Abstract
The genome can be divided into two spatially segregated compartments, A and B, which partition active and inactive chromatin states. While constitutive heterochromatin is predominantly located within the B compartment near the nuclear lamina, facultative heterochromatin marked by H3K27me3 spans both compartments. How epigenetic modifications, compartmentalization, and lamina association collectively maintain heterochromatin architecture remains unclear. Here we develop Lamina-Inducible Methylation and Hi-C (LIMe-Hi-C) to jointly measure chromosome conformation, DNA methylation, and lamina positioning. Through LIMe-Hi-C, we identify topologically distinct sub-compartments with high levels of H3K27me3 and differing degrees of lamina association. Inhibition of Polycomb repressive complex 2 (PRC2) reveals that H3K27me3 is essential for sub-compartment segregation. Unexpectedly, PRC2 inhibition promotes lamina association and constitutive heterochromatin spreading into H3K27me3-marked B sub-compartment regions. Consistent with this repositioning, genes originally marked with H3K27me3 in the B compartment, but not the A compartment, remain largely repressed, suggesting that constitutive heterochromatin spreading can compensate for H3K27me3 loss at a transcriptional level. These findings demonstrate that Polycomb sub-compartments and their antagonism with lamina association are fundamental features of genome structure. More broadly, by jointly measuring nuclear position and Hi-C contacts, our study demonstrates how compartmentalization and lamina association represent distinct but interdependent modes of heterochromatin regulation.