Roseman, S. A. | bioRxiv Science

bioRxiv2023

DNA methylation insulates genic regions from CTCF loops near nuclear speckles

The insulator protein CTCF is essential for mediating chromatin loops and regulating gene expression. While it is established that DNA methylation hinders CTCF binding, the impacts of this methylation-sensitive CTCF binding on chromatin architecture and transcription are poorly defined. Here, we used a selective DNMT1 inhibitor (DNMT1i) to investigate the characteristics and functions of DNMT1i-specific CTCF peaks resulting from global DNA demethylation. We found that DNMT1i-specific peaks preferentially form chromatin loops on gene bodies and interact with highly-looping partner peaks located in regions of active chromatin. Notably, both DNMT1i-specific CTCF peaks and their highly-looping partners are enriched near nuclear speckles, condensate bodies implicated in transcription and splicing. Utilizing targeted protein degradation, we specifically depleted CTCF and nuclear speckles to elucidate their functional interplay. By degrading CTCF upon DNMT1 inhibition, we revealed that CTCF is important for DNMT1i-dependent interactions between chromatin and speckle proteins. Moreover, we found that CTCF promotes the activation of genes near speckles upon DNMT1 inhibition. Conversely, acute depletion of nuclear speckles revealed that they influence RNA abundance but do not maintain CTCF binding or looping. Collectively, our study suggests a model wherein DNA methylation prevents spurious CTCF occupancy and interactions with regulatory elements near nuclear speckles, yet CTCF looping is robust towards the loss of speckles.

genomics↗

bioRxiv2022

Polycomb-lamina antagonism partitions heterochromatin at the nuclear periphery

The genome can be divided into two spatially segregated compartments, A and B,1,2 which broadly partition active and inactive chromatin states, respectively. Constitutive heterochromatin is predominantly located within the B compartment and comprises chromatin that is in close contact with the nuclear lamina.3-5 By contrast, facultative heterochromatin marked by H3K27me3 can span both compartments.2-5 How epigenetic modifications, A/B compartmentalization, and lamina association collectively maintain heterochromatin architecture and function remains unclear.6,7 Here we developed an approach termed Lamina-Inducible Methylation and Hi-C (LIMe-Hi-C) that jointly measures chromosome conformation, DNA methylation, and nuclear lamina positioning. Through this approach, we identified topologically distinct A/B sub-compartments characterized by high levels of H3K27me3 and differing degrees of lamina association. To study the regulation of these sub-compartments, we inhibited Polycomb repressive complex 2 (PRC2), revealing that H3K27me3 is an essential factor in sub-compartment segregation. Unexpectedly, PRC2 inhibition also elicited broad gains in lamina association and constitutive heterochromatin spreading into H3K27me3-marked B sub-compartment regions. Consistent with repositioning to the lamina, genes originally marked with H3K27me3 in the B compartment, but not in the A compartment, remained largely repressed, suggesting that constitutive heterochromatin spreading can compensate for loss of H3K27me3 at a transcriptional level. These findings demonstrate that Polycomb sub-compartments and their antagonism with nuclear lamina association are fundamental organizational features of genome structure. More broadly, by jointly measuring nuclear position and Hi-C contacts, our study demonstrates how dynamic changes in compartmentalization and nuclear lamina association represent distinct but interdependent modes of heterochromatin regulation.

genomics↗