Nuclear N-glycosylation maintains H3K9me3 heterochromatin and genomic stability.

Polysaccharides are known to be synthesized by enzymes in the endoplasmic reticulum and Golgi apparatus and transported through the secretory pathway to the cell surface or extracellular space, where they mediate essential biological processes. While classical localization and functions of polysaccharides are well established, their presence and potential roles in the nucleus remain unclear. Here we demonstrate that N-glycans, a type of polysaccharides, modify inner nuclear membrane (INM) proteins and are present in the cell nucleus across diverse cell types-a modification referred to as N-linked glycosylation (N-glycosylation). N-glycosylation is enriched in chromatin regions marked by H3K9me3 and long interspersed nuclear element-1 (LINE-1) retrotransposons. N-glycosylation inhibition and INM protein N-glycosylation site mutation both downregulate H3K9me3 within lamina-associated domains and lead to genomic instability. Mechanistically, N-glycosylation regulates the interaction between the histone H3K9 methyltransferase SETDB1 and INM proteins, promotes the association of SETDB1 with the INM, and maintains H3K9me3. Moreover, we reveal that canonical N-glycan biosynthetic machinery in the endoplasmic reticulum contributes to the N-glycosylation of INM proteins. These findings uncover a previously unrecognized nuclear role for polysaccharides, broadening our understanding beyond their traditional subcellular distributions and functional profiles.