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 screen and identified 101 genes critical for perinuclear LAD localization, with a significant enrichment for RNA-binding proteins. Among these, hnRNPK emerged as a key regulator, required for the perinuclear positioning of approximately 70% of LADs genome-wide. Loss of hnRNPK led to their repositioning away from the nuclear periphery without altering global heterochromatin state, yet resulted in misexpression of genes within these domains. Notably, hnRNPK-sensitive LADs are uniquely enriched for H3K27me3, distinguishing them from hnRNPK-insensitive LADs that are predominantly marked by H3K9me3. These findings reveal mechanistically distinct classes of LADs and suggest that specialized pathways underlie their spatial organization. Our results uncover a pivotal role for hnRNPK in regulating chromatin architecture and highlight the broader diversity of LAD tethering mechanisms.