Loss of CTCF disrupts chromatin organization and disease stress-gene response

The three-dimensional chromatin organization regulates transcription by bringing regulatory elements such as enhancers into close spatial proximity to target genes and conversely, by insulating against promiscuous interactions. This organization is essential for cell-type and locus-specific gene expression, but how it regulates complex disease-state changes remains to be fully explored. CCCTC-binding factor (CTCF) is a DNA binding protein integral to chromatin organization. Heart failure is a complex disease characterized by hallmark gene expression changes in cardiomyocytes (CMs). We set out to map the cardiac chromatin architecture and used conditional Ctcf knockout to investigate the role of chromatin organization in the heart disease stress-gene response. Gene expression changes were correlated with changes in local chromatin interactions of adult ventricular CMs from healthy and diseased hearts, whereas large-scale genome organization showed a high degree of similarity. Conditional Ctcf knockout resulted in cardiac dilatation, with extensive misregulated gene expression. The majority of these occurred in genes with CTCF binding at or near transcriptional start sites (TSS), but CTCF loss also led to TAD boundary disruption, increased cross-TAD interaction, and transcriptional interference across CTCF boundaries. To exclude the possibility that changes were simply secondary to disease progression, we imposed further surgical stress on Ctcf-knockout mice and found a failure to mount the complete stress-gene response. Using Nppa as a stress-gene model, we validated an example of an upstream regulatory element whose gene-interaction critically relied on CTCF-directed loop formation. Thus, we show evidence of the relationship between chromatin organization and gene expression in CMs. Changes in gene expression between healthy and disease cell-states require the appropriate rewiring of enhancer interactions, governed by CTCF-anchored chromatin loops.