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 toward the loss of speckles.