Mechanistic and Epigenetic Partitioning of Lamina-Associated Chromatin Revealed by a Genome-Wide Imaging Screen

The nuclear periphery is a key site for heterochromatin organization in eukaryotic cells, where lamina-associated domains (LADs) promote transcriptional repression and genome stability. Despite their importance, the mechanisms governing LAD positioning in human cells remain poorly understood. To this end, we performed a genome-wide imaging-based siRNA screen and identified over 100 genes critical for perinuclear LAD localization, with a striking enrichment for RNA-binding proteins. Among these, hnRNPK emerged as a key regulator, required for the perinuclear positioning of approximately two-thirds of LADs genome-wide. Loss of hnRNPK led to LAD repositioning away from the nuclear periphery without altering their heterochromatin state, yet resulted in misexpression of genes within these domains. Notably, hnRNPK-sensitive LADs are uniquely marked by both H3K9me2 and H3K27me3, distinguishing them from hnRNPK-insensitive LADs that are enriched for H3K9me2 and H3K9me3. These findings reveal at least two mechanistically and epigenetically distinct LAD classes, suggesting that specialized pathways underlie their spatial organization. Our results uncover a pivotal role for hnRNPK in regulating the spatial organization of chromatin and highlight the broader diversity of LAD localization mechanisms.