MORC2 cause chromatin remodeling and how it is regulated by phosphorylation, thereby, providing molecular basis of chromatin remodelling activities of the MORC family, contributing to our comprehension of epigenetic regulation.