Heterochromatin diversity modulates genome compartmentalization and loop extrusion barriers

Abstract

Two dominant processes organizing chromosomes are loop extrusion and the compartmental segregation of active and inactive chromatin. The molecular players involved in loop extrusion during interphase, cohesin and CTCF, have been extensively studied and experimentally validated. However, neither the molecular determinants nor the functional roles of compartmentalization are well understood. Here, we distinguish three inactive chromatin states using contact frequency profiling, comprising two types of heterochromatin and a previously uncharacterized inactive state exhibiting a neutral interaction preference. We find that heterochromatin marked by long continuous stretches of H3K9me3, HP1α and HP1β correlates with a conserved signature of strong compartmentalization and is abundant in HCT116 colon cancer cells. We demonstrate that disruption of DNA methyltransferase activity dramatically remodels genome compartmentalization as a consequence of the loss of H3K9me3 and HP1 binding. Interestingly, H3K9me3-HP1α/β is replaced by the neutral inactive state and retains late replication timing. Furthermore, we show that H3K9me3-HP1α/β heterochromatin is permissive to loop extrusion by cohesin but refractory to CTCF, explaining a paucity of visible loop extrusion-associated patterns in Hi-C. Accordingly, CTCF loop extrusion barriers are reactivated upon loss of H3K9me3-HP1α/β, not as a result of canonical demethylation of the CTCF binding motif but due to an intrinsic resistance of H3K9me3-HP1α/β heterochromatin to CTCF binding. Together, our work reveals a dynamic structural and organizational diversity of the inactive portion of the genome and establishes new connections between the regulation of chromatin state and chromosome organization, including an interplay between DNA methylation, compartmentalization and loop extrusion.HighlightsThree inactive chromatin states are distinguishable by long-range contact frequencies in HCT116, respectively associated with H3K9me3, H3K27me3 and a H3K9me2 state with neutral contact preferences.H3K9me3-HP1α/β heterochromatin has a high degree of homotypic affinity and is permissive to loop extrusion but depleted in extrusion barriers.Disrupting DNA methylation causes widespread loss of H3K9me3-HP1α/β and dramatic remodeling of genome compartmentalization.H3K9me3-HP1α/β is replaced by the neutral inactive state, which gains CTCF loop extrusion barriers and associated contact frequency patterns.DNA methylation suppresses CTCF binding via two distinct mechanisms.

Polycomb-lamina antagonism partitions heterochromatin at the nuclear periphery

“…At a global level, EZH2 and DNMT1 inhibition led to largely contrasting changes in compartmentalization with DNMT1 inhibition impacting compartmentalization to a larger degree than EZH2 inhibition ( Fig 3a-c, Fig S3i ). In agreement with prior studies, DNMT1 inhibition reduced genome compartmentalization on a global scale, 35,50,51 which is reflected by the decreased ratio of intra-to inter-compartment interactions as well as broad compartment shifts, with B regions becoming more ‘A-like’ and vice versa ( Fig 3a ). At the sub-compartment level, these shifts included strengthened interactions between Core-B and both A sub-compartments ( Fig 3b, Fig 3d ).…”

“…Furthermore, PcG-B regions that gained the most H3K9me3 lost the most H3K27me3 (Fig 5c). Spreading of H3K9me3 into former H3K27me3 domains was also observed at the borders of B compartments and LADs as well as at Polycomb domains near the HOXD and HOXA loci (Fig 5d-e, Fig S6a-b).Altogether, these results support a model where H3K27me3 at compartment borders prevents constitutive heterochromatin and lamina association from spreading into neighboring facultative heterochromatic regions 51.…”

“…Altogether, these results support a model where H3K27me3 at compartment borders prevents constitutive heterochromatin and lamina association from spreading into neighboring facultative heterochromatic regions. 51…”

Generation of dynamic three-dimensional genome structure through phase separation of chromatin

“…In type-A chains, various functional complexes should block the cohesin movement [30, 31] as found in the large NCI (Fig. 1), while the cohesin movement is less disturbed in type-B chains [32]. We modeled such difference in the cohesin movement along chromatin chains in the 1-kb resolution model.…”

“…These intra-domain contacts may arise from DNA-protein complexes organized for transcription or replication [28,29], and cohesin molecules off the CTCFbound sites can associate with these complexes [30,31] to reinforce contacts and enhance NCI. A type-u region represents either the region showing the intermediate feature between euchromatin and heterochromatin as was identified by clustering the Hi-C contact data [32] or the mosaic of type-A and B regions averaged over a 100-kb interval.…”